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BackgroundPreclinical evidence supports combining checkpoint inhibition (CPI) with T cell costimulatory agonism to improve the breadth and durability of anti-tumor responses relative to CPI alone. Currently, there are a number of therapeutic approaches combining costimulatory receptor agonists (e.g. CD28, 4-1BB, OX40L, etc.) with tumor targeting agents and/or CPI. Identification of a pharmacodynamically-justified therapeutic dose can be challenging because traditional duration of target occupancy does not necessarily correlate with immunological activity in the case of costimulatory molecules, and an ‘always on’ dose risks immune exhaustion. ALPN-202, a variant CD80 vIgD-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints, is in development for the treatment of multiple advanced malignancies. To assess clinical CD28 agonism in the context of ALPN-202 treatment, we developed a novel, ex vivo whole blood target-dependent costimulation (TDC) assay.MethodsA TDC assay was developed using clinical samples from NEON-1 (NCT04186637), an ongoing dose escalation and expansion clinical trial of ALPN-202 for patients with advanced malignancies. The assay uses patient blood stimulated with paraformaldehyde-fixed, artificial antigen presenting cells (aAPC) expressing both cell-surface anti-CD3 and PD-L1. Pre-dose and end-of-infusion (EOI) blood was drawn from trial participants and co-cultured for 24 hours with the aAPCs in a pre-made assay plate. Plasma was collected and secreted IL-2 was quantified and used as a measure of PD-L1-dependent CD28 costimulation. Nonlinear regression was used to calculate area under the curve (AUC) for each condition, and sample AUC values were compared to a positive control (pre-dose blood stimulated with a fixed concentration of ALPN-202). Serum concentration of ALPN-202 and CD28 target saturation analyses were conducted concurrently to evaluate the exposure-response relationship.ResultsUsing the ex vivo TDC assay, ALPN-202 demonstrated PD-L1-dependent T cell costimulation at all dose levels tested to date in the NEON-1 clinical trial, consistent with preclinical assay development data. Similarly, CD28 target saturation levels on circulating T cells correlated with serum concentration of ALPN-202.ConclusionsWe have developed a novel ex vivo assay to assess induction of PD-L1-dependent CD28 costimulation in a therapeutic setting. This assay has been successfully employed to monitor controlled CD28 costimulation by the CD28 agonist therapeutic candidate ALPN-202, helping to establish a PK/PD relationship that is consistent with preclinical data. More broadly, this type of cell-based, ex vivo TDC assay could be adapted to assess costimulatory receptor engagement, particularly target-dependent costimulation, for other therapeutic agonists in clinical development.Ethics ApprovalThis study was approved by WCG IRB’s Human Subjects Review, approval number: 20211877.

ALPN‐101 (ICOSL vIgD‐Fc) is an Fc fusion protein of a human inducible T cell costimulatory ligand (ICOSL) variant immunoglobulin domain (vIgD) designed to inhibit the cluster of differentiation 28 (CD28) and inducible T cell costimulator (ICOS) pathways simultaneously. A first‐in‐human study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ALPN‐101 in healthy adult subjects. ALPN‐101 was generally well‐tolerated with no evidence of cytokine release, clinically significant immunogenicity, or severe adverse events following single subcutaneous (SC) doses up to 3 mg/kg or single intravenous (IV) doses up to 10 mg/kg or up to 4 weekly IV doses of up to 1 mg/kg. ALPN‐101 exhibited a dose‐dependent increase in exposure with an estimated terminal half‐life of 4.3–8.6 days and SC bioavailability of 60.6% at 3 mg/kg. Minimal to modest accumulation in exposure was observed with repeated IV dosing. ALPN‐101 resulted in a dose‐dependent increase in maximum target saturation and duration of high‐level target saturation. Consistent with its mechanism of action, ALPN‐101 inhibited cytokine production in whole blood stimulated by Staphylococcus aureus enterotoxin B ex vivo, as well as antibody responses to keyhole limpet hemocyanin immunization, reflecting immunomodulatory effects upon T cell and T‐dependent B cell responses, respectively. In conclusion, ALPN‐101 was well‐tolerated in healthy subjects with dose‐dependent PK and PD consistent with the known biology of the CD28 and ICOS costimulatory pathways. Further clinical development of ALPN‐101 in inflammatory and/or autoimmune diseases is therefore warranted.

