hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications.

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.

INTRODUCTION: Immune-stimulating antibody conjugates (ISACs) consist of tumor-targeting antibodies conjugated to immune stimulants and are designed to activate the innate and adaptive immune systems against tumor cells following systemic administration. PD-L1 is an immune checkpoint that regulates anti-tumor T cell responses and is expressed on tumor cells as well as tumor-infiltrating immune cells across many tumor types. Here we show that PD-L1-targeted TLR7/8 ISACs elicit robust myeloid cell activation and can act through PD-L1 on either tumor or immune cells to improve anti-tumor responses compared to anti-PD-L1 treatment in preclinical tumor models. METHODS: A panel of proprietary anti-PD-L1 ISACs was evaluated for target-dependent in vitro myeloid cell activation by co-culturing PD-L1 expressing tumor cells with cDC-enriched primary myeloid cells. Anti-tumor efficacy of anti-PD-L1 ISACs was evaluated in vivo in both syngeneic and xenograft tumor models. MB49-PD-L1 KO cells were generated by knocking-out PD-L1 gene using CRISPR/Cas9 system. RESULTS: Anti-PD-L1 antibodies induced robust ADCP by myeloid effector cells and PD-L1/PD-1 blockade in vitro and in vivo. The conjugated PD-L1 ISACs induced robust, target-dependent activation of myeloid cells when co-cultured with tumor cells expressing PD-L1 at physiological levels, as measured by increased secretion of such cytokines as IL-12p70, TNFα, and IFNγ. Systemically administered surrogate PD-L1 ISACs were well tolerated in mice and showed improved anti-tumor efficacy over anti-PD-L1 antibodies, with significant tumor growth delay or complete responses frequently observed in syngeneic (e.g., MB49, MC38-hPD-L1) as well as xenograft (e.g., HCC1954-hPD-L1) tumor models. The improved in vivo efficacy of PD-L1 ISACs was sustained even in the absence of PD-L1 expression on tumor cells in syngeneic MB49-PD-L1 KO model suggesting that PD-L1 ISAC can induce its mechanism of action also through PD-L1 on myeloid cells. No tumor growth was observed after rechallenging mice previously cured with PD-L1 ISACs indicating development of immunological memory following the ISAC treatment. CONCLUSIONS: These preclinical data demonstrate the potential of a PD-L1-targeted ISAC as a novel multifunctional therapeutic that may improve efficacy of PD-L1/PD-1 inhibition by combining three mechanisms of action into a single molecule: TLR-mediated myeloid cell activation, T cell activation through immune-checkpoint inhibition as well as ADCP. Citation Format: Justin A. Kenkel, Rishali Gadkari, Po Y. Ho, Lisa K. Blum, Romas Kudirka, Karla A. Henning, William G. Mallet, Jennifer E. Melrose, Ganapathy Sarma, Steven J. Chapin, Matthew Zhou, Suprit Deol, Cindy Kreder, Yuyi Shen, Bruce Hug, Puneet Anand, Arthur Lee, Hai Li, Shelley E. Ackerman, Brian S. Safina, David Dornan, Michael N. Alonso, Marcin Kowanetz. PD-L1-targeted ISAC combines myeloid cell activation, immune-checkpoint inhibition and ADCP to improve anti-tumor efficacy over anti-PD-L1 antibodies 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 4252.

BackgroundPD-L1 is an immune checkpoint that regulates anti-tumor T cell responses and is expressed on tumor cells as well as tumor-infiltrating immune cells across many tumor types. Immune-stimulating antibody conjugates (ISACs) consist of tumor-targeting antibodies conjugated to immune stimulants and are designed to activate the innate and adaptive immune systems against tumor cells following systemic administration. Here we show that PD-L1-targeted TLR7/8 ISACs elicit robust myeloid cell activation which leads to improved anti-tumor responses compared to anti-PD-L1 treatment in pre-clinical tumor models.MethodsA panel of proprietary anti-PD-L1 antibodies was identified through a phage display screen and subsequently tested for PD-L1 binding affinity and specificity, PD-L1/PD-1 blocking, antibody-dependent cellular phagocytosis (ADCP) by myeloid cells, and anti-tumor efficacy. Lead antibodies were conjugated to proprietary TLR7/8 agonists, and the resulting PD-L1 ISACs were evaluated for in vitro myeloid cell activation and in vivo efficacy against syngeneic and xenograft tumors.ResultsAnti-PD-L1 antibodies induced robust ADCP by myeloid effector cells and medium to strong PD-L1/PD-1 blockade in vitro. Selected antibodies inhibited the growth of syngeneic MC38-hPD-L1 tumors in vivo, confirming efficient immune-checkpoint blockade. The conjugated PD-L1 ISACs induced robust, target-dependent activation of myeloid cells when co-cultured with PD-L1-expressing tumor cells, as measured by increased secretion of such cytokines as IL-12p70, IFN-alpha, and TNF-alpha. Importantly, myeloid activation was observed following co-culture with tumor cells having various levels of endogenous PD-L1 expression that was within the range of PD-L1 expression observed in human tumors. Systemically administered surrogate PD-L1 ISACs were well tolerated in mice and showed improved anti-tumor efficacy over anti-PD-L1 antibodies, with significant tumor growth delay or complete responses frequently observed in syngeneic (e.g. MB49, MC38-hPD-L1) as well as xenograft (e.g. HCC1954-hPD-L1) tumor models.ConclusionsThese data demonstrate the potential of a PD-L1-targeted ISAC as a multifunctional therapeutic that may improve efficacy of PD-L1/PD-1 inhibition by combining three mechanisms of action into a single molecule: TLR-mediated myeloid cell activation, T cell activation through immune-checkpoint inhibition as well as ADCP.Ethics ApprovalAll animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols.