Erythromer (EM), a Nanoscale Bio-Synthetic Artificial Red Cell: Proof of Concept and In Vivo Efficacy Results

Abstract BACKGROUND: There is need for an artificial oxygen (O2) carrier for use when: stored blood is unavailable or undesirable. To date, efforts to develop hemoglobin (Hb) based oxygen carriers (HBOCs) have failed, because of design flaws which do not preserve physiologic interactions of Hb with: O2 (they capture O2 in lungs, but do not release O2 effectively to tissue) and nitric oxide (NO) (they trap NO, causing vasoconstriction). EM design surmounts these weaknesses by: encapsulating Hb, controlling O2 capture/release with a novel 2,3-DPG shuttle and attenuating NO uptake through shell properties. METHODS: The EM prototype and its lyophilized form were analyzed: (1) structurally (dynamic light scattering (DLS), transmission electron microscopy (TEM) and atomic force microscopy (AFM)), as well as for: (2) payload retention (Drabkin), (3) biocompatibility (ex vivo complement activation), (4) O2 affinity (p50, Hill n, Adair), (5) rheology (cone and plate viscometer in rabbit plasma), (6) NO consumption (chemiluminescence), (7) pharmacokinetic (PK) profile (tracking 99mTc-labeled EM in rats), and (8) in vivo O2 delivery (two rodent models: hemorrhagic shock [rats, instrumented for tissue pO2] and hemodilution [bioluminescent HIF-1α reporter mice]). RESULTS: EM was structurally stable (size: 175±10 nm; polydispersity: 0.26±0.0 by DLS, confirmed by TEM and AFM; zeta potential: 12±2 mV). After 3 months storage, we observed nominal change (<10%) in size, zeta potential, or polydispersity. CH50 (complement activation) results were indistinguishable from negative controls and we observed no impact on plasma viscosity (1:10 and 1:5 dilution). p50 was calculated to be 21.46±2.75 Torr (control RBC p50: 23.63±1.84); EM Hill & Adair also similar to control RBC. Two compartment PK modeling in rats resulted in good fit, with distribution t1/2=26.2±3.6 min and elimination t1/2=300±12 min (R2>0.96); which is likely to translate to a t1/2 in humans of ~ 3h. EM NO sequestration varied as a function of shell crosslinking and was below the rate observed for RBCs. In our hemorrhagic shock model in fully instrumented SD Rats (400g), 40% blood volume was removed; animals were then resuscitated with an equal volume of EM (N=6) or normal saline (N=6). EM was suspended at 40 wt/vol%, [Hb]=4mM. EM infusion rapidly stabilized hemodynamics. During the 1st hour, we observed resolution of both lactic acidosis (3.2±1.5 v 8.2±2.1 mM) [for EM and NS, respectively, throughout] and elevated AV O2 difference (24±11 v 67±23%) as well as improved brain pO2 (30.5±1.4 v 17.2±1.3 Torr); p<0.05, RMANOVA, for all. Hemodilution model:Un-instrumented, HIF-1α (ODD) luciferase mice underwent hemodilution (70% v/v) with pentastarch, fresh blood (autotransfusion controls), or EM [N=6, all groups];Hb target nadir was reached (5 mg/dL). To detect whole body luciferase expression, D-luciferin (50 mg/kg, IP) was injected, then serial images were obtained (IVIS, Living Image). HIF-luc radiance was significantly higher in the HES group than in autotransfusion and EM groups, which did not differ (p<0.01, RMANOVA). CONCLUSIONS: The ErythroMer prototype has passed rigorous initial ex vivo and in vivo "proof of concept" testing and bench testing, which suggests this design surmounts prior challenges (by HBOCs) in emulating normal RBC physiologic interactions with O2 and NO. In models of major bleeding/anemia, EM reconstitutes normal hemodynamics and O2 delivery, observed at the system, tissue, and cellular level. EM potential for extended ambient dry storage has significant implications for portability and use. Next steps include formulation scaling, detailed study of pharmacokinetics, biodistribution and safety, as well as evaluation in large animal models of hemorrhagic shock. Disclosures Pan: KaloCyte, Inc.: Equity Ownership; Children's Discovery Institute: Research Funding; National Institutes of Health: Research Funding. Spinella:KaloCyte, Inc.: Equity Ownership; Children's Discovery Institute: Research Funding; National Institutes of Health: Research Funding. Hare:Children's Discovery Institute: Research Funding. Lanza:KaloCyte, Inc.: Membership on an entity's Board of Directors or advisory committees; National Institutes of Health: Research Funding. Doctor:KaloCyte, Inc.: Equity Ownership; Children's Discovery Institute: Research Funding; National Institutes of Health: Research Funding.

