Antitumor Effects In Preclinical Models Research Articles

Abstract 5707: Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies

Abstract KRLS-017 is a potent, selective, and reversible inhibitor of Cyclin Dependent Kinase 7 (CDK7). Its molecular formula is C24H35N5O2Si and its molecular weight is 453.65 Daltons. KRLS-017 is being developed for the treatment of patients with advanced solid tumor malignancies. To differentiate KRLS-017 from other CDK7 inhibitors, we created a reversible inhibitor to allow for treatment schedule optimization and maximization of therapeutic index in clinic. High selectivity of KRLS-017 for CDK7 was confirmed using a panel of 313 recombinant human kinases. The IC50 of KRLS-017 against recombinant human CDK7 was determined to be 16 nM, and no other kinases in the panel showed IC50 values less than 300 nM. Antiproliferative activity of KRLS-017 was investigated in vitro using a broad panel of human tumor cell lines. KRLS-017 showed potent antiproliferative effect across a wide variety of both solid and hematologic tumor types, producing GI50 values <200 nM in 258/302 human tumorcell lines tested. Antitumor activity of KRLS-017 was investigated in vivo using multiple mouse xenograft tumormodels at dose levels ranging from 25 mg/kg to 200 mg/kg using both continuous (daily) andintermittent dosing schedules. Statistically significant tumor growth inhibition was seen at doselevels of 50 mg/kg and higher, with tumor regression observed at 100 mg/kg and 200 mg/kg. Better in vivo tolerability at efficacious doses was observed using intermittent dosing schedules in mouse xenograft models, which led to adoption of an intermittent schedule in the proposed first-in-human phase 1 trial. KRLS-017 is orally bioavailable in mice, rats, and dogs and plasma drug exposure increased in a proportional manner with dose. Comparing first dose and 28th dose in the dog, minimal plasma accumulation was observed, suggesting once daily dosing in humans would be initially tested. Repeat dose 28 day GLP toxicology studies in rat and dog showed adverse effects in highly proliferative bone marrow and gastrointestinal tissues which is consistent with the known mechanism of action for CDK7 and is considered to be monitorable and manageable in the setting of oncology. The clinical development plan for KRLS-017 includes an initial Phase 1 dose escalation study (KRLS-017-101) in patients with refractory solid tumor malignancies to assess pharmacokinetics/pharmacodynamics and to determine an optimal dose level and treatment schedule to test in expansion cohorts. Given the potential of CDK7 inhibitors in the treatment of hormone receptor positive breast cancer, combination work with other agents (such as selective estrogen receptor degraders) is on-going. Citation Format: Alison L. Hannah, Steve Ritland, Thomas Steele, Steven Smith, Ashwin Ram, BT Slingsby, Kapil Dhingra, James Chrisman, Rajesh Dua, Yasunori Tokunaga, Shigeyuki Kono, Yasuhiro Aga. Preclinical evaluation of KRLS-017, a potent, highly selective and reversible CDK7 inhibitor with broad antitumor effect in preclinical models, in preparation for a Phase 1 clinical trial in advanced solid tumor malignancies [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 5707.

Abstract 6734: Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC

Abstract Background: Stimulator of interferon genes (STING) agonists elicit potent innate immune enhancement and mobilize robust adaptive immune responses. Given the potential risk of immunotoxicity from associated cytokine release due to STING agonist treatment, there is a need to identify mitigation strategies that minimize immune-related toxicity while offering a therapeutic window for STING-mediated anti-tumor effects. This strategy is particularly relevant for myeloid-targeted immunotherapies, as cytokine release syndrome (CRS) can be myeloid cell-mediated. We utilized preclinical models to evaluate the pharmacological impact of pretreatment with agents routinely used for management of CRS in advance of delivering TAK-500, a novel, systemic, immune-cell directed STING agonist antibody drug conjugate (iADC) that selectively targets CCR2+ myeloid cells. Methods: Dexamethasone and anti-IL-6R antibodies were tested at clinically relevant doses 1 hour prior to treatment with TAK-500 or mTAK-500, a murine surrogate, to study immunomodulatory and anti-tumor effects in preclinical models. Induction of innate and adaptive immune responses and cytokine release was evaluated in human, ex vivo, and in tumor-bearing mouse models to characterize potential impact of premedication strategies on drug pharmacology and anti-tumor efficacy. Results: TAK-500 triggered dose-dependent monocyte activation and cytokine secretion ex vivo. Monocyte activation was minimally impacted by dexamethasone or anti-IL-6R pretreatment. Suppression of IL-6 and IFN-ƴ and increase in IL-10 were observed following dexamethasone pretreatment, whereas anti-IL-6R maintained a more proinflammatory milieu. In syngeneic mouse models, pretreatment with dexamethasone and anti-mouse IL-6R had minimal impact on mTAK-500 anti-tumor efficacy. Key pharmacodynamic effects, including innate immune cell activation in periphery and tumor-draining lymph node and intratumoral CD8+ T cell accumulation, were minimally impacted by either pretreatment agent. Serum cytokine analyses demonstrated suppression of IL-6, IFN-ƴ and increases in IL-10 with dexamethasone, but not with anti-IL-6R, similar to ex vivo findings. Conclusion: Premedication of TAK-500 and mTAK-500 with dexamethasone or anti-IL-6R antibodies does not substantially impact STING-mediated innate immune activation, mobilization of adaptive immune responses or anti-tumor efficacy, despite altered cytokine profiles compared to (m)TAK-500 without premedication. Clinically, these premedication strategies have been effective in mitigating immunotherapy-associated CRS with T cell therapeutics, and here resulted in maintained anti-tumor activity when used in combination with TAK-500, suggesting a viable potential strategy for mitigation of STING-induced immunotoxicities in patients. Citation Format: Emily Rosentrater, Michelle L. Ganno, Tiquella Hatten, Henry Mack, Arjun Reddy Bollampalli, Dong Mei Zhang, Alex Parent, Jeffrey Raizer, Richard C. Gregory, Neil Lineberry, Vicky Appleman. Preclinical impact of pretreatment with CRS mitigation agents on pharmacodynamic response to TAK-500, a systemically delivered CCR2-targeted STING agonist iADC [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 6734.

