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Introduction: Squamous cell carcinoma of the head and neck (SCCHN) is the seventh most common cancer worldwide, accounting for ~ 4% of all cancers in the United States. Although PD-1-directed therapy has efficacy in SCCHN, 85-95% of patients progress following initial response. B7-H3 is a member of the B7 family of immunomodulatory molecules, is overexpressed in SCCHN, and correlates with disease severity and poor clinical outcome. Furthermore, consistent with its putative coinhibitory function, B7-H3 expression in SCCHN is inversely correlated with the number of tumor infiltrating CD8+ T-cells. We are developing therapeutics targeting B7-H3, including enoblituzumab, an Fc-engineered anti-B7-H3 monoclonal antibody, and MGC018, a duocarmycin-based B7-H3 ADC, both of which are currently being evaluated in clinical studies. Here the potential of MGC018 was explored in preclinical models as a proof of concept for targeting B7-H3 in SCCHN. Methods: Immunohistochemistry studies were performed to define the expression of B7-H3 in SCCHN tissue microarrays (TMA). Single and repeat-dose in vivo efficacy studies were conducted in CD-1 nude mice with cell line-derived SCCHN human tumor xenografts to explore the relationship between Cmax, exposure and antitumor activity, and to define the minimal efficacious dose in these models. Based on results in these cell-derived xenograft (CDX) studies, in vivo efficacy studies were extended to a panel of SCCHN patient-derived xenograft (PDX) models, which more closely mimic the biological characteristics of the patient tumor and exhibit heterogenous expression of B7-H3. Results: Analysis of B7-H3 expression on a SCCHN TMA confirmed and extended previously reported expression of B7-H3 in SCCHN. Of the SCCHN samples evaluated, 90% (36/40) of the tumor samples were positive for B7-H3: 35% (14/40) had H-scores greater than 200, with the remaining 22 samples equally distributed between the H-score range of 101-200 and 1-100 (~ 28% each). MGC018 demonstrated specific, dose-dependent in vitro cytotoxicity toward SCCHN human tumor cell lines. The in vitro cytotoxicity translated to potent antitumor activity in vivo against SCCHN CDX models, with a single administration of 3 mg/kg resulting in complete responses in 7/7 mice in the FaDu model. In the PDX setting (H-scores 120-283), repeat dose administration every week or two weeks with MGC018 at 3 mg/kg/dose, led to regressions and/or stable disease in 10/18 models, and a delay in tumor growth in 5 additional models. Conclusion: B7-H3 is frequently overexpressed in SCCHN. At clinically relevant dose levels, MGC018 demonstrated potent antitumor activity in vivo toward SCCHN CDX mouse models and the majority of SCCHN PDX mouse models examined. These results support SCCHN as a potential indication that may be responsive to ADC-based treatments directed toward B7-H3. Citation Format: Juniper A. Scribner, Francine Z. Chen, Ying Li, Michael Chiechi, Thomas Son, Jeff Hooley, Scott Koenig, Paul A. Moore, Ezio Bonvini, Chet Bohac, Deryk Loo. Targeting B7-H3 in squamous cell carcinoma of the head and neck: Preclinical proof-of-concept with the investigational anti-B7-H3 antibody-drug conjugate, MGC018 [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 950.

Background: Margetuximab (M) has demonstrated anti-tumor activity in patients with advanced HER2+ gastric cancer and PFS superiority to trastuzumab (T) in pre-treated metastatic HER2+ breast cancer patients (1). Similar to T, M inhibits HER2 signaling; additionally, M enhances engagement with the activating Fcγ receptor (FcR)IIIA, while diminishing interaction with the inhibitory FcRIIB. Given the role of FcRs in coupling innate and adaptive immune responses, clinical studies of M in combination with PD-1 or PD-1 plus LAG-3 blockade have been initiated (2,3). The combination of M with tebotelimab, an investigational bispecific DART® molecule blocking PD-1 and LAG-3, has demonstrated encouraging early evidence of anti-tumor activity in patients with advanced HER2+ malignancies, including those with PD-L1-negative tumors (3). Here we present data supporting the potential mechanisms underlying the anti-tumor activity elicited by M and tebotelimab combination therapy. Methods: PBMCs were challenged with HER2-expressing tumor cell lines in the presence of M or T. Immune modulation was assessed by analysis of immune cell phenotype, gene expression profiles, and cytokine secretome. Functional activity of immune cells following exposure to the combination of M with tebotelimab was evaluated. Results: M induced greater upregulation relative to T of both co-stimulatory (eg 4-1BB) and/or checkpoint molecules (eg PD-L1 and LAG-3) on NK cells and/or monocytes in PBMCs cocultured with HER2-expressing tumor cells. Compared with T, M also induced greater proinflammatory cytokine release, including IFNγ that in turn upregulates PD-L1 and MHC class I expression on co-cultured tumor cells. These effects were not observed with an Fc-null version of M or were inhibited by the addition of FcR-blocking Abs, demonstrating Fc:FcR dependency of M-induced immune activation. Interestingly, CD8 T cells, which lack FcR, also exhibited LAG-3 upregulation in the above cultures, suggesting the effects of M can also be mediated via a cascade mechanism. An IFNγ neutralizing mAb reduced tumor cell-associated PD-L1 and MHC class I upregulation, but not LAG-3 induction on T-cells. PBMCs, pre-treated with a combination of M and tebotelimab in the presence of HER2-expressing tumor cells, demonstrated enhanced cytolytic activity against both the canonical NK cell target, K562, or M-opsonized HER2-positive tumor cells. Conclusions: M, through an Fc-dependent mechanism, can upregulate checkpoint molecules on NK-cells, CD8 T cells and tumor cells, thereby sensitizing them to immune checkpoint blockade. In turn, PD-1 and LAG-3 blockade by tebotelimab enhances M-mediated NK cell cytolytic activity in vitro. References: 1. Rugo et al., 2019 ASCO oral presentation 2. Catenacci et al., 2020 Lancet Oncology 21(8):1066-76 3. Luke et al., 2020 ASCO oral presentation Citation Format: Xiaoyu Zhang, Haiquan Li, Yinhua Yang, Qihong Xu, Tom Kaufman, Doug Smith, Jeffrey Nordstrom, Ezio Bonvini, Paul Moore. Enhanced HER2-dependent immune activation by margetuximab, an investigational Fc-engineered anti-HER2 mAb, supports combination with checkpoint blockade [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 1555.

Summary Combination immunotherapy with antibodies directed against PD-1 and CTLA-4 shows improved clinical benefit across cancer indications compared to single agents, albeit with increased toxicity. Leveraging the observation that PD-1 and CTLA-4 are co-expressed by tumor-infiltrating lymphocytes, an investigational PD-1 x CTLA-4 bispecific DART molecule, MGD019, is engineered to maximize checkpoint blockade in the tumor microenvironment via enhanced CTLA-4 blockade in a PD-1-binding-dependent manner. In vitro, MGD019 mediates the combinatorial blockade of PD-1 and CTLA-4, confirming dual inhibition via a single molecule. MGD019 is well tolerated in non-human primates, with evidence of both PD-1 and CTLA-4 blockade, including increases in Ki67+CD8 and ICOS+CD4 T cells, respectively. In the ongoing MGD019 first-in-human study enrolling patients with advanced solid tumors (NCT03761017), an analysis undertaken following the dose escalation phase revealed acceptable safety, pharmacodynamic evidence of combinatorial blockade, and objective responses in multiple tumor types typically unresponsive to checkpoint inhibitor therapy., Graphical Abstract, Highlights PD-1 and CTLA-4 are co-expressed by TILs but not healthy lymphocytes MGD019 is designed to block PD-1 and deliver enhanced CTLA-4 blockade in TME MGD019 is safe in NHP while demonstrating biomarkers of PD-1 and CTLA-4 inhibition Encouraging activity in tumors traditionally unresponsive to checkpoint blockade, Co-blockade of PD-1 and CTLA-4 increases benefits in cancer immunotherapy but also its toxicity. Berezhnoy et al. constructed a bispecific DART molecule to deliver safer and potentially more effective co-blockade by targeting CTLA-4 inhibition to tumors. MGD019 demonstrates encouraging activity in tumors traditionally unresponsive to checkpoint blockade.

Introduction: B7-H3, a member of the B7 family of immunomodulatory molecules, is overexpressed in a wide range of solid tumors. B7-H3 tumor overexpression has been correlated with disease severity and poor outcome. MGC018 is a duocarmycin-based antibody-drug conjugate (ADC) targeting B7-H3. MGC018 exhibits a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, and potent antitumor activity toward B7-H3-expressing tumor xenografts. With the emergence of immune-checkpoint blockade as a promising treatment for cancer, interest has grown in understanding the potential of cytotoxic agents to promote immune surveillance or stimulate immune responses to dying cancer cells, leading to immunological memory. ADCs bearing tubulin and DNA modifying cytotoxic payloads have been reported to induce immunological cell death (ICD), mediate antitumor immunity in immunocompetent mouse models, and synergistically combine with checkpoint inhibitors to deliver enhanced antitumor responses. Based on those results, we investigated the immunomodulatory potential of MGC018 and the prospect to combine with checkpoint blockade to enhance antitumor responses. Methods: Syngeneic mouse models expressing human B7-H3 were employed to investigate the antitumor activity of MGC018 in an immune competent setting. Studies were conducted to assess the role of the immune system in the MGC018-mediated antitumor responses, whether MGC018 could impart antitumor memory responses in vivo, and the potential to enhance antitumor responses by combining MGC018 with PD-1 blockade. Results: MGC018 demonstrated specific, dose-dependent in vivo antitumor activity toward human B7-H3-bearing tumors in immunocompetent syngeneic mouse models. Depletion of CD8+ T cells led to reduced antitumor responses, indicating that CD8+ T cells contributed to MGC018-mediated antitumor activity. Antitumor activity in these models was enhanced when MGC018 was combined with anti-PD-1. Treatment with MGC018 alone, or in combination with anti-PD-1, led to complete antitumor responses, and the majority of mice rejected subsequent tumor rechallenge. Conclusion: MGC018, a clinical-stage therapeutic comprised of a humanized antibody targeting B7-H3, conjugated to a duocarmycin-based DNA alkylating payload, exhibits a favorable preclinical profile. Results from these syngeneic model studies support the hypothesis that the antitumor activity of the duocarmycin-based MGC018 ADC (1) mediates immunomodulatory activity, (2) is enhanced by combination with checkpoint blockade, and (3) induces immunological memory. Our findings support a clinical strategy that combines MGC018 with checkpoint blockade for the treatment of B7-H3-expressing solid cancers. Citation Format: Juniper A. Scribner, Michael Chiechi, Pam Li, Thomas Son, Jeff Hooley, Ying Li, Anushka De Costa, Peter Lung, Nicholas Yee-Toy, Francine Chen, Bhaswati Barat, Christina Wolff, Valentina Ciccarone, James Tamura, Scott Koenig, Chet Bohac, Jon Wigginton, Paul A. Moore, Ezio Bonvini, Deryk Loo. MGC018, a duocarmycin-based antibody-drug conjugate targeting B7-H3, exhibits immunomodulatory activity and enhanced antitumor activity in combination with checkpoint inhibitors [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 5203.

Background: Tregs are suppressive cells whose presence in the tumor microenvironment (TME) has been correlated with tumor progression and may be a major impediment in the effectiveness of tumor immunotherapy. Removal of Tregs has been shown to induce cancer regression in animal models; however, selective depletion of tumor-infiltrating Tregs (TI-Tregs) is complicated as they share phenotypic markers with Tregs in healthy tissues as well as with tumor-infiltrating effector cells. To overcome cross-reactivity and achieve enhanced targeting specificity, we employed a bispecific strategy to simultaneously target two cell surface antigens (CD25 and CTLA-4) with overlapping, high level expression on TI-Tregs. Furthermore, we incorporated Fc engineering to enable target-cell depletion via enhanced Fc-mediated effector function. Methods: CD25 x CTLA-4 DART proteins were constructed based on CD25 and CTLA-4 mAbs and incorporating an Fc domain with enhanced binding to the activating Fc-gamma RIIIa receptor (CD16A) and reduced binding to the inhibitory Fc-gamma RIIb receptor (CD32B). Depletion of FoxP3-positive cells ex vivo was monitored by flow cytometry. Immunomodulatory effects of the DART proteins were evaluated in cultured PBMC or dissociated tumor cells in various in vitro immune assays, including mixed lymphocytes reaction (MLR). Results: Consistent with their design, we observed increased binding of CD25 x CTLA-4 DART molecules to cells when both target antigens were engaged compared to single antigen engagement. The bispecific DART molecule depleted in vitro induced FoxP3+ CD4+ TI-like Tregs cells by an Fc-dependent mechanism, with minimal effect on the viability of FoxP3-negative T cells. A molecule bearing a null Fc was ineffective. The extent of FoxP3+ T-cell depletion by the Fc-enhanced CD25 x CTLA-4 DART molecule was greater than that exhibited by the combination of its monovalent components, with the depleting activity requiring both arms of the bispecific molecule. The DART molecule had only minor effect on the capacity of effector T cells to secrete IL-2 and interferon-gamma upon restimulation or to exert cytotoxic function. In contrast the combination of CD25 and CTLA-4 mAbs reduced the ability of effector T cells to produce cytokines in a restimulation assay. An increased response in the presence of the Fc-enhanced CD25 x CTLA-4 DART molecule was also observed in various immune assays, consistent with Treg suppression and preservation of T-cell effector function. Conclusions: A CD25 x CTLA-4 DART molecule was effective in depleting activated FoxP3+ CD4+ Tregs in vitro, with reduced effects on resting Tregs or activated FoxP3- effector cells. Targeting CD25 and CTLA-4 in a bispecific molecular format with enhanced Fc-dependent effector function may be suitable for specifically depleting TI-Tregs. Citation Format: Alexey Berezhnoy, Kalpana Shah, Daorong Liu, Rillema Jill, Claudia Fieger, Douglas Smith, Francine Chen, James Tamura, Ezio Bonvini, Gundo Diedrich, Paul A. Moore. Investigational CD25 x CTLA-4 bispecific DART® molecule for depletion of tumor infiltrating Tregs via an enhanced Fc-dependent effector mechanism [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 2288.