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Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽

10.1182/blood.v120.21.580.580 ◽

2012 ◽

Vol 120 (21) ◽

pp. 580-580

Author(s):

Mark Wunderlich ◽

Mahesh Shrestha ◽

Lin Kang ◽

Eric Law ◽

Vladimir Jankovic ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Employment Equity ◽

Cell Product ◽

Equity Ownership ◽

Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Blood ◽

10.1182/blood.v128.22.1587.1587 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1587-1587

Author(s):

Giulia Agnello ◽

Susan Alters ◽

Joseph Tyler ◽

Jinyun Liu ◽

Peng Huang ◽

...

Keyword(s):

Research Funding ◽

Hematological Malignancies ◽

Ex Vivo ◽

Redox Balance ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Equity Ownership ◽

Antioxidant Mechanisms

Abstract Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

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Evaluating the Antitumor Activity of MLN9708 in a Disseminated Mouse Model of Double Transgenic iMyc Ca/Bcl-XL Plasma Cell Malignancy.

Blood ◽

10.1182/blood.v114.22.3835.3835 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3835-3835 ◽

Cited By ~ 1

Author(s):

Michael Fitzgerald ◽

Yueying Cao ◽

Bret Bannerman ◽

Zhi Li ◽

Olga Tayber ◽

...

Keyword(s):

Antitumor Activity ◽

Mouse Model ◽

Lymph Nodes ◽

Plasma Cell ◽

Research Funding ◽

Ex Vivo ◽

Employment Equity ◽

Equity Ownership

Abstract Abstract 3835 Poster Board III-771 Introduction The first generation proteasome inhibitor VELCADE® (bortezomib) is indicated for the treatment of patients with multiple myeloma (MM), a form of plasma cell malignancy (PCM). MLN9708 is our novel proteasome inhibitor that selectively and reversibly binds to, and potently inhibits the b5 site of the 20s proteasome in preclinical studies. We have recently demonstrated that MLN9708 significantly prolongs tumor-free survival of double transgenic iMycCa/Bcl-XL mice, a genetically-engineered mouse model of de novo PCM. Here we describe the in vivo evaluation of cell lines derived from double transgenic iMycCa/Bcl-XL mice and the antitumor activity of MLN9708 in a disseminated mouse model of iMycCa/Bcl-XL PCM. Materials MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Double transgenic iMycCa/Bcl-XL mice develop de novo PCM, in which neoplastic plasma cell development is driven by the targeted expression of the oncoprotein Myc and anti-apoptotic Bcl-XL (J. Clin. Invest. 113:1763-1773, 2004). DP54 and DP42 are plasma cell tumor cell lines isolated from the bone marrow and lymph nodes, respectively, of syngeneic mice previously inoculated with iMycCa/Bcl-XL tumors (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 and DP42 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and have gene expression profiles very similar to human MM. Methods Cell viability studies were performed to determine the antiproliferative effects of MLN2238 in DP54 and DP42 cells in vitro. To evaluate DP54 and DP42 cells in vivo, these cells were aseptically inoculated into the tail vein of NOD-SCID mice. Progressions of the resultant PCM were monitored and tumor burdens were evaluated by magnetic resonance imaging (MRI), ex vivo mCT imaging, and histopathology. Mouse plasma samples were collected at the end of the studies and levels of immunoglobulin were assessed. To establish a preclinical disseminated mouse model of iMycCa/Bcl-XL PCM, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically inoculated into the tail vein of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated with vehicle, bortezomib (at 0.7mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (at 11 mg/kg IV BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. Results In vitro, both DP54 and DP42 cells were sensitive to MLN2238 treatment (LD50 values of 14 and 25 nM, respectively). In vivo, NOD-SCID mice rapidly succumbed to PCM after being inoculated with DP54 and DP42 cells (25 and 14 days post-inoculation, respectively), where the disease was accompanied by marked elevation of plasma immunoglobulins. MRI scans revealed the presence of multiple lesions and several abnormalities were found including: cranial deformation, bowel distortion, splenomegaly and renal edema. Tumor infiltrates, ranging from minor to extensive, were identified in multiple organ compartments (brain<kidney<liver<lymph nodes<spleen<bone marrow) by histopathological analysis. Ex vivo mCT imaging has also revealed signs of bone erosion in the cranial sagittal sutures. Dissemination of DP54-Luc cells after tail vein inoculations was detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were identified in the spleen, kidneys, liver, intestine, lymph nodes, spinal bone and cranium. To assess the antitumor activity of MLN2238, an efficacy study was performed using the DP54-Luc disseminated model. Tumor burden (bioluminescence), skeletal malformation (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. Conclusion The DP54-Luc disseminated mouse model of double transgenic iMycCa/Bcl-XL PCM recapitulated several key features of human MM and provided real-time assessment of novel MM therapy preclinically. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures: Cao: Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Li:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Silverman:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Kupperman:Milllennium: Employment. Manfredi:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership.

