Background SGN-B7H4V is a novel investigational vedotin antibody–drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex.Methods B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model.Results Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent.Conclusion The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.

Ligation of toll-like receptors 7 and 8 (TLR7/8) can potently activate innate immune cells, including tumor-associated macrophages, to prime downstream T cell activation and drive potent, lasting anti-tumor immunity. TLR7/8 agonists have long been pursued as an anti-cancer therapeutic because of their potential in re-programing the immune system but the clinical utility has been limited by systemic toxicity. Several means of decreasing this systemic toxicity have been investigated, including intra-tumoral administration, nanoparticle encapsulation and conjugation to a tumor-targeting antibody (antibody-drug conjugates, ADCs). ADCs are a clinically validated technology designed to target drugs to disease tissues to reduce the systemic toxicity of highly potent payloads and improve anti-tumor activity. We have developed an imidazoquinoline-based dual TLR7 and TLR8 small molecule agonist that has been specifically designed as an ADC payload. The initial compound was chosen from a set of imidazoquinoline-based small molecules using HEK293 reporter cells expressing either TLR7 or TLR8. This newly identified TLR7/8 agonist potently reactivated immunosuppressive macrophages to produce inflammatory cytokines, increased phagocytosis of tumor cells and enhanced T cell activation and proliferation. A lead payload candidate based on a modified version of the initial TLR7/8 agonist was designed to decrease drug permeability to minimize non-targeted systemic immune activation. The lead TLR7/8 agonist was shown to be significantly less potent than the first-generation compound as a small molecule but demonstrated enhanced immune-stimulating capability when conjugated to a direct immune-targeting antibody. The increased potency as an ADC is hypothesized to be driven by enhanced intracellular retention of the less cell permeable payload and/or a higher binding affinity to the TLR7 or 8 receptors, which was predicted via modeling. The greater in vitro potency of the lead payload was also observed in vivo in an MC38 syngeneic tumor model where 33% complete tumor cures were observed with the lead versus only tumor delay with the permeable version. Drug linkers employing the lead payload were evaluated by varying payload linkage chemistry, drug linker hydrophobicity, and drug release mechanisms using in vivo anti-tumor activity in different syngeneic mouse tumor models as a readout. The optimized drug linker design increased the in vitro and in vivo potency up to 4-fold when compared with the initial linker/payload. These data clearly demonstrate the potency of TLR7/8 agonists as immune stimulants in anti-cancer therapies and outline how their optimization through chemical modification can make them suitable payloads for ADCs. Citation Format: Kung-Pern Wang, Chris Neumann, Angela Epp, Weiping Zeng, Thomas Griffith, David Ferguson, Shyra Gardai, Alyson J. Smith. Generation of an antibody-drug conjugate-optimized TLR 7/8 agonist payload [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1542.

SGN-B7H4V is a novel investigational antibody-drug conjugate composed of a B7-H4-directed human monoclonal antibody conjugated to the validated vedotin drug linker, which incorporates the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linkage. This vedotin drug linker system has been clinically validated by multiple ADC programs, including brentuximab vedotin, enfortumab vedotin, tisotumab vedotin, and polatuzumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumor types, including breast, ovarian, and endometrial tumors. SGN-B7H4V is designed to bind and internalize the B7-H4/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. The antitumor activity of SGN-B7H4V may be multimodal as SGN-B7H4V can induce tumor cell death through several mechanisms, including MMAE-mediated direct cytotoxicity and bystander killing as well as antibody-mediated functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Previously, vedotin ADCs have been described to elicit antitumor immune responses in part through induction of immunogenic cell death (ICD) mediated by the MMAE payload. These immunomodulatory effects potentially position vedotin ADCs to uniquely synergize with checkpoint inhibitors, supported by recent clinical activity observed when vedotin ADCs are paired with anti-PD1 agents. Here, we characterize SGN-B7H4V-mediated ICD and subsequent immunomodulatory activity. We also evaluate the contribution of SGN-B7H4V-induced immune activation to antitumor activity in combination with an anti-PD1 agent in an immunocompetent mouse model. In vitro, tumor cells treated with SGN-B7H4V showed several hallmarks of immunogenic cell death, including calreticulin exposure and release of ATP. In vivo, treatment of B7-H4-expressing tumors with SGN-B7H4V led to immune changes in the tumor microenvironment, including recruitment of macrophages and T cells. Finally, SGN-B7H4V drove robust, curative activity in an immunocompetent tumor model as a monotherapy and paired well with an anti-PD1 agent. Altogether, these data support the evaluation of SGN-B7H4V as a monotherapy in a first-in-human phase 1 clinical study and potential future clinical combinations with immunotherapies. Citation Format: Elizabeth E. Gray, Michelle Ulrich, Angela Epp, Patrick Younan, Kelly Hensley, Sean Allred, Julie Hahn, Kristen Gahnberg, Piper M. Treuting, John J. Gosink, Robert Thurman, Alyson J. Smith, Jason Schrum, Natalya Nazarenko, Shyra J. Gardai. SGN-B7H4V shows immunomodulatory activity through induction of immunogenic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1281.