3004 Background: MGD013 is an investigational, first-in-class, Fc-bearing bispecific tetravalent DART molecule designed to bind PD-1 and LAG-3 and sustain/restore the function of exhausted T cells. MGD013 demonstrates ligand blocking properties consistent with anti-PD-1 and anti-LAG-3 benchmark molecules, and improves T cell responses beyond that observed with benchmark or component antibodies alone or in combination. Methods: This study characterizes the safety, tolerability, dose-limiting toxicities, maximum tolerated dose (MTD), PK/PD, and antitumor activity of MGD013 in patients (pts) with advanced solid and hematologic malignancies. Sequential single-pt cohorts were treated with escalating flat doses of MGD013 (1-1200 mg IV every 2 weeks), followed by a 3+3 design. Tumor-specific expansion cohorts are being treated at the recommended Phase 2 dose of 600 mg. Results: At data-cutoff, 50 pts (46% checkpoint-experienced) were treated in Dose Escalation, and 157 pts (32% checkpoint-experienced) in Cohort Expansion. No MTD was defined. Treatment-related adverse events (TRAEs) occurred in 146/207 (70.5%) pts, most commonly fatigue (19%) and nausea (11%). The rate of Grade ≥ 3 TRAEs was 23.2%. Immune-related AEs were consistent with events observed with anti-PD-1 antibodies. Mean half-life was 11 days; peripheral blood flow cytometry analyses confirmed full and sustained on-target binding during treatment at doses ≥ 120 mg. Among 41 response-evaluable [RE] dose escalation pts, 3 confirmed partial responses [cPRs] (triple negative breast cancer [TNBC], mesothelioma, gastric cancer) per RECIST 1.1 were observed, while 21 pts had stable disease [SD]. Among select expansion cohorts, PRs have been observed in epithelial ovarian cancer (n=2; both cPRs, and 7 with SD among 15 RE pts) and TNBC (n=2; 1 cPR, 1 unconfirmed PR [uPR], and 5 with SD among 14 RE pts). In a cohort of pts with HER2+ tumors treated with MGD013 in combination with margetuximab (investigational anti-HER-2 antibody), 3 PRs have been observed (breast [n=2], colorectal [n=1]; 1 cPR, 2 uPRs) and 2 pts with SD among 6 RE pts. Objective responses have been observed in several pts after prior anti-PD-1 therapy. Investigations into potential correlative biomarkers including LAG-3 and PD-1 are ongoing. Conclusions: MGD013, a novel molecule designed to coordinately block PD-1 and LAG-3, has demonstrated an acceptable safety profile and encouraging early evidence of anti-tumor activity. Clinical trial information: NCT03219268 .

e15086 Background: Concomitant blockade of LAG-3 and PD-1 has been shown to restore T-cell activation and enhance anti-tumor immunity in preclinical models. Clinical trials evaluating anti-LAG-3/anti-PD-1 mAb combinations or bispecific molecules are ongoing. Establishing the tumor expression profile of LAG-3 and its relationship to PD-1 will aid in patient selection and interpretation of clinical responses to combined inhibition. Methods: LAG-3 Ab clone EPR4392(2) (Abcam) and PD-1 Ab clone NAT105 (Ventana) were developed as individual or combined dual IHC assays on the Ventana Discovery Ultra platform. LAG-3 IHC was performed on panels of commercially available tissues: 33 normal human tissues (2 each) and various tumor types including anal (n = 39), cervical cancer (40), cholangiocarcinoma (11), DLBCL (40), gastric cancer (34), HCC (18), SCCHN (39), HER2-positive breast cancer (48), mesothelioma (40), NSCLC (39), ovarian cancer (47), SCLC (48), and TNBC (40). LAG-3 IHC expression was confirmed by in situ hybridization (ACD RNAScope). Positivity was defined as at least one LAG-3+ve or PD-1+ve tumor-infiltrating lymphocyte (TIL) per 40x magnification hot spot field (HSF); high LAG-3 expression was defined as > 15 LAG-3+ve TILs per HSF. Results: LAG-3-specific staining in normal tissues was limited to mononuclear cells in lymphoid organs and GALT, with no staining observed in normal parenchyma. Across various tumor types, the frequency of LAG-3+ve tumor samples and high expression, respectively (%, %), were as follows: cervical (100; 57.5), anal (97.4; 30.8), DLBCL (95; 75), NSCLC (92.3; 25.6), TNBC (90; 22.5), gastric (88.2; 44.1), SCCHN (87.2; 56.4), HER2-positive breast (85.4; 39.6), HCC (83.3; 22.2), cholangiocarcinoma (81.8; 9.1), ovarian (70.2; 21.3), SCLC (50; 14.6) and mesothelioma (25; 10). In addition to TILs, LAG-3 expression was observed on tumor cells in DLBCL and a fraction of anal, breast, cervical, gastric, hepatocellular, SCCHN, NSCLC and ovarian cancers. LAG-3/PD-1 double IHC staining revealed dual positivity across 92.3% of NSCLC samples, with approximately 60% demonstrating TIL co-expression of PD-1 and LAG-3. Conclusions: LAG-3 expression was detected on TILs across a broad range of solid tumors and DLBCL, with varying level of intensity or association with PD-1 expression. Correlative assessments of LAG-3/PD-1 expression with clinical responses to MGD013, an investigational bispecific DART(R) molecule targeting LAG-3/PD-1 (NCT03219268), will be undertaken.

Many patients with B-cell malignancies can be successfully treated, although tumor eradication is rarely achieved. T-cell-directed killing of tumor cells using engineered T-cells or bispecific antibodies is a promising approach for the treatment of hematologic malignancies. We investigated the efficacy of CD19xCD3 DART bispecific antibody in a broad panel of human primary B-cell malignancies. The CD19xCD3 DART identified 2 distinct subsets of patients, in which the neoplastic lymphocytes were eliminated with rapid or slow kinetics. Delayed responses were always overcome by a prolonged or repeated DART exposure. Both CD4 and CD8 effector cytotoxic cells were generated, and DART-mediated killing of CD4+ cells into cytotoxic effectors required the presence of CD8+ cells. Serial exposures to DART led to the exponential expansion of CD4 + and CD8 + cells and to the sequential ablation of neoplastic cells in absence of a PD-L1-mediated exhaustion. Lastly, patient-derived neoplastic B-cells (B-Acute Lymphoblast Leukemia and Diffuse Large B Cell Lymphoma) could be proficiently eradicated in a xenograft mouse model by DART-armed cytokine induced killer (CIK) cells. Collectively, patient tailored DART exposures can result in the effective elimination of CD19 positive leukemia and B-cell lymphoma and the association of bispecific antibodies with unmatched CIK cells represents an effective modality for the treatment of CD19 positive leukemia/lymphoma.

Approximately 40% of patients (pts) with newly diagnosed (AML) either fail to achieve complete remission with intensive induction therapy or experience disease recurrence after a short remission duration (< 6 months). These pts, herein considered to have primary refractory disease, are an extremely challenging population to treat, with only 14% achieving remission following conventional chemotherapy and with subsequent salvage attempts being nearly universally ineffective (1). Increased immune infiltration of the tumor microenvironment (TME) and high CD123 expression on AML blasts have been associated with primary induction failure and poor prognosis (2, 3). Flotetuzumab (FLZ), a CD123 x CD3 bispecific DART molecule, is currently being tested in a phase 1/2 study in pts with either relapsed or refractory (R/R) AML. We have previously reported FLZ activity in primary refractory AML (4); herein, we provide additional scientific rationale supporting the investigation of FLZ in this patient population. The recommended Phase 2 dose (RP2D) of FLZ identified in an ongoing Phase 1/2 study is 500 ng/kg/day administered as a 7 -day/week continuous infusion. Pts receive a lead-in dose during week (W) 1, followed by 500 ng/kg/day during W2-4 of Cycle 1, and a 4-day on/3-day off schedule for Cycle 2 and beyond. Disease status was assessed by modified IWG criteria; bone marrow (BM) samples were collected to investigate biomarkers, including CD123 receptor density (RD), and gene expression profiling using the NanoString PanCancer IO 360™ panel, which measures the expression of 770 genes, including 14 immune cell types and 32 immuno-oncology biological signatures. Gene expression comparisons are presented at fold change (FC) and a t-test was used for statistical analysis. Fifty pts with R/R AML received FLZ at the RP2D. Thirty (60%) pts had primary refractory AML: 24 failed ≥2 induction attempts and 6 recurred after remission of 30% decrease in BM blasts) was associated with increased baseline immune gene signatures, including significantly higher IFNγ scores (1.8 FC, p=0.0212; AUROC=0.74), and an increased tumor inflammation signature (TIS) score (1.658 FC, p=0.00827, AUROC=0.847 compared to non-responders. FLZ was well tolerated, with no increased cytokine release syndrome events in primary refractory pts (30% G1, 67% G2, 3% G3) compared to relapse pts (26% G1, 58% G2, 16% G3), notwithstanding the increased CD123 RD in the former. In conclusion, we show that FLZ elicits clinical response in heavily treated patients with poor response rates to primary therapy. We also show that increased IFNγ signaling gene expression scores in baseline BM appears to associate with response to FLZ therapy. Enrollment has been expanded to further define FLZ's activity specifically in pts with primary refractory AML, and candidate biomarkers to enable identification of pts more likely to respond to FLZ. Estey E, et al. Blood (1996), 88 (2) 756Vadakekolathu J, et al. Blood (2017) 130:3942Vergez F, et al. Haematologica (2011), 96(12):1792-8Uy GL, et al. Blood (2018) 132:764Duong V, et al. Leukemia (2013),13(6):711-5 Disclosures Uy: Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Aldoss:Agios: Consultancy, Honoraria; AUTO1: Consultancy; Jazz Pharmaceuticals: Honoraria, Other: travel/accommodation/expenses, Speakers Bureau; Helocyte: Consultancy, Honoraria, Other: travel/accommodation/expenses. Foster:Bellicum Pharmaceuticals, Inc: Research Funding; Daiichi Sankyo: Consultancy; MacroGenics: Research Funding; Celgene: Research Funding. Sallman:Celgene: Research Funding, Speakers Bureau; Celyad: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Jazz: Research Funding; Novartis: Speakers Bureau; Abbvie: Speakers Bureau. Sweet:Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Stemline: Consultancy; Pfizer: Consultancy; Jazz: Speakers Bureau; Incyte: Research Funding. Rizzieri:AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; Stemline: Research Funding. Advani:Amgen: Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Emadi:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Research Funding; Genentech: Consultancy, Honoraria; KinaRx: Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and Scientific Advisor, Patents & Royalties; Jazz Pharmaceuticals: Research Funding. Wieduwilt:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen, Leadiant, Merck, Servier: Research Funding; Reata Pharmaceuticals: Equity Ownership. Vey:Novartis: Consultancy, Honoraria; Janssen: Honoraria. Church:NanoString Technologies, Inc.: Employment, Equity Ownership. Rettig:WashU: Patents & Royalties: Patent Application 16/401,950. Arellano:Gilead: Consultancy. Löwenberg:Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees. Ravandi:Selvita: Research Funding; Xencor: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Macrogenix: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cyclacel LTD: Research Funding. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Tran:MacroGenics: Employment. Timmeny:MacroGenics, Inc.: Employment, Other: Stock Ownership. Topp:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Guo:Macrogenics: Employment. Zhao:MacroGenics, Inc.: Employment. Wigginton:MacroGenics, Inc.: Employment, Equity Ownership; Western Oncolytics: Other: clinical advisory board. Bonvini:MacroGenics, Inc.: Employment, Equity Ownership. Rutella:NanoString Technologies, Inc.: Research Funding; MacroGenics, Inc.: Research Funding. Walter:Seattle Genetics: Research Funding; Race Oncology: Consultancy; Pfizer: Consultancy, Research Funding; New Link Genetics: Consultancy; Kite Pharma: Consultancy; Agios: Consultancy; Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership. DiPersio:Amphivena Therapeutics: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau; Magenta Therapeutics: Equity Ownership; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Celgene: Consultancy; Incyte: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Karyopharm Therapeutics: Consultancy.