BACKGROUND: Checkpoint inhibitors (CPI) targeting the PD-1 and CTLA-4 pathways have demonstrated significant clinical benefit in multiple tumors, but the majority of patients exhibit or develop resistance, at least in part due to insufficient activation and/or excessive exhaustion of tumor-specific T cells. Strong preclinical rationale exists to combine CPIs with T cell costimulatory agonists, but such approaches have not yet translated into significantly improved clinical benefit. The majority of these approaches, though, have not targeted CD28, a critical T cell costimulator recently recognized as a key target of checkpoint inhibition. ALPN-202 is a variant CD80 vIgD-Fc fusion that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints. As monotherapy, ALPN-202 has demonstrated superiority to CPI-only therapies in tumor models, while demonstrating a favorable safety profile in preclinical toxicology studies. METHODS: This is a cohort-based, open-label dose escalation and expansion study in adults with advanced solid tumors or lymphoma (NCT04186637). The objectives of this study are to evaluate the safety, efficacy, pharmacokinetics, anti-drug antibodies, and pharmacodynamics of ALPN-202. Subjects who are refractory or resistant to standard therapy (including approved CPIs), or without available standard or curative therapy are eligible. ALPN-202 is administered by IV infusion. After two single-subject cohorts (0.001 mg/kg, 0.01 mg/kg), a standard 3+3 dose escalation (0.1-20 mg/kg) has been implemented with two dose schedules in parallel, Q1W and Q3W. Thereafter, expansion cohorts are planned to enroll subjects at a selected dose regimen(s) in selected indications. Biomarkers include tumor measurements of PD-L1, CD28, CD80 and CD86 to explore the possible relationship between baseline expression and outcomes. Other pharmacodynamic analyses include target saturation, inflammatory cytokines and immunophenotyping of circulating leukocytes. As of January 2021, the trial is continuing as planned; no DLTs have been observed through the 0.3 mg/kg cohorts (dose level 4). Citation Format: Mark Voskoboynik, Justin C. Moser, Nehal Lakhani, Michael Millward, Diwakar Davar, Wael A. Harb, Jing Yang, Gary Means, Kristi Manjarrez, Almudena Tercero, Hany Zayed, Jan Hillson, Christine Dela Cruz, Graciela Perez, Stanford L. Peng. NEON-1: a first-in-human phase I open-label study of ALPN-202, a conditional CD28 costimulator and dual checkpoint inhibitor, in advanced malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT213.

