CTX-8573, an Innate-Cell Engager Targeting BCMA, is a Highly Potent Multispecific Antibody for the Treatment of Multiple Myeloma

Introduction: Current therapies for multiple myeloma (MM), such as immunomodulatory agents, proteasome inhibitors, stem-cell transplantation, and monoclonal antibodies against tumor-associated antigens have greatly improved patient survival. However, MM remains an incurable disease as most patients will eventually relapse. Recent advances in targeted T-cell therapies have shown promise in clinical trials but the adaptive immune system may be insufficient to eradicate all MM clones. In contrast, treatments harnessing the innate immune system have been relatively underdeveloped in MM despite evidence suggesting a role of innate immunity in the efficacy of existing therapies. Innate or innate-like cells, such as NK and γδ T cells, have the potential to display strong anti-tumor activity, and strategies aimed to improve or re-direct their cytotoxicity represent a new opportunity for cancer immunotherapies and a complementary approach to existing therapies. Here we describe the preclinical characterization of CTX-8573, a novel multispecific antibody that targets B-cell maturation antigen (BCMA) on tumor cells and promotes potent cytotoxicity by NK and γδ T cells through engagement of the activating receptors NKp30 and CD16a. Method: Bispecific constructs were generated by appending two common-light chain compatible anti-NKp30 Fab fragments to the C-terminus of an anti-BCMA IgG1 antibody containing an afucosylated Fc for enhanced CD16a engagement. To test the effects of targeting NKp30 alone, variants were expressed with an aglycosylated Fc to eliminate CD16a binding. In-vitro assays were performed with primary NK or γδ T cells to determine innate-cell activation, cytokine production, proliferation, and target-cell cytotoxicity against tumor cell lines with a range of BCMA expression levels. In-vivo efficacy studies were performed in multiple humanized mouse models and pharmacokinetics and safety were evaluated in Cynomolgus monkeys. Results: CTX-8573 is highly expressed in CHO cells with minimal aggregation and displays stability, solubility, and binding to BCMA and NKp30 equivalent to the parental monoclonal antibodies. By engaging NKp30 and CD16a, CTX-8573 promotes potent cytotoxicity of BCMA expressing target cells by NK and γδ T cells with >100 fold reduced EC50 compared to the corresponding BCMA monoclonal antibody control. CTX-8573 also demonstrates robust killing of low BCMA expressing cell lines including RPMI-8226 where monoclonal BCMA antibodies lack activity. An aglycosylated variant of CTX-8573 lacking CD16a binding maintains cell killing activity, demonstrating that engagement of NKp30 alone is sufficient to promote innate cell activation and cytotoxicity, although activity is enhanced by CD16A engagement. Furthermore, CTX-8573 maintains its cytotoxic activity in presence of soluble BCMA or BCMA ligands APRIL and BAFF. CTX-8573 does not induce innate cell activation, cytokine production, or killing in the absence of BCMA expressing target cells, supporting a wide therapeutic window. Additionally, unlike daratumumab, CTX-8573 does not induce NK-cell fratricide. In-vivo, CTX-8573 demonstrates anti-tumor efficacy in multiple humanized mouse models including killing of low BCMA expressing cell lines. In Cynomolgus monkeys, CTX-8573 displays standard biphasic pharmacokinetics with a 16 day β-phase half-life and has no evidence of systemic immune activation as measured by C-reactive protein levels. Lastly, NKp30 expression is maintained on bone marrow NK cells from MM patients including the presence of a significant NKp30+CD16a- subpopulation. Conclusion: CTX-8573 represents a novel class of bispecific antibodies that promote potent tumor cell killing by NK and γδ T-cells through engagement of the activating receptors NKp30 and CD16a. CTX-8573 demonstrates strong anti-tumor efficacy in vitro and in vivo, a wide therapeutic window with no evidence of systemic toxicity, and monoclonal-like pharmacokinetics and manufacturability. Together, these data highlight the potential of CTX-8573 as a novel treatment for MM either alone or as a complement to existing therapies. Disclosures Watkins-Yoon: Compass therapeutics LLC: Employment, Equity Ownership. Guzman:Compass therapeutics LLC: Employment, Equity Ownership. Oliphant:Compass therapeutics LLC: Employment, Equity Ownership. Haserlat:Compass therapeutics LLC: Employment, Equity Ownership. Leung:Compass therapeutics LLC: Employment, Equity Ownership. Chottin:University of Louisiana at Lafayette: Employment. Ophir:Compass therapeutics LLC: Employment, Equity Ownership. Vekeria:Compass therapeutics LLC: Employment, Equity Ownership. Nanjappa:Compass therapeutics LLC: Employment, Equity Ownership. Markrush:Compass therapeutics LLC: Employment, Equity Ownership. McConaughy:Compass therapeutics LLC: Employment, Equity Ownership. Wang:Compass therapeutics LLC: Employment, Equity Ownership. Schilling:Compass therapeutics LLC: Employment, Equity Ownership. Kim:Compass therapeutics LLC: Employment, Equity Ownership. Wu:Compass Therapeutics LLC: Employment, Equity Ownership. Liu:Compass therapeutics LLC: Employment, Equity Ownership. Rogers:University of Louisiana at Lafayette: Employment. Villinger:University of Louisiana at Lafayette: Employment. Gong:Compass therapeutics LLC: Employment, Equity Ownership. Hamilton:Compass therapeutics LLC: Employment, Equity Ownership. Bobrowicz:Compass therapeutics LLC: Employment, Equity Ownership. Schuetz:Compass therapeutics LLC: Employment, Equity Ownership. Schmidt:Compass therapeutics LLC: Employment, Equity Ownership. Draghi:Compass therapeutics LLC: Employment, Equity Ownership.