Background: Cytokine release syndrome (CRS) management in acute myeloid leukemia (AML) patients treated with flotetuzumab, an investigational CD123xCD3 bispecific DART® molecule for T cell redirected therapy. CRS is a hallmark of T cell activating therapy and can be correlated with efficacy, specifically, with CAR-T cells(1). Identification of patients at risk for high grade CRS will help guide CRS management. Flotetuzumab (MGD006) is anovel CD123xCD3 bispecific DART® molecule in Phase 1/2 testing in patients with relapsed/ refractory AML. Several strategies have been successfully employed to mitigate CRS severity, some have been previously reported (2, 3). Here we report on further refinement of CRS management and subsequent investigation of potentiel predictive biomarkers of severity. Methods: The recommended phase 2 dose (RP2D) of flotetuzumab is 500ng/kg/d CIV. Week 1 comprises a step-wise lead-in dose (LID) (1-step: 100 ng/kg/day days 1-4; 2-step: 30ng/kg/d for 3days, 100ng/kg/d for 4days, or multi-step (MS) LID at 30, 60, 100, 200, 300, 400 and 500 ng/kg/day each for 24 hours) in order to improve flotetuzumab tolerability. Tocilizumab usage recommended early in CRS management. The relationships between immune cells (T-cell subsets, monocytes) and tumor burden (percent CD123+ AML blasts, CD123 expression) were further interrogated as potential determinants of CRS. Results: 50 patients have been treated at the RP2D. While almost all patients experienced IRR/CRS events, the majority of these patients experienced IRR/CRS that were mild-moderate in severity (28% Grade(G)1, 62% G2, and 8% G3), of short duration (median 1 day for G1, 2 days G2, 2.5 days G3), and resolved completely with no clinical sequalae reported. Most CRS events occured in the first week of treatment (38.3%) and gradually decreased with continuous dosing (24.8%, 7.4%, and 4.3% during weeks 2-4, respectively). Several key interventions have helped mitigate CRS severity. Sequential increment in steps of LID schedules (1 step, 2-step or multi-step LID) have successfully decreased CRS severity and incidence. For example, CRS mean grade±SEM for week 1 was 2.0±0.26 vs 1.4±0.72 vs 1.5±0.63 and for week 4, 0.67±0.42 vs 0.2 ±0.50 vs 0.1 ±0.50 (1 step, 2-step or multi-step LID, respectively). Moreover, LID improved overall tolerability. Introduction of early use of tocilizumab has helped forestall CRS development; 27 patients received tocilizumab (10 doses for G1, 27 for G2, and 2 for G3 events), only 5 pts have required steroids (4 for G2 and 1 for G3), and no pts have required vasopressor support. Blunting of CRS events did not impact antileukemic activity. CRS severity showed a relationship with baseline frequency of circulating CD4+ cells (mean 0.2 K/µL in patients with no CRS vs. 1.0 K/µL in G1 vs 1.6 K/µL in G ≥2, p < 0.000.1), and peak CRS grade in week 1. Conclusion: Like other T-cell activating therapies, flotetuzumab is associated with CRS. Several mitigating factors have helped to blunt the severity of CRS, including lead-in dosing and early tocilizumab usage. Circulating CD4+ cells at baseline continues to be associated with CRS risk, and may be a helpful marker to identify patients at increased risk for CRS. 1. Maude, SL. et al. Managing Cytokine Release Syndrome Associated With Novel T Cell-Engaging Therapies. Cancer J. 2014; 20(2): 119-122. 2. Jacobs, K, et al.Lead-in Dose Optimization to Mitigate Cytokine Release Syndrome in AML and MDS Patients Treated with Flotetuzumab, a CD123 x CD3 Dart® Molecule for T-Cell Redirected Therapy. Blood 2017 130:3856. 3. Jacobs, K, et al.Management of Cytokine Release Syndrome in AML Patients Treated with Flotetuzumab, a CD123 x CD3 Bispecific Dart® Molecule for T-Cell Redirected Therapy. Blood 2018 132:2738. Disclosures Bakkacha: Macrogenics,Inc: Employment, Equity Ownership. Uy:Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Aldoss:Helocyte: Consultancy, Honoraria, Other: travel/accommodation/expenses; AUTO1: Consultancy; Jazz Pharmaceuticals: Honoraria, Other: travel/accommodation/expenses, Speakers Bureau; Agios: Consultancy, Honoraria. Foster:Bellicum Pharmaceuticals, Inc: Research Funding; Daiichi Sankyo: Consultancy; MacroGenics: Research Funding; Celgene: Research Funding. Sallman:Celyad: Membership on an entity's Board of Directors or advisory committees. Sweet:Pfizer: Consultancy; Incyte: Research Funding; Agios: 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; Celgene: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Consultancy; Jazz: Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Rizzieri:Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding. Advani:Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Emadi:Genentech: Consultancy, Honoraria; KinaRx: Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and Scientific Advisor, Patents & Royalties; NewLink Genetics: Research Funding; Jazz Pharmaceuticals: Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Wieduwilt:Reata Pharmaceuticals: Equity Ownership; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen, Leadiant, Merck, Servier: Research Funding. Vey:Novartis: Consultancy, Honoraria; Janssen: Honoraria. Arellano:Gilead: Consultancy. Löwenberg:Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Frame Pharmaceuticals: Equity Ownership; Hoffman-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherland: Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; CELYAD: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ravandi:Cyclacel LTD: Research Funding; Menarini Ricerche: Research Funding; Selvita: Research Funding; Xencor: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Macrogenix: Consultancy, Research Funding. Tran:MacroGenics: Employment. Muth:MacroGenics, Inc.: Employment, Equity Ownership. Baughman:MacroGenics, Inc.: Employment, Equity Ownership. Timmeny:MacroGenics, Inc.: Employment, Other: Stock Ownership. Topp:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Guo:Macrogenics: Employment. Zhao:MacroGenics, Inc.: Employment. Wigginton:macrogenics: Employment, Equity Ownership; western oncolytics: Consultancy, Other: consultancy. Bonvini:MacroGenics, Inc.: Employment, Equity Ownership. Walter:Daiichi Sankyo: Consultancy; Amgen: Consultancy; Agios: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Boehringer Ingelheim: Consultancy; Pfizer: Consultancy, Research Funding; Race Oncology: Consultancy; Seattle Genetics: Research Funding; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Consultancy; New Link Genetics: Consultancy. Davidson:Macrogenics,Inc: Employment, Equity Ownership. DiPersio:Incyte: Consultancy, Research Funding; Celgene: Consultancy; Karyopharm Therapeutics: Consultancy; Bioline Rx: Research Funding, Speakers Bureau; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Equity Ownership; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Amphivena Therapeutics: Consultancy, Research Funding; NeoImmune Tech: Research Funding; Macrogenics: Research Funding, Speakers Bureau. Jacobs:Macrogenics,Inc: Employment, Equity Ownership.

Background Acute myeloid leukemia (AML) blasts and leukemic stem and progenitor cells typically express higher levels of CD123 than their normal hematopoietic stem cell counterparts, making CD123 an attractive target. Leukemic CD123 expression is associated with poor prognosis, high-risk disease, and increased risk of induction failure (Vergez et al 2011). Single-agent flotetuzumab, an investigational CD123 x CD3 bispecific DART protein, has shown evidence of clinical activity in a Phase 1 study of relapsed/refractory (R/R) acute myeloid leukemia (AML). In this study, flotetuzumab led to T-cell activation which in turn was associated with PD-1 induction on T lymphocytes, enhanced IFNɣ secretion, and upregulation of PD-L1 expression by AML blasts (ASH 2018 Rutella, ASH 2018 Uy). In vitro studies have shown synergistic T-cell mediated cytotoxicity of an AML cell line (KG1A) with flotetuzumab in the presence of PD-1/PD-L1 axis blockade compared to flotetuzumab alone. MGA012, also known as INCMGA00012, is an investigational anti-PD-1 antibody that has shown clinical activity in a Phase 1 study (SITC 2018 Mehnert). We hypothesize that combined checkpoint inhibition with MGA012 together with redirected T‐cell killing of CD123+ cells with flotetuzumab may show enhanced activity over flotetuzumab alone. Methods This is a Phase 1 dose escalation study designed to characterize the safety, tolerability, dose-limiting toxicities, maximum tolerated dose (MTD) or maximum administered dose (if no MTD is defined), pharmacokinetics, and preliminary anti-leukemic activity of flotetuzumab in combination with MGA012, each administered intravenously (IV) in patients with R/R AML. Response evaluation will be determined by modified ELN 2017 criteria. Activity is measured by complete response (CR) (complete remission [CR], or CR with partial hematologic recovery [CRh], or CR with incomplete hematological recovery [CRi], or morphologic leukemia-free state [MLFS]) rate, relapse-free survival, or mortality rate at 1 and 3 months. The impact of flotetuzumab/MGA012 combination on overall survival and event-free survival will be explored. Eligible patients will consist of adults with relapsed or refractory AML (any subtype except acute promyelocytic leukemia) who have exhausted standard of care options. In Induction Cycle 1, patients will be treated with a step-up lead in dose of flotetuzumab, followed by continuous infusion flotetuzumab, starting at week 2 of Cycle 1 and continuing through each 28-day cycle. MGA012 will be administered every two weeks. Depending on response, patients will transition to either consolidation or second induction. Eligible patients who achieve CR/CRh/CRi can receive maintenance MGA012 alone for up to 12 months. Disclosures Wei: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. Fong:Novartis: Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Astellas: Consultancy. Montesinos:Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Research support; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau. Gil:Jazz Pharmaceuticals: Honoraria; Gilead: Honoraria; Daiichi-Sankyo: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Abbvie: Honoraria. Perez De Oteyza:Celgene: Speakers Bureau. Rowe:BioSight: Consultancy. Wolach:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Speaker; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Speaker. Sun:MacroGenics, Inc.: Employment, Equity Ownership. Baughman:MacroGenics, Inc.: Employment, Equity Ownership. McNulty:MacroGenics, Inc.: Employment, Equity Ownership. Bonvini:MacroGenics, Inc.: Employment, Equity Ownership. Wigginton:Western Oncolytics: Other: clinical advisory board; MacroGenics, Inc.: Employment, Equity Ownership. Davidson-Moncada:MacroGenics, Inc.: Employment, Equity Ownership.

Dysregulation of ADAM9, a member of the ADAM (a disintegrin and metalloproteinase) family of proteases, has been implicated in tumor progression and metastasis, as well as pathological neovascularization. ADAM9 overexpression correlates with poor prognosis in multiple cancers. We have shown that ADAM9 is overexpressed in multiple solid tumor indications and that anti-ADAM9 antibodies are efficiently internalized and degraded by tumor cell lines making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe IMGC936, the first ADAM9-targeting ADC to enter preclinical development. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable peptide linker at a drug-antibody ratio of two. To maximize the potential for IMGC936 activity, the M252Y/S254T/T256E (YTE) mutation was introduced into the CH2 domain of the antibody to increase in vivo plasma half-life and exposure. In vitro studies demonstrated targeted cytotoxicity of IMGC936 across a panel of ADAM9-positve tumor cell lines with activity at least 2 logs greater than a non-targeting conjugate. Consistent with the in vitroactivity, an anti-ADAM9-DM21 conjugate displayed compelling anti-tumor activity in multiple xenograft models representing non-small cell lung, gastric and colorectal cancers. For example, in the EBC-1 non-small cell lung cancer subcutaneous xenograft model with only moderate ADAM9 expression (H-score of 130), anti-ADAM9-DM21 not only induced tumor growth delay but produced complete and durable remissions in 6/6 mice following a single intravenous dose of 8.6 mg Ab/kg (100 ug DM21/kg). IMGC936 demonstrated a favorable pharmacokinetic profile with good conjugate stability in non-human primates. Importantly, IMGC936 was well-tolerated following repeat dosing in cynomolgus monkeys with no ADAM9 target-related toxicities identified at doses exceeding the levels required for anti-tumor activity in murine xenograft models. Based on the totality of the preclinical data, IMGC936 represents a promising therapeutic candidate to target a wide range of ADAM9-expressing tumors. Citation Format: Stuart Hicks, Deryk Loo, Kerstin Sinkevicius, Juniper Scribner, Bhaswati Barat, Nicholas Yoder, Christopher Espelin, Marian Themeles, Francine Chen, Jacquelynn Lucas, Jennifer Brown, Bahar Matin, Megan Fuller, Jenny Lee, Paulin Salomon, Juliet Costoplus, Sadiqa Yancey, Gundo Diedrich, Sergey Gorlatov, Thomas Son, Michael Chiechi, Pam Li, Michael Spliedt, Valentina Ciccarone, Jeff Hooley, Nadia Gantt, James Tamura, Kerry Donahue, Paul Moore, Syd Johnson, Thomas Chittenden, Richard Gregory, Ezio Bonvini. IMGC936, a first-in-class ADAM9-targeting antibody-drug conjugate, demonstrates promising anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1533.

