Abstract Background: Humanized immune system (HIS) mice are critical immuno-oncology tools for in vivo evaluation of therapeutic target engagement and efficacy. HIS mice require a human donor source of CD34+ hematopoietic stem cells to provide the human immune cell system. However, variance across donors complicates study design and hypothesis testing. To determine if parameters critical to drug discovery are consistent across batches of HIS mice from the same donor, we evaluated two independent cohorts of human tumor xenograft-bearing mice made using a common set of five different CD34+ cell donors. Our study included evaluation of tumor growth kinetics, human immune reconstitution and phenotype, and the pharmacokinetics and anti-tumor efficacy of a therapeutic monoclonal antibody directed against a human target expressed on tumor-associated macrophages (TAMs). Method: HIS NOG-EXL (huNOG-EXL) mice were created using CD34+ cells from five donors in two independent cohorts (~12 weeks apart). Baseline immune reconstitution in blood was assessed at 10 weeks post engraftment. MDA-MB-231 cells were inoculated bilaterally and mice were randomized into groups for immune profiling, pharmacokinetics, and efficacy studies. Body weight, clinical observations, endogenous hIgG levels, and tumor growth were measured. For efficacy, tumors grew to ~100 mm3 and mice were dosed intraperitoneally every five days (6 doses total). Separate cohorts of mice were used for immune phenotyping and the single-dose PK study. Results: Donor consistency was seen for some of the parameters assessed but not all. The overall health outcomes for a given donor appeared to have an impact on the consistency of important parameters (e.g. efficacy). Donors that had poor health outcomes (body condition scores [BCS] <3) showed less consistency than those that experienced good (BCS=3) outcomes. The health trajectory, peripheral human immune cell engraftment, and PK profiles were similar across experiments for a given donor. Total human immune cell infiltration in tumors was similar across experiments, but there were notable shifts in immune cell composition. Whereas mice in the first cohort showed a dominant TAM phenotype within the tumor (~>50% of all hCD45+ cells), mice across donors in the second cohort had reduced TAMs and corresponding increases in several T cell subsets. Efficacy was consistent in donors with good health outcomes (Donors 1 and 4) and was inconsistent in those with poor outcomes (Donors 3 and 5). Conclusion: Our study demonstrates that a return-to-donor approach can provide information that is reproducible across cohorts to help build a donor profile. An understanding of this profile should afford greater predictability and facilitate study design for drug discovery applications. Citation Format: Janell R. Richardson, Megan M. MacBride, Esther Andino, Emily Sack, Nicholas Smith, Po Y. Ho, Fang Xiao, Katelynn A. McEachin, David T. Omstead, Rachel Grgich, Michael N. Alonso, Justin A. Kenkel, Shelley E. Ackerman. Profiling of human CD34+ donors in a myeloid-supportive HIS model provides increased predictability and facilitates study design for evaluation of immuno-oncology therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5337.

Background Immune-stimulating antibody conjugates (ISACs) are comprised of immune stimulants conjugated to tumor-targeting antibodies. Trastuzumab-T785 ISAC is a murine surrogate of BDC-1001, a HER2-targeting ISAC currently under evaluation in Phase 2 studies. Trastuzumab-T785 consists of trastuzumab conjugated with a non-cleavable linker to a TLR7/8 agonist. Trastuzumab-T785 elicits myeloid activation and tumor eradication in trastuzumab-resistant HER2 IHC3+ models. Given that the activity of trastuzumab-T785 is dependent on FcgR-mediated phagocytosis, we hypothesized that trastuzumab-T785 and pertuzumab, which binds a distinct HER2 epitope from trastuzumab, would enhance anti-tumor efficacy by increasing Fc clustering and promoting phagocytosis. Methods The effect of combining pertuzumab with ISAC therapy was explored in nine different xenograft tumor models. SCID/beige mice bearing HER2-expressing tumors (IHC3+, 2+, or 1+) were treated with trastuzumab-T785 at multiple dose levels administered Q5Dx4 in combination with pertuzumab or an isotype control; tumor growth inhibition (TGI) was assessed approximately 20 days after the initial treatment. Intra-tumoral cytokines and chemokines were measured using multiplex ELISA to examine myeloid activation. The role of phagocytes was assessed through in vivo depletion with an anti-CSF1R antibody. The requirement of Fc-mediated effector function was explored using a pertuzumab variant containing Fc effector-attenuating substitutions. Results Trastuzumab-T785 ISAC monotherapy improved TGI compared to trastuzumab and pertuzumab combination, and trastuzumab-T785 and pertuzumab combination further enhanced TGI across multiple tumor models and dose levels. Moreover, the addition of pertuzumab lowered the quantity of ISAC required for efficacy in the JIMT-1 HER2 IHC2+ model. Anti-tumor efficacy depended on phagocytes, as depletion with an anti-CSF1R antibody significantly reduced TGI by ~50%. Treatment with ISAC and a pertuzumab variant with a non-functional Fc region also reduced TGI by ~50%, demonstrating a functional Fc was required for optimal efficacy. The combination of pertuzumab with trastuzumab-T785 ISAC significantly increased the amplitude of the cytokine and chemokine response relative to ISAC monotherapy or antibody combination therapy, indicating enhanced myeloid activation in the tumor. Conclusions The combination of trastuzumab-T785 and pertuzumab significantly enhanced efficacy in multiple HER2-expressing tumors, including those with lower HER2 expression. ISAC therapy utilizes phagocytosis to initiate anti-tumor responses, and pertuzumab serves as an additional source of 'eat me' signals that likely enhance phagocytosis and deepen anti-tumor efficacy. These studies suggest that this combination may enhance the clinical activity of trastuzumab-based ISACs. This hypothesis is being assessed in a randomized Phase 2 clinical trial with BDC-1001 and pertuzumab in patients with HER2+ breast cancer post trastuzumab deruxtecan (BBI-20231001).

Tumor-associated macrophages (TAMs) are the largest immune cell population in many cancers and play a key role in establishing the immunosuppressive tumor microenvironment (TME) that enables tumor progression. However, TAMs are phenotypically plastic and have the potential to be reprogrammed into immunostimulatory cells that enhance innate and adaptive anti-tumor immunity. To this end, we developed BDC-3042, an agonistic antibody targeting an immune-activating receptor expressed on TAMs known as Dectin-2 (CLEC6A). Dectin-2 is a C-type lectin receptor (CLR) known best for its role in pathogen recognition and induction of protective immune responses against fungi and other microbes. We previously demonstrated that Dectin-2 agonism with natural ligands stimulates pro-inflammatory cytokine secretion and antigen presentation by TAMs, resulting in robust CD8+ T cell-mediated anti-tumor immunity in syngeneic mouse models. Here we present our preclinical studies demonstrating the therapeutic potential of the Dectin-2 agonistic antibody, BDC-3042, as a novel TAM-directed immunotherapy for diverse human cancers. BDC-3042 exhibits strong binding to Dectin-2-expressing macrophages generated in vitro and to primary human TAMs from a range of solid tumor types. In contrast, BDC-3042 binds weakly to peripheral monocytes and minimally to other immune cells in blood and tumor tissues. Macrophages exposed to cytokines and growth factors commonly found in the TME exhibit increased Dectin-2 expression and BDC-3042 binding. Treatment with BDC-3042 activates in vitro-generated macrophages and primary TAMs to produce an array of pro-inflammatory cytokines and chemokines associated with anti-tumor immunity. Consistent with low target expression, BDC-3042 elicits little to no activation of peripheral monocytes or cells in whole blood. BDC-3042 activity is dependent on both Dectin-2 and FcγRs, as indicated through studies utilizing Fc variants with enhanced or attenuated effector function. In mice with humanized immune systems, BDC-3042 elicits activation of TAMs, as evidenced by modulation of key activation markers. Systemically administered BDC-3042 mediates anti-tumor efficacy as a monotherapy, and combination with checkpoint blockade therapy enhances anti-tumor efficacy. The data presented demonstrate the therapeutic potential of targeting Dectin-2 expressed by TAMs with the agonistic antibody BDC-3042 as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy. Citation Format: Justin A. Kenkel, Fang Xiao, Po Y. Ho, Jess L. Nolin, Rishali K. Gadkari, Laughing Bear Torrez, David T. Omstead, Katelynn A. McEachin, Jason Ptacek, Rachel Grgich, Cecelia I. Pearson, Stefan Chun, Cindy L. Kreder, Karla A. Henning, Han K. Kim, Lu Xu, Steven J. Chapin, Michael N. Alonso, Shelley E. Ackerman. Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042 [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 2964.