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Minimally Ex Vivo Manipulated Gene-Modified T Cells Display Enhanced Tumor Control

Blood ◽

10.1182/blood.v128.22.4549.4549 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4549-4549 ◽

Cited By ~ 2

Author(s):

Saba Ghassemi ◽

Patel Prachi ◽

John Scholler ◽

Selene Nunez-Cruz ◽

David M. Barrett ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Low Dose ◽

Ex Vivo ◽

University Of Pennsylvania ◽

Car T Cells ◽

Car T ◽

Equity Ownership

Abstract Adoptive cell therapy employing T cells equipped with a chimeric antigen receptor (CAR) containing a single chain antibody fragment fused to T cell signaling domains 4-1BB and CD3zeta (CTL019) has shown great potency against various hematopoietic malignancies, e.g. B cell acute lymphoblastic leukemia (ALL). However, it has not shown the same response rate in other malignancies such as chronic lymphocytic leukemia (CLL). We recently demonstrated that the in vivo expansion and persistence of CAR T cells is an important predictor of response to CTL019 in CLL (PMID: 26333935) and ALL (Thudium et al., ASH 2016; Fraietta et al., ASH 2016). Furthermore, it is well known that prolonged culture of T cells negatively impacts the in vivo expansion of the adoptively transferred cells. We therefore hypothesized that minimizing the ex vivo manipulation of T cells would improve the efficacy of CAR T cells. We tested this hypothesis by generating CART19 cells using our standard 9-day manufacturing process plus two abbreviated versions. Cells from normal donors (n=9) and from patients with adult ALL (n=6) were stimulated on day 0 followed by transduction with the CAR19-encoding lentiviral vector on day 1. Cells were harvested on days 3, 5, and 9. Cryopreserved aliquots were evaluated for T cell differentiation using polychromatic flow cytometry, cytokine secretion profile using Luminex, cytolytic ability against a leukemia cell line (NALM6), proliferative ability upon restimulation with CD19-expressing target cells, and in vivo control of our well-established xenogeneic ALL model employing NALM6 as the target. Our data show that all cultures contain a substantial proportion (40%-80%) of na•ve-like CD45RO-CCR7+ T cells that progressively differentiate leading to the accumulation of predominantly (60%-90%) central memory T cells by the end of expansion. Comparative assessment of the CART19 cells at all three time points demonstrated that the cells from the shorter cultures displayed a superior in vitrocytolytic activity, and proliferative response compared to the standard process. In addition,the cells from our standard and shortened cultures all secreted comparable levels of type I cytokines (i.e. IFN-g, IL-2, and TNF-α). Importantly, we investigated the therapeutic potential of cells harvested at day 3 versus later time points. We treated NALM6 xenograftmice with a low dose (0.5 x106 CAR+ T cell I.V.) or standard dose (3 x106 CAR+ T cell I.V.).We demonstrate that day 3 CART19 cells show superior anti-leukemic activity compared to day 5 or day 9 cells. Additionally, we show that mice treated at a low dose with day 3 cells exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia (Figure 1). Our preclinical findings provide evidence that extended ex vivo manipulation of T cells negatively affects their in vivo potency.In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction provides the most efficacious T cells for adoptive T cell immunotherapy. Figure 1 Figure 1. Disclosures Ghassemi: Novartis: Research Funding. Scholler:Novartis: Patents & Royalties; University of Pennsylvania: Patents & Royalties: FAP-CAR US Patent 9,365,641 for targeting tumor microenvironment. Nunez-Cruz:Novartis: Research Funding. Barrett:Novartis: Research Funding. Bedoya:Novartis: Patents & Royalties. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. June:Johnson & Johnson: Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership. Milone:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding.

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T Cells Educated By DC/AML Fusions in the Context of 4-1BB Costimulation As a Potent Strategy for Adoptive Cellular Therapy

Blood ◽

10.1182/blood-2019-131491 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2673-2673

Author(s):

Jessica Liegel ◽

Dina Stroopinsky ◽

Haider Ghiasuddin ◽

Adam Morin ◽

Marzia Capelletti ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Cellular Therapy ◽

Ex Vivo ◽

Advisory Board ◽

Adoptive Cellular Therapy ◽

Advisory Committees ◽

Equity Ownership

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (>120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy.