2547 Background: Strong preclinical rationale has emerged for combining checkpoint inhibition (CPI) with T cell costimulatory agonists, particularly CD28, a critical T cell costimulatory molecule recently recognized as a key target of checkpoint inhibition. ALPN-202 is a variant CD80 vIgD-Fc fusion that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints. It has demonstrated superiority to CPI-only therapies in tumor models, while demonstrating favorable safety in preclinical toxicology studies. Methods: This is a cohort-based, open-label dose escalation and expansion study of ALPN-202 in adults with advanced solid tumors or lymphoma (NCT04186637). Subjects with cancers refractory to standard therapies (including approved CPIs), or cancers without available standard or curative therapy are eligible. After two planned single-subject cohorts, a standard 3+3 dose escalation has been implemented with two dose schedules in parallel, Q1W and Q3W. Objectives include evaluation of safety and tolerability, PK, PD and preliminary anticancer activity of ALPN-202. Disease assessments are evaluated by RECIST v1.1 for solid tumors or by Lugano Classification for lymphoma. Results: As of January 2021, 20 subjects with advanced malignancies have received ALPN-202. Dose-dependent PK and target saturation have been preliminarily observed. So far, ALPN-202 has been well tolerated at dose levels ranging from 0.001 to 1 mg/kg weekly, with no DLTs. Low-grade skin toxicities (grade 1-2 rash) have been observed in 4 subjects (20%). Among 11 evaluable subjects, an unconfirmed partial response has been observed in one subject with colorectal carcinoma, while stable disease has been observed in 5 subjects with colorectal carcinoma, mesothelioma (2), cholangiocarcinoma, and renal cell carcinoma -- for a preliminary clinical benefit (PR+SD) rate of 100% (4/4) at dose levels of 0.3 mg/kg and higher, or 54% (5/11) overall (table). The meeting presentation will update this data, which is expected to include the conclusion of Q1W dose escalation, as well as immune correlates. Conclusions: First-in-human dose escalation with ALPN-202 has been well tolerated at doses capable of engaging CD28 costimulation in vivo in association with dual PD-L1/CTLA-4 checkpoint inhibition, with early signs of anti-tumor activity. These findings suggest that CD28 agonism can be safely achieved in humans, and further suggest that dose expansion with ALPN-202 is warranted to assess the relevance of controlled CD28 costimulation as a novel approach to cancer immunotherapy. Clinical trial information: NCT04186637. [Table: see text]

Background: Checkpoint inhibitors (CPI) targeting the PD-(L)1 and CTLA-4 pathways have demonstrated significant clinical benefit in many cancers, but the majority of patients unfortunately still fail to demonstrate objective responses and/or develop resistance, at least in part due to insufficient activation and/or excessive exhaustion of tumor-specific T cells. Strong preclinical rationale has emerged for combining CPIs with T cell costimulatory agonists, but such approaches have not yet translated into significantly improved clinical benefit. The majority of these approaches, though, have not targeted CD28, a well-known, critical T cell costimulatory pathway. ALPN-202 is a variant CD80 vIgD-Fc fusion that mediates PD-L1-dependent CD28 costimulation and inhibits the PD-L1 and CTLA-4 checkpoints. As monotherapy, ALPN-202 has demonstrated superiority to CPI-only therapies in preclinical tumor models, while demonstrating a favorable safety profile in rat and monkey toxicology studies. It is in development for the treatment of multiple advanced malignancies. Methods: This is a cohort-based, open-label dose escalation and expansion study in adults with advanced solid tumors or lymphoma (NCT04186637). The objectives of this study are to evaluate the safety, efficacy, pharmacokinetics (PK), anti-drug antibodies (ADA), and pharmacodynamics (PD) of ALPN-202. Subjects who are refractory or resistant to standard therapy (including approved CPIs), or without available standard or curative therapy are eligible. ALPN-202 will be administered by IV infusion. Dose escalation will initially use single-subject cohorts as an accelerated titration strategy to minimize the number of patients that will receive dose levels with low likelihood to elicit clinically meaningful PD activity. At dose levels predicted to elicit clinically meaningful PD activity, a standard 3+3 strategy will be used in two arms studying different dose schedules–Q1W and Q3W. Thereafter, an expansion cohort(s) will enroll subjects at a selected dose(s) and selected indication(s). Outcomes include safety assessments such as adverse events and dose-limiting toxicities. Tumor responses will be categorized by RECIST v1.1 for solid tumors, and by Lugano Classification for lymphoma. Biomarkers will include tumor measurements of PD-L1, CD28, CD80 and CD86 to explore the possible relationship between baseline expression and outcomes. Other PD will include target saturation, inflammatory cytokines and immunophenotyping of circulating leukocytes. Citation Format: Jing Yang, Gary Means, Almudena Tercero, Kristi Manjarrez, Jan Hillson, Stanford L. Peng. NEON-1: A first-in-human phase I open-label study of ALPN-202, a conditional CD28 costimulator and dual checkpoint inhibitor, in advanced malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT245.

