Shyra J. Gardai

Abstract 2469: Brentuximab vedotin-mediated immunogenic cell death

Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate (ADC) directed against CD30. It consists of an anti-CD30 monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a microtubule-disrupting agent. Brentuximab vedotin is approved for the treatment of relapsed Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). Brentuximab vedotin antitumor activity is due to the binding of the ADC to CD30-expressing cells, followed by internalization, and release of MMAE after proteolytic cleavage resulting in apoptotic cell death. While Brentuximab vedotin induced cell death has been extensively studied, its potential immune modulatory activity has yet to be explored. Normal apoptosis is non-immunogenic, however multiple chemotherapeutic agents have been shown to induce a unique form of cell death termed Immunogenic Cell Death (ICD). ICD is characterized by exposure of danger-associated molecular patterns (DAMPs), many of which are Toll-like receptor ligands, which can reinitiate the immune responses suppressed by the tumor microenvironment. To test whether Brentuximab vedotin mediated tumor cell death induced ICD the phenotypic characteristics of Brentuximab vedotin-killed CD30+ HL tumor cell lines was examined. Brentuximab vedotin, dose-dependently induced surface exposure of the hallmark ICD markers calreticulin and HSP90 to similar levels induced by oxaliplatin, a chemotherapeutic agent known to mediate ICD. Since ICD is downstream of the ER stress response, Brentuximab vedotin was assessed for its ability to induce ER stress. Brentuximab vedotin treatment upregulated the apoptotic ER sensor C/EBP homologous protein (CHOP) and induced the cleavage and activation of ATF6, a transcription factor required for induction of the ER stress response. ER stress response occurred concurrently with induction of ICD markers and preceded the appearance of active caspase 3/7. These results indicate that Brentuximab vedotin mediated disruption of the microtubule network, in addition to perturbing the cell division cycle and mitosis, also induces ER stress contributing to ICD-dependent cellular toxicity. The functional consequence of ICD is activation of an immune reaction. Thus, exposure of dendritic cells to Brentuximab vedotin-killed tumor cells evoked an inflammatory phenotype including an increase in co-stimulatory markers CD86 and MHC Class II antigens, and activation of NFkB, an intermediate of inflammatory signaling pathways. Taken together, this report suggests that CD30-expressing tumor cells killed by Brentuximab vedotin may potentially activate the innate immune system to initiate antitumor immune response. It also provides a rationale for exploring therapeutic strategies that combine Brentuximab vedotin with other immune stimulatory regimens. Citation Format: Shyra J. Gardai, Angela Epp, Che-Leung Law. Brentuximab vedotin-mediated immunogenic cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2015-2469

Abstract 2472: SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities

SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. SEA-CD40 is derived from dacetuzumab, a humanized IgG1 previously developed and studied for B-lineage malignancies. Glycosylation of the antibody Fc is essential for Fc receptor-mediated activity and non-fucosylated antibodies show improved efficacy, particularly via increased binding to low affinity FcγRIIIa. Enhanced functionality of SEA-CD40 was determined through FcγRIIIa binding affinity, antibody-dependent cellular cytotoxicity (ADCC) activity, activation of the immune response, and induction of antigen-specific T-cells. While SEA-CD40 and the parent antibody dacetuzumab bind to CD40 with similar affinity, the non-fucosylated SEA-CD40 binds equally well to the low (158F) and high (158V) affinity versions of FcγRIIIa with higher affinity than dacetuzumab. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent ADCC activity against a CD40+ lymphoma B cell line and improved agonistic signaling to antigen presenting cells (APCs). SEA-CD40 treatment of human PBMCs elicits a robust immune response as measured by increased cytokine production and up-regulation of maturation markers on APCs with maintained activity at antibody concentrations as low as 10 ng/ml. The immune stimulatory properties of SEA-CD40 were observed in vivo as increased activity in xenograft and syngenic tumor models as well as induction of cytokine production in cynomolgus monkeys. Both in-vitro and in-vivo activity of SEA-CD40 was significantly greater than with dacetuzumab. The increased functionality of SEA-CD40 occurs through the non-fucosylated Fc domain as a F(ab’)2 version loses the ability to induce ADCC, stimulate cytokines, or up-regulate APC activation markers. SEA-CD40 induction of antigen specific T-cells was assessed using human peripheral blood mononuclear cells (PBMCs) exposed to influenza antigen. In the presence of SEA-CD40 influenza specific T-cells mount a robust antigen-specific response characterized by tetramer staining and elevated production of IFNγ. SEA-CD40 activity on PBMCs from donors with melanoma, pancreatic, or breast cancer was assessed and tumor antigen specific T-cell proliferation and IFNγ production was observed. SEA-CD40 is a non-fucosylated agonistic anti-CD40 antibody that shows enhanced binding to FcγRIIIa resulting in amplified cytokine production, co-stimulatory molecule up regulation, and ultimately stimulation of antigen specific T-cell responses to viral and tumor antigens. Citation Format: Shyra J. Gardai, Angela Epp, Germein Linares, Lori Westendorf, May Sutherland, Haley Neff-LaFord, Stanford L. Peng, Che-Leung Law. SEA-CD40, a sugar engineered non-fucosylated anti-CD40 antibody with improved immune activating capabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2472. doi:10.1158/1538-7445.AM2015-2472