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Inducible MyD88/CD40 Allows AP1903-Dependent Costimulation to Control Proliferation and Survival of Chimeric Antigen Receptor-Modified T Cells

Blood ◽

10.1182/blood.v124.21.1121.1121 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1121-1121 ◽

Cited By ~ 1

Author(s):

Aaron Foster ◽

Aruna Mahendravada ◽

Peter Chang ◽

Nicholas Shinners ◽

Kevin Slawin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

T Cell Activation ◽

Cell Activation ◽

First Generation ◽

Employment Equity ◽

Co Activation ◽

Equity Ownership

Abstract Introduction: Adoptive transfer of T cells genetically engineered to express chimeric antigen receptors (CARs) has begun to show impressive clinical results. The efficacy of T cell therapy is dependent not only on tumor recognition, but also on the survival and expansion of T cells following infusion. T cells modified with CAR constructs encoding costimulatory domains such as CD28 or 4-1BB have the capacity to rapidly proliferate in vivo, but severe toxicities have been observed due to unchecked T cell activation. Thus, strategies to regulate T cell activation in vivowould allow physicians to prevent toxicities and maximize anti-tumor efficacy. Here, we describe a novel T cell costimulation switch, inducible MyD88/CD40 (iMC), that can be activated by a small molecule chemical inducer of dimerization, AP1903, to enhance survival and drive T cell proliferation. Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a biscistronic retrovirus encoding myristolated tandem AP1903 binding domains (FKBPv36), cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and truncated CD19 to identify transduced T cells (SFG-iMC.2A.ΔCD19). Control vectors without signaling elements, or with only MyD88 or cytoplasmic CD40 were also used to generate gene-modified T cell lines. iMC activation was measured by treating T cells with and without AP1903 and measuring cytokine production by ELISA, and assessing cell surface activation markers by flow cytometry. Co-activation of T cells through CD3ζ in combination with iMC was accomplished using anti-CD3 antibodies, or by co-transducing T cells with first generation CAR constructs recognizing prostate stem cell antigen or CD19 (PSCA.ζ or CD19.ζ, respectively), and coculturing T cells with PSCA+ (Capan-1) or CD19+ tumor cell lines (Raji, Daudi and Nalm-1) with and without AP1903. Efficacy of iMC-modified CAR T cells were assessed using NOD scid gamma (NSG) immune deficient mice engrafted with tumor cell lines followed by intravenous injection of T cells. The iMC costimulatory molecule was subsequently activated in vivo by intraperitoneal injection of AP1903 (5 mg/kg). Tumor burden was assessed and T cell expansion in vivowas measured by bioluminescent imaging using an IVIS instrument. Results: T cells transduced with iMC produce cytokines (e.g. IFN-γ, TNF-α, IL-6) in response to AP1903. iMC activation permits T cell survival in the absence of growth cytokines, such as IL-2, but by itself is not sufficient to induce IL-2 production or autonomous growth. Interestingly, AP1903-induction of MyD88 or cytoplasmic CD40 alone showed minimal T cell activation, suggesting potential synergy of the two signaling molecules. However, co-activation of the T cell receptor (TCR) with soluble anti-CD3 and iMC with AP1903 upregulated CD25 expression, induced IL-2 production and promoted T cell expansion. Importantly, endogenous TCR signaling could be substituted by a PSCA-specific CAR linked to the CD3 ζ endodomain (PSCA.ζ CAR), where co-activation of iMC by AP1903, and CAR by tumor cells expressing PSCA (Capan-1) induced high levels of IL-2 secretion, CD25 upregulation and rapid T cell proliferation. Similar results were achieved using T cells transduced with iMC-enabled CD19 CAR (SFG-iMC.2A.CD19.ζ) when cocultured with CD19+lymphoma cell lines. Treatment of tumor bearing immunodeficient mice with T cells modified with iMC and PSCA.ζ CAR showed enhanced antitumor efficacy when mice were administered with AP1903 dimerizer. Bioluminescence imaging also demonstrated marked proliferation and persistence of iMC-transduced T cells in response to AP1903 administration. Following AP1903 withdrawal, T cell levels declined, consistent with the requirement for costimulation in combination with CAR activation. Summary: Inducible MyD88/CD40 represents a novel activation switch that can be used to provide a controllable costimulatory signal to T cells transduced with a first generation CAR. The separation of the cytolytic signal 1 (CD3 ζ) domain from signal 2 costimulation (iMC) provides a unique mechanism by which T cells can be expanded only in response to both AP1903 and tumor antigen, or reduced in number by withdrawal of AP1903-induced iMC costimulation. Disclosures Foster: Bellicum Pharmaceuticals: Employment, Patents & Royalties. Mahendravada:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