Introduction: CD137 (4-1BB) signaling provides co-stimulation of CD8 or NK cells following antigen or FcγR engagement, respectively. Efforts to leverage CD137 co-stimulation via agonistic monoclonal antibodies (mAbs) have been thwarted by limited clinical efficacy or unacceptable toxicity. Bispecific targeting strategies linking CD137 activation to a tumor-targeting moiety provides an approach to localize CD137 activation to the tumor microenvironment. Here we evaluate a panel of Fc-bearing HER2 x CD137 bispecific molecules incorporating different valency and geometry to define the format providing optimal CD137 co-stimulation in a tumor-cell anchor-dependent manner. Methods: An anti-HER2 mAb specificity that does not cross compete with margetuximab, trastuzumab or pertuzumab and a proprietary anti-CD137 mAb were utilized to assemble a set of HER2 x CD137 bispecific molecules in bivalent and tetravalent DART® or trivalent TRIDENTTM configurations. The resulting molecules were compared in binding, signaling and co-stimulation assays in the presence or absence of tumor cells expressing HER2. Combination studies were performed in vitro and in immune deficient mice reconstituted with human PBMCs. Results: TRIDENT molecules bearing bivalent CD137 and monovalent HER2 binding achieve optimal HER2-dependent tumor-cell anchored CD137 immune cell co-stimulation. CD137 co-stimulation increases proportionally with the level of HER2 expression as observed with HER2 1+ (MCF7 breast), HER2 2+ (JIMT1 breast) and 3+ (N87 gastric) tumor cells and was paralleled with increased HER2+/CD137+ cell association. No CD137 activation is observed in the absence of HER2-expressing tumor cells. HER2 x CD137 bispecifics enhance NK-cell proliferation and IFN-γ release induced by margetuximab, an Fc-optimized anti-HER2 mAb that up-regulates CD137 expression on NK-cells concomitant with enhanced ADCC against HER2-positive cells. Similarly, HER2 x CD137 bispecific molecules enhanced the in vitro activity of orlotamab, a B7-H3 x CD3 bispecific DART molecule that up-regulates CD137 during T-cell redirected killing. Finally, in vivo mouse studies demonstrate the ability of HER2 x CD137 molecules to expand tumor-associated CD8 cells when co-administered with a tumor targeted CD3 bispecific molecule and support enhance anti-tumor activity. Conclusion: An optimal HER2 x CD137 bispecific format providing maximal CD137 activation in a HER2- dependent manner was identified as a trivalent TRIDENT molecule bearing bivalent CD137 and monovalent HER2 binding. Combinatorial activity with HER2 x CD137 bispecifics was observed with both a HER2-directed therapeutic mAb and a CD3-engaging tumor-targeted bispecific molecule. HER2 x CD137 TRIDENT molecules have therapeutic potential and provide a structural template for incorporating alternate tumor- and/or co-stimulatory-targeting arms. Citation Format: Liqin Liu, Chia-Ying K. Lam, Ralph Alderson, Vatana Long, Yinhua Yang, Robert Burns, Lusiana Widjaja, Jonathan Li, Christina Wolf, Valentina Ciccarone, James Tamura, Gundo Diedrich, Ezio Bonvini, Syd Johnson, Paul A. Moore. Selection of a bispecific trivalent HER2 x CD137 TRIDENT format providing optimal tumor-anchored immune co-stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1560.

Introduction: Trophoblast glycoprotein 5T4 is expressed on the cell surface of multiple cancers yet sparingly on normal adult tissues. CD137 (4-1BB) is a co-stimulatory molecule expressed by activated T and NK cells that support cell activation, proliferation and survival. Previously we described bispecific tumor associated antigen x CD137 DART® molecules limiting CD137 mediated immunostimulation to the tumor microenvironment (AACR2017) as an approach to reduce the unwanted systemic CD137 effects associated with agonistic anti-CD137 mAbs. We subsequently identified a bispecific TRIDENT format bearing bivalent CD137 and monovalent tumor antigen engagement that provides maximal CD137 activation in a tumor antigen anchored manner. Here we apply that format to generate a 5T4 x CD137 x CD137 TRIDENT molecule that promotes CD137 activation in proximity to 5T4-expressing tumor cells leading to enhanced T-cell co-stimulation, proliferation and tumor cell killing activity when combined with CD3-engaging tumor-targeting bispecific DART molecules. Methods: TRIDENT molecules were constructed comprising bivalent CD137 binding and monovalent 5T4 binding. Binding properties were evaluated by surface plasmon resonance (SPR) and flow cytometry. Signaling was assessed using a NF-κB luciferase reporter cell line expressing CD137. Co-stimulatory activity was characterized with primary human or cynomolgus monkey T cells. T cells were incubated with or without antigen-expressing cells and submaximal concentrations of either anti-CD3 bead or CD3-engaging tumor-targeting bispecific DART molecules. Results: SPR and flow cytometry analyses demonstrate that 5T4 x CD137 x CD137 TRIDENT molecule binds human and monkey target antigens. The 5T4 x CD137 x CD137 TRIDENT molecule induces CD137 signaling in a reporter cell line and promotes cytokine release in primary human & cynomolgus monkey T-cell assays in the presence of 5T4+ tumor cells. In the absence of 5T4+ tumor cells, the TRIDENT molecule lacks agonistic activity. Furthermore, 5T4 x CD137 x CD137 TRIDENT molecule enhances T-cell proliferation and tumor cell cytolysis induced by CD3-targeted DART molecules. Consistent with preferential induction of CD137 by CD8 T-cells, 5T4 x CD137 x CD137 TRIDENT increases the fraction of CD8+ central memory and effector memory T cells in the presence 5T4+ tumor cells. Conclusions: 5T4 x CD137 x CD137 TRIDENT molecule promotes T-cell co-stimulation in a tumor antigen-dependent manner offering an opportunity to target CD137 immunostimulation, while potentially limiting non-specific systemic T-cell activation and related side effects. Citation Format: Liqin Liu, Ling Huang, Vatana Long, Yinhua Yang, Robert Burns, Jonathan Li, Jennifer DiChiara, Qihong Xu, Arin Whiddon, Chia-Ying K Lam, Jim Tamura, Valentina Ciccarone, Syd Johnson, Ezio Bonvini, Paul Moore. Tumor-antigen 5T4-dependent activation of the CD137 costimulatory pathway by bispecific 5T4 x CD137 x CD137 TRIDENT™ molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 554.

1030 Background: Previous studies have shown that 44-71% of trastuzumab (T)-treated pts develop HER2-specific immunity (Clin Cancer Res 2007, 13:5133; Cancer Res 2016, 76:3702; Breast Cancer Res 2018, 20:52). M is an Fc-engineered mAb that shares similar HER2 binding and antiproliferative activity as T. The Fc region of M has been engineered for increased affinity to the activating FcγRIIIA (CD16A) and lower binding to the inhibitory FcγRIIB (CD32B), attributes that may enhance the mAb’s immune function, such as antigen presentation. Methods: HER2+ cancer pts who progressed on prior therapy received M (0.1-6 mg/kg QW for 3 of every 4 weeks [N = 34]; or 10-18 mg/kg Q3W [N = 32]) in phase 1 trial NCT01148849. PBMC and plasma were collected pre-dose and 50 days post-dose for 46 pts and > 4 years for 3 pts on long-term treatment. Response to HER2 or control antigens (Ag) was assessed by IFNγ ELISpot and antibody (Ab) ELISA. In 14 pts, T-cell antigen receptor (TCR) repertoire was assessed by immunosequencing. Results: Following M treatment, mean frequencies of IFNγ-producing T cells specific for intra- or extracellular fragments of HER2 increased by 2.5 to 6-fold (p < 0.0027, paired t test). Most (95%) of subjects responded to ≥2 of 6 (median = 5) HER2 Ag. Mean HER2-specific Ab concentration increased by 19-54% (p < 0.0001), with all subjects responding to ≥2 (median = 5) of the 6 Ag. A small 1.6-fold increase in IFNγ response to control CMV/EBV/Flu (but not tetanus or cyclin D1) peptides was observed; no increase in Ab response to control Ag was noted. Subsets of HER2-specific T-cell and Ab responses persisted during long-term treatment. Median TCR clonality increased by 54% (p = 0.003), with an average of 125 unique clones expanding, while overall TCR diversity remained unchanged (p = 0.19). Conclusions: Treatment of HER2+ cancer with M was associated with enhanced HER2-specific T-cell and Ab responses together with increased TCR clonality, indicative of a more focused T-cell repertoire. The high frequency of HER2-specific immunity in M-treated patients ( > 95%) is consistent with its enhanced Fc region contributing to linkage of innate and adaptive immune responses.

Protege was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC andgt;0.2 nmol/L, those randomized 6 weeks after diagnosis, HbA(1c) andlt;7.5% (58 mmol/mol), insulin use andlt;0.4 units/kg/day, and 8-17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose. Funding Agencies|MacroGenics, Inc (Rockville, MD)||Johnson Johnson||Tolerx||MacroGenics||JDRF|2008-502|Le Bonheur Foundation (Memphis, TN)

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

Fc receptors are a critical component of the innate immune system responsible for the recognition of cross-linked antibodies and the subsequent clearance of pathogens. However, in autoimmune diseases, these receptors play a role in the deleterious action of self-directed antibodies and as such are candidate targets for treatment. GMA161 is an aglycosyl, humanized version of the murine antibody 3G8 that targets the human low-affinity Fcγ receptor III (CD16). As CD16 expression and sequence have high species specificity, preclinical assessments were conducted in mice transgenic for both isoforms of human CD16, CD16A, and CD16B. This transgenic mouse model was useful in transitioning into phase I clinical trials, as it generated positive efficacy data in a relevant disease model and an acceptable single-dose safety profile. However, when GMA161 or its murine parent 3G8 were dosed repeatedly in transgenic mice having both human CD16 isoforms, severe reactions were observed that were not associated with significant cytokine release, nor were they alleviated by antihistamine administration. Prophylactic dosing with an inhibitor of platelet-activating factor (PAF), however, completely eliminated all signs of hypersensitivity. These findings suggest that (1) GMA161 elicits a reaction that is target dependent, (2) immunogenicity and similar adverse reactions were observed with a murine version of the antibody, and (3) the reaction is driven by the atypical hypersensitivity pathway mediated by PAF.

Summary Background Findings of small studies have suggested that short treatments with anti-CD3 monoclonal antibodies that are mutated to reduce Fc receptor binding preserve β-cell function and decrease insulin needs in patients with recent-onset type 1 diabetes. In this phase 3 trial, we assessed the safety and efficacy of one such antibody, teplizumab. Methods In this 2-year trial, patients aged 8–35 years who had been diagnosed with type 1 diabetes for 12 weeks or fewer were enrolled and treated at 83 clinical centres in North America, Europe, Israel, and India. Participants were allocated (2:1:1:1 ratio) by an interactive telephone system, according to computer-generated block randomisation, to receive one of three regimens of teplizumab infusions (14-day full dose, 14-day low dose, or 6-day full dose) or placebo at baseline and at 26 weeks. The Protege study is still underway, and patients and study staff remain masked through to study closure. The primary composite outcome was the percentage of patients with insulin use of less than 0·5 U/kg per day and glycated haemoglobin A 1c (HbA 1C ) of less than 6·5% at 1 year. Analyses included all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00385697. Findings 763 patients were screened, of whom 516 were randomised to receive 14-day full-dose teplizumab (n=209), 14-day low-dose teplizumab (n=102), 6-day full-dose teplizumab (n=106), or placebo (n=99). Two patients in the 14-day full-dose group and one patient in the placebo group did not start treatment, so 513 patients were eligible for efficacy analyses. The primary outcome did not differ between groups at 1 year: 19·8% (41/207) in the 14-day full-dose group; 13·7% (14/102) in the 14-day low-dose group; 20·8% (22/106) in the 6-day full-dose group; and 20·4% (20/98) in the placebo group. 5% (19/415) of patients in the teplizumab groups were not taking insulin at 1 year, compared with no patients in the placebo group at 1 year (p=0·03). Across the four study groups, similar proportions of patients had adverse events (414/417 [99%] in the teplizumab groups vs 98/99 [99%] in the placebo group) and serious adverse events (42/417 [10%] vs 9/99 [9%]). The most common clinical adverse event in the teplizumab groups was rash (220/417 [53%] vs 20/99 [20%] in the placebo group). Interpretation Findings of exploratory analyses suggest that future studies of immunotherapeutic intervention with teplizumab might have increased success in prevention of a decline in β-cell function (measured by C-peptide) and provision of glycaemic control at reduced doses of insulin if they target patients early after diagnosis of diabetes and children. Funding MacroGenics, the Juvenile Diabetes Research Foundation, and Eli Lilly.

We describe the application of a novel, bispecific antibody platform termed dual affinity retargeting (DART) to eradicate B-cell lymphoma through coengagement of the B cell–specific antigen CD19 and the TCR/CD3 complex on effector T cells. Comparison with a single-chain, bispecific antibody bearing identical CD19 and CD3 antibody Fv sequences revealed DART molecules to be more potent in directing B-cell lysis. The enhanced activity with the CD19xCD3 DART molecules was observed on all CD19-expressing target B cells evaluated using resting and prestimulated human PBMCs or purified effector T-cell populations. Characterization of a CD19xTCR bispecific DART molecule revealed equivalent potency with the CD19xCD3 DART molecule, demonstrating flexibility of the DART structure to support T-cell/B-cell associations for redirected T cell–killing applications. The enhanced level of killing mediated by DART molecules was not accompanied by any increase in nonspecific T-cell activation or lysis of CD19− cells. Cell-association studies indicated that the DART architecture is well suited for maintaining cell-to-cell contact, apparently contributing to the high level of target cell killing. Finally, the ability of the CD19xTCR DART to inhibit B-cell lymphoma in NOD/SCID mice when coadministered with human PBMCs supports further evaluation of DART molecules for the treatment of B-cell malignancies.

