The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. These receptors are promising targets for vaccine development and cancer immunotherapy. However, currently available agonists are heterogeneous glycoconjugates and polysaccharides from natural sources. Herein, we designed and synthesized the first chemically defined ligands for dectin-1 and dectin-2. They comprised glycopolypeptides bearing mono-, di-, and trisaccharides and were built through polymerization of glycosylated N-carboxyanhydrides. Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. We identified structures that elicit a pro-inflammatory response through dectin-1 or dectin-2 in antigen-presenting cells. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications.

BackgroundImmune-stimulating antibody conjugates (ISACs) covalently attach immune stimulants to tumor-targeting antibodies such as trastuzumab. We have shown that HER2-targeted TLR7/8 ISACs elicit robust myeloid activation and tumor eradication in a TLR- and Fc-dependent manner in trastuzumab-resistant and HER2-low models. Upon treatment with ISACs, T cell-mediated immunological memory extends to tumor antigens beyond HER2.1 Here we describe the ISAC mechanism of action in vivo that leads to eradication of tumors in mice.MethodsEstablished syngeneic rHER2- or xenograft HER2-expressing tumors treated with anti-HER2 ISACs or appropriate controls were assessed for gene expression by NanoString Pan-Cancer Immune Profiling panel comprising 750 genes related to tumor immune biology. Tumor cytokines were measured using MesoScale Discovery (MSD) technology, and immune cell infiltrates were assessed by immunohistochemistry (IHC). Anti-tumor efficacy was assessed after depletion of CD8+ T cells and phagocytes.ResultsWithin 24 hours of administration, HER2-directed ISACs induced robust, target-dependent activation of the immune system. In a syngeneic tumor model, 34% of the measurable genes were significantly upregulated after treatment with the rHER2-targeted ISAC vs 0.1% with the non-binding ISAC control. Similarly, 13% vs 0% of genes were upregulated in a xenograft model after HER2-targeted vs control ISAC treatment. In both models anti-HER2 ISAC treatment led to activation of pathways indicative of TLR7/8 agonism (e.g. IRF-7; type 1 interferons), and FcgR engagement (e.g. NF-kappaB associated genes). Cytokines and chemokines, including myeloid chemokines CCL2/3/4 and T cell chemokines CXCL9/10/11 were specifically upregulated in the tumors at the gene and protein level, indicating robust activation of myeloid cells following anti-HER2 ISAC treatment. Furthermore, in syngeneic tumors T cell activation markers (e.g. Granzyme B; IFN-gamma) were induced within 24 hours post treatment with an anti-rHER2 ISAC, and IHC at day 6 showed a 5-fold increase in CD11c+ cells. Control-treated tumors had sparse CD8+ T cells, whereas rHER2-targeted ISAC treatment led to ~3.5-fold increase in T cell frequency that shifted the tumor microenvironment from immunologically cold to hot. The recruitment of both phagocytes and CD8+ T cells was consequential, as depletion of either abrogated anti-tumor efficacy of the rHER2-targeted ISAC. Systemically delivered ISACs were well-tolerated.ConclusionsIn contrast to other immune therapies, such as anti-PDL1/PD1 and anti-CD40, systemically administered ISACs locally engage both the innate and adaptive arms of the immune system to eradicate tumors. The molecular and cellular phenotype associated with ISAC-mediated activation is being evaluated in the on-going BDC-1001 Phase I/II clinical trial.2ReferenceAckerman Set al, Poster# P756, SITC 20192. Phase 1/2 Study of BDC-1001 as a Single Agent and in Combination With Pembrolizumab in Patients With Advanced HER2-Expressing Solid Tumors; ClinicalTrials.gov (NCT04278144)