Background Claudin (CLDN) 18.2 is a transmembrane tight junction protein that is expressed in stomach epithelia. CLDN18.2 expression is significantly elevated in gastric and pancreatic adenocarcinomas. Loss of cell polarity in tumors results in CLDN18.2 localization to surfaces that are more readily accessible to biologics and effector cells. This expression pattern makes it an excellent target for immune stimulating antibody conjugate (ISACs), which combine the specificity of a tumor-targeting antibody with potent immune stimulation. The delivery of ISACs to the tumor microenvironment triggers the innate and adaptive immune system to attack CLDN18.2-expressing tumors. T cell priming following phagocytosis of CLDN18.2-expressing tumor cells in the context of immune stimulation results in epitope spreading and the targeting of CLDN18.2-negative tumors cells with durable immunologic memory. These mechanisms differ from other cytotoxic payloads, which rely on the induction of apoptosis or cell death to kill tumor cells. Herein, we describe the development of a Claudin 18.2 ISAC with a TLR7/8 linker-payload. Methods For human in vitro assessment of ISACs, PBMCs or myeloid APCs were isolated from human healthy donor blood and activation was measured by flow cytometry, cytokine-bead array, and other ELISA-based methods. In vivo assessment of antitumor activity was performed using the PATU-8889s (endogenously expressing CLDN18.2) and MC38-muCLDN18.2 (engineered to express mouse CLDN18.2). Tolerability of a mouse CLDN18.2 binding ISAC was performed in healthy C57BL/6 mice. Results CLDN18.2 ISACs elicit robust tumor antigen-dependent activation of the immune system as measured by the secretion of proinflammatory cytokines TNFa and IL-12p70. Furthermore, a CLDN18.2 ISAC significantly inhibited tumor growth in a syngeneic model with CLDN18.2 expression levels consistent with those measured in the clinical setting. Interestingly, tumor regression was also observed in a model where only approximately 15% of tumor cells expressed CLDN18.2. Furthermore, the CLDN18.2 ISAC elicited T cell-dependent immunological memory with epitope spreading, as evidenced by a lack of tumor growth upon rechallenge with the original tumor cell line lacking CLDN18.2 expression. An exploratory mouse toxicity study revealed that the CLDN18.2 ISAC was well tolerated following two doses at 60 mg/kg, and the MTD was not reached in this study. Conclusions We believe that this is the first reported CLDN18.2 ISAC that demonstrates potent anti-tumor activity, induction of immunologic memory with epitope spreading, and an acceptable safety profile in preclinical studies. A CLDN18.2 ISAC may offer benefits beyond other ADCs in development.

Background Tumor-associated macrophages (TAMs) are a major component of the immune infiltrate in most cancers and play a key role in establishing the immunosuppressive tumor microenvironment (TME) that enables tumor progression. However, TAMs are phenotypically plastic and have the potential to be reprogrammed into immunostimulatory cells that enhance innate and adaptive anti-tumor immunity. BDC-3042 is a novel agonistic antibody targeting an immune-activating receptor expressed on TAMs known as Dectin-2 (CLEC6A).1 Dectin-2 is a C-type lectin receptor best known for its role in pathogen recognition and induction of protective immune responses against fungi and other microbes. We have previously demonstrated that Dectin-2 agonism stimulates pro-inflammatory cytokine secretion and antigen presentation by TAMs, resulting in robust CD8+ T cell-mediated anti-tumor immunity in syngeneic mouse models.2 Differential gene expression of Dectin-2 has been found in a wide range of solid tumor-associated macrophages compared to non-malignant tissues. Nonclinical studies with BDC-3042 have demonstrated its potential to reprogram TAMs and elicit anti-tumor activity as a novel immunotherapeutic approach for diverse human cancers.2 A phase 1/2, four-part, first-in-human, dose-escalation and dose-expansion study of BDC-3042 as a single agent and in combination with pembrolizumab in subjects with advanced malignancies has been initiated. Methods This dose-escalation and dose-expansion study is enrolling approximately 185 subjects with advanced malignancies, including triple-negative breast cancer, renal cell carcinoma, colon cancer, head and neck cancer, non-small cell lung cancer, and ovarian cancer. Primary objectives of the dose-escalation phase are to define safety and tolerability and to determine the recommended phase 2 dose (RP2D) of BDC-3042 as a monotherapy (Part 1) and in combination with pembrolizumab (Part 2). Part 2 is planned to start once single agent BDC-3042 safety data are available. Part 1 will be the dose escalation. The dose-expansion phase of the study will evaluate preliminary anti-tumor activity of BDC-3042 monotherapy (Part 3) and in combination with pembrolizumab (Part 4). Secondary objectives will evaluate pharmacokinetic parameters and pharmacodynamic biomarkers in tumor tissue and in peripheral blood associated with drug exposure. Exploratory analyses will also be conducted to assess BDC-3042's ability to reprogram TAMs and identify biomarkers associated with BDC-3042 biological activity with and without pembrolizumab. This study is being conducted in the US and is currently recruiting patients. References Kenkel JA, F Xiao, PY Ho, JL Nolin, et al. 'Abstract 2964: Targeting tumor-associated macrophages to enhance anti-tumor immunity with the Dectin-2 agonistic antibody BDC-3042.' Cancer Research 2023;83(7_Supplement):2964. Kenkel J, P Ho, S Kongara, K Henning, et al. '862 Dectin-2, a novel target for tumor macrophage reprogramming in cancer immunotherapy.' Journal for ImmunoTherapy of Cancer 2021;9(Suppl 2):A903-A903. Ethics Approval Protocols, protocol amendments, and informed consents are approved by Institutional review boards or independent ethics committees of participating sites. The study will be conducted in compliance with the Declaration of Helsinki and International Conference on Harmonization Guidelines for Good Clinical Practice. All patients will provide written informed consent.