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Extracellular Tyrosyl-tRNA Synthetase Is a Potent Stimulator of Thrombocytopoiesis

Blood ◽

10.1182/blood.v128.22.1476.1476 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1476-1476

Author(s):

Sachiko Kanaji ◽

Taisuke Kanaji ◽

My-Nuong Vo ◽

Alessandro Zarpellon ◽

Ryan Shapiro ◽

...

Keyword(s):

Research Funding ◽

Ex Vivo ◽

Trna Synthetase ◽

Mouse Bone Marrow ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Platelet Production ◽

Equity Ownership

Abstract Aminoacyl-tRNA synthetases (aaRSs) are enzymes with a key role in the first step of protein synthesis by catalyzing the esterification of a specific cognate amino acid or its precursor to one of all its compatible cognate tRNAs to form an aminoacyl-tRNA. During evolution, eukaryotic aaRSs have acquired additional domains and motifs conferring non-canonical functions beyond translation, such as expressing multiple cytokine activities. Repurposing aaRSs often requires an activation step and the first reported example was for human tyrosyl-tRNA synthetase (YRS), which is abundant in platelets and released from their α-granules upon thrombin or arachidonic acid stimulation. As shown by previous work, activated YRS (YRSACT) - created by natural proteolysis, alternative splicing, or rational mutagenesis - can express the activity of different cytokines. In the current study, we demonstrate that recombinant YRSACT rendered active by the gain-of-function mutation Tyr341Ala exhibits a previously unrecognized role in megakaryocytopoiesis and thrombocytopoiesis. When administered in vivo in C57BL/6 wild type (WT) mice, recombinant YRSACT caused platelet increase both under baseline conditions as well as in a model of immune-mediated thrombocytopenia in which mice are made thrombocytopenic by injection of rat anti-mouse glycoprotein (GP) Ib monoclonal IgG. When WT mouse bone marrow (BM) cells were cultured ex vivo for 3 days, YRSACT treatment increased the number of megakaryocytes by 3.0-fold, particularly of megakaryocytes with 16N ploidy. This effect was independent of thrombopoietin (TPO) signaling because YRSACT could support the expansion of c-mpl-/- (TPO receptor knock-out) mouse megakaryocytes. YRSACT had no effect on purified mouse CD41+ or Sca1+ hematopoietic progenitor cells, indicating that YRS-dependent stimulation likely required the contribution of other cells present in BM cultures. When mouse BM cells were stimulated with different doses of YRSACT, the number of F4/80+ monocyte/macrophages as well as of megakaryocytes increased in a dose-dependent manner. Mechanistic analysis revealed YRSACT targets the Toll-like receptor (TLR) pathway signaling through MyD88 in monocyte/macrophages, thereby enhancing release of cytokines that influence megakaryocyte development. In vitro binding assay showed that YRSACT is capable of binding to TLR2 and TLR4. The effect of YRSACT was attenuated in the BM cells derived from TLR2-/- mice and was abolished in MyD88-/- mice. Among the cytokines with synthesis induced by YRSACT, IL-6 plays a pivotal role in megakaryocyte development. Thus, we tested the effect of YRSACT on megakaryocytes obtained by culturing BM cell derived from IL-6-/- mice and found that no effect was apparent. The stimulatory effect of YRSACT on megakaryocytopoiesis was confirmed with human CD41+ megakaryocyte progenitors differentiated from CD34+ hematopoietic stem cells derived from peripheral blood. In conclusion, we have documented a previously unrecognized activity of YRSACT that results in enhanced megakaryocytopoiesis and platelet production. These studies document a mechanistically distinct aaRS-directed hematological activity that highlights new potential approaches to stimulating platelet production for treating thrombocytopenia and for improving ex vivo preparation of platelet concentrates for transfusion. Disclosures Belani: aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties. Do:aTyr Pharma: Employment, Equity Ownership, Patents & Royalties. Yang:aTyr Pharma: Consultancy, Patents & Royalties, Research Funding. Schimmel:aTyr Pharma: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

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Targeting of JAK/STAT Signaling to Reverse Stroma-Induced Cytoprotection Against BCL2 Antagonist Venetoclax in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.32.32 ◽

2016 ◽

Vol 128 (22) ◽

pp. 32-32

Author(s):

Riikka Karjalainen ◽

Mihaela Popa ◽

Minxia Liu ◽

Mika Kontro ◽

Mireia Mayoral Safont ◽

...