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GRP78 Is Expressed on the Cell Surface of Pediatric AML Blasts and Can be Targeted with GRP78-CAR T Cells without Toxicity to Hematopoietic Progenitor Cells

Blood ◽

10.1182/blood-2020-141671 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 12-12 ◽

Cited By ~ 1

Author(s):

Nikhil Hebbar ◽

Rebecca Epperly ◽

Abishek Vaidya ◽

Sujuan Huang ◽

Cheng Cheng ◽

...

Keyword(s):

T Cells ◽

Cell Surface ◽

Cell Lines ◽

Target Cells ◽

Advisory Committees ◽

Car T Cells ◽

Car T ◽

Pediatric Aml

Finding the ideal immunotherapy target for AML has proven challenging and is limited by overlapping expression of antigens on hematopoietic progenitor cells (HPCs) and AML blasts. Intracellular Glucose-regulated-protein 78 (GRP78) is a key UPR regulator, which normally resides in the endoplasmic reticulum (ER). GRP78 is overexpressed and translocated to the cell surface in a broad range of solid tumors and hematological malignancies in response to elevated ER stress, making it an attractive target for immune-based therapies with T cells expressing chimeric antigen receptors (CARs). The goal of this project was to determine the expression of GRP78 on pediatric AML samples, generate GRP78-CAR T cells, and evaluate their effector function against AML blasts in vitro and in vivo. To demonstrate overexpression of GRP78 in AML, we performed gene expression analysis by RNAseq on a cohort of cord blood CD34+ cell samples (N=5) and 74 primary AML samples. Primary AML samples included RUNX1-RUNX1T1 (N=7), CBFB-MYH11(N=17), KMT2A rearrangement (N=28) and NUP98 (N=22). Analysis showed increased GRP78 expression in AML samples, especially in KMT2A- and NUP98-rearranged AML. To demonstrate surface expression of GRP78, we performed flow cytometry of AML (Kg1a, MOLLM13, THP-1, MV4-11) cell lines as well as 11 primary AML samples and 5 PDX samples; non transduced (NT) T cells served as control. All AML samples, including cell lines, primary AML blasts, and PDX samples, showed increased expression of GRP78 on their cell surface in comparison to NT T cells We then designed a retroviral vector encoding a GRP78-CAR using a GRP78-specific peptide as an antigen recognition domain, and generated GRP78-CAR T cells by retroviral transduction of primary human T cells. Median transduction efficiency was 82% (± 5-8%, N=6), and immunophenotypic analysis showed a predominance of naïve and terminal effector memory subsets on day 7 after transduction (N=5). To determine the antigen specificity of GRP78-CAR T cells, we performed coculture assays in vitro with cell surface GRP78+ (AML cell lines: MOLM13, MV-4-11, and THP-1 and 3 AML PDX samples) or cell surface GRP78- (NT T cells) targets. T cells expressing CARs specific for HER2-, CD19-, or a non-functional GRP78 (DGRP78)-CAR served as negative controls. GRP78-CAR T cells secreted significant amounts of IFNg and IL-2 only in the presence of GRP78+ target cells (N=3, p<0.005); while control CAR T cells did not. GRP78-CAR T cells only killed GRP78+ target cells in standard cytotoxicity assays confirming specificity. To test the effects of GRP78-CAR T cells on normal bone marrow derived HPCs, we performed standard colony forming unit (CFU) assays post exposure to GRP78-CAR or NT T cells (effector to target (E:T) ratio 1:1 and 5:1) and determined the number of BFU-E, CFU-E, CFU-GM, and CFU-GEMM. No significant differences between GRP78-CAR and NT T cells were observed except for CFU-Es at an E:T ratio of 5:1 that was not confirmed for BFU-Es. Finally, we evaluated the antitumor activity of GRP78-CAR T cells in an in vivo xenograft AML model (MOLM13). Tumor growth was monitored by serial bioluminescence imaging. A single intravenous dose of GRP78-CAR T cells induced tumor regression, which resulted in a significant (p<0.001) survival advantage in comparison to mice that had received control CAR T cells. In conclusion, GRP78 is expressed on the cell surface of AML. GRP78-CAR T cells have potent anti-AML activity in vitro and in vivo and do not target normal HPCs. Thus, our cell therapy approach warrants further active exploration and has the potential to improve outcomes for patients with AML. Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Vaidya:St. Jude: Patents & Royalties. Gottschalk:TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties. Velasquez:St. Jude: Patents & Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees.