Acute myeloid leukemia (AML) blasts and leukemia stem cells highly express the α chain of the IL-3 receptor (CD123), compared to normal hematopoietic stem cells. CD123 expression is associated with high-risk features, increased risk of induction failure and poor prognosis (Vergez F, et al. Haematologica 2011; 96: 1792-8). Flotetuzumab (FLZ), a CD123 × CD3 bispecific DART molecule, is being tested in a phase 1/2 study in patients with relapsed/refractory (R/R) AML. The recommended Phase 2 dose (RP2D) of FLZ is 500 ng/kg/day (d) administered as a 7-day/week continuous infusion. Patients receive a lead-in dose (30 ng/kg/d x 3d then 100 ng/kg/d x 4d) during week (W) 1, followed by 500 ng/kg/d during W2-4 of cycle 1, and a 4d on/3 d off schedule for cycle 2 and beyond. Disease status was assessed by modified IWG criteria; samples were collected to investigate candidate biomarkers, including CD123 receptor density/cell (RD), and gene expression profiling using the NanoString® PanCancer IO 360™ assay. This platform was used to assess the expression of 770 genes, including 14 immune cell types and 32 immuno-oncology biological signatures in bone marrow (BM) samples from patients treated with FLZ. Thirty patients with R/R AML, median age 64.5 years, received FLZ at the RP2D. Most patients enrolled had primary refractory disease (60% [18/30; 14 to cytotoxic chemotherapy (refractory to ≥ 2 induction attempts, or first CR with initial CR In conclusion, among this initial cohort of patients treated at the RP2D (500 ng/kg/day), FLZ demonstrates antileukemic activity with an acceptable safety profile, in particular, among refractory patients, a difficult-to-treat patient population that represents a significant area of unmet medical need. Enrollment to the current study has now been expanded to further define the antileukemic activity of FLZ in patients with refractory AML, and investigate candidate biomarkers to enable identification of patients more likely to respond to FLZ. In addition, studies are now being initiated to investigate opportunities to expand the antileukemic activity of FLZ via combined administration with either concurrent or sequenced anti-PD-1 checkpoint blockade. Disclosures Uy: GlycoMimetics: Consultancy; Curis: Consultancy. Rettig:Novimmune: Research Funding; Amphivena Therapeutics: Research Funding. Foster:Celgene: Research Funding; Macrogenics: Research Funding; Pfizer: Research Funding; Shire: Honoraria. Rizzieri:Arog: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy; Teva: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees. Arellano:Cephalon: Research Funding. Topp:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Boehringer Ingelheim: Research Funding; Regeneron Pharmaceuticals, Inc.: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sweet:Phizer: Consultancy; BMS: Honoraria; BMS: Honoraria; Agios: Consultancy; Jazz: Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy; Astellas: Consultancy; Jazz: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Agios: Consultancy; Celgene: Honoraria, Speakers Bureau; Phizer: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau. Ravandi:Abbvie: Research Funding; Xencor: Research Funding; Astellas Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Jazz: Honoraria; Jazz: Honoraria; Macrogenix: Honoraria, Research Funding; Sunesis: Honoraria; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Orsenix: Honoraria; Macrogenix: Honoraria, Research Funding; Amgen: Honoraria, Research Funding, Speakers Bureau; Xencor: Research Funding; Orsenix: Honoraria; Astellas Pharmaceuticals: Consultancy, Honoraria; Sunesis: Honoraria; Abbvie: Research Funding. Church:NanoString Technologies: Employment. Rutella:NanoString Technologies: Research Funding. Sun:MacroGenics: Employment. Yang:MacroGenics: Employment. Baughman:MacroGenics: Employment. Curtis:MacroGenics: Employment. Timmeny:MacroGenics: Employment. Cali:MacroGenics: Employment. Tran:MacroGenics: Employment. Muth:MacroGenics: Employment. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Poirot:Servier: Employment. Pallis:Servier: Employment. Cesano:NanoString Technologies: Employment. Bonvini:MacroGenics: Employment, Equity Ownership. Wigginton:MacroGenics: Employment. Lowenberg:Astex: Consultancy; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity's Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity's Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity's Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity's Board of Directors or advisory committees; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity's Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity's Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity's Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity's Board of Directors or advisory committees; Editorial Board "European Oncology & Haematology": Membership on an entity's Board of Directors or advisory committees. Davidson-Moncada:MacroGenics: Employment.

Background: CD3-engaging DART molecules direct T cells to tumor-expressed antigens. Flotetuzumab, a CD123 x CD3 bispecific DART molecule that targets the differential expression of the IL-3 receptor alpha chain (CD123) on acute myeloid leukemia (AML) blasts and leukemic stem cells, is currently being investigated in a phase 1 study in relapsed/refractory AML and myelodysplastic syndrome (MDS), with initial evidence of clinical activity1. A common adverse event in redirected T-cell killing interventions is cytokine release syndrome (CRS), a Cmax-associated event that may limit their therapeutic window. Owing to its short circulating half-life, flotetuzumab is administered as continuous infusion, which affords prolonged exposure with limited Cmax excursions. In contrast, long-acting Fc-bearing, CD3-engaging molecules would eliminate the need for continuous dosing, but would be especially impacted by CRS, given the high Cmax levels required to maintain adequate trough concentrations over several days or weeks. We previously reported the development of a CD3-engaging DART molecule with reduced affinity for CD3 that maintained maximal target cell killing and T-cell proliferation, albeit at higher concentrations than its wild-type (WT) counterpart, but with reduced cytokine release2. We report here further optimization and validation of the approach. Methods: Anti-CD3 variants spanning a range of affinities were formatted as Fc-bearing DART molecules with CD123- or CD19-targeting arms and evaluated for cell binding as well as for antigen-dependent induction of T-cell proliferation, cytolytic activity and cytokine release. A therapeutic index (TI) was determined from the ratio of in vitro cell killing activity to that of target cell-induced cytokine release. In vivo anti-tumor activity was assessed in human immune cell-reconstituted mouse tumor models or in mice expressing the epitope recognized by the CD3 arm of the DART proteins (human CD3 k/i mice). Safety, pharmacokinetic (PK) and biological activity of selected molecules was evaluated in cynomolgus monkeys. Results: Six Fc-bearing DART molecules (from a panel of 23 CD123 x CD3 DART variants) were selected for characterization. These molecules spanned a relatively small interval of low CD3 affinities, but varied greatly in activity, from inactive at all concentrations tested to being capable of lysing tumor cells, including primary leukemic and MDS blasts, to the same maximal level of killing observed with the parental WT DART molecule. Higher concentrations of the active variants were needed, owing to a deliberate design incorporating decreased CD3 binding. At levels capable of complete tumor cell lysis, active DART variants showed greatly reduced pro-inflammatory cytokine release (e.g., IL-2, IFN-γ and TNF-α) in vitro and in human PBMC-reconstituted mice compared to the WT version. Molecules were ranked based on their TI and a version with a highly favorable index was engineered as a B-cell targeting CD19 x CD3 DART molecule to conveniently ascertain its biological activity in cynomolgus monkeys. The CD19 x CD3 DART variant demonstrated good tolerability at single doses up to 30 mg/kg. B-cell depletion in the circulation and lymphoid tissues occurred at 1 mg/kg and achieved maximal depletion at ≥10 mg/kg, equivalent to the degree of reduction observed with 0.1 mg/kg of the WT molecule. However, only minimal circulating pro-inflammatory cytokines (including IL-6) were observed at all doses of the DART variant, while a substantial increase in IL-2, IL-6 and IFN-γ (among others) was observed with 0.1 mg/kg of the WT counterpart. The PK profile of the DART variant in cynomolgus monkeys appeared linear in the 1-30 mg/kg interval, with a half-life of 5 to 10 days, consistent with possible clinical dosing at weekly or longer intervals. Conclusions: A next-generation CD3-engaging Fc-bearing DART platform was generated through CD3 arm affinity modulation. These DART variants feature extended PK and an expanded TI suitable for potential clinical development in hematological malignancies. 1. Uy et al, Blood 2017, vol. 130, Suppl 1 637 2. Huang et al, Keystone Symposia: Antibodies as Drugs, 2/25-3/1/2018, Whistler, BC, Canada Disclosures Bonvini: MacroGenics: Employment, Equity Ownership. La Motte-Mohs:MacroGenics: Employment, Equity Ownership. Huang:MacroGenics, Inc.: Employment, Equity Ownership. Lam:MacroGenics, Inc.: Employment, Equity Ownership. Kaufman:MacroGenics, Inc.: Employment, Equity Ownership. Liu:MacroGenics, Inc.: Employment, Equity Ownership. Alderson:MacroGenics, Inc.: Employment, Equity Ownership. Stahl:MacroGenics, Inc.: Employment, Equity Ownership. Brown:MacroGenics, Inc.: Employment, Equity Ownership. Li:MacroGenics, Inc.: Employment, Equity Ownership. Sharma:MacroGenics, Inc.: Employment, Equity Ownership. Tamura:MacroGenics, Inc.: Employment, Equity Ownership. Johnson:MacroGenics, Inc.: Consultancy, Equity Ownership. Moore:MacroGenics, Inc.: Employment, Equity Ownership.

Introduction MGD007 (glycoprotein A33 x CD3), a DART protein designed to redirect T cells to target gpA33-expressing colon cancer, is presently undergoing clinical evaluation (NCT02238805). The gpA33 target was selected based on its universal expression profile across primary and metastatic CRC, including expression on putative cancer stem cell (CSC) populations. MGD007 activity in CRC cell cytolysis and its prolonged PK in non-human primates have previously been reported (Cancer Res 2014;74(19 Suppl): Abstract nr 669.1). Here we further characterize MGD007 cellular mechanisms associated with redirected T-cell killing, cytokine responses and modulation with steroids. Method Redirected killing assays were performed using luciferase labelled gpA33+ve Colo205 or RECA0201-GF colorectal cancer stem-like cells (CSLC) with freshly isolated PBMC or fractionated T-cell populations; Treg cells (CD4+, CD127lo, CD25+) were expanded for 14 days in presence of IL-2 and rapamycin and confirmed to be suppressive; steroids (budesonide and dexamethasone) were evaluated at pharmacologically relevant concentrations; multi-parameter FACS and ELISA were performed to determine cell surface marker expression and cytokine levels respectively. Results MGD007 displays potent redirected T-cell killing of gpA33+ve CRC cells, including complete lysis of CRC stem cell-like models. Importantly, MGD007 mediated cytolysis can be supported by various T-cell populations, including Treg cells. Following prolonged in vitro exposure to MGD007 and gpA33+ve tumor cells, expanded T cells acquire a memory phenotype and retain potent CTL activity when challenged with fresh gpA33+ve target cells; however, much decreased cytokine release was observed compared to that observed following initial T-cell exposure. The addition of dexamethasone or budesonide to freshly isolated effector cells and gpA33+ve target cells also reduces cytokine release levels to baseline in the presence of MGD007, with minimal impact observed on MGD007-mediated killing. Discussion MGD007 supports targeted lysis of CRC, including CSC subpopulations, and can leverage suppressive Tregs in addition to conventional T cells for cytolytic activity. Biological activity modulation is also feasible through induction of cytolytic Tmem cells with diminished cytokine release potential via sequential exposure to MGD007 or the simultaneous exposure to low-dose steroids. These data support further clinical development of MGD007 for the treatment of CRC patients. Citation Format: Paul A. Moore, Yinhua Yang, Kalpana Shah, Daorong Liu, Vatana Long, Douglas H. Smith, Zhuangyu Pan, Ralph Alderson, Liqin Liu, Syd Johnson, Ezio Bonvini. Immunotherapy of colorectal cancer by the T-cell targeted DART® protein MGD007: Cellular mechanisms of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1772.

TPS2601Background: T cells naturally undergo activation-induced upregulation of co-inhibitory pathways, which may limit the antitumor immune response. Blocking these inhibitory pathways may enhance...

The majority of human subjects who receive subcutaneous allergen immunotherapy (IT) develop decreased sensitivity to their allergens. Multiple factors may explain the efficacy of IT, some evidence support a role for allergen specific IgG antibodies. There is controversy whether such antibodies act by blocking allergen binding to IgE or initiation of active inhibitory signaling through low affinity IgG receptors (FcgammaRIIB) on mast cells and basophils. In this study, we addressed this question using peripheral blood from cat non-allergic, cat allergic, and immunotherapy-treated cat allergic subjects. Blood from subjects who received IT contain IgG antibodies that mediate inhibition of basophil activation by a mechanism that is blocked by antibodies specific for the inhibitory IgG receptor FcgammaRIIB. Surprisingly, inhibition was also blocked by aglycosylated, putatively non-FcR binding, antibodies that are specific for the FcgammaRIIA, suggesting a contribution of this receptor to the observed effect. Consistent with a cooperative effect, ex vivo basophils were found to express both IgG receptors. In other studies we found that basophils from subjects who were both chronically exposed to allergen and were producing both cat allergen specific IgE and IgG, are hyporesponsive to allergen. These studies confirm that IgG antibodies produced during IT act primarily by stimulation of inhibitory signaling, and suggest that FcgammaRIIA and FcgammaRIIB function cooperatively in activation of inhibitory signaling circuit. We suggest that under normal physiologic conditions in which only a small proportion of FcepsilonRI are occupied by IgE of a single allergen specificity, FcgammaRIIA co-aggregation may, by providing activated Lyn, be required to fuel activation of inhibitory FcgammaRIIB function.

Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor FcγRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcγRIIB in NIH3T3IRcells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain–containing inositol 5′-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via FcγRIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes.

Despite advances in therapy, many patients with systemic light-chain amyloidosis (AL) die within 3 years from diagnosis. The humanized 2B6 monoclonal antibody (MoAb) is specific for the low-affinity IgG Fc receptor CD32B and effective in a human CD32B+ B-cell lymphoma murine xenograft model. Because MoAb therapy could improve outcomes in AL, we studied CD32B expression by clonal plasma cells obtained from 48 patients with AL. Transcript profiling showed that expression of CD32B was significantly higher than expression of all other Fc receptor family members. Reverse-transcriptase polymerase chain reaction (RT-PCR) using double-enriched CD138+ plasma cells showed uniform expression of the stable cell surface CD32B1 isoform at diagnosis and relapse, and flow cytometry showed intense CD32B cell surface staining on 99% of CD138+ plasma cells at diagnosis and relapse. These data provide a rationale for the novel therapeutic targeting of CD32B using the humanized 2B6 MoAb in patients with systemic AL-amyloidosis.

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) is a key enzyme that governs cellular functions such as gene transcription, secretion, proliferation, motility, and development. Here, we show that PLC-gamma1 is regulated via a novel autoinhibitory mechanism involving its carboxy-terminal Src homology (SH2C) domain. Mutation of the SH2C domain tyrosine binding site led to constitutive PLC-gamma1 activation. The amino-terminal split pleckstrin homology (sPHN) domain was found to regulate the accessibility of the SH2C domain. PLC-gamma1 constructs with mutations in tyrosine 509 and phenylalanine 510 in the sPHN domain no longer required an intact amino-terminal Src homology (SH2N) domain or phosphorylation of tyrosine 775 or 783 for activation. These data are consistent with a model in which the SH2C domain is blocked by an intramolecular interaction(s) that is released upon cellular activation by occupancy of the SH2N domain.

Current therapies for acute myeloid leukemia (AML) are largely ineffective, and AML patients may benefit from targeted immunotherapy approaches. MGD006 is a bispecific CD3xCD123 dual-affinity re-targeting (DART) molecule that binds T lymphocytes and cells expressing CD123, an antigen up-regulated in several hematological malignancies including AML. MGD006 mediates blast killing in AML samples, together with concomitant activation and expansion of residual T cells. MGD006 is designed to be rapidly cleared, and therefore requires continuous delivery. In a mouse model of continuous administration, MGD006 eliminated engrafted KG-1a cells (an AML-M0 line) in human PBMC (peripheral blood mononuclear cell)-reconstituted NSG/β2m(-/-) mice at doses as low as 0.5 μg/kg per day for ~7 days. MGD006 binds to human and cynomolgus monkey antigens with similar affinities and redirects T cells from either species to kill CD123-expressing target cells. MGD006 was well tolerated in monkeys continuously infused with 0.1 μg/kg per day escalated weekly to up to 1 μg/kg per day during a 4-week period. Depletion of circulating CD123-positive cells was observed as early as 72 hours after treatment initiation and persisted throughout the infusion period. Cytokine release, observed after the first infusion, was reduced after subsequent administrations, even when the dose was escalated. T cells from animals with prolonged in vivo exposure exhibited unperturbed target cell lysis ex vivo, indicating no exhaustion. A transient decrease in red cell mass was observed, with no neutropenia or thrombocytopenia. These studies support clinical testing of MGD006 in hematological malignancies, including AML.

Human CD32B (FcγRIIB), the low-affinity inhibitory receptor for IgG, is the predominant Fc receptor (FcR) present on B cells. Immunohistochemical and expression studies have identified CD32B expression in a variety of B-cell malignancies, suggesting that CD32B is a potential immunotherapeutic target for B-cell malignancies. A high-affinity monoclonal antibody (mAb 2B6), from a novel panel of anti–human CD32B–specific mAbs, was chimerized (ch2B6) and humanized (hu2B6-3.5). Both ch2B6 and hu2B6-3.5 were capable of directing cytotoxicity by peripheral blood mononuclear cells and monocyte-derived macrophages against B-lymphoma lines in vitro. In a human B-cell lymphoma mouse xenograft model, administration of ch2B6 or hu2B6-3.5 reduced tumor growth rate and improved tumor-free survival. Both the in vitro and in vivo activities of 2B6 required an intact Fc, suggesting an FcR-mediated mechanism of action. These data support the hypothesis that CD32B is a viable target for mAb treatment of B-cell lymphoproliferative disorders.

Human monocyte-derived DCs (moDCs) and circulating conventional DCs coexpress activating (CD32a) and inhibitory (CD32b) isoforms of IgG Fc gamma receptor (Fc gamma R)II (CD32). The balance between these divergent receptors establishes a threshold of DC activation and enables immune complexes to mediate opposing effects on DC maturation and function. IFN-gamma most potently favors CD32a expression on immature DCs, whereas soluble antinflammatory concentrations of monomeric IgG have the opposite effect. Ligation of CD32a leads to DC maturation, increased stimulation of allogeneic T cells, and enhanced secretion of inflammatory cytokines, with the exception of IL-12p70. Coligation of CD32b limits activation through CD32a and hence reduces the immunogenicity of moDCs even for a strong stimulus like alloantigen. Targeting CD32b alone does not mature or activate DCs but rather maintains an immature state. Coexpression of activating and inhibitory Fc gamma Rs by DCs reveals a homeostatic checkpoint for inducing tolerance or immunity by immune complexes. These findings have important implications for understanding the pathophysiology of immune complex diseases and for optimizing the efficacy of therapeutic mAbs. The data also suggest novel strategies for targeting antigens to the activating or inhibitory Fc gamma Rs on human DCs to generate either antigen-specific immunity or tolerance.

The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis.

We investigated the structural requirements for c-Cbl-mediated inhibition of Ag receptor-induced PLCgamma1 activation. Analysis of site-specific c-Cbl mutants indicated that tyrosine phosphorylation of c-Cbl was required for down-regulation of the PLCgamma1/Ca2+ pathway. Coprecipitation experiments indicated that c-Cbl and PLCgamma1 constitutively interact through a PLCgamma1 SH3 domain-dependent mechanism and that c-Cbl and PLCgamma1 can inducibly interact through the SH2(C) domain of PLCgamma1. Additional data indicate that the SH3 domain of PLCgamma1 binds to both canonical and noncanonical SH3 domain-binding sites in the proline-rich region of c-Cbl. Overexpression of c-Cbl in a PLCgamma-deficient B cell line, P10-14, stably reconstituted with wild-type PLCgamma1 led to a significant decrease in B cell receptor-induced NF-AT-dependent transcription, a PLCgamma- and Ca(2+)-dependent event. In contrast, c-Cbl overexpression in P10-14 cells reconstituted with a PLCgamma1 SH3 domain mutant had little effect on receptor-induced NF-AT activation. These data suggest that c-Cbl-mediated regulation of PLCgamma1 requires an interaction between c-Cbl and PLCgamma1 that is primarily mediated by the SH3 domain of PLCgamma1. The interaction of c-Cbl with PLCgamma1 may negatively effect events required for PLCgamma1 activation.

The oncoprotein 70Z/3 Cbl signals in an autonomous fashion or through blockade of endogenous c-Cbl, a negative regulator of signaling. The mechanism of 70Z/3 Cbl-induced signaling was investigated by comparing the molecular requirements for 70Z/3 Cbl- and TCR-induced phospholipase C gamma 1 (PLC gamma 1) activation. 70Z/3 Cbl-induced PLC gamma 1 tyrosine phosphorylation required, in addition to the PLC gamma 1 N-terminal SH2 domain, the C-terminal SH2 and SH3 domains that were dispensable for TCR-induced phosphorylation. Deletion of the leucine zipper of 70Z/3 Cbl did not eliminate 70Z/3 Cbl-induced PLC gamma 1 phosphorylation, suggesting that blockage of c-Cbl via dimerization with 70Z/3 Cbl cannot fully explain 70Z/3 Cbl activating characteristics. The complete elimination of PLC gamma 1 phosphorylation required deleting the SH3 domain-binding region of 70Z/3 Cbl, consistent with 70Z/3 Cbl binding the PLC gamma 1 SH3 domain. 70Z/3 Cbl-induced PLC gamma 1 phosphorylation required Zap-70, as for the TCR, and the tyrosine kinase binding domain of 70Z/3 Cbl, which binds Zap-70, but did not require PLC gamma 1 binding to Lat, a crucial interaction in TCR-induced PLC gamma 1 phosphorylation. Furthermore, 70Z/3 Cbl-induced activation of NFAT, a PLC gamma 1/Ca(2+)-dependent transcriptional event, required Zap-70, but was independent of Slp-76, an adapter required for TCR-induced NFAT activation. These results suggest that 70Z/3 Cbl and PLC gamma 1 form a TCR-, Lat- and Slp-76-independent complex that leads to PLC gamma 1 phosphorylation and activation.

Background: Acute myeloid leukemia (AML) blast and leukemic stem cells highly express CD123, which is associated with high-risk disease and disease progression. CD123 expression on normal hematopoietic stem cells is minimal, enabling a strategy of preferential ablation of AML with a CD123-targeted approach. Flotetuzumab (MGD006/S80880) is a novel T-cell redirecting (CD123 x CD3) bispecific DART® protein being tested in a phase 1 study in patients with relapsed/refractory (R/R) acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Methods: This phase 1 dose-escalation study is designed to define the safety profile, maximum tolerated dose and schedule (MTDS), and preliminary anti-leukemic activity of flotetuzumab. Relapsed/refractory (R/R) AML or intermediate-2/high-risk MDS patients (pts) are treated on 28-day cycles at doses from 3-1000ng/kg/day on one of 2 dosing schedules (4-day on/3-day off or a continuous 7-day on schedule). To mitigate cytokine-release syndrome (CRS), a one-step lead-in dose (LID) (100ng/kg/day for 4 days) or two-step LID (30 ng/kg/day for 3 days followed by 100ng/kg/day for 4 days) was instituted during Cycle 1/Week 1 (C1W1), followed by the cohort target dose (300-1000ng/kg/day) on either of the dosing schedules on W2-4. Cycle 2 and beyond, all pts were treated on a 4-day on/3-day off schedule at the cohort target dose for a maximum of 12 cycles, with 2 cycles after a complete response or complete remission with incomplete blood count recovery. Steroid-sparing, anti-cytokine therapy was used, if clinically indicated, to manage CRS symptoms. Disease status was assessed by International Working Group (IWG) criteria. Samples were collected for pharmacokinetics, anti-drug antibodies (ADA) and cytokine analysis, including IL-2, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma and GM-CSF. Results: 45 pts with R/R AML/MDS (89% AML and 11% MDS) have been treated with flotetuzumab, median age of 64 (29-84), and 44% female. The MTDS has been reached at 500ng/kg/day. Overall flotetuzumab has demonstrated manageable toxicity; drug-related adverse events ≥G3 were observed in 20/45 (44%) pts; infusion-related reaction/CRS (IRR/CRS), the most common toxicity, was observed in 34/45 (76%) pts (G3 in 6/45, 13%). The most frequent CRS symptoms were pyrexia (15), chills (10), tachycardia (10), and hypotension (4). Cytokine levels were higher in pts with CRS than in pts without (median IL-6, 116.2 vs. 67.9 pg/mL; IL-8, 191.1 vs. 144.6 pg/mL; IL-10, 867.6 vs. 348.7 pg/mL) and were generally higher with increasing CRS grade. A two-step LID during week 1 appeared to decrease the severity of CRS grade (mean grade reduction 0.54) compared to pts that received a one-step LID during cycle 1. In addition, lower median peak cytokine levels are observed with two-step LID during W1 and after achieving W2 target dose. Fourteen pts treated at the threshold 500 ng/kg/day dose cohort and beyond (700ng/kg/day dose cohort) have completed at least one cycle of treatment and had a post-treatment bone marrow (BM) biopsy. Anti-leukemic activity was documented in 57% (8/14) pts, 6/14 reached IWG criteria (3 CR, 1 CRi, 1 MLF, 1 PR) for an overall response rate (ORR) of 43%, and 2 pts had stable disease and bone marrow (BM) blast reduction of 20 and 25% from baseline, respectively. Blast reduction occurred rapidly, often within one cycle of therapy, and extended beyond flotetuzumab discontinuation. Multispectral immunohistochemistry analysis of BM samples showed flotetuzumab in situ with a significant increase (in CD-8 T cells (1.58-fold increase, p=0.0013). Consistent with T-cell activation, CD-25, CD-69 and PD-1 upregulation on both CD-4 and CD-8 T-cells was also observed in peripheral blood samples. Conclusions: Flotetuzumab in R/R AML and MDS demonstrated evidence of anti-leukemic activity (ORR 43%) with a manageable safety profile. This program advances an immune-activating agent in treating AML and continues to enroll patients in cohort expansion (24 AML and 24 MDS patients at the MTDS) in the US and Europe. clinicaltrials.gov NCT02152956. Disclosures Uy: Boehringer Ingelheim: Consultancy; GlycoMimetics: Consultancy; Novartis: Consultancy, Other: Travel Suppport. Foster: Macrogenics: Research Funding; Shire: Honoraria; Pfizer: Research Funding; Amgen: Honoraria; Incyte: Honoraria; Celgene: Research Funding; Celator: Research Funding. Arellano: Cephalon Oncology: Research Funding. Rizzieri: Shire: Research Funding; Erytech: Research Funding. Topp: Roche: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel, Research Funding; Macrogenics: Consultancy, Research Funding; Celgene: Other: Travel; Regeneron: Consultancy, Honoraria, Research Funding. Martinelli: Incyte, Pfizer, MSD, Abbvie, J&J, Seattle Genetics, Jazz Pharmaceuticals, Astellas: Consultancy, Other: Advisory Board, Speakers Bureau. Ciceri: GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding. Lelièvre: Institut de recherches international Servier: Employment. La Motte-Mohs: Sunnybrook Health Sciences Centre: Patents & Royalties; MacroGenics: Employment, Equity Ownership, Patents & Royalties. Sun: Macrogenics Inc: Employment, Equity Ownership. Baughman: MacroGenics, Inc.: Employment. Shannon: MacroGenics, Inc.: Employment. Fox: Bristol Myers-Squibb: Consultancy, Research Funding; AstraZeneca/MedImmune: Consultancy, Research Funding; PerkinElmer: Consultancy, Research Funding; Janssen/Johnson and Johnson: Consultancy, Research Funding; Argos: Consultancy; Bayer: Consultancy; Definiens: Consultancy; OncoSec: Consultancy, Research Funding; PrimeVax: Consultancy, Equity Ownership; Peregrine: Consultancy; UbiVac: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder and managing Member of LLC; Ventana/Roche: Consultancy; Viralytics: Consultancy, Research Funding. Bonvini: MacroGenics, Inc.: Employment, Equity Ownership, Research Funding. Wigginton: MacroGenics: Employment, Equity Ownership. Davidson-Moncada: MacroGenics: Employment, Equity Ownership.