Keyword(s):

Board Of Directors ◽

Stromal Cells ◽

Research Funding ◽

Stromal Cell ◽

Ex Vivo ◽

Intrinsic Resistance ◽

Advisory Committees ◽

Equity Ownership ◽

Refractory Aml

Abstract Several promising new, targeted agents are being developed for the treatment of AML. The BH3 mimetic venetoclax (ABT-199) is a specific inhibitor of BCL2, with results from a phase 2 study showing transient activity of venetoclax in relapsed/refractory AML (Konopleva et al, 2014). The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy and we showed earlier that conditioned medium (CM) from BM stromal cells applied to AML patient cells conferred resistance to venetoclax, which could be reversed by the addition of the JAK1/2 inhibitor ruxolitinib (Karjalainen et al, 2015). Here, we investigated the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib. To identify the soluble factor(s) contributing to stroma-induced protection of BCL2 inhibition, we analyzed the cytokine content of 1) CM from the human BM stromal cell line HS-5, 2) CM from BM mesenchymal stromal cells (MSCs) isolated from AML patients, 3) supernatants from BM aspirates collected from AML patients, and 4) supernatants from BM aspirates collected from healthy donors. Although expression levels varied, the cytokines detected were similar among the different samples. In HS-5 CM, IL-6, IL-8 and MIP-3α were among the most abundant cytokines. In addition, gene expression analysis showed the receptors for these cytokines were expressed in AML patient samples. IL-6, IL-8 and MIP-3α were added individually to mononuclear cells collected from AML patients, which were then treated with venetoclax. However, none of the cytokines alone could mimic the reduced sensitivity to venetoclax conferred by the HS-5 CM suggesting that stromal cell induced cytoprotection is likely multi-factorial. Next we tested the effect of AML-derived BM MSCs on the ex vivo response of AML patient samples (n=8) to ruxolitinib or venetoclax alone or in combination in a co-culture setting. Apoptosis assays showed negligible effects of ruxolitinib at a concentration of 300 nM, while venetoclax at a dose of 100 nM induced reduction in the percentage of CD34+ AML cells. Co-treatment with venetoclax and ruxolitinib demonstrated synergistic effects in 6 out of 8 samples and significantly reduced the number of CD34+ AML cells. Mechanistic studies showed that ruxolitinib treatment inhibited the BM stromal medium-induced expression of BCL-XL mRNA on AML cells and the drugs in combination down-regulated BCL2, MCL1 and BCL-XL protein expression, which was in correlation with sensitivity to the drugs. To further evaluate the ability of the venetoclax and ruxolitinib combination to eradicate leukemic cells in vivo we used an orthotopic xenograft model of AML. NSG mice were injected with genetically engineered MOLM-13luc cells and after engraftment treated with venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) or both and imaged once per week for 4 weeks. At the end of the treatment period bioluminescent imaging showed significantly reduced leukemia burden in the ruxolitinib and venetoclax co-treated mice compared to controls demonstrating superior anti-tumor efficacy than either agent alone (Figure 1). In summary, our data demonstrate that the combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax is synergistic in ex vivo co-culture models and in vivo in an AML mouse model. The addition of ruxolitinib was able to overcome intrinsic resistance to venetoclax by reducing expression of MCL1, a known escape mechanism of BCL2 inhibition. These results support further clinical investigation of this combination, particularly for relapsed/refractory AML. Disclosures Porkka: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Pfizer: Research Funding. Gjertsen:BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Heckman:Celgene: Research Funding; Pfizer: Research Funding.

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Pan-Mutant-IDH1 Inhibitor Bay-1436032 Is Highly Effective Against Human IDH1 Mutant Acute Myeloid Leukemia In Vivo

Blood ◽

10.1182/blood.v128.22.745.745 ◽

2016 ◽

Vol 128 (22) ◽

pp. 745-745 ◽

Cited By ~ 6

Author(s):

Michael Heuser ◽

Lena Herbst ◽

Stefan Pusch ◽

Lara Klett ◽

Ramya Goparaju ◽

...

Keyword(s):