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Differential Effects of Milatuzumab On Human Antigen-Presenting Cells in Comparison to Malignant B Cells.

Blood ◽

10.1182/blood.v114.22.2744.2744 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2744-2744

Author(s):

Xiaochuan Chen ◽

Rhona Stein ◽

Chien-Hsing Chang ◽

David M. Goldenberg

Keyword(s):

T Cell ◽

B Cells ◽

Cell Lines ◽

Hairy Cell ◽

Employment Equity ◽

Cross Presentation ◽

Equity Ownership ◽

Antigen Presenting

Abstract Abstract 2744 Poster Board II-720 Introduction: The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, is in clinical evaluation as a therapeutic mAb for non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma after preclinical evidence of activity in these tumor types. In addition to its expression in malignant cells, CD74 is also expressed in normal B cells, monocytes, macrophages, Langerhans cells, follicular and blood dendritic cells. A question therefore arises whether milatuzumab is toxic to or affects the function of these immune cells. This has important implications, not only for safe therapeutic use of this mAb, but also for its potential application as a novel delivery modality for in-vivo targeted vaccination. Methods: We assessed the binding profiles and functional effects of milatuzumab on human antigen-presenting cell (APC) subsets. Studies on the effect of milatuzumab on antigen presentation and cross-presentation are included. In addition, binding and cytotoxicity on a panel of leukemia/lymphoma cell lines and CLL patient cells were tested to demonstrate the range of malignancies that can be treated with this mAb. Results: Milatuzumab bound efficiently to different subsets of blood dendritic cells, including BDCA-1+ myeloid DCs (MDC1), BDCA-2+ plasmacytoid DCs (PDC), BDCA-3+ myeloid DCs (MDC2), B lymphocytes, monocytes, and immature DCs derived from human monocytes in vitro, but not LPS-matured DCs, which correlated well with their CD74 expression levels. In the malignant B-cells tested, milatuzumab bound to the surface of 2/3 AML, 2/2 mantle cell (MCL), 4/4 ALL, 1/1 hairy cell leukemia, 2/2 CLL, 7/7 NHL, and 5/6 multiple myeloma cell lines, and cells of 4/6 CLL patient specimens. Significant cytotoxicity (P<0.05) was observed in 2/2 MCL, 2/2 CLL, 3/4 ALL, 1/1 hairy cell, 2/2 NHL, and 2/2 MM cell lines, and 3/4 CD74-positive CLL patient cells, but not in the AML cell lines following incubation with milatuzumab. In contrast, milatuzumab had minimal effects on the viability of DCs or B cells that normally express CD74. The DC maturation and DC-mediated T-cell functions were not altered by milatuzumab treatment, which include DC-induced T-cell proliferation, CD4+CD25+FoxP3+ Treg expansion, and CD4+ naïve T-cell polarization. Moreover, milatuzumab had little effect on CMV-specific CD8- and CD8+ T cell interferon-g responses of peripheral blood mononuclear cells stimulated in vitro with CMV pp65 peptides or protein, suggesting that milatuzumab does not influence antigen presentation or cross-presentation. Conclusion: These results demonstrate that milatuzumab is a highly specific therapeutic mAb against B-cell malignancies with potentially minimal side effects. It also suggests that milatuzumab may be a promising novel delivery mAb for in vivo targeted vaccinations, given its efficient binding, but lack of cytotoxicity and functional disruption on CD74-expressing normal APCs. (Supported in part by NIH grant PO1-CA103985.) Disclosures: Chang: Immunomedics Inc.: Employment, Equity Ownership, Patents & Royalties. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

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Coltuximab Ravtansine (SAR3419) Demonstrates Enhanced Activity in Combination with Bendamustine, Gemcitabine and Novel Targeted Agents Such As PI3K Inhibitors in Pre-Clinical Models of Relapsed and/or Refractory Non-Hodgkins Lymphoma (NHL)

Blood ◽

10.1182/blood.v126.23.5125.5125 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5125-5125

Author(s):

Callum M Sloss ◽

Katie O'Callaghan ◽

Jutta Deckert ◽

Jenny Tsui ◽

Leanne Lanieri ◽

...