Background SGN-B7H4V is an investigational vedotin antibody-drug conjugate (ADC) directed to B7-H4, an immune checkpoint ligand with elevated expression on multiple solid tumor types, including breast, ovarian, and endometrial tumors.1 SGN-B7H4V is composed of an anti-B7-H4 human monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linker. This vedotin drug linker system has been clinically validated in multiple ADC programs, including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. SGN-B7H4V is designed to bind the immune checkpoint ligand B7-H4, internalize the ligand/ADC complex from the surface of B7-H4-expressing tumor cells and release the cytotoxic payload, MMAE, within the cell. SGN-B7H4V demonstrates strong antitumor activity in preclinical models through multiple potential mechanisms including direct MMAE-mediated cytotoxicity as well as antibody-mediated functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) in vitro.1 Vedotin ADCs can elicit MMAE-mediated immunomodulatory changes to the tumor microenvironment (TME) via induction of immunogenic cell death 2–4 which may position vedotin ADCs to uniquely synergize with checkpoint inhibitors. This is supported by clinically meaningful responses observed when vedotin ADCs are paired with anti-PD1 agents.5,6 Here, we use an immunocompetent mouse model to characterize SGN-B7H4V-mediated immunomodulatory activity along with antitumor activity and induction of immune memory in combination with an anti-PD1 agent. Methods Immunomodulatory changes in SGN-B7H4V-treated tumors were characterized by RNAseq and immunohistochemistry. The antitumor activity of SGN-B7H4V as a monotherapy and in combination with an anti-PD1 agent was evaluated using a murine B7-H4-expressing Renca syngeneic tumor model. Finally, tumor rechallenge experiments were performed to evaluate immune memory. Results Treatment of B7-H4-expressing syngeneic tumors with SGN-B7H4V led to immunomodulatory changes in the TME, including recruitment of multiple immune cell types and upregulation of immune-related genes that have been previously associated with response to anti-PD(L)1 agents. Moreover, SGN-B7H4V drove robust antitumor activity as well as durable immune memory as a monotherapy and in combination with an anti-PD1 agent. Conclusions In preclinical models, SGN-B7H4V demonstrates robust antitumor activity accompanied by immunomodulatory changes in the TME. Moreover, SGN-B7H4V in combination with an anti-PD1 agent led to improved antitumor activity and elicited durable immune memory. Altogether, these nonclinical data further support the evaluation of SGN-B7H4V as a monotherapy in the ongoing Phase 1 Study of SGN-B7H4V in Advanced Solid Tumors (NCT05194072) and potential future clinical combinations with immunotherapies. References Gray E, et al. 854 SGN-B7H4V, a novel, investigational vedotin antibody-drug conjugate directed to the T cell checkpoint ligand B7-H4, shows promising activity in preclinical models. Journal for ImmunoTherapy of Cancer 2021;9:A895–A895. Pusztai LL, Hale H, Grosse-Wilde C, Specht A, Modi J, Han S, Cortes H, Oliveira J, Garfin H, Wang P, Onsum ZM. Systemic administration of ladiratuzumab vedotin alone or in combination with pembrolizumab results in significant immune activation in the tumor microenvironment in metastatic breast cancer patients. Journal for ImmunoTherapy of Cancer 2020;8. Liu BAOD, Snead K, Gosink J, Tenn E-T, Zaval M, Cao A, Sahetya D, Nesterova A, Hensley K, Cochran J, Gardai S, Lewis TS. Enfortumab vedotin, an anti-Nectin-4 ADC demonstrates bystander cell killing and immunogenic cell death anti-tumor activity mechanisms of action in urothelial cancers. Cancer Research, 2020;80(16_Supplement):5581. Gray EHK, Allred S, Trueblood E, Gosink J, Thurman R, Smith K, Jacquemont C, Bieda M; Gow J, Harris J, Brady L, Soumaoro I, Jain S, Nicacio L, Gardai S. Tisotumab vedotin shows immunomodulatory activity through induction of immunogenic cell death. Journal for ImmunoTherapy of Cancer 2020;8. Rosenberg JEF, Friedlander TW; Milowsky MI, Srinivas S, Petrylak DP, Merchan JR, Bilen MA, Carret A-S, Yuan N, Sasse C, Hoimes CJ. Study EV-103: Preliminary durability results of enfortumab vedotin plus pembrolizumab for locally advanced or metastatic urothelial carcinoma. Journal of Clinical Oncology 2020;38(6):441–441. Advani RH, et al. Brentuximab vedotin in combination with nivolumab in relapsed or refractory Hodgkin lymphoma: 3-year study results. Blood 2021;138(6):427–438. Ethics Approval All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen or the external testing facility that conducted the studies.