Peripheral Blood ◽

Myeloid Leukemia ◽

Research Funding ◽

Ex Vivo ◽

Leukemic Cells ◽

End Of Treatment ◽

Equity Ownership ◽

Human Leukemic Cells ◽

Mutant Idh1

Abstract Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are frequently found in patients with acute myeloid leukemia (AML) and several other tumors. Mutant IDH1 produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, and leads to a block in differentiation to promote tumorigenesis. Methods: We developed a novel, highly active oral pan-IDH1 inhibitor, BAY-1436032, for clinical evaluation. Its inhibitory potency was evaluated in primary human AML cells in vitro for the five major IDH1R132 mutation types and in two patient derived AML xenograft (PDX) models in vivo, in which BAY-1436032 cleared leukemic blasts in peripheral blood and prolonged survival by induction of differentiation and inhibition of leukemia stem cell self-renewal. Results: R-2HG production by mutant IDH1 was effectively inhibited in patient derived AML cells with all reported IDH1R132 mutations ex vivo by BAY-1436032 with an IC50 between 3 to 16 nM. AML cells cultured ex vivo showed morphologic differentiation and marked upregulation of the myeloid differentiation markers CD14 and CD15. For in vivo experiments, human AML cells from two patients were transplanted into sublethally irradiated NSG mice. After stable engraftment at 17 (PDX1) or 90 (PDX2) days post transplantation, mice were treated with BAY-1436032 orally every day at a dose of 150 mg/kg or vehicle for 100-150 days (n=10 per group). The R/S-2HG ratio in serum was reduced to near normal levels by BAY-1436032. Leukemic cell counts in peripheral blood constantly increased in control mice, while leukemic cells declined from day 30 of BAY-1436032 treatment onwards with morphologic and immunophenotypic evidence of differentiation (Figure). Importantly, all BAY-1436032 treated PDX1 mice survived until the end of treatment at 150 days. In contrast, vehicle-treated mice died with a median latency of 91 days (range 70-95, P<.001). In an independent second model (PDX2) 6 of 10 BAY-1436032 treated mice survived until the end of treatment at day 100 with a median of 15% leukemic cells in peripheral blood, while all vehicle-treated mice suffered from high leukemic burden and died from leukemia with a median survival of 62 days (P=.014). Early mortality was increased with 4 mice dying in the BAY-1436032 group reminiscent of clinical differentiation syndrome in AML patients treated with the IDH1 inhibitor AG-120. To assess the effect of BAY-1436032 on leukemic stem cell self-renewal we treated PDX1 mice with 150 mg/kg BAY-1436032 or vehicle for 4 weeks and performed a limiting dilution transplantation experiment in secondary recipient mice. LSC frequency was 100-fold lower in BAY-1436032 treated compared to control mice. Gene expression profiling showed that stemness associated genes were downregulated, while genes associated with myeloid differentiation like PU.1 and CEBPA were upregulated upon treatment with BAY-1436032. In addition, cell cycle progression was slowed and E2F transcription factors concomitantly inhibited. In accordance with gene expression profiling results, methylation of the PU.1 promoter decreased, while E2F1 promoter methylation increased upon treatment with BAY-1436032. Finally, histone trimethylation levels at residues H3K4, H3K9, H3K27, and H3K36 decreased in both IDH1R132C and IDH1R132H mutant AML cells but not in IDH1 wildtype cells upon BAY-1436032 treatment. Conclusion: In summary, the novel oral pan-mutant IDH1 inhibitor BAY-1436032 is active against all IDH1R132 mutation types and shows strong anti-leukemic activity in two independent AML PDX mouse models. Clinical development is ongoing with a first in man study with BAY-1436032 in IDH1 mutant solid tumors. * M. Heuser and L. Herbst contributed equally to this article #A. Krämer and A. Chaturvedi share senior authorship Figure Human leukemic cells in peripheral blood of mice treated with BAY-1436032. ** P<.001, ns, not significant. Figure. Human leukemic cells in peripheral blood of mice treated with BAY-1436032. ** P<.001, ns, not significant. Disclosures Heuser: Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Research Funding. Pusch:German Cancer Research Center: Patents & Royalties: WO2013/127997A1. Kaulfuss:Bayer Pharma AG: Employment. Panknin:Bayer Pharma AG: Employment. Zimmermann:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210 . Toschi:Bayer Pharma AG: Employment. Neuhaus:Bayer Pharma AG: Employment, Patents & Royalties: WO2015/121210. Haegebarth:Bayer Pharma AG: Employment, Equity Ownership. Rehwinkel:Bayer Pharma AG: Employment, Equity Ownership, Patents & Royalties: WO2015/121210. Hess-Stumpp:Bayer Pharma AG: Employment. Bauser:Bayer Pharma AG: Employment. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. von Deimling:German Cancer Research Center: Patents & Royalties: IDH1R132H mutant specific antibody H09; BRAF V600E mutant specific antibody VE1; BAY-1436032 patent.

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Transient Inhibition of Transforming Growth Factor-β1 in Human Blood-Derived CD34+ Cells Enhances Survival and Proliferation in Vitro and Vascular Reparative functions In Vivo.