Keyword(s):

Cell Lines ◽

Single Agent ◽

Targeted Agents ◽

Employment Equity ◽

Pi3k Inhibitors ◽

Xenograft Models ◽

Equity Ownership ◽

Chemotherapy Agents

Abstract Introduction: Relapsed/refractory B-cell NHL remains an area of significant medical need. CD19 is broadly expressed on B-cell malignancies making it an ideal target for antibody-drug conjugate (ADC) based therapy. Coltuximab ravtansine is a CD19-targeting ADC consisting of a CD19-targeting antibody conjugated to the maytansinoid anti-mitotic DM4. In preclinical studies, coltuximab ravtansine has shown potent, targeted activity against NHL cell lines and xenograft models. In early clinical trials, it has been well tolerated and has shown promising signs of efficacy as both a single agent and in combination with rituximab. In the STARLYTE Phase 2 trial coltuximab ravtansine monotherapy resulted in an ORR of 44% in R/R-DLBCL that included an ORR of 21% in hard-to-treat primary refractory patients (NCT01472887). Here we describe studies aimed at the identification of combination partners for coltuximab ravtansine to further optimize clinical benefit to R/R-NHL patients. We are employing a dual approach where we investigate combination of coltuximab ravtansine with multiple, novel targeted therapy partners whilst in parallel also investigating the combination of coltuximab ravtansine with chemotherapies commonly used in the late stage R/R-NHL setting. Methods: Coltuximab ravtansine and the DM4 payload were evaluated in a high throughput screen both as single agents and in combination with a selection of novel, emerging targeted agents across a panel of twenty NHL cell lines. The combinations were evaluated in a dose-response matrix and a statistical method was used to identify combination synergies significantly superseding baseline additivity values. The in vivo efficacy of coltuximab ravtansine was additionally assessed in combination with various clinically relevant chemotherapy agents in subcutaneous xenograft models of NHL. Results: Coltuximab ravtansine and DM4 both showed potent single agent activity against the entire panel of NHL cell lines with median GI50's of 770pM and 100pM, respectively. We observed a significant correlation in the cell line sensitivity of the two compounds suggesting that sensitivity to coltuximab ravtansine is driven, at least in part, by inherent sensitivity of cells to the cytotoxic effects of the DM4 payload. In vitro combination studies for coltuximab ravtansine were performed to identify targets or pathways that result in the most prominent combination effects across the cell line panel. Analysis of the in vitro combination dose-matrix revealed particularly strong synergy between coltuximab ravtansine and various inhibitors of the PI3K/AKT/mTOR axis. Studies to examine the synergism between coltuximab ravtansine and PI3K inhibitors in in vivo models of NHL are ongoing. In order to further determine the utility of coltuximab ravtansine as part of a potential combination regimen for the treatment of R/R-NHL, we assessed the combination of coltuximab ravtansine with the chemotherapy agents bendamustine and gemcitabine in vivo. As gemcitabine is typically used in combination we assessed the efficacy of a coltuximab ravtansine with rituximab and gemcitabine in vivo. In both cases the combination with coltuximab ravtansine was significantly more efficacious than the standard-of-care alone arms. Conclusions: Coltuximab ravtansine demonstrates synergistic activity in combination with multiple PI3K pathway inhibitors across a large panel of NHL cell lines. Additionally, we have shown that combination of coltuximab ravtansine with clinically relevant late stage treatments such as bendamustine and rituximab + gemcitabine is more efficacious than the chemotherapy regimens alone. These results support the continued development of coltuximab ravtansine in R/R-NHL in combination with chemotherapy regimens and suggest that a combination of coltuximab ravtansine with PI3K inhibitors may also be of interest in the clinical setting. Disclosures Sloss: ImmunoGen, Inc.: Employment, Equity Ownership. O'Callaghan:ImmunoGen, Inc.: Employment, Equity Ownership. Deckert:ImmunoGen, Inc.: Employment, Equity Ownership. Tsui:ImmunoGen, Inc.: Employment, Equity Ownership. Lanieri:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

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Late-Stage Preclinical Characterization of Switchable CD123-Specific CAR-T for Treatment of Acute Leukemia

Blood ◽

10.1182/blood-2018-99-113288 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 964-964 ◽

Cited By ~ 3

Author(s):

Simon Loff ◽

Jan-Erik Meyer ◽

Josephine Dietrich ◽

Johannes Spehr ◽

Riewaldt Julia ◽

...