Antibody-drug conjugates (ADC) leverage the specificity of antibodies to direct the delivery of potent cytotoxic agents to cancer cells. To fully leverage this specificity, targets with significant differential tumor versus normal tissue expression are ideal for ADC development. Placental alkaline phosphatases, ALPP and ALPPL2, are proteins present during fetal development but are found in several tumor types, making them an attractive ADC target. These cell membrane-attached phosphatases form homo- and heterodimers and play key roles in nucleotide recycling. SGN-ALPV is a novel investigational vedotin ADC comprised of a humanized IgG1 monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linker that has been clinically validated in multiple ADC programs. Here we characterize the target antigens, ALPP and ALPPL2, and evaluate SGN-ALPV antitumor activity in preclinical models. Immunohistochemistry and genomic data mining showed that ALPP and ALPPL2 have a highly restricted normal tissue expression yet high expression in several solid tumor types including ovarian, endometrial, germ cell, non-small cell lung and gastric carcinomas. Importantly, normal tissue expression is restricted to placenta and reproductive tissue, with low levels in lung tissue. SGN-ALPV utilizes a humanized antibody, h12F3, that is highly specific for both human and cynomolgus monkey ALPP and ALPPL2 proteins, but not other related phosphatases. In vitro, upon binding to SGN APLV, the antigens, ALPP and ALPPL2, are internalized to lysosomal vesicles releasing the MMAE payload, whose cytotoxic features drive mitotic arrest, apoptosis, and the induction of immunogenic cell death. Additionally, SGN-ALPV mediates antibody dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in vitro but lacks complement-dependent cytotoxicity. In preclinical studies, SGN-ALPV exhibits robust antitumor activity in cell line- and patient-derived xenograft models of ovarian, lung, pancreatic, and gastric carcinoma including models with both homogenous and heterogeneous expression of ALPP and ALPPL2, consistent with robust bystander activity of vedotin ADCs. Although, the contribution of ADCC and ADCP in vivo is currently unknown, it is expected that the antitumor activity of SGN-ALPV is mediated by MMAE cytotoxicity. SGN-ALPV was well tolerated in non-human primates (NHP) and exhibited linear pharmacokinetic characteristics, with a toxicity profile consistent with other vedotin-based ADCs. In summary, differential expression of ALPP and ALPPL2 in the tumor versus normal tissue, antibody specificity, antitumor activity, and tolerability of SGN-ALPV provide a strong rationale for the initiation of a planned first-in-human Phase 1 clinical study. Citation Format: Sarah Anderson, Nanna Hansen, Robert Lawrence, Aroon T. Chande, David Ortiz, Christopher Carosino, Esther Trueblood, Nicole Stevens, Kerry Klussman, Angela Epp, Bill Arthur, Shyra Gardai, Hector Rincon. SGN-ALPV a novel, investigational vedotin ADC demonstrates highly effective targeting of oncofetal phosphatases ALPP and ALPPL2 in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1766.