Blood ◽

10.1182/blood.v114.22.3546.3546 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3546-3546

Author(s):

Stephen Bartelmez ◽

Ashay Bhatwadekar ◽

Patrick Iversen ◽

Francis W Ruscetti ◽

Maria Grant

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Ex Vivo ◽

Cxcr4 Expression ◽

Employment Equity ◽

Advisory Committees ◽

Cd34 Cells ◽

Equity Ownership ◽

Tgf Β1

Abstract Abstract 3546 Poster Board III-483 CD34+ cells from diabetic patients demonstrate reduced vascular reparative function due to decreased proliferation as well as diminished migration prowess which is largely due to lower levels of bioavailable nitric oxide (NO). We asked whether a transient TGF-β1 blockade in CD34+ cells from diabetics would improve their reparative ability given that TGF-β is a key factor modulating stem cell quiescence. Peripheral blood lin-CD34+ cells or lin-CD34+CD38+/− cells were treated ex vivo with antisense phosphorodiamidate morpholino oligomers (TGF-β1 -PMO), demonstrated to inhibit TGF-β1 protein expression in stem cells. Cells were then analyzed for cell surface TGF-β Receptor 2 (TGF-β R2) and CXCR4 expression, their ability to generate NO, their ability to migrate toward SDF-1, their ability to survive in the absence of added growth factors, and tested in vivo for their vascular reparative ability. After TGF-β1-PMO treatment, healthy and diabetic CD34+CD38+ and - cells downregulated TGF-βR2, upregulated CXCR4 expression, survived in the absence of added growth factors ex vivo and migrated more efficiently to SDF-1 compared to controls. TGF-β1-PMO treated diabetic CD34+ cells restored NO production to non-diabetic levels. In contrast, TGF-β1-PMO did not enhance NO generation in CD34+ cells from healthy subjects. Using an in vivo retinal ischemia reperfusion model, we observed that TGF-β1-PMO treatment increased the ability of both healthy and diabetic CD34+ cells to home to injured capillaries compared to control PMO treated cells. As also observed in our current study, a reduction of TGF-β1 levels in murine hematopoietic stem cells correlates with a reduction in TGF-βR2 expression which may induce proliferation in vivo. We also show that both diabetic and healthy lin-CD34+CD38+ cells express TGF-βR2 by FACS. In contrast, only healthy lin-CD34+CD38- cells expressTGF-βR2 while diabetic lin-CD34+CD38 - cells express essentially no cell surface TGF-βR2 (<5 % of cells are TGF-βR2+). Our results suggest that a transient blockade of TGF-β1 may represent a promising therapeutic strategy in restoring vascular reparative function in diabetic CD34+ cells. Disclosures: Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Head, SRB, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Iversen:AVI-Biopharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Preclinical Activity of IRAK4 Kinase Inhibitor CA-4948 Alone or in Combination with Targeted Therapies and Preliminary Phase 1 Clinical Results in Non-Hodgkin Lymphoma

Blood ◽

10.1182/blood-2018-99-117383 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4168-4168 ◽

Cited By ~ 1

Author(s):

Robert N Booher ◽

Grzegorz S. Nowakowski ◽

Krish Patel ◽

Matthew A. Lunning ◽

Maria Elena S. Samson ◽

...

Keyword(s):