Abstract DDT02-02: SGN-2FF: A novel small molecule inhibitor of fucosylation with preclinical antitumor activity through multiple immune mechanisms

Increased fucosylation is associated with tumor progression and metastasis, and targeting fucosylation is a novel strategy in cancer therapy. 2-Fluorofucose (SGN-2FF) has been shown to inhibit cellular fucosylation by depletion of the fucosylation substrate GDP-fucose, as well as by direct inhibition of fucosyltransferases, leading to the production of afucosylated glycoproteins including antibodies. SGN-2FF has antitumor activity in multiple mouse tumor models, showing substantial tumor growth delay. SGN-2FF also enhanced the protective effect of a lymphoma vaccine in a syngeneic mouse model (1). This protection was determined to be immune dependent since depletion of CD4 and CD8 T cells reduced the SGN-2FF/vaccine activity. In vitro, SGN-2FF has been shown to activate human T cells in an antigen-dependent manner. Therefore, by inhibiting fucosylation SGN-2FF can potentially work through multiple mechanisms, including but not limited to effects on immune cells, tumor cells, and the tumor microenvironment. A first-in-human, phase 1, multicenter dose-escalation study of SGN-2FF is ongoing to investigate the safety, pharmacokinetics, and antitumor activity of SGN-2FF given orally to patients with advanced solid tumors (NCT# 02952989). Pharmacodynamic effects, including markers of fucosylation status, will also be evaluated to help determine the optimal biologic dose. This presentation reviews the preclinical activity data of SGN-2FF and describes the design of the phase 1 study, showing how demonstrated preclinical pharmacodynamic effects on fucosylation status informed how activity and pharmacodynamics will be monitored and evaluated in the phase 1 study. Reference 1. Okeley NM, Alley SC, Anderson ME, Boursalian TE, Burke PJ, Emmerton KM, et al. Development of orally active inhibitors of protein and cellular fucosylation. Proc Natl Acad Sci U S A 2013;110:5404-9. Citation Format: Stephen C. Alley, Megan O9Meara, Shyra J. Gardai, Nicole M. Okeley. SGN-2FF: A novel small molecule inhibitor of fucosylation with preclinical antitumor activity through multiple immune mechanisms [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 DDT02-02. doi:10.1158/1538-7445.AM2017-DDT02-02

Abstract 4005: Understanding the mechanism of 2FF-induced immune modulation

Fucosylation is the process of adding a fucose sugar to a glycan chain. The fucose analog, 2-fluorofucose (2FF), has been shown to inhibit cellular fucosylation by depletion of the fucosylation substrate, GDP-fucose, as well as direct inhibition of fucosyltransferases. 2FF has shown promising activity in a number of tumor models. For example 2FF enhanced the protective effect of a lymphoma vaccine (Okeley et al, PNAS 110, 2013), and this protection was determined to be immune dependent since depletion of CD4 and CD8 T-Cells reduced the 2FF/vaccine activity. Due to the complex nature of immune interactions in vivo we established an experimental system using human PBMCs to assess how 2FF-mediated immune changes may contribute to 2FF anti-tumor activity. Human PBMCs were matured in the presence of 2FF for 10 days and phenotypic analysis was performed. Using lectin binding, we found that 2FF dose-dependently decreased cell surface fucosylation which correlated with decreased levels of GDP-fucose and formation of GDP-2FF. 2FF-treatment alone had minimal effect on immune cell phenotype and no significant changes in activation or lineage markers were identified. Recently receptor fucosylation has been shown to be critical for BCR antigen recognition and antibody production (Li et al, J Immunol 194, 2015) as well as TLR recognition and signaling through the scavenger receptor DC-SIGN (Gringhuis et al, Nat Commun 5, 2014). As the TCR is reported to be fucosylated and basal signaling and activation of this receptor is regulated by a galectin-glycoprotein lattice we hypothesized that afucosylation following 2FF treatment may affect TCR functionality. During TCR engagement and activation the galectin-glycoprotein lattice is disrupted. 2FF-treated T cells show decreased galectin-3 levels and when activated have increased and more sustained TCR signaling, shown by increased levels of phosphorylated TCR associated proteins. Additionally, 2FF-treated T cells show increased tetramer specific binding. These data indicate that alteration in surface fucosylation on T cells impacts the regulatory glycoprotein lattice that negatively influences T cell action. Following up on this observation, in co-cultures 2FF-treated T cells activate dendritic cells in a contact dependent and antigen specific manner, shown by upregulation of maturation markers MHCII, CD83, CD86 and CD40. Overall we hypothesize that 2FF-treated T cells are more easily activated due to decreased avidity of galectin-3 binding to glycoproteins which lowers the threshold of lattice disruption by peptide-MHC resulting in easier TCR engagement, increased TCR signaling, and dendritic cell maturation. Citation Format: Jessica J. Field, Nicole M. Okeley, Weiping Zeng, Che-Leung Law, Peter D. Senter, Shyra J. Gardai. Understanding the mechanism of 2FF-induced immune modulation. [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 4005.

Abstract 4914: Auristatin-based antibody drug conjugates activate multiple ER stress response pathways resulting in immunogenic cell death and amplified T-cell responses

Brentuximab vedotin (ADCETRIS®) is an antibody-drug conjugate (ADC) directed against CD30. This ADC consists of monoclonal antibodies conjugated to the auristatin monomethyl auristatin E (MMAE) a microtubule-disrupting agent. ADC antitumor activity is thought to be primarily the result of intracellular payload release leading to mitotic arrest and apoptotic cell death. We have recently demonstrated that brentuximab vedotin induces surface expression of calreticulin and HSP70 as well as activation of the transcription factor ATF6 which are classical hallmarks of immunogenic cell death (ICD) associated with unfolded protein/ER stress response. In this study, we provide evidence that additional ER stress response pathways are activated. Treatment of CD30+ Hodgkin lymphoma (HL) cells or xenografts with brentuximab vedotin resulted in the phosphorylation of the ER-associated, unfolded-protein response transmembrane protein inositol-requiring transmembrane kinase/endonuclease 1 (IRE1). Activation of IRE1 was confirmed by the increase in phosphorylation of the downstream effector Jun N-terminal kinase (JNK). Kinetics of tubulin disruption, ER stress induction, evidence of ICD, mitotic arrest, and apoptosis was assessed. Onset of ICD occurred concurrently with induction of ER stress response pathways including induction of pIRE1 and pJNK. Interestingly, induction of ER stress response and ICD preceded mitotic arrest and caspase activation. These results indicate that brentuximab vedotin-mediated disruption of the microtubule network contributes to ER stress, which maybe an apoptotic mechanism mediated by auristatin-ADCs in addition to mitotic arrest at G2/M phase of the cell cycle. Induction of ICD may also be a class effect of the auristatin-based ADCs, as the small molecule payload MMAE, was able to activate IRE1 and JNK and induced surface expression of calrecutilin on target cells. Previously, brentuximab vedotin-killed HL cells were shown to induce innate immune cell activation in vitro and in vivo. Addition of ADC-killed tumor-like cells to autologous PBMCs results in the expansion of INFa-secreting cytotoxic T-cells. Via induction of ER stress, tumor cells killed by auristatin-based ADCs may initiate an antitumor immune response and provides a rationale for exploring therapeutic strategies that combine ADCs with other immune stimulatory regimens. Citation Format: Anthony Cao, Ryan Heiser, Che-Leung Law, Shyra J. Gardai. Auristatin-based antibody drug conjugates activate multiple ER stress response pathways resulting in immunogenic cell death and amplified T-cell responses. [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 4914.