Supplementary Figure 1. Standard curves for quantification of cellular integrin expression; Supplementary Figure 2. Competition binding to U87MG tumor cell surface integrins; Supplementary Figure 3. Inhibition of tumor cell proliferation. U87MG, A2780 and MB-468 cells were incubated with varying concentrations of 2.5F-Fc-MMAF, and CTRL-Fc-MMAF;Supplementary Figure 4. Growth inhibition of subcutaneous U87MG tumors implanted into Nu/Nu mice; Supplementary Figure 5. Weight monitoring.

Tumor-associated macrophages (TAMs), an abundant immune cell population in most cancers, support tumor progression through their immunosuppressive effects. We discovered that TAMs express the pattern recognition receptor Dectin-2 (Clec4n/CLEC6A), an activating C-type lectin receptor (CLR) that binds to high-mannose glycans on fungi and other microbes and stimulates immune responses against infectious disease. Dectin-2 is selectively expressed by myeloid cells, and upon ligation, mediates enhanced phagocytosis, antigen processing and presentation, and proinflammatory cytokine production. Given these properties, we evaluated the therapeutic potential of targeting Dectin-2 to reprogram TAMs using natural ligands as well as our novel agonistic antibodies. We show that Dectin-2 agonists can convert TAMs into immunostimulatory cells in vitro and in vivo, and drive robust anti-tumor immunity. Dectin-2 gene expression is minimal in normal human tissues but elevated across many tumor types, including breast, colon, lung, ovarian, and kidney cancers. We found that Dectin-2 is strongly expressed by macrophages differentiated in vitro and on primary TAMs from various solid tumors. Treatment of tumor-bearing mice with mannan, a natural Dectin-2 ligand derived from S. cerevisiae, mediated tumor regression in multiple syngeneic tumor models, with high rates of tumor clearance in the MB49 bladder cancer model. These effects were Dectin-2 dependent, as efficacy was not observed when a Dectin-2-blocking antibody was co-administered or in knockout mice lacking Dectin-2 signaling components. Depletion of either macrophages or CD8+ T cells impaired efficacy, suggesting that Dectin-2-stimulated TAMs augment anti-tumor CD8+ T cell responses. Based on these data, we developed novel Dectin-2-targeted agonist antibodies capable of activating both in vitro-generated and primary human TAMs to produce an array of proinflammatory cytokines and chemokines akin to tumor-destructive “M1” macrophages. Furthermore, systemically administered Dectin-2 agonist antibodies activated TAMs and mediated anti-tumor effects in immunodeficient mice engrafted with human CD34+ HSCs. The data presented demonstrate the therapeutic potential of Dectin-2 agonist antibodies as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy. Citation Format: Justin A. Kenkel, Po Y. Ho, Karla A. Henning, Cindy L. Kreder, Jess L. Nolin, Sameera Kongara, Steven J. Chapin, Amreen Husain, Marcin Kowanetz, Edgar G. Engleman, Michael N. Alonso, David Dornan, Shelley E. Ackerman. Dectin-2 agonist antibodies reprogram tumor-associated macrophages to drive anti-tumor immunity [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 2883.