Whole Blood ◽

Research Funding ◽

Ex Vivo ◽

Employment Equity ◽

Response Biomarker ◽

Xenograft Models ◽

Equity Ownership ◽

Anti Tumor Activity ◽

Myd88 L265p

Abstract Background: IRAK4 kinase activity is required for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in a variety of myeloid and lymphoid cell types. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein. The MYD88-L265P activating mutation is prevalent in DLBCL (~30% in ABC subtype) and WM (>90%). MYD88- L265P leads to constitutive activation of NF-κB signaling that is associated with worse prognosis. In MCL, dysregulation of B-cell receptor (BCR) and TLR pathway components correlate with constitutive NF-κB signaling. CA-4948 is a small molecule inhibitor of IRAK4 kinase that modulates the TLR and IL-1R signaling cascades. CA-4948 is being developed as a novel agent for the treatment of hematologic cancers with dysregulated IRAK4 signaling and is currently in a Ph1 trial for R/R NHL (clinicaltrials.gov NCT03328078). In preclinical studies, CA-4948 demonstrates pharmacodynamic and antitumor activity in in vitro and in vivo models with MYD88 alterations, and was previously shown to have a synergistic anti-tumor activity when combined with venetoclax in vivo. To further guide CA-4948's clinical development in NHL, we report here nonclinical studies exploring a twice-daily dosing schedule in DLBCL xenograft models. We also investigated the use of an ex-vivo whole-blood TLR-stimulation assay as a surrogate PD response biomarker. Additionally, we tested the efficacy of CA-4948 alone or in combination with the BTK inhibitor ibrutinib in DLBCL and MCL tumor models. Furthermore, preliminary PK and PD data from the first-in-human Ph1 trial are presented. Methods: Mice bearing DLBCL PDX tumors were orally administered CA-4948 twice-daily (BID) with 37.5 or 75 mg/kg doses and once-daily (QD) with 75 or 150 mg/kg doses. The ex-vivo whole blood assay involved TLR-stimulation of blood isolated at various time-points after CA-4948 administration. For the drug combination studies, mice bearing subcutaneous tumors of a MYD88-L265P DLBCL cell line or six MCL cell lines were treated. Results: (1) CA-4948 exhibited dose-dependent tumor growth inhibition in two DLBCL PDX xenograft tumor models with BID dosing showing equal or enhanced efficacy as compared to the equivalent total daily QD dose. The BID schedule was well tolerated with only a slight body weight loss as compared to the equivalent total QD dose schedule. (2) Overall, in mouse, the ex-vivo blood assay showed a time and exposure dependent relationship with the level of cytokine production after TLR-stimulation. A similar CA-4948 dose-dependent inhibition of TLR-stimulated cytokine production was observed in healthy human whole blood samples in which CA-4948 was spiked into the blood sample. Based on these findings, CA-4948 exposure levels capable of inhibiting TLR-stimulation are anticipated to be readily achievable in clinical studies. This was also supported by preliminary clinical PD data showing post treatment, on-target, reduced release of NF-κB-associated cytokines in 2 of 4 patients treated so far. (3) In xenograft efficacy studies using MCL models, single agent CA-4948 and ibrutinib exhibited anti-tumor activity and showed an additive effect when combined in the majority of the models known to have BCR-driven constitutive canonical NF-kB signaling (REC-1, MINO, and JeKo-1). Interestingly, neither CA-4948, ibrutinib, nor the combination had anti-tumor activity in Z-138 and GRANTA-591 xenograft models, consistent with these cell lines having activated NF-κB through the alternative NIK signaling pathway. (4) The human QD PK data (n=4) demonstrated that CA-4948: was rapidly absorbed, Tmax 1-3 hr, and t1/2 of 3.6 -6.8 hr. The bioavailability and exposure, as assessed by Cmax and AUC, is within the expected range compared to non-clinical PK and did not show any evidence of accumulation after QD dosing for 15 consecutive days. Conclusion: These results provide a rationale for CA-4948 BID dosing and incorporating the use of an ex-vivo whole-blood TLR-stimulation assay as a surrogate PD response biomarker, the former of which will be evaluated in the current Ph1 dose escalation soon and the latter of which is currently being implemented in the Ph1 trial for patients with advanced NHL. The murine xenograft results further support exploration of CA-4948 as monotherapy and in combination with canonical and alternative NF-κB pathway-targeted agents in DLBCL and MCL. Disclosures Booher: Curis, Inc: Employment, Equity Ownership. Patel:Juno Therapeutics: Consultancy; Pharmacyclics/Janssen: Speakers Bureau; Genentech: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; Sunesis Pharmaceuticals: Consultancy. Lunning:Celgene: Consultancy; AbbVie: Consultancy; Astra-Zeneca: Consultancy; Bayer: Consultancy; Genentech: Consultancy; Genzyme: Consultancy; Genentech: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Juno: Consultancy; Kite: Consultancy; Portola: Consultancy; Seattle Genetics: Consultancy; Spectrum: Consultancy; TG Therapeutics: Consultancy; Verastem: Consultancy. Samson:Curis, Inc: Employment, Equity Ownership. Atoyan:Curis, Inc: Employment, Equity Ownership. Ma:Curis, Inc: Employment, Equity Ownership. Xu:Curis, Inc: Employment, Equity Ownership. Dellarocca:Curis, Inc: Employment, Equity Ownership. Modafferi:Curis, Inc: Employment, Equity Ownership. Borek:Curis, Inc: Employment, Equity Ownership. Zhang:Curis, Inc: Employment, Equity Ownership. Parker:Curis, Inc: Employment, Equity Ownership. Whitney:Curis, Inc: Employment, Equity Ownership. Wang:Curis, Inc: Employment, Equity Ownership. Tuck:Curis, Inc: Employment, Equity Ownership. Younes:Merck: Honoraria; Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Pharmacyclics: Research Funding; Celgene: Honoraria; Abbvie: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Incyte: Honoraria; Bayer: Honoraria; BMS: Honoraria, Research Funding; J&J: Research Funding; Novartis: Research Funding; Genentech: Research Funding; Astra Zeneca: Research Funding; Curis: Research Funding.

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