Abstract 3647: Therapeutic activity of effector function-enhanced, non-fucosylated anti-CD40 antibodies in preclinical immune-competent rodent tumor models

Recent success of immune checkpoint inhibitors (CPIs) in the clinic underscores the role of cancer immune surveillance and the therapeutic potential of targeting immune receptors. Unlike CPIs, many members of the TNF receptor superfamily (TNFRSF), e.g., CD40, 4-1BB, OX-40, and GITR deliver agonistic signals to immune cells that may directly activate antitumor immune responses and, in combination with CPIs, may improve outcomes of active immunotherapy. Sugar-Engineered Antibody (SEA)-CD40 is a non-fucosylated, humanized IgG1 anti-human CD40 monoclonal antibody currently being evaluated in a phase 1 clinical trial in solid tumors and lymphoma. It binds FcγR3A with higher affinity and demonstrates enhanced immune stimulatory activity compared to its parent dacetuzumab. An SEA version of an anti-mouse CD40 antibody (mouse IgG2a), SEA-1C10, has been generated. As expected, SEA-1C10 bound to mouse FcγRIV, the functional homolog of human FcγR3A in mice, at a higher affinity than parent 1C10. Enhanced FcγRIV engagement by SEA-1C10 translated into stronger antitumor activity in the CD40 (-) B16F10 melanoma and CD40 (+) A20 lymphoma models. In the A20 model, tumor free mice that survived the initial A20 tumor implantation and SEA-1C10 treatment were re-challenged with live A20 cells. Twenty nine out of thirty one such tumor free mice rejected A20 tumor re-engraftment, suggesting that a strong memory immune response was elicited by SEA-1C10 treatment. The SEA-1C10/anti PD-1 combination also showed greater antitumor activity compared to SEA-1C10 or anti-PD-1 alone, supporting the hypothesis that stimulation of antigen-presenting cells by agonistic CD40 signaling can complement immune checkpoint inhibition. In addition, a transgenic (TG) mouse strain has been generated in which the endogenous Cd40 locus is inactivated by the targeted insertion of a human-mouse chimeric CD40 gene. This allows for incorporation of the extracellular domain of human CD40 while keeping the transmembrane and cytoplasmic domains of mouse CD40. Lineage-restricted expression of the CD40 TG on B cells and monocytes was confirmed. CD40 TG cells responded to SEACD40-mediated agonistic signaling in vitro by secreting CD40 signature cytokines and up-regulating the costimulatory molecules CD86 and MHC class II. Treatment of CD40 TG animals with SEA-CD40 resulted in cytokine induction and B-cell depletion. Similar to studies in human PBMCs and non-human primates, SEA-CD40 induced more robust cytokine production and immune cell activation than its fucosylated parent dacetuzumab. These experimental systems will be further applied to address clinically important questions including mechanism(s) of anti-tumor effects, biomarkers indicating immune activation, dosing strategies, and timing of combinatorial regimens with CPIs. Citation Format: Shyra J. Gardai, Weiping Zeng, Che-Leung Law. Therapeutic activity of effector function-enhanced, non-fucosylated anti-CD40 antibodies in preclinical immune-competent rodent tumor models [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 3647. doi:10.1158/1538-7445.AM2017-3647

Abstract 5588: Brentuximab vedotin-driven immunogenic cell death enhances antitumor immune responses, and is potentiated by PD1 inhibition in vivo

Brentuximab vedotin (BV) is an antibody-drug conjugate directed against CD30 consisting of a monoclonal antibody conjugated to monomethyl auristatin E (MMAE), a microtubule-disrupting agent. BV antitumor activity is thought to be primarily the result of intracellular payload release leading to mitotic arrest and apoptotic cell death. We have demonstrated that BV drives apoptosis in a manner consistent with immunogenic cell death (ICD) including activation of the unfolded protein/ER stress response with a concomitant increase in surface expression of calreticulin and HSP70. In this study, we provide evidence that in vivo administration of BV leads to directed proinflammatory immune responses against the tumor and this activity is further potentiated by anti-PD-1 therapy. To examine the immunomodulatory effects of BV-induced ICD in vivo, we employed 3 murine model systems. In the first model, BV treatment induced ICD in subcutaneously-engrafted CD30+ L540cy cells, generated strong chemotactic responses detectable within the tumor and in circulation, and increased infiltration of dendritic cells into the tumor microenvironment. To assess whether BV-driven ICD could confer specific antitumor immunity, we used a model in which ICD was induced in BV-treated human CD30-expressing A20 lymphoma cells in vitro. These cells were used to immunize wild-type BALB/c mice followed by live A20 challenge. Immunization with BV-treated cells, undergoing ICD, delayed tumor growth and improved survival compared to mice immunized with flash-frozen cells. Furthermore, administering anti-PD-1 to mice immunized with BV-treated cells displayed marked combinatorial effects, leading to improved tumor clearance compared to either treatment alone. Additionally, T cells isolated from mice immunized with BV-treated cells and transferred into tumor-bearing immunodeficient mice resulted in tumor regression and survival, demonstrating robust T cell memory. Lastly, we employed a humanized tumor model pairing CD30+ PDL1+ PDL2+ lymphoblastoid cell line (LCL) xenografts with adoptively transferred autologous PBMC. In this setting, mice treated with suboptimal doses of BV showed greatly enhanced cytotoxic T cell and NK cell accumulation in LCL tumors resulting in accelerated immune-mediated tumor clearance. Tumor regression was further accelerated by treating mice with a combination of BV and a human PD1 inhibitor, demonstrating complementary modes of action for these agents. Together, these data indicate that targeted treatment with the MMAE antibody drug conjugate brentuximab vedotin drives an immunogenic form of tumor cell death that enhances innate and adaptive antitumor immunity. Combination of BV with PD1 inhibitors resulted in greater antitumor activity than either agent alone. Multiple clinical trials are ongoing to evaluate the efficacy of this treatment pairing. Citation Format: Anthony T. Cao, Che-Leung Law, Shyra J. Gardai, Ryan A. Heiser. Brentuximab vedotin-driven immunogenic cell death enhances antitumor immune responses, and is potentiated by PD1 inhibition in vivo [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 5588. doi:10.1158/1538-7445.AM2017-5588