CD33 is a valid target for acute myeloid leukemia (AML) but has proven challenging for antibody-drug conjugates. Herein, we investigated the cellular determinants for the activity of the novel CD33/CD3-directed bispecific T-cell engager antibody, AMG 330. In the presence of T cells, AMG 330 was highly active against human AML cell lines and primary AML cells in a dose- and effector to target cell ratio-dependent manner. Using cell lines engineered to express wild-type CD33 at increased levels, we found a quantitative relationship between AMG 330 cytotoxicity and CD33 expression; in contrast, AMG 330 cytotoxicity was neither affected by common CD33 single nucleotide polymorphisms nor expression of the adenosine triphosphate-binding cassette (ABC) transporter proteins, P-glycoprotein or breast cancer resistance protein. Unlike bivalent CD33 antibodies, AMG 330 did not reduce surface CD33 expression. The epigenetic modifier drugs, panobinostat and azacitidine, increased CD33 expression in some cell lines and augmented AMG 330-induced cytotoxicity. These findings demonstrate that AMG 330 has potent CD33-dependent cytolytic activity in vitro, which can be further enhanced with other clinically available therapeutics. As it neither modulates CD33 expression nor is affected by ABC transporter activity, AMG 330 is highly promising for clinical exploration as it may overcome some limitations of previous CD33-targeted agents.

Background: Cluster of differentiation 28 (CD28) and inducible T cell costimulator (ICOS) provide costimulatory signals required for optimal T cell activation when bound to their respective ligands, CD80 (B7-1) and CD86 (B7-2), and ICOS ligand (ICOSL). CD28 is involved in initiation of the pathogenic process in GVHD and recent studies suggest therapeutic utility of CD28 pathway inhibitors for prophylaxis and treatment. However, CD28 pathway inhibition alone appears insufficient to control established disease in most patients. In this context, it is recognized that following initial activation, CD28 is often down-regulated while ICOS, its most closely related family member, is upregulated, providing additional T cell costimulation that may sustain GVHD including gastrointestinal manifestations (Adom D et al. Blood. 2018; 132:355). In murine models of aGVHD, combined blockade of CD28 and ICOS was significantly superior to isolated blockade of the CD28 or ICOS pathways alone. ALPN-101 is an Fc fusion protein of a human ICOSL variant immunoglobulin domain (vIgDTM) designed to inhibit both the CD28 and ICOS costimulatory pathways. Nonclinical studies of ALPN-101 demonstrate high affinity binding to CD28 and ICOS, potent inhibition of T cell activation, and suppression of disease activity in a human xenogeneic model of GVHD in mice, after only a single dose (Dillon SR et al. Blood. 2018; 132:2037). ALPN-101 is in development as a novel and potentially transformative approach for GVHD. A first-in-human study of ALPN-101 in healthy subjects is ongoing. This clinical study will assess the safety, efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of ALPN-101 in subjects with aGVHD. Study Design and Methods: Adults with Grade II-IV aGVHD per Mount Sinai Acute GVHD international Consortium (MAGIC) criteria (Harris et al. 2016) that is resistant or refractory to glucocorticoids will receive a single intravenous dose of ALPN-101. This study will be conducted in two parts including dose escalation and dose expansion. Dose escalation will proceed by using an accelerated titration design. Thereafter, a selected dose level(s) will be evaluated in an expansion cohort of 10 subjects; if ≥ 25% (n=3) subjects achieve response, 15 additional subjects will be enrolled (Simon two-stage design). Background Therapy:At the investigator's discretion, subjects may continue therapies administered for prophylaxis, continue or increase glucocorticoids, and/or add another salvage therapy. Responders will be considered for glucocorticoid taper. Endpoints: Safety will be assessed based on the incidence, severity, and seriousness of adverse events. Efficacy endpoints include the objective response rate, duration of response, failure-free survival, event-free survival, non-relapse mortality, malignancy relapse/progression, overall survival, and glucocorticoid use. The incidence and titer of anti-drug antibodies will be assessed. Serum concentrations of ALPN-101 will be measured and PK parameters will be estimated. PD endpoints include target saturation and immunophenotyping of circulating leucocytes, and may include quantification of circulating cytokines, immunoglobulins, acute phase reactants, and soluble forms of the targeted pathway receptor, evaluation of changes in mRNA expression in circulating leucocytes, evaluation of risk alleles, and correlates of response. Disclosures Yang: Alpine immune sciences: Employment, Equity Ownership. Hillson:Alpine Immune Sciences: Employment, Equity Ownership. Manjarrez:Alpine Immune Sciences: Employment, Equity Ownership. Wiley:Alpine Immune Sciences: Employment, Equity Ownership. Means:Alpine Immune Sciences: Employment, Equity Ownership. Dillon:Alpine Immune Sciences: Employment, Equity Ownership. Peng:Alpine Immune Sciences: Employment, Equity Ownership.