Introduction: CD137 (4-1BB) is a co-stimulatory molecule expressed by activated T and NK cells that, upon interaction with its CD137 ligand, further supports cell activation, proliferation and survival. Activation via CD137 holds great promise for cancer immunotherapy; however, current CD137 agonistic interventions are associated with systemic safety concerns. To develop a therapeutic modality that reduces the potential for systemic CD137 effects, we applied the DART® bispecific platform to generate proteins that can induce tumor-antigen dependent T-cell activation. Methods: DART molecules were constructed containing anti-CD137 variable regions together with either anti-HER2 or anti-EphA2 variable regions. DART binding properties were evaluated by ELISA or flow cytometry; signaling responses assessed using a NF-κB luciferase reporter cell line expressing CD137. Co-stimulatory activity was characterized with primary human T cells in the presence or absence of tumor target antigen-expressing cells. Results: ELISA and flow cytometry analysis demonstrated that both HER2 x CD137 and EphA2 x CD137 DART molecules bind their respective target antigens. Co-culturing of a CD137/NF-κB reporter cell line with tumor lines expressing HER2 or EphA2 revealed tumor antigen-dependent CD137 pathway activation by HER2 x CD137 and EphA2 x CD137 DART molecules, respectively. To evaluate the effects of HER2 x CD137 and EphA2 x CD137 DART molecules on T-cell responses, co-stimulation T-cell assays were performed. In the presence of the relevant antigen-positive cell line, each respective DART molecule was able to promote T-cell proliferation and cytokine release in a HER2 or EphA2-dependent manner. No T-cell co-stimulation was observed by either DART molecule in the absence of antigen-expressing tumor cells. Furthermore, the level of tumor antigen-dependent co-stimulation supported by the DART molecules correlated with the level of tumor target expression. Consistent with the preferential induction of CD137 by the CD8 T cell subset, CD137-based DART proteins induced a substantial increase in the fraction of CD8+ central memory and effector memory T cells in the presence of the proper tumor antigen expressing cells. Conclusions: HER2 x CD137 and EphA2 x CD137 DART proteins promote T-cell co-stimulation in a tumor antigen-dependent manner and may provide an opportunity to target the CD137 co-stimulatory pathway for cancer immunotherapy, while limiting systemic T-cell activation and related side effects. Citation Format: Liqin Liu, Chia-Ying K. Lam, Vatana Long, Lusiana Widjaja, Yinhua Yang, Kalpana Shah, Doug Smith, Joanna Pan, Syd Johnson, Ezio Bonvini, Paul Moore. Tumor-antigen expression-dependent activation of the CD137 costimulatory pathway by bispecific DART® proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3642. doi:10.1158/1538-7445.AM2017-3642

Introduction: 5T4 (trophoblast glycoprotein), an oncofetal antigen involved in embryo development, is expressed on the cell surface of multiple cancers. Several 5T4-directed interventions have been reported, including a cancer vaccine and an antibody-drug conjugate. A superantigen-driven redirected cell killing modality was also developed, but obstacles, such as immunogenicity, remain to a successful 5T4 therapeutic molecule. We have developed an Fc-bearing 5T4 x CD3 DART® bispecific protein designed to redirect T cells to target 5T4-expressing tumors. Methods: 5T4 x CD3, a humanized Fc-bearing DART molecule, was stably expressed in CHO cells and purified to homogeneity via a standard antibody-purification platform. In vitro characterization and functional studies were performed with 5T4-positive tumor cell lines and human T cells. In vivo studies were performed in immune-deficient tumor-bearing mice co-implanted with activated human T cells or reconstituted with human peripheral blood mononuclear cells (PBMCs). Pharmacokinetic studies were performed in both human FcRn transgenic mice and cynomolgus monkeys. Results: 5T4 x CD3 demonstrated bispecific binding properties to both human and cynomolgus monkey antigens. In redirected cytolysis studies, 5T4 x CD3 mediated lysis of 5T4-positive pancreatic, lung, renal, triple-negative breast, and ovarian cancer cell lines with EC50 values ranging between 0.03 and 0.08 ng/mL. 5T4 x CD3 displayed favorable pharmacokinetics with a prolonged circulating half-life in human FcRn transgenic mice and cynomolgus monkeys. Tumor clearance studies in NOD/SCID mice implanted subcutaneously with activated human T cells and tumor cells demonstrated robust inhibition of tumor growth upon intravenous administration of 5T4 x CD3 at doses as low as 0.8 μg/kg, but not with a CD3-binding control DART protein. In addition, human PBMC-reconstituted NOD/SCID/IL2 gamma-chain null mice were implanted intradermally with renal and pancreatic tumor cell lines or orthotopically with a triple-negative breast cancer line and, after tumor establishment, were treated with 5T4 x CD3. Anti-tumor activity was observed at doses as low as 4 μg/kg. Immunohistochemical analysis of tumor xenografts confirmed tumor clearance was associated with T-cell recruitment into the tumor mass. Conclusions: In summary, 5T4 x CD3 displays robust antitumor activity against several cancer cell lines in vitro and in vivo together with a favorable pharmacokinetic profile and merits further consideration as a potential treatment for 5T4-positive cancers. Citation Format: Ling Huang, Gurunadh Chichili, Ralph Alderson, Francine Chen, Jennifer Brown, Hua Li, Valentina Ciccarone, Jim Tamura, Daorong Liu, Liqin Liu, Syd Johnson, Ezio Bonvini, Paul Moore. A 5T4 x CD3 bispecific DART® molecule with extended half-life for T-cell immunotherapy of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4608. doi:10.1158/1538-7445.AM2017-4608

Introduction: Immunotherapy with the combination of monoclonal antibodies (mAbs) that block PD-1 (nivolumab) and CTLA-4 (ipilimumab) has shown clinical benefit beyond that observed with either mAb alone. We have developed PD-1 x CTLA-4 bispecific proteins aimed at inducing antitumor immunity through simultaneous blockade of both checkpoint molecules. Two proteins, a tetravalent PD-1 x CTLA-4 bispecific DART® protein (bivalent for both PD-1 and CTLA-4) and a trivalent PD-1 x CTLA-4 TRIDENT™ protein (bivalent for PD-1 but monovalent for CTLA-4) were engineered; the TRIDENT protein was designed to promote CTLA-4 blockade through avidity when bound to PD-1 on cells co-expressing both receptors, thus favoring PD-1 over CTLA-4 blockade in cells that do not co-express both molecules. Methods: Proteins were engineered from humanized PD-1 and CTLA-4 mAb sequences and demonstrated favorable biophysical properties. Binding assays were performed by ELISA or flow cytometry. Co-ligation of PD-1 and CTLA-4 was assessed in an enzyme-fragment complementation assay (DiscoverX). T-cell activation was tested in reporter cells, staphylococcus enterotoxin B-stimulated PBMCs or MLR assays. Results: Both DART and TRIDENT proteins showed equivalent potency in binding immobilized recombinant PD-1 or PD-1-expressing cell lines, inhibition of PD-1 interaction with PD-L1 or PD-L2 as well as reversal of PD-1/PD-L1 mediated T-cell signal inhibition. In all assays, both formats showed activities that were comparable to those of the precursor PD-1 mAb. With respect to CTLA-4, the DART protein showed a minor potency loss in binding to CTLA-4 expressing cells, inhibition of CTLA-4/B7 interaction and reversal of T-cell signal inhibition compared to the precursor mAb. The TRIDENT protein, however, showed substantial lower potency than the DART protein in all CTLA-4 assays, consistent with the monovalent nature of the CTLA-4 arm. Importantly, in cells that co-express both receptors, DART and TRIDENT proteins show comparable co-engagement of PD-1 and CTLA-4, as shown by enzyme-fragment complementation, suggesting that anchoring through PD-1 compensates for the decreased CTLA-4 avidity of the TRIDENT molecule when both target receptors are expressed. Similarly, in T-cell co-activation assays, both DART and TRIDENT proteins showed a comparable increase in IFN-gamma response that recapitulated that observed with the combination of the individual parental mAbs. Conclusion: Both PD-1 x CTLA-4 DART and TRIDENT bispecific molecules block PD-1 and CTLA-4 pathways, with the TRIDENT protein demonstrating a PD-1-biased binding preference, consistent with its design intended to reduce CTLA-4 blockade in the absence of PD-1 co-expression. Both molecules showed comparable T-cell activation activity. Further development of bispecific PD-1 x CTLA-4 molecules for cancer treatment is warranted. Citation Format: Gurunadh R. Chichili, Kalpana Shah, Ross La Motte-Mohs, Alex Berezhnoy, Daorong Liu, Jessica Hill, Leilei He, Christine Shoemaker, Jennifer Brown, Kurt Stahl, Hua Li, Jonathan Li, Sergey Gorlatov, Jim Tamura, Valentina Ciccarone, Ralph Alderson, Ezio Bonvini, Paul A. Moore, Syd Johnson. Co-targeting of PD-1 and CTLA-4 inhibitory pathways with bispecific DART® and TRIDENT™ molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3637. doi:10.1158/1538-7445.AM2017-3637

Introduction: B7-H3, a member of the B7 family of immunomodulatory molecules, is overexpressed in a wide range of solid cancers. B7-H3 overexpression has been correlated with disease severity and poor outcome in several cancer types. Proof-of-concept studies targeting B7-H3 demonstrated that auristatin-based B7-H3 antibody-drug conjugates (ADCs) exhibited potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines. Based on these preliminary results, we undertook preclinical development of a B7-H3 ADC comprised of a humanized B7-H3 mAb conjugated to a potent DNA alkylating payload. Methods: Chimeric B7-H3 mAbs were conjugated to vc-seco-DUocarmycin-hydroxyBenzamide Azaindole (DUBA) (ADC conjugated and provided by Synthon Biopharmecuticals B.V). In vitro and in vivo activity studies were conducted with tumor cell lines that overexpress B7-H3. Based on the potency analysis, together with the biophysical properties and immunohistochemistry (IHC) profiles of the candidates, a lead mAb was selected for preclinical development. The mAb was humanized via CDR grafting and conjugated to DUBA to yield the development candidate MGC018. In vitro and in vivo studies were then conducted with MGC018 to confirm and extend the results with the chimeric ADCs. Results: Confirming our previous data and consistent with a growing body of literature, B7-H3 mAbs exhibited strong reactivity toward carcinoma cells and the vasculature of solid cancers. Chimeric B7-H3-DUBA ADCs demonstrated specific, dose-dependent cytotoxicity toward B7-H3-positive tumor cell lines in vitro and potent antitumor activity in vivo. The humanized ADC development candidate, MGC018, retained the favorable biophysical properties and the normal tissue-versus-tumor IHC profile of the parental mAb. MGC018 displayed cytotoxicity toward B7-H3-positive tumor cell lines in vitro, with IC50 values in the sub-nM range, and potent antitumor activity in vivo, resulting in tumor stasis and tumor regression in mice bearing B7-H3-positive human tumor xenografts, representing breast, lung and ovarian cancers. Conclusion: MGC018, a preclinical candidate comprised of a humanized mAb targeting B7-H3 conjugated to the potent DNA alkylating payload DUBA via a cleavable peptide linker, exhibited a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, potent cytotoxicity in vitro and antitumor activity in vivo toward a range of B7-H3-expressing tumor cell lines representing several cancer types. Our findings support further preclinical development of MGC018 to evaluate its potential as an ADC therapeutic for B7-H3-expressing solid cancers. Citation Format: Thomas Son, Juniper A. Scribner, Jeff Hooley, Michael Chiechi, Pam Li, Timothy E. Hotaling, Anushka De Costa, Yan Chen, Francine Chen, Bhaswati Barat, Valentina Ciccarone, Timur Gaynutdinov, James Tamura, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo. Preclinical development of a duocarmycin-based antibody-drug conjugate targeting B7-H3 for solid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 42. doi:10.1158/1538-7445.AM2017-42