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.

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.

Background: To date, anti-HER2 monoclonal antibodies are the only immune therapies approved for treating pts with HER2-over-expressing cancers. Intratumoral delivery of immunostimulatory adjuncts such as toll-like receptor (TLR) 7/8 agonists can activate tumor resident antigen-presenting cells (APCs) to promote antitumor immunity. To optimize intratumoral delivery while leveraging favorable preclinical biology we developed a novel, systemically delivered ISAC (BDC-1001). BDC-1001 consists of an investigational trastuzumab biosimilar chemically conjugated to a TLR 7/8 agonist (payload) with an intervening non-cleavable linker. BDC-1001 activates human myeloid APCs and retains antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity/phagocytosis. Our trastuzumab-based ISACs seem to elicit potent and durable immune-mediated antitumor efficacy including tumor regression in a TLR- and Fc receptor-dependent manner in xenograft and syngeneic tumor resistant models (Ackerman et al. Nat Cancer 2020). BDC-1001 did not induce interstitial lung disease, cytokine release syndrome, or thrombocytopenia in non-human primate studies. A four-part phase 1/2, first-in-human study was initiated in 2020 to evaluate BDC-1001 with or without (±) pembro in pts with HER2-overexpressing or amplified advanced solid tumors. Methods: This phase 1/2 dose-escalation/expansion study will enroll up to 390 pts with HER2-overexpressing (IHC2+ or 3+) or HER2-amplified (by in situ hybridization or next-generation sequencing) advanced solid tumors. Patients may have received prior anti-HER2 therapies. Primary objectives of dose-escalation are safety and tolerability, and establishing a recommended phase 2 dose of BDC-1001 alone administered IV q3w (Part 1) and combined with pembro (Part 2). Primary endpoints of Parts 1 & 2 include assessment of safety and tolerability; dose-limiting immune-related toxicities in a 3+3 design. The dose-expansion phase 2 portion will evaluate preliminary antitumor activity of BDC-1001 alone (Part 3) and with pembro (Part 4) using RECIST v1.1 and iRECIST. The primary endpoint of the phase 2 is best\ overall response rate; with secondary endpoints of duration of response, disease control rate, and progression-free survival. Exploratory objectives include pharmacokinetic parameters and pharmacodynamic biomarkers associated with drug exposure to help elucidate mechanism of action and identify biomarkers to improve selection of pts most likely to benefit from the single therapy or combination. Recruitment is ongoing (NCT04278144). Citation Format: Manish R. Sharma, Richard D. Carvajal, Daniel Catenacci, Leisha A. Emens, Glenn J. Hanna, Dejan Juric, Yoon-Koo Kang, Jeeyun Lee, Keun-Wook Lee, Bob T. Li, Kathleen Moore, Mark D. Pegram, Paula R. Pohlmann, Drew Rasco, Alexander Spira, Arielle L. Heeke, Ding Wang, Lawrence Garbo, Sudhir Manda, Jasgit Sachdev, Shelley E. Ackerman, Heidi LeBlanc, David Dornan, Marcin Kowanetz, Michael N. Alonso, Amreen Husain, Edith A. Perez, Ecaterina Ileana Dumbrava. Phase 1/2 study of a novel HER2 targeting TLR7/8 immune-stimulating antibody conjugate (ISAC), BDC-1001, alone and in combination with pembrolizumab (pembro) in patients (pts) with HER2-expressing advanced solid tumors [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 CT218.

Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2− parental tumor. These results provide a strong rationale for the clinical development of ISACs. Alonso and colleagues develop immune-stimulating antibody conjugates capable of specific delivery of TLR7/8 agonists to tumors, which induces durable antitumor immunity.