Keyword(s):

T Cells ◽

Acute Leukemia ◽

Patient Treatment ◽

Malignant Cells ◽

Employment Equity ◽

Car T ◽

Equity Ownership ◽

Plasma Half Life

Abstract Application of autologous T cells genetically engineered to express CD19-specific chimeric antigen receptors (CAR-T) is highly effective in the treatment of B cell malignancies. To this date, application of CAR-T therapy beyond CD19 remains challenging due to the inability to control CAR-T reactivity in patients and the lack of tumor-associated antigens exclusively expressed by malignant cells. The interleukin-3 receptor alpha chain (CD123) is a promising immunotherapeutic target and associated with leukemia-initiating compartments in myeloid- or lymphoid derived diseases. However, in contrast to CD19, CD123 is a precarious target due to its prevalent expression on healthy hematopoietic stem and progenitor cells (HSPC) as well as endothelial cells. Thus, CAR-T lacking any fine-tuned control mechanisms are at risk to cause life threatening toxicities or can only act as bridging therapy to an allogeneic stem cell transplantation. To extend application of CAR-T therapy and safely redirect CAR-engineered T cells to challenging targets such as CD123, a switch-controllable universal CAR-T platform (UniCAR) was recently introduced. The UniCAR system consists of two components: (1) a non-reactive inducible second generation CAR with CD28/CD3ζ stimulation for an inert manipulation of T cells (UniCAR-T) and (2) soluble targeting modules (TM) enabling UniCAR-T reactivity in an antigen-specific manner. Here we provide late stage pre-clinical data for UniCAR-T in combination with a CD123-specific TM (TM123) for treatment of acute leukemia. Primary patient-derived CD123-positive leukemic blasts were efficiently eradicated by TM123-redirected clinical-grade manufactured UniCAR-T in vitro and in vivo. Activation, cytolytic responses and cytokine release were proven to be strictly switch-controlled. Moreover, anti-leukemic responses of UniCAR-T were demonstrated to be comparable to conventional CD123-specific CAR-T in vitro. In contrast to conventional CD123 CAR-T, TM123-redirected UniCAR-T discriminate between CD123high malignant cells and CD123low healthy cells with negligible toxicity towards HSPC in vivo. As 4-1BB mediated co-stimulation is known to enhance CAR-T activity in vivo, a novel CD123-specific targeting module bearing a covalently bound trimeric 4-1BB ligand (4-1BBL) was developed and characterized for co-stimulation at the leukemic site in trans. Specific binding of TM123-4-1BBL was demonstrated against native 4-1BB as well as CD123-positive leukemic blasts. In long-term tumor eradication models, TM123-4-1BBL ameliorated the killing capability of UniCAR-T in vitro. Additionally, the increased hydrodynamic radius of trimeric 4-1BBL-coupled TM123 prolonged plasma half-life and enhanced bioavailability in vivo. In conclusion, UniCAR-T maintain high anti-leukemic efficacy, while adding a sophisticated mechanism for immediate control to improve safety and versatility of CD123-directed CAR-T therapy. Moreover, switching between several TMs from short to moderate plasma half-life allows for an individualized treatment of various leukemic settings while minimizing potential adverse effects. Disclosures Loff: GEMoaB Monoclonals GmbH: Employment. Meyer:Cellex Patient Treatment GmbH: Employment. Dietrich:Cellex Patient Treatment GmbH: Employment. Spehr:Cellex Patient Treatment GmbH: Employment. Julia:Cellex Patient Treatment GmbH: Employment. Gründer:GEMoaB Monoclonals GmbH: Employment. Franke:GEMoaB Monoclonals GmbH: Employment. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership. Ehninger:Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership. Ehninger:GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Cellex Gesellschaft fuer Zellgewinnung mbH: Equity Ownership. Cartellieri:Cellex Patient Treatment GmbH: Employment.

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Prophylactic Itacitinib (INCB039110) for the Prevention of Cytokine Release Syndrome Induced By Chimeric Antigen Receptor T-Cells (CAR-T-cells) Therapy

Blood ◽

10.1182/blood-2019-128288 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1934-1934 ◽

Cited By ~ 3

Author(s):

Eduardo Huarte ◽

Roddy S O'Connor ◽

Melissa Parker ◽

Taisheng Huang ◽

Michael C. Milone ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cytokine Release ◽

Employment Equity ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.

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TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽

10.1182/blood-2019-123226 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4070-4070

Author(s):

Harbani Malik ◽

Ben Buelow ◽

Udaya Rangaswamy ◽

Aarti Balasubramani ◽

Andrew Boudreau ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Cytokine Secretion ◽

Employment Equity ◽

Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

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A T Cell-Engaging CD3 Recruit-Tandab Potently Kills CD19+ Tumor B Cells

Blood ◽

10.1182/blood.v120.21.3721.3721 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3721-3721

Author(s):