BACKGROUND Biomarker assays are often conducted on whole blood samples in the course of drug development studies. Because bacterial lipopolysaccharide (LPS) (endotoxin) contamination is known to cause spontaneous cytokine production by monocytes, contamination of blood collection tubes may interfere with biomarker assay results. METHODS Whole blood from healthy donors was collected into plastic or glass sodium (Na+)-heparin Vacutainer™ blood collection tubes and heparinized syringes. Samples were analyzed for phosphoprotein response, cytokine production, and RNA expression. Tubes were tested for endotoxin contamination by use of the limulus amoebocyte lysate assay. RESULTS Results of phospho-flow cytometry, branched DNA (bDNA), and ELISA assays indicated that a specific lot (#5339582) of plastic Na+-heparin Vacutainer tubes was highly contaminated with an endotoxinlike substance, and contamination was confirmed by the limulus amoebocyte lysate assay. Analysis of multiple-analyte panels revealed that analytes whose changed expression was predictive of LPS stimulation were increased when whole blood was incubated in contaminated tubes for 6 or 18 h. Two additional lots of plastic tubes tested had detectable amounts of endotoxin sufficient to strongly alter phospho-flow cytometry analyses, as determined by the fold change in phosphorylation of p38 mitogen-activated protein kinase in response to tumor necrosis factor α and LPS. In contrast, 3 lots of glass tubes had substantially lower levels of spontaneous blood activation. CONCLUSIONS Endotoxin contamination associated with tubes from 3 lots of a particular type of plastic Na+-heparin Vacutainer tube dramatically affected biomarker assay measurements. Prescreening these tubes is suggested before their use in clinical sample analysis.

The importance of the analysis of signaling pathways has been proven for many years by the elucidation of key signaling molecules. However, in most cases these pathways tend to represent a rather narrow view of the biological state under investigation. Clearly a more detailed understanding of the complexities of cross-talk between signaling pathways is required to further our knowledge of normal and disease processes. The tools that provide the framework for this increased understanding of biology, those that enable identification, characterization, and quantitation of sites of phosphorylation in proteins, have advanced over the past 25 years. This review will present a brief overview of the history of the tools used in phosphorylation analysis and the latest technologies that are being applied in this field, such as mass spectrometry (for broad-based discovery efforts) and flow cytometry (for translation to clinical applications).

IL-15 is a proinflammatory cytokine that plays an important role in the development and activation of NK cells and is a potential target for inflammatory disease therapy. Studies conducted in IL-15- and IL-15R knockout mice identified IL-15 as an important cytokine for NK cell homeostasis. Consistent with this information derived from genetically modified mice, we demonstrated that neutralizing IL-15 with a mouse anti-mouse IL-15 mAb (M96) depletes C57BL/6 mouse NK cells. An mAb directed against macaque IL-15 (Hu714MuXHu) was manufactured and demonstrated to block IL-15–induced activation of nonhuman primate (NHP) NK cells in vitro. Neutralization of macaque IL-15 by parenteral administration of Hu714MuXHu reduces (>95%) circulating NK cell counts in NHPs. A blocking mAb directed against human IL-15 (huIL-15; AMG 714) was manufactured. Unexpectedly, when human subjects were treated with the blocking anti–IL-15 Ab AMG 714 in clinical trials, no reductions in circulating NK cell counts were observed despite achieving significantly higher exposures than the levels of Hu714MuXHu needed to cause NK cell count reductions in NHPs in vivo. Both AMG 714 and Hu714MuXHu are able to block huIL-15 activity in a human T cell blast proliferation and IFN-γ production assay. Both Abs block huIL-15–mediated Stat5 activation and CD69 expression in human NK cells. Collectively, these results demonstrate that NK cell homeostasis is obligatorily dependent upon IL-15 in both mice and NHPs, but that IL-15 is dispensable for maintenance of circulating human NK cells.