SGN-B7H4V is a novel, investigational vedotin antibody drug conjugate (ADC) directed to B7-H4, a member of the B7 family of immune checkpoint ligands. B7-H4 expression is elevated on a variety of solid tumors including breast, ovarian, and endometrial tumors.1 SGN-B7H4V is composed of a fully human IgG1 anti-B7-H4 monoclonal antibody (mAb) conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linker. SGN-B7H4V is designed to bind and internalize the immune checkpoint ligand B7-H4/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. This ”vedotin” drug linker system has been clinically validated by multiple ADC programs, including brentuximab vedotin, enfortumab vedotin, and polatuzumab vedotin.2–4 Here, we characterize the target antigen B7-H4 and evaluate SGN-B7H4V activity in preclinical models.B7-H4 expression was characterized by RNA expression and immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the tolerability of SGN-B7H4V was assessed in rodent and non-human primate toxicology studies.Immunohistochemistry confirmed expression of B7-H4 across multiple solid tumor types, including ovarian and breast tumors. In vitro, upon binding to SGN-B7H4V, the immune checkpoint ligand B7-H4 was rapidly internalized and delivered the cytotoxic payload MMAE. Moreover, SGN-B7H4V killed B7-H4-expressing tumor cells in vitro by MMAE-mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In vivo, SGN-B7H4V demonstrated strong anti-tumor activity in multiple xenograft models, including ovarian and breast cancer models. Activity was observed in models with both uniformly high and heterogeneous expression of B7-H4, consistent with robust bystander activity of vedotin ADCs. Finally, SGN-B7H4V was tolerated in both rat and non-human primate (NHP) toxicology studies at doses consistent with approved vedotin ADCs.B7-H4 is a promising ADC target expressed by several solid tumor types. SGN-B7H4V demonstrates robust anti-tumor activity in preclinical models through multiple potential mechanisms and is tolerated in rat and NHP toxicity studies. Altogether, these data support further evaluation of SGN-B7H4V in a planned, first-in-human phase 1 clinical study.We would like to thank Kellie Spahr for conjugation support and Martha Anderson for in vivo biology support.Leong SR, Liang WC, Wu Y, Crocker L, Cheng E, Sampath D, et al. An anti-B7-H4 antibody-drug conjugate for the treatment of breast cancer. Mol Pharm 2015;12(6):1717–29. Epub 2015/04/09. doi: 10.1021/mp5007745. PubMed PMID: 25853436.Rosenberg JE, O’Donnell PH, Balar AV, McGregor BA, Heath EI, Yu EY, et al. Pivotal trial of enfortumab vedotin in urothelial carcinoma after platinum and anti-programmed death 1/programmed death ligand 1 therapy. J Clin Oncol 2019;37(29):2592–600. Epub 2019/07/30. doi: 10.1200/JCO.19.01140. PubMed PMID: 31356140; PubMed Central PMCID: PMC.Senter PD, Sievers EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat Biotechnol 2012;30(7):631–7. Epub 2012/07/12. doi: 10.1038/nbt.2289. PubMed PMID: 22781692.Tilly H, Morschhauser F, Bartlett NL, Mehta A, Salles G, Haioun C, et al. Polatuzumab vedotin in combination with immunochemotherapy in patients with previously untreated diffuse large B-cell lymphoma: an open-label, non-randomised, phase 1b-2 study. Lancet Oncol 2019;20(7):998–1010. Epub 2019/05/19. doi: 10.1016/S1470-2045(19)30091–9. PubMed PMID: 31101489.All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen or the external testing facility that conducted the studies.