Eugene Zhukovsky ◽

Uwe Reusch ◽

Carmen Burkhardt ◽

Stefan Knackmuss ◽

Ivica Fucek ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

B Cell ◽

T Cell Activation ◽

Cell Activation ◽

Target Cells ◽

Cytotoxicity Assays

Abstract Abstract 3721 Background: CD19 is expressed from early B cell development through differentiation into plasma cells, and is an attractive alternative to CD20 as a target for the development of therapeutic antibodies to treat B cell malignancies. T cells are potent tumor-killing effector cells that cannot be recruited by native antibodies. The CD3 RECRUIT-TandAb AFM11, a humanized bispecific tetravalent antibody with two binding sites for both CD3 and CD19, is a novel therapeutic for the treatment of NHL that harnesses the cytotoxic nature of T cells. Methods: We engineered a bispecific anti-CD19/anti-CD3e tetravalent TandAb with humanized and affinity-matured variable domains. The TandAb's binding properties, T cell-mediated cytotoxic activity, and target-mediated T cell activation were characterized in a panel of in vitro assays. In vivo efficacy was evaluated in a murine NOD/scid xenograft model reconstituted with human PBMC. Results: AFM11 mediates highly potent CD19+ tumor cell lysis in cytotoxicity assays performed on a panel of cell lines (JOK-1, Raji, Nalm-6, MEC-1, VAL, Daudi) and primary B-CLL tumors: EC50 values are in the low- to sub-picomolar range and do not correlate with the expression density of CD19 on the target cell lines. The cytotoxic activity of tetravalent AFM11 is superior to that of alternative bivalent antibody formats possessing only a single binding site for both CD19 and CD3. High affinity binding of AFM11 to CD19 and to CD3 is essential for efficacious T cell recruitment. Both CD8+ and CD4+ T cells mediate cytotoxicity however the former exhibit much faster killing. We observe that AFM11 displays similar cytotoxic efficacy at different effector to target ratios (from 5:1 to 1:5) in cytotoxicity assays; this suggests that T cells are engaged in the serial killing of CD19+ target cells. In the absence of CD19+ target cells in vitro, AFM11 does not elicit T cell activation as manifested by cytokine release (from a panel of ten cytokines associated with T cell activation), their proliferation, or their expression of activation markers. AFM11 activates T cells exclusively in the presence of its targets and mediates lysis of CD19+ cells while sparing antigen-negative bystanders. In the absence of CD19+ target cells, AFM11 concentrations in excess of 500-fold over EC50 induce down-modulation of the CD3/TCR complex. Yet, AFM11-treated T cells can be re-engaged for target cell lysis. All of these features of AFM11-induced T cell activation may contribute additional safety without compromising its efficacy. In vivo AFM11 demonstrates a robust dose-dependent inhibition of subcutaneous Raji tumors in mice. At 5 mg/kg AFM11 demonstrates a complete suppression of tumor growth, and even at 5 ug/kg tumor growth is reduced by 60%. Moreover, we observe that a single administration of AFM11 produces inhibition of tumor growth similar to that of 5 consecutive administrations. Conclusions: In summary, our in vitro and in vivo experiments with AFM11 demonstrate the high potency and efficacy of its anti-tumor cytotoxicity. Thus, AFM11 is a novel highly efficacious drug candidate for the treatment of B cell malignancies with an advantageous safety profile. Disclosures: Zhukovsky: Affimed Therapeutics AG: Employment, Equity Ownership. Reusch:Affimed Therapeutics AG: Employment. Burkhardt:Affimed Therapeutics AG: Employment. Knackmuss:Affimed Therapeutics AG: Employment. Fucek:Affimed Therapeutics AG: Employment. Eser:Affimed Therapeutics AG: Employment. McAleese:Affimed Therapeutics AG: Employment. Ellwanger:Affimed Therapeutics AG: Employment.

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Expression of Bcl-2 Pro-Survival Family Proteins Predicts Pharmacological Responses to ABT-199, a Novel and Selective Bcl-2 Antagonist, in Multiple Myeloma Models

Blood ◽

10.1182/blood.v120.21.5028.5028 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5028-5028 ◽

Cited By ~ 1

Author(s):