In flow cytometry, different cell types are usually selected or “gated” by a series of 1- or 2-dimensional geometric subsets of the measurements made on each cell. This is easily accomplished in commercial flow cytometry packages but it is difficult to work computationally with the results of this process. The ability to retrieve the results and work with both them and the raw data is critical; our experience points to the importance of bioinformatics tools that will allow us to examine gating robustness, combine manual and automated gating, and perform exploratory data analysis. To provide this capability, we have developed a Bioconductor package called flowFlowJo that can import gates defined by the commercial package FlowJo and work with them in a manner consistent with the other flow packages in Bioconductor. We present this package and illustrate some of the ways in which it can be used.

TPS3097 Background: The bispecific CD19-directed CD3 T-cell engager (BiTE(R)) blinatumomab was recently approved by FDA as single-agent immunotherapy for patients with Philadelphia chromosome-negat...

Numerous proteins are cleaved or "shed" from their membrane-bound form. One such protein, tumour necrosis factor alpha (TNF-alpha), is synthesized as a type 2 transmembrane protein. Recently, a human protease responsible for this shedding, the TNF-alpha converting enzyme (TACE/ADAM17), was isolated. TACE/ADAM17 is a member of the adamalysin class of zinc-binding metalloproteases or ADAM (a disintegrin and metalloprotease). We report the isolation and characterization of the mouse TACE/ADAM17 cDNA and gene. Mouse TACE/ADAM17 has a 92% amino-acid identity with the human protein and was ubiquitously expressed. A recombinant form of the protease is found to cleave a peptide representing the cleavage site of precursor mouse TNF-alpha. An alternatively spliced form of mouse TACE/ADAM17 was found that would produce a soluble protein. The gene for TACE/ADAM17 is approximately 50 kb and contains 19 exons. Chromosomal mapping places TACE/ADAM17 on mouse chromosome 12 and human chromosome 2p25.

Introduction The phosphoinositide 3-kinase p110δ isoform (PI3Kδ) is expressed primarily in hematopoietic cells and is essential in B-cell receptor (BCR) signaling. PI3Kδ is expressed in lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). AMG 319 is an investigational, highly selective, small molecule inhibitor of PI3Kδ that blocks B cell proliferation following BCR stimulation both in vitro and in vivo, inhibits basal AKT phosphorylation, and inhibits proliferation in lymphoid tumor cells. This study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of AMG 319. Methods Patients (pts) with relapsed or refractory CLL or NHL were eligible. Key eligibility criteria included ≥ 18 years old, ECOG ≤ 2, and adequate organ function. AMG 319 was administered once per day (QD; except day 2 for PK) until disease progression or unacceptable toxicity. Doses of 25, 50, 100, 200, 300, and 400 mg were administered in sequential cohorts (3 pts per cohort). Dose-limiting toxicities (DLT) were defined during the first 28 days. Response assessments for CLL and NHL were done using IWCLL 2008 and IWG 2007 criteria, respectively. CT scans for CLL pts were done at baseline and week 8. Results 28 pts received AMG 319 (6 at 25 mg; 3 at 50 mg, 100 mg, 200 mg, and 300 mg; and 10 at 400 mg). Demographics: median age 68 years, 71% men, ECOG 0/1 93%. 