Abstract 4605: Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody

B cell maturation antigen (BCMA) has recently emerged as an attractive therapeutic target in multiple myeloma. BCMA has restricted expression on plasma cells with little to no expression on other normal tissues, but is upregulated on the surface of multiple myeloma cells. BCMA can regulate proliferation and survival of myeloma cells via binding to its ligands APRIL and BAFF and induce downstream signaling pathways. Thus, several approaches to target BCMA are currently under clinical investigation, including chimeric antigen receptor (CAR) T cell therapies, bispecific antibodies and antibody drug conjugates. The Antibody-Coupled T cell Receptor (ACTR) technology is a universal, engineered T cell therapy consisting of the extracellular domain of human CD16 and the intracellular T cell co-stimulatory and signaling domains. ACTR is designed to engage the Fc domain of therapeutic antibodies opsonized to target cells to mediate anti-tumor activity. Previous work has demonstrated ACTR T cell activity in combination with rituximab, trastuzumab, and hu14.18 K322A against CD20, Her2, and GD2 expressing cell lines, respectively (Kudo et al. Cancer Res 2014; 74:93-103). Currently ACTR is being evaluated in Phase I clinical trials with rituximab to treat relapsed refractory B cell lymphoma. Here we demonstrate a humanized afucosylated anti-BCMA antibody, SEA-BCMA, binds to ACTR expressing T cells with high affinity and mediates T cell activation, potent cytotoxicity, cytokine release and proliferation across a wide range of BCMA expressing myeloma cells. ACTR activity was specific to SEA-BCMA - opsonized target cells, dose dependent and had no activity on BCMA negative tumor lines. Furthermore, the SEA-BCMA antibody has additional properties that might contribute to a therapeutic effect, including blocking the binding of ligands to BCMA and driving natural killer cell mediated ADCC effects. These preclinical studies demonstrate a promising multi-faceted activity of ACTR T cells in combination with the anti-BCMA antibody, SEA-BCMA, for clinical consideration in multiple myeloma patients. Citation Format: Tooba Cheema, Taylor Hickman, Katie O9Callaghan, Lori Westendorf, Luke Manlove, Shyra Gardai, Allison Nelson, Ryan Boomer, Kathleen McGinness, Birgit Schultes, Seth Ettenberg, Django Sussman, Heather Huet. Efficient targeting of BCMA-positive multiple myeloma cells by antibody-coupled T-cell receptor (ACTR) engineered autologous T cells in combination with an anti-BCMA antibody [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 4605. doi:10.1158/1538-7445.AM2017-4605

Abstract 4994: SEA-CD40: from bench to bedside

SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40, a co-stimulatory receptor of the TNF receptor superfamily. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent immune stimulatory activity. CD40 receptor ligation induces multiple pathways; to pave the road for identification of a specific activity signature in the clinical setting, in vitro preclinical assays were developed to monitor the immune modulatory activity of SEA-CD40. Human PBMCs stimulated with increasing concentrations of SEA-CD40 were assessed for immune changes including cytokine production, cellular activation, and modulation of cellular subsets. SEA-CD40 PBMC stimulation elicited a unique set of cytokines including MIP-1β, MCP-1, and IL-8. In addition to inducing cytokines, specific immune cell changes were also observed including up-regulation of stimulatory molecules on monocyte/ macrophages, activation of NK cells, and changes in cellular subsets such as deletion of B-cells. While some of these changes were common across the other CD40 therapeutic antibodies being tested in the clinic, SEA-CD40-specific changes were identified. These changes included a reduction in the immune dampening cytokine IL-10, induction of Th1 CXCR3 positive cells, and reduction of T-regulatory cells, all potentially contributing to an antitumor immune response. The specific in vitro SEA-CD40 signature was also observed in vivo in cynomolgus monkeys. SEA-CD40 treatment induced the same signature cytokines observed in vitro and elicited the same cellular changes including depletion of B-cells, and activation of CD8+ T-cells. A CD40 receptor occupancy assay was also created to correlate receptor engagement with activity. Interestingly, while SEA-CD40 is rapidly cleared from plasma, it is detectable on the surface of antigen-presenting cells for up to 3 weeks. The initial starting dose for SEA-CD40 clinical trials was calculated using the minimal anticipated biological effect level (MABEL). SEA-CD40 cytokine induction was the most sensitive preclinical marker of biologic activity and was, therefore, used for MABEL dose calculation. In the ongoing phase 1 First-In-Human clinical trial, this preclinical SEA-CD40 activity signature is being monitored in adult patients with advanced solid tumors (study NCT02376699). Establishing a clear immune biomarker strategy from pre-clinical research to clinical trials is vital for tracking the activity of our immuno-oncology drugs in patients and for identifying a safe and efficacious regimen. Citation Format: Shyra J. Gardai, Haley Neff-LaFord, Angela Epp, Jing Yang, Thomas Manley, Che-Leung Law. SEA-CD40: from bench to bedside. [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 4994.