Deficient anti-tumor immunity often results from an immunosuppressive tumor microenvironment (TME) that renders antigen presenting cells (APCs) unable to effectively stimulate T cells. Recent studies indicate that local delivery of immunostimulatory adjuvants can activate tumor resident APCs, driving uptake, processing and presentation of tumor neoantigens to T cells that mediate anti-tumor immunity. To overcome challenges associated with intratumoral delivery of such adjuvants, we developed a novel class of TLR immune-stimulating antibody conjugates (TAC) that comprise a TLR7/8 agonist conjugated to tumor-targeting monoclonal antibodies. In vitro co-cultures with human cancer cell lines and leukocytes revealed that TACs potently activate primary APCs, leading to increased co-stimulatory molecule expression (e.g. CD40, CD86) and secretion of pro-inflammatory cytokines (e.g. TNFα). The TACs also enhanced antibody-mediated effector functions such as ADCC and ADCP. Surprisingly, these constructs also induced dendritic cell (DC) differentiation from monocytes, as measured by changes in cellular morphology and DC surface markers (e.g. CD14 downregulation). CyTOF-based analysis of intracellular signaling in human leukocytes revealed a unique signaling signature of the conjugate compared to a mixture of its components, suggesting a novel biological mechanism by which the conjugate stimulates APCs. Finally, we demonstrated in vivo efficacy in syngeneic tumor models in which TAC treatment led to tumor clearance and development of immunologic memory. These results provide a strong rationale for this technology as a platform for cancer immunotherapy. Citation Format: Shelley E. Ackerman, Joseph C. Gonzalez, Josh D. Gregorio, Jason C. Paik, Felix J. Hartmann, Justin A. Kenkel, Arthur Lee, Angela Luo, Cecelia I. Pearson, Murray L. Nguyen, Benjamin Ackerman, Lauren Y. Sheu, Richard P. Laura, Steven J. Chapin, Brian S. Safina, Sean C. Bendall, David Dornan, Edgar G. Engleman, Michael N. Alonso. TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation leading to enhanced effector function and anti-tumor immunity in pre-clinical models [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 1559.

Chronic inflammation in adipose tissue, possibly related to adipose cell hypertrophy, hypoxia, and/or intestinal leakage of bacteria and their metabolic products, likely plays a critical role in the development of obesity-associated insulin resistance (IR). Cells of both the innate and adaptive immune system residing in adipose tissues, as well as in the intestine, participate in this process. Thus, M1 macrophages, IFN-γ-secreting Th1 cells, CD8+ T cells, and B cells promote IR, in part through secretion of proinflammatory cytokines. Conversely, eosinophils, Th2 T cells, type 2 innate lymphoid cells, and possibly Foxp3+ Tregs protect against IR through local control of inflammation.

L'invention concerne un immunoconjugue qui comprend une construction d'anticorps comportant un domaine de liaison a l'antigene et un domaine Fc, un fragment d'adjuvant et un lieur, chaque fragment d'adjuvant etant lie de maniere covalente a l'anticorps par l'intermediaire du lieur. L'invention concerne egalement des methodes de traitement du cancer au moyen des immunoconjugues de l'invention.

Abstract Antibody–drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F–Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvβ3, αvβ5, and α5β1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin–Fc–drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F–Fc–MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F–Fc or MMAF alone or added in combination. As a single agent, 2.5F–Fc–MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F–Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL–Fc–MMAF, showed a modest but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications. Mol Cancer Ther; 15(6); 1291–300. ©2016 AACR.

Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature's repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.

Tumors of the central nervous system are challenging to treat due to the limited effectiveness and associated toxicities of chemotherapy and radiation therapy. For tumors that can be removed surgically, extent of malignant tissue resection has been shown to correlate with disease progression, recurrence, and survival. Thus, improved technologies for real-time brain tumor imaging are critically needed as tools for guided surgical resection. We previously engineered a novel peptide that binds with high affinity and unique specificity to αVβ3, αVβ5, and α5β1 integrins, which are present on tumor cells, and the vasculature of many cancers, including brain tumors. In the current study, we conjugated this engineered peptide to a near infrared fluorescent dye (Alexa Fluor 680), and used the resulting molecular probe for non-invasive whole body imaging of patient-derived medulloblastoma xenograft tumors implanted in the cerebellum of mice. The engineered peptide exhibited robust targeting and illumination of intracranial medulloblastoma following both intravenous and intraperitoneal injection routes. In contrast, a variant of the engineered peptide containing a scrambled integrin-binding sequence did not localize to brain tumors, demonstrating that tumor-targeting is driven by specific integrin interactions. Ex vivo imaging was used to confirm the presence of tumor and molecular probe localization to the cerebellar region. These results warrant further clinical development of the engineered peptide as a tool for image-guided resection of central nervous system tumors.