25 pts had CLL, 3 had NHL, including 2 mantle cell and 1 marginal zone. The median number of prior treatment regimens was 4 (range 1-9) for CLL and 7 (range 5-9) for NHL. Central cytogenetic analysis of CLL pts prior to dosing (n=24) revealed 10 with del 17p (42%), 3 with del 11 and not 17p (13%), 1 with trisomy 12 alone (4%), 9 normal (38%), and 1 del 13q alone (4%). Doses up to 400 mg QD were explored without reaching a maximum tolerated dose. There was 1 DLT: grade 3 hemolytic anemia at 25 mg in a CLL pt after 1 dose, considered as possibly related to AMG 319. AMG 319 was well absorbed and exhibited linear PK with mean plasma half-lives of 3.8 to 6.6 hours across dose cohorts. Dose-dependent coverage of BCR-induced pAKT in CLL samples (ex-vivo IgD stimulated) was observed in all samples with an inducible signal (60%); near complete inhibition for 24 hours was seen at 400 mg. 75% of pts had ≥1 treatment-related adverse event (AE, 25% grade ≥3). Grade ≥3 treatment-related AEs (n>1) were colitis 3 (10%), anemia 3 (10%), leukocytosis 2 (7%), infection 2 (7%), and hemolysis 2 (7%). The colitis cases occurred at 400 mg between days 40 and 60; 2 were successfully re-challenged at a lower dose, while the third was diagnosed with C Diff colitis and discontinued. Grade 3 transaminitis was observed in 1 pt (4%) at 50 mg on day 29, which resolved with holding of drug and did not recur with re-challenge. Subset analysis of lymphocytes revealed no changes in % of T, B, or NK cells. Baseline % of T-regulatory cells (as a function of total CD4) was elevated in CLL pts (14.4% ± 7.6%). Elevated T regulatory cells (>10% of CD4+) tended to normalize during treatment (14/19 pts), suggesting immune restoration. Response data are available for 24 CLL pts (pt with DLT removed after 1 dose) and 3 NHL pts. Lymphocyte counts in CLL were highly variable, with some pts experiencing marked and early lymphocytosis (at all doses) and others showing gradual decline. Consistent nodal regression was observed at all doses by CT and physical exam. Early CT imaging (21 pts) at week 8 revealed lymph node (LN) reduction in all pts, and >50% decrease was seen in 7 pts; all 7 were dosed at 400 mg including 4 with del 17p or del 11q. Two pts at week 8 met IWCLL response criteria for partial response. By physical exam, all 20 evaluable pts had >50% LN reduction as a best response, with 15 (75%) having >90%. Response was present in all cytogenetic subtypes. At the 200-400 mg dose levels, 13/15 CLL pts remain on study after a median follow-up of 30 weeks (range 14-65). 1 pt with mantle cell NHL had 46% shrinkage while on therapy but progressed after 26 weeks; the 2 other NHL pts progressed by the first evaluation. Conclusions AMG 319 exhibited linear absorption and was tolerated at doses up to 400 mg QD. This degree of inhibition provided complete blockade of ex-vivo stimulated pAKT for 24 hours in CLL. Anti-tumor activity was observed early in CLL pts and included high-risk cytogenetic subgroups. While activity was noted at all dose levels, an apparent dose response was observed, with deeper and faster responses in CLL pts at higher doses. Disclosures: Lanasa: Amgen: Consultancy. Glenn:Amgen: Research Funding; Sanofi Aventis: Research Funding. Mato:Amgen: Honoraria. Wong:Amgen: Employment, Equity Ownership. Amore:Amgen: Employment, Equity Ownership. Means:Amgen: Employment, Equity Ownership. Stevens:Amgen: Employment, Equity Ownership. Yan:Amgen: Employment, Equity Ownership. Friberg:Amgen: Employment, Equity Ownership. Goy:Amgen Inc: Research Funding.