BackgroundCEA (CEACAM5) is a well-validated cell-surface antigen that is highly expressed in multiple solid tumors. Bolt's immune-stimulating antibody conjugates (ISACs) direct a TLR7/8 agonist into tumors to activate tumor-infiltrating myeloid cells and initiate a broad innate and adaptive anti-tumor immune response.1 The favorable properties of CEA, including robust cell surface expression, low internalization rate, and limited normal tissue expression, suggest that the antigen may be a suitable ISAC target. We are evaluating an anti-CEA ISAC, BDC-2034, as a multi-functional approach to treat CEA-expressing cancers.MethodsAnti-CEA antibodies were tested for binding affinity and specificity, CEA-targeted antibody-dependent cellular phagocytosis (ADCP), and myeloid-mediated tumor cell killing. Selected antibodies were conjugated to proprietary TLR7/8 agonists, and the resulting CEA ISACs were evaluated for in vitro myeloid activation and in vivo efficacy against xenograft tumors.ResultsAntibody CEA1 binds to the CEA protein with high affinity (EC50 = 0.25 nM), binds selectively to CEA-positive tumor cell lines, and mediates ADCP more efficiently than a reference anti-CEA antibody, labetuzumab (figure 1). We generated BDC-2034 by conjugating a potent TLR7/8 agonist to CEA1. BDC-2034 tumor cell binding drives myeloid effector cell ADCP, agonist delivery to TLR7 and TLR8 in endosomes, and secretion of cytokines critical for innate and adaptive immunity (including IL-12p70, CXCL10, and TNFa). In the HPAF II + cDC co-culture model, IL-12p70 is induced with EC50 = 1.2 nM, and the level of induction is at least ten-fold higher than with ISACs using labetuzumab (figure 2). Potent cellular activity is strictly dependent on tumor cell CEA expression; in whole blood, in the absence of CEA-expressing tumor cells, cytokine induction was only observed at approximately 100-fold higher concentrations. BDC-2034 inhibits the growth of HPAF II xenograft tumors in SCID/beige mice with a minimal efficacious dose (MED) of 1 mg/kg, demonstrating anti-tumor activity solely through innate immune activation (figure 3). The TLR7/8 agonist in BDC-2034 has relatively poor activity in mice; a surrogate CEA1 ISAC with a mouse TLR7-activating agonist achieved MED = 0.5 mg/kg in the HPAF II model, with eradication of all tumors at the 5 mg/kg dose.ConclusionsThese pre-clinical data demonstrate the potential of BDC-2034 to treat CEA-expressing human cancers. Most importantly, the antigen-dependent induction of immune-stimulating cytokines promises a robust immune response that combines the activation of innate and adaptive arms.ReferenceAckerman S, Pearson C, Gregorio J. Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nature Cancer 2021;2:18–33. https://doi.org/10.1038/s43018-020-00136-xAbstract 784 Figure 1ADCP. Anti-CEA antibody CEA1 is an efficient inducer of ADCP of Raji/CEA cells by M-CSF differentiated monocyte-derived macrophagesAbstract 784 Figure 2Tumor-dependent dendritic cell activation. BDC-2034 induces IL-12p70 secretion from primary dendritic cells (cDC); native CEA1 antibody and reference anti-CEA ISAC are ineffectiveAbstract 784 Figure 3Efficacy against xenograft tumors. BDC-2034 inhibits the growth of HPAF II tumors in SCID/beige mice; native CEA1 antibody and isotype ISAC are ineffective

Introduction: Immune-stimulating antibody conjugates (ISACs) direct a TLR7/8 agonist into tumors through engagement of cell surface antigens to activate tumor-associated myeloid cells and initiate a broad innate and adaptive anti-tumor immune response. We are developing the ISAC BDC-2034 to target the tumor antigen, CEA (CEACAM5), which is expressed in many solid tumors. BDC-2034 comprises our proprietary antibody, CEA1, covalently conjugated to a potent TLR7/8 agonist via a non-cleavable linker. The strong pro-phagocytic capacity and slow internalization rate of CEA1 makes it an ideal antibody for the ISAC approach. Methods: The ability of BDC-2034 to induce the secretion of pro-inflammatory cytokines was evaluated in vitro using co-cultures of tumor cells and primary immune cells (including monocytes and dendritic cells). Anti-tumor efficacy was demonstrated in vivo with xenograft and syngeneic mouse tumor models. In parallel studies, the mechanism of BDC-2034 action was assessed by quantifying intratumoral immune infiltration, cytokine levels, and transcript profile. Results: In co-cultures of CEA-expressing tumor cells and immune cells, BDC-2034 induced the secretion of cytokines and chemokines that are essential for a broad anti-tumor immune response, including IL-12p70, CXCL10, and TNFα. These effects were accompanied by myeloid cell activation as demonstrated by elevation in costimulatory surface markers such as CD40. In vivo, BDC-2034 inhibited tumor growth in multiple xenograft and syngeneic mouse tumor models having CEA expression comparable to human cancers. Consistent with the proposed mechanism of action, BDC-2034 induced intratumoral immune cell infiltration, inflammatory cytokine secretion, and myeloid re-programming in a dose-dependent and CEA-dependent fashion. The constellation of BDC-2034 effects translated to durable therapeutic activity. Conclusions: These preclinical findings demonstrate the potential of BDC-2034 to generate anti-tumor activity in CEA-expressing cancers through direct innate immune activation and the induction of adaptive anti-tumor immunity. Citation Format: Lisa K. Blum, Cecelia I. Pearson, Laughing Bear Torrez Dulgeroff, Rishali Gadkari, Angela Luo, Andrew Luo, Jennifer E. Melrose, Jess L. Nolin, Hai Li, Arthur Lee, Matthew N. Zhou, Puneet Anand, Ganapathy Sarma, Karla A. Henning, Steven J. Chapin, Shelley E. Ackerman, Romas Kudirka, Yuyi Shen, Bruce Hug, Edith A. Perez, Marcin Kowanetz, Michael N. Alonso, Brian S. Safina, David Dornan, William G. Mallet. The CEA-targeted ISAC, BDC-2034, shows preclinical efficacy associated with innate immune activation, phagocytosis, and myeloid reprogramming [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 2911.

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.