Abstract Targeting glyco-epitopes with antibody-drug conjugates (ADCs) provides a unique advantage as multiple cell surface proteins expressing the same carbohydrate can be harnessed to deliver drug. Sialyl-Thomsen nouveau (STn) is a tumor associated carbohydrate antigen that has historically been difficult to target with specificity. Using glycan array screening, h2G12 was identified as a highly specific human IgG1 antibody, that exclusively binds the STn glyco-epitope independent of protein backbone. Here we introduce SGN-STNV, an investigational antibody-drug conjugate (ADC) targeting monomethyl auristatin E (MMAE) to STn expressing tumor cells with the clinically validated vedotin linker technology. STn has restricted normal tissue expression and is expressed on various solid tumors including ovarian, non-small cell lung, gastric, and endometrial carcinomas. Our work shows correlation of enzyme ST6GALNAC1 expression with STn expression on tumors. Given the frequent O-glycosylation of mucins, we also confirmed high STn expression in mucinous subtypes of ovarian, pancreatic, colorectal, and lung adenocarcinomas. Previous reports have confirmed STn is found on mucins MUC1, MUC5A, and MUC16 (CA-125), as well as cell surface receptors CD44 and integrin β1. These proteins are known to play functional roles in tumor progression and, importantly for ADC delivery, they internalize well to deliver cytotoxic payload. We sought to identify other proteins that may contribute to cytotoxic activity of SGN-STNV and determined that additional surface receptors are tagged with STn. These receptors are internalized and likely contribute to SGN-STNV activity. After binding to STn, SGN-STNV is internalized into cells and releases the microtubule inhibitor MMAE to drive mitotic arrest, apoptosis, and to induce immunogenic cell death. SGN-STNV further mediates anti-tumor response through Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In preclinical xenograft studies, SGN-STNV monotherapy treatment leveraged h2G12 specificity and ability to target STn on multiple tumor associated proteins to drive durable tumor regressions. SGN-STNV was well tolerated in non-human primates (NHP), with no concerning target-mediated toxicities and a maximum tolerated dose similar to other vedotin-platform ADCs. In summary, the antibody specificity, unique mechanism of targeting a carbohydrate, anti-tumor activity, and tolerability provide a strong rationale for initiation of a phase I study to investigate the therapeutic potential of SGN-STNV. Citation Format: Alyssa Schwartz, Hector Rincon, Nanna Hansen, Robert Lawrence, Sarah Anderson, Nicole Blesie, Kerry Klussman, Angela Epp, Shyra Gardai, William Arthur. Targeting Sialyl-Thomsen nouveau (STn) antigen with the SGN-STNV antibody-drug conjugate is effective in preclinical studies [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 50.

Abstract 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.

Abstract 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 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

3074 Background: SEA-CD40 is a non-fucosylated, humanized IgG1 monoclonal antibody directed against human CD40. It is derived from dacetuzumab, a humanized IgG1 previously developed for B-lineage malignancies. Antibody glycosylation is essential for Fc receptor-mediated activity and nonfucosylated antibodies may show improved efficacy via increased binding to FcγRIIIa (CD16). Methods: Enhanced functionality of SEA-CD40 was determined through FcγRIIIa binding, immune activation, and induction of antigen-specific T-cells Results: While SEA-CD40 and the parent antibody dacetuzumab bind to CD40 with similar affinity, the non-fucosylated SEA-CD40 has a higher affinity for both low (158F) and high (158V) affinity FcγRIIIa. The consequence of enhanced SEA-CD40/FcγRIIIa binding is potent ADCC activity and improved agonistic signaling to antigen presenting cells (APCs). SEA-CD40 treatment of human PBMCs elicits a robust immune response consisting of proinflammatory cytokine production, APC maturation and up-regulation of co-stimulatory receptors on APCs with activity at antibody concentrations as low as 10 ng/ml. Utilizing a surrogate antibody against mouse CD40, the immune stimulatory properties of nonfucosylated anti-CD40 were confirmed in vivo in syngeneic tumor models. SEA-CD40 induction of antigen specific T-cells was assessed using human peripheral blood mononuclear cells (PBMCs) exposed to the M1 influenza antigen. SEA-CD40 stimulated the expansion of influenza specific T-cells and elevated their production of IFNγ. Likewise, SEA-CD40 also stimulated T-cell proliferation and IFNγ production in PBMCs from melanoma, pancreatic, or breast cancer patients in response to a mixture of the tumor-associated antigens MAGE-A1/A3. Interestingly, antigen-specific T-cell responses to both the influenza and tumor antigens were enhanced in the presence of blocking antibodies to CTLA4 or PD1. Conclusions: These observations demonstrate the potential of combining the non-fucosylated agonistic SEA-CD40 with immune check point inhibitors to generate more effective adaptive antitumor immune responses.