Introduction: A target-unbiased approach based on intact cell immunizations with fetal progenitor cells and cancer stem cells, followed by an immunohistochemistry (IHC) screen for cancer-specific candidates, led to the identification of anti-ADAM9 (a disintegrin and metalloproteinase) mAbs with highly differential tumor-versus-normal tissue binding. ADAM9 is a cell surface protein over-expressed in multiple tumors, with a possible role in promotion and progression of cancer through multiple mechanisms, including modulation of adhesion and migration as well as processing of tumorigenic and pro-angiogenic factors. In this preclinical study, we performed target/mAb validation and evaluated the therapeutic potential of anti-ADAM9 antibody-drug conjugates (ADCs) toward ADAM9-expressing solid cancers. Methods: IHC was performed with anti-ADAM9 mAbs to confirm and extend available data of human normal and tumor tissue expression. Epitope mapping studies were conducted to define epitope-specificity. mAbs were also screened to identify those that efficiently internalized into tumor cells. In vitro cellular processing studies were performed to further evaluate the mAbs as ADC candidates. Selected mAbs were converted to ADCs via chemical conjugation to potent anti-microtubule (DM4) or DNA alkylating (DGN549) agents; in vitro cytotoxicity studies were conducted with tumor cell lines representing human cancer types that overexpress ADAM9. A lead mAb was then selected for humanization and affinity maturation to yield a development candidate. Results: Anti-ADAM9 mAbs exhibited strong reactivity toward the tumor epithelium of solid cancers, including pancreatic, kidney, prostate, bladder, breast, colon, lung, and ovarian cancer, but limited reactivity toward normal tissues. Anti-ADAM9 mAbs were efficiently internalized and processed by tumor cell lines, including lines with only modest ADAM9 expression. Anti-ADAM9 ADCs exhibited specific, dose-dependent cytotoxicity toward ADAM9-positive cancer cell lines in vitro, with IC50 values in the sub-nanomolar range. Humanization and affinity maturation of the lead mAb yielded a development candidate that retains potent antitumor activity toward ADAM9-positive tumor cell lines and equivalent, high affinity binding to both human and cynomolgus monkey ADAM9. Conclusion: ADAM9 is a cell surface antigen that is over-expressed on a wide range of solid cancers. Anti-ADAM9 mAbs that were strongly reactive with representative tumors exhibited high affinity for the antigen and were efficiently internalized and processed by ADAM9-bearing tumor cells. Anti-ADAM9 ADCs demonstrated dose-dependent cytotoxicity in vitro toward a panel of ADAM9-positive tumor cell lines. Our findings demonstrate that an ADC targeting ADAM9 may serve as a potential therapeutic for ADAM9-expressing solid tumors. Citation Format: Juniper A. Scribner, Bhaswati Barat, Stuart W. Hicks, Nicholas C. Yoder, Thomas Son, Lusiana Widjaja, Gundo Diedrich, Sergey Gorlatov, Jeff Hooley, Ann Easton, Peter Lung, Anushka De Costa, Francine Chen, Michael Chiechi, Pam Li, Monica Licea, Timothy E. Hotaling, Michael Spliedt, Valentina Ciccarone, Nadia Gantt, James Tamura, Megan E. Fuller, Molly McShea, Scott Koenig, Syd Johnson, Paul A. Moore, Ezio Bonvini, Deryk Loo. Target validation, antibody discovery and preclinical data supporting ADAM9 as an antibody-drug conjugate therapeutic target for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 38. doi:10.1158/1538-7445.AM2017-38

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML.

Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cgamma-1 (PLCgamma1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCgamma1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal. Raft-targeted PLCgamma1 was constitutively tyrosine phosphorylated and induced constitutive NF-AT-dependent transcription and interleukin-2 secretion in Jurkat cells. Tyrosine phosphorylation of raft-targeted PLCgamma1 did not require Zap-70 or the interaction with the adapters Lat and Slp-76, molecules that are necessary for TCR signaling. In contrast, the Src family kinase Lck was required. Coexpression in HEK 293T cells of PLCgamma1-HA with Lck or the Tec family kinase Rlk resulted in preferential phosphorylation of raft-targeted PLCgamma1 over wild-type PLCgamma1. These data show that localization of PLCgamma1 in lipid rafts is sufficient for its activation and demonstrate a role for lipid rafts as microdomains that dynamically segregate and integrate PLCgamma1 with other signaling components.

Studying apoptosis induced by T cell receptor (TCR) cross-linking in the T cell hybridoma, 3DO, we found both neutral sphingomyelinase activation and production of ceramide upon receptor engagement. Pharmacological inhibition of ceramide production by the fungal toxin, fumonisin B1, impaired TCR-induced interleukin (IL)-2 production and programmed cell death. Addition of either exogenous ceramide or bacterial sphingomyelinase reconstituted both responses. Moreover, specific inactivation of neutral sphingomyelinase by antisense RNA inhibited IL-2 production and mitogen-activated protein kinase activation after TCR triggering. These results suggest that ceramide production by activation of neutral sphingomyelinase is an essential component of the TCR signaling machinery.

In mature T cells, limited synthesis of the TCR-ζ subunit is primarily responsible for regulating surface expression of TCRs. Transcription of ζ is directed by a complex promoter that includes two potential binding sites for the Ets family of transcription factors at −52 (zEBS1) and −135 (zEBS2). Mutation of these two sites results in a marked reduction of transcription from this promoter. Using electrophoretic mobility shift analysis, Elf-1 was demonstrated to be the Ets family member that binds to these sites. One site, zEBS1, matches the optimal Elf-1 consensus sequence in eight of nine bases, making it the best match of any known mammalian Elf-1 binding site. A role for Elf-1 in TCR-ζ trans-activation was confirmed by ectopic expression of Elf-1 in COS-7 cells. This resulted in an increase in TCR-ζ promoter activity that mapped to zEBS1 and zEBS2. Additional support for the involvement of Elf-1 in TCR-ζ trans-activation derives from the finding that a GAL4-Elf-1 fusion protein trans-activated TCR-ζ promoter constructs that had been modified to contain GAL4 DNA binding sites. These results demonstrate that Elf-1 plays an essential role in the trans-activation of a constitutively expressed T cell-specific gene, and that trans-activation occurs in the context of the native promoter in both lymphoid and nonlymphoid cells. Taken together with the existing literature, these data also suggest that the requirement for inducible factors in Elf-1-mediated trans-activation may decrease as the affinity and number of Elf-1 sites increase.

TCR engagement activates phospholipase Cγ1 (PLCγ1) via a tyrosine phosphorylation-dependent mechanism. PLCγ1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLCγ1 SH2 domains in PLCγ1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLCγ1 in response to TCR/CD3 perturbation. In addition, the PLCγ1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36–38), which has previously been recognized to interact with PLCγ1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLCγ1. Furthermore, binding of p36–38 to PLCγ1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLCγ1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLCγ1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLCγ1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLCγ1 with a tyrosine kinase.

The functional role of Cbl in regulating T cell receptor (TCR)-mediated signal transduction pathways is unknown. This study uses Cbl overexpression in conjunction with a Ras-sensitive AP1 reporter construct to examine its role in regulating TCR-mediated activation of the Ras pathway. Cbl overexpression in Jurkat T cells inhibited AP1 activity after TCR ligation. However, AP1 induction by 4β-phorbol 12-myristate 13-acetate, which up-regulates Ras activity in a protein kinase C-dependent, TCR/tyrosine kinase-independent manner, was not affected by Cbl overexpression. Cbl overexpression also did not affect AP1 induction by an activated Ras protein or a membrane-bound form of the guanine nucleotide exchange factor Sos. In addition, activation of the mitogen-activated protein kinase Erk2 was decreased by Cbl overexpression. Therefore, Cbl regulates events that are required for full TCR-mediated Ras activation, and data are presented to support a model whereby Cbl regulates events required for Ras activation via its association with Grb2.

TPS219 Background: Prognosis for advanced HER2+ GEJ and G cancers remains poor, with median survival just beyond one year. Trastuzumab (T) in combination with chemotherapy is the initial treatment of choice, but therapeutic options targeting HER2 beyond T are poorly defined. M is an Fc-enhanced monoclonal antibody (Mab) to HER2 that recognizes with similar affinity the same epitope as T and whose Fc domain, compared to T, binds with increased affinity to the activating CD16A Fc-receptor (FcR) and decreased affinity to the inhibitory CD32B FcR. Preliminary data shows that M monotherapy has clinical activity against HER2+ tumors in GEJ and G cancer patients previously treated with T or other anti-HER2 agents. P is a Mab that blocks the interaction of the immune checkpoint molecule, PD-1, with its ligands, facilitating tumor cell elimination by releasing tumor-associated T cells from exhaustion. Monotherapy P has demonstrated remarkable and durable clinical activity in a Phase I study. Safety profiles of M and P are acceptable and non-overlapping. Methods: This study advances a chemotherapy free combination of M + P treatment for advanced HER2+ GEJ and G cancer patients. Enrolled patients will have relapsed/refractory HER2+ GEJ or G adenocarcinoma with measurable disease that has progressed on T plus first line chemotherapy. HER2+ (IHC 3+ or ISH+) will be confirmed by central review. Two dose levels of M (10mg/kg and 15mg/kg) and a fixed dose of P (200mg) will be evaluated for safety and tolerability. Patients will receive combination treatment once every 21 days for up to 24 months, until confirmed disease progression or intolerable toxicity. Dose expansion will enroll up to 60 patients, with 20 undergoing pre- and on-treatment biopsy. Response will be assessed every 6 weeks for the first 6 months and every 12 weeks thereafter per RECIST v1.1 and immune RECIST to account for response patterns observed with immunotherapies. Primary endpoint is ORR and duration of response, and secondary endpoints include PFS and OS. The study was initiated on January 2016 and is ongoing in North America and Asia. Clinical trial information: NCT02689284.

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with

Introduction: EphA2 is a receptor tyrosine kinase that plays a critical role in cancer progression through both ligand-dependent and independent mechanisms. The broad overexpression in tumors but limited normal tissue expression of EphA2 makes it an attractive therapeutic target amenable for redirected T-cell killing via an EphA2 x CD3 Dual-Affinity Re-Targeting (DART®) molecules designed to co-engage cytotoxic T cells with EphA2 expressing tumor cells. Methods: Anti-EphA2 monoclonal antibodies (mAbs) were identified by target-specific screening of a library generated by whole-cell immunization with proprietary cancer cell lines, including models of cancer stem cells. The binding and signaling properties of the antibodies were characterized by ELISA, SPR, flow cytometry and phosphorylation assays. Receptor binding regions were determined by ELISA based competition assays and by utilizing human-mouse chimeric EphA2 molecules. Immunohistochemistry (IHC) was performed on frozen normal and tumor tissues. In vitro functional studies were performed with various cancer as well as transfected cell lines, and primary human T cells or PBMCs. In vivo activity was evaluated in xenograft models in immune deficient mice. Results: EphA2 mAbs encompassing diversity in binding kinetics and effects on receptor phosphorylation were classified in 5 discrete binding groups. The majority interacted with the N-terminal ligand-binding domain of EphA2 and most mAbs within that group interfered with ligand binding. The majority of mAbs displayed little IHC reactivity with normal tissue, while strong staining of cancer tissues was observed, including colon, lung, pancreas, ovary, bladder and breast cancers. Seven mAbs recognizing independent epitopes were selected for conversion into EphA2 x CD3 DART molecules that showed a range of potency in redirecting T cells to kill EphA2-expressing target cells. A lead EphA2 x CD3 DART molecule was selected based on potency and cross-reactivity with the cynomolgus monkey ortholog; this lead was engineered with a human Fc domain to confer an extended circulating half-life. The resulting Fc-bearing EphA2 x CD3 DART molecule demonstrated in vivo anti-tumor activity at doses as low as 20 mcg/kg in NOD/SCID/IL-2 gamma chain null mice co-implanted with activated human T cells and MDA-MB231 breast cancer cells. Conclusion: EphA2 is a potential cancer target for redirected T-cell killing applications independent of ligand mediated mechanisms. Further preclinical assessment of EphA2 x CD3 DART molecules as a strategy for targeting EphA2-expressing malignancies is warranted. Citation Format: Claudia B. Fieger, Kalpana Shah, Gurunadh Chichili, Jonathan Li, Thomas Son, Jeffrey Hooley, Francine Chen, Sergey Gorlatov, Steve Burke, Valentina Ciccarone, Ralph Alderson, Deryk Loo, Syd Johnson, Ezio Bonvini, Paul Moore. Evaluation of EphA2 as a therapeutic target for redirected T-cell killing by DART® bispecific molecules. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 583.