Abstract 4964 Immune receptors such as the B cell receptor (BCR) require key signaling intermediate phosphatidylinositol-3 kinase delta (PI3Kδ) for normal immune cell survival, development and function. PI3Kδ is a class IA lipid kinase, is expressed primarily within the hematopoietic system and is composed of a catalytic subunit p110δ and a regulatory subunit p85. Recently, deregulated BCR-PI3Kδ signaling has been reported to play a role in B-cell malignancies such as chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) by mediating abnormal B-cell growth and survival. Indeed, the constitutive phosphorylation of downstream signaling intermediate AKT is associated with poor prognosis in several B cell malignancies. Here, we have investigated the potential of a novel small molecule inhibitor of PI3Kδ, AMG 319, to suppress PI3K signaling in human B cell lines and assessed the subsequent effects on viability as a single agent and in combination with chemotherapeutic drugs in preclinical models. Small molecule AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent preclinical pharmacokinetic (PK) properties. AMG 319 was found to potently inhibit PI3Kδ in enzyme assays (IC50 5000 fold). Furthermore, AMG 319 was considered inactive at 10 μM on non-PI3K class I kinases in a broader kinome screen of 402 kinases. In preclinical PK studies, AMG 319 had low systemic clearance, T1/2 range of 2–4 hr, oral bioavailability of >45% and unbound fractions in plasma of 5–19%. Here, we have investigated the potential for AMG 319 to inhibit constitutive PI3K mediated signaling and effects on human B cell line viability. In a broad screen of >20 cell lines derived from B cell malignancies, the majority of lines were found to express PI3Kδ protein, all cells lines expressed the PI3Kα and β isoforms and variable levels of constitutive pAKTS473 were detected. AMG 319 was found to potently suppress constitutive pAKTS473 in the cell lines with IC50 in the low single to double digit nM range. Cellular viability was inhibited by AMG 319 though lines were variably sensitive to drug (range low double digit nM to μM IC50). As cell lines were variably sensitive to AMG 319 as a single agent, we examined if AMG 319 could enhance the efficacy of chemotherapeutic agents in vitro and in vivo. These studies focused on a DLBCL cell line HT which was relatively insensitive to AMG 319 as a single agent (IC50 ∼10 μM) in viability assays even though pAKTS473 was potently suppressed (IC50 ∼ 0.030 μM). Treatment with AMG 319 was found to synergize with the effects of vincristine to reduce cell viability in vitro using a 72 hr viability assay. Next we examined whether the enhanced cytotoxicity using these drugs in combination could be observed in vivo. Using the human B-cell lymphoma HT xenograft model, we found that AMG 319 in combination with vincristine enhanced tumor growth inhibition above that observed with either agent alone. Taken together, these findings suggest that the inhibition of PI3Kδ with AMG 319 may enhance the effects of chemotherapeutic agents in B cell malignancies. In conclusion, AMG 319 is a potent and selective inhibitor of PI3Kδ with excellent PK properties. AMG 319 inhibited constitutive pAKTS473, reduced the viability of B cell lines and synergized with vincristine in vitro and in vivo. The safety, PK and preliminary efficacy of AMG 319 are currently being investigated in a Phase I trial in patients with relapsed or refractory lymphoid malignancies. Disclosures: Sinclair: Amgen: Employment, Stock and Options. Metz:Amgen, Inc: Employment, Stock and Options. Cushing:Amgen, Inc: Employment, Stock and Options. Liu:Amgen, Inc: Employment, Stock and Options. Brake:Amgen, Inc: Employment, Stock and Options. Starnes:Amgen, Inc: Employment, Stock and Options. Means:Amgen, Inc: Employment, Stock and Options. Henne:Amgen, Inc: Employment, Stock and Options. Archibeque:Amgen: Employment, Stock and Options. Mattson:Amgen, Inc: Employment, Stock and Options. Drew:Amgen, Inc: Employment, Stock and Options. Busse:Amgen, Inc: Employment, Stock and Options. Wang:Amgen, Inc: Employment, Stock and Options. Al-Assaad:Amgen, Inc: Employment, Stock and Options. Molineux:Amgen: Employment, Stock and Options.