Deepak Sampath ◽

Elizabeth Punnoose ◽

Erwin R. Boghaert ◽

Lisa Belmont ◽

Jun Chen ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Cell Lines ◽

Single Agent ◽

Employment Equity ◽

Bone Marrow Biopsies ◽

Xenograft Models ◽

Equity Ownership

Abstract Abstract 5028 Multiple myeloma (MM) is a hematological malignancy of the bone marrow caused by the dysregulated proliferation of monoclonal antibody producing plasma cells. A hallmark feature of cancer is the ability to evade cell death signals induced by stress response cues. The Bcl-2 family of proteins regulates the intrinsic apoptosis pathways and consists of pro-apoptotic (Bax, Bak, Bad, Bim, Noxa, Puma) and pro-survival (Bcl-2, Bcl-xL, Mcl-1); the balance of which dictates the life or death status of MM tumor cells. Thus, there is a strong rationale to target members of the Bcl-2 proteins for the treatment of MM. ABT-199 is a potent BH3-only mimetic that selectively antagonizes Bcl-2 and is currently in phase I clinical trials for the treatment of hematological malignancies. Therefore, we evaluated the efficacy of ABT-199 as a single agent and in combination with standard of care drugs such as Velcade (bortezomib) in preclinical models of MM. A panel of 21 human MM cell lines was evaluated in vitro for to sensitivity to ABT-199. ABT-199 potently inhibited cell viability in a sub-set of MM cell lines (7/21) with EC50 values less than 1 μM. Expression of Bcl-2, Bcl-xL, Mcl-1, Bim and other Bcl-2 family proteins were evaluated by protein and mRNA. Cell line modeling identified thresholds for expression of Bcl-2, Bcl-xL and Mcl-1 that best predicted sensitivity and resistance to ABT-199 and the dual Bcl-2/Bcl-xL antagonist, navitoclax. Consistent with the target inhibition profile of these drugs, we found that MM lines that were Bcl-2high/Bcl-xLlow/Mcl-1low are the most sensitive to ABT-199 treatment. Whereas cell lines that are Bcl-xLhigh remain sensitive to navitoclax but not ABT-199. MM cell lines that are Mcl-1high are less sensitive to both ABT-199 and navitoclax, suggesting that Mcl-1 is a resistance factor to both drugs. Utilizing a novel Mesoscale Discovery based immunoassay we determined that levels of Bcl-2/Bim complexes also correlated with sensitivity of ABT-199 in the MM cell lines tested. In addition, the t(11;14) status in these cell lines associated with sensitivity to ABT-199. The clinical relevance of the Bcl-2 pro-survival expression pattern in MM cell lines, was determined by a collection of bone marrow biopsies and aspirates (n=27) from MM patients by immunohistochemistry for prevalence of Bcl-2 and Bcl-xL. Similar to our in vitro observations, the majority (75%) of the MM bone marrow biopsies and aspirates had high Bcl-2 levels whereas 50% had high Bcl-xL expression. Therefore, a subset of patient samples (33%) were identified with a favorable biomarker profile (Bcl-2high/Bcl-xLlow) that may predict ABT-199 single agent activity. ABT-199 synergized with bortezomib in decreasing cell viability in the majority of MM cell lines tested in vitro based on the Bliss model of independence analyses (Bliss score range = 10 to 40). However the window of combination activity was reduced due to high degree of sensitivity to bortezomib alone. Therefore, the combination efficacy of ABT-199 and bortezomib was further evaluated in vivo in MM xenograft models that expressed high levels of Bcl-2 protein (OPM-2, KMS-11, RPMI-8226, H929 and MM. 1s). Bortezomib treatment alone at a maximum tolerated dose resulted in tumor regressions or stasis in all xenograft models tested. ABT-199 at a maximum tolerated dose was moderately efficacious (defined by tumor growth delay) as a single agent in xenograft models that expressed high protein levels of Bcl-2 but relatively lower levels of Bcl-xL. However, the combination of ABT-199 with bortezomib significantly increased the overall response rate and durability of anti-tumor activity when compared to bortezomib, resulting in increased cell death in vivo. Treatment with bortezomib increased levels of the pro-apoptotic BH3-only protein, Noxa, in MM xenograft models that expressed high levels of Mcl-1. Given that the induction of Noxa by bortezomib results in neutralization of Mcl-1 pro-survival activity in MM models [Gomez-Bougie et al; Cancer Res. 67:5418–24 (2007)], greater efficacy may be achieved when Bcl-2 is antagonized by ABT-199 thereby inhibiting pro-survival activity occurring through either Bcl-2 or Mcl-1 and increasing cell death. Thus, our preclinical data support the clinical evaluation of ABT-199 in combination with bortezomib in MM patients in which relative expression of the Bcl-2 pro-survival proteins may serve as predictive biomarkers of drug activity. Disclosures: Sampath: Genentech: Employment, Equity Ownership. Punnoose:Genentech: Employment, Equity Ownership. Boghaert:Abbott Pharmaceuticals: Employment, Equity Ownership. Belmont:Genentech: Employment, Equity Ownership. Chen:Abbott Pharmaceuticals: Employment, Equity Ownership. Peale:Genentech: Employment, Equity Ownership. Tan:Genentech: Employment, Equity Ownership. Darbonne:Genentech: Employment, Equity Ownership. Yue:Genentech: Employment, Equity Ownership. Oeh:Genentech: Employment, Equity Ownership. Lee:Genentech: Employment, Equity Ownership. Fairbrother:Genentech: Employment, Equity Ownership. Souers:Abbott Pharmaceuticals: Employment, Equity Ownership. Elmore:Abbott Pharmaceuticals: Employment, Equity Ownership. Leverson:Abbott Pharmaceuticals: Employment, Equity Ownership.

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Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.237.237 ◽

2013 ◽

Vol 122 (21) ◽

pp. 237-237 ◽

Cited By ~ 2

Author(s):

Michael P. Rettig ◽

Matthew Holt ◽

Julie Prior ◽

Sharon Shacham ◽

Michael Kauffman ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Nuclear Export ◽

Employment Equity ◽

Advisory Committees ◽

Ic50 Values ◽

Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

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