Abstract 5618: Evaluating the combination of datopotamab deruxtecan (Dato-DXd) with AZD5305, a highly potent, PARP1-selective inhibitor, -in preclinical models

Abstract Background: Dato-DXd is a TROP2-directed antibody drug conjugate comprised of a topoisomerase I inhibitor (an exatecan derivative, DXd) conjugated to datopotamab, a humanized anti-TROP2 IgG1 antibody, via a cleavable plasma-stable tetrapeptide-based linker. Topoisomerase I inhibitors stabilize DNA-topoisomerase cleavage complexes (Topo1cc) that lead to double-strand breaks and activation of DNA damage response (DDR). Poly(ADP-ribose) polymerase 1 (PARP1) mediates signal transduction in the DDR as an important regulator. AZD5305 is a highly potent and selective inhibitor of PARP1. Since PARP1 is a key component driving the repair of trapped Topo1cc, we aimed to investigate if combinations of Dato-DXd with AZ5305 lead to synergistic anti-tumor effects in preclinical models. Methods: We evaluated the cytotoxic effect of the combination of Dato-DXd with AZD5305 in a panel of six non-small cell lung cancer (NSCLC) and five triple negative breast cancer (TNBC) cell lines in a 7-day viability assay. We evaluated PARylation inhibition and gH2AX response by Western blotting. Topo1cc accumulation in response to combination treatment was evaluated by immunofluorescence. This combination was evaluated in vivo in TROP2+, HRD-negative gastric cancer (GC) N87 cell line xenograft at varying concentrations of AZD5305 and in a TROP2+, PARPi-resistant Ovarian cancer patient derived xenograft (PDX) model (CTG-3718). Results: The in vitro combination screen demonstrated enhanced cell killing by the Dato-DXd and AZD5305 combination over either single agent in both NSCLC and TNBC cell lines. Combination benefit was observed at all doses regardless of homologous recombination deficiency (HRD) status. We observed dose-dependent increase in PARylation inhibition and enhanced induction of gH2AX in the combination treatment. In a N87 tumor model, while Dato-DXd and AZD5305 provided 84% and 31% tumor growth inhibition (TGI) as monotherapies respectively, the addition of AZD5305 to Dato-DXd resulted in 95% TGI. This effect was observed with multiple dose levels of AZD5305. In a TROP2+, ovarian cancer PDX model, AZD5305 demonstrated limited monotherapy activity (<10% TGI) and Dato-DXd demonstrated robust monotherapy activity with 72% TGI. Additional anti-tumor activity was observed for the combination treatment compared to either monotherapy with 88% TGI. All treatment groups were well tolerated in vivo. Conclusion: Dato-DXd combination with AZD5305 increased cytotoxic activity and pharmacodynamic response in vitro. This combination led to superior tumor growth inhibition in a GC xenograft model and a PARP1i-resistant Ovarian PDX model in vivo. These pre-clinical findings support the ongoing clinical evaluation of Dato-DXd as a monotherapy and in combination with AZD5305 in patients with various advanced solid tumor types (NCT05489211, NCT04644068). Citation Format: Gopi Patel, Joe’l Owusu, Chetan Rane, Alan Rosen, Matthew Griffin, Matthew Sung. Evaluating the combination of datopotamab deruxtecan (Dato-DXd) with AZD5305, a highly potent, PARP1-selective inhibitor, -in preclinical models [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 5618.

Abstract 4322: Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer

Abstract Approximately 30% of lung cancers are driven by mutationally activated KRAS. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, a major obstacle in treating lung cancer is resistance to current therapeutic treatments. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagy in response to treatment with sotorasib. Currently, the only therapeutics in clinical use to inhibit autophagy are lysosomal inhibitors (hydroxy)chloroquine. High concentrations of these compounds are needed to achieve modest inhibition of autophagy, suggesting that the potency of these compounds may limit clinical responses. ULK kinases are serine/threonine kinases that are necessary for the initiation of autophagy. DCC-3116 is a potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy. Treatment with sotorasib and DCC-3116 led to superior anti-tumor effects in preclinical models of lung cancer. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in the regulation of nutrient sensing and autophagy. In other KRAS-driven cancers, the LKB1-AMPK-ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data demonstrates that LKB1 is dispensable for sotorasib-induced autophagy, suggesting the mechanism(s) of autophagy induction in KRASG12C-driven lung cancer may be different from those detected in other KRAS mutated cancers. Consequently, we have generated genetically engineered mouse models (GEMMs) of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. We have treated KRASG12C-driven GEMMs with sotorasib and/or DCC-3116 to test the sensitivity of lung tumors to these treatment options. Citation Format: Phaedra Ghazi, Kayla O'Toole, Sanjana Boggaram, Michael Scherzer, Madhumita Bogdan, Bryan Smith, Dan Flynn, Conan Kinsey, Martin McMahon. Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer [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 4322.

BGB-A445, a novel non-ligand-blocking agonistic anti-OX40 antibody, exhibits superior immune activation and antitumor effects in preclinical models.

OX40 is a costimulatory receptor that is expressed primarily on activated CD4+, CD8+, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.

Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

BackgroundIrradiation (IR) and immune checkpoint inhibitor (ICI) combination is a promising treatment modality. However, local and distance treatment failure and resistance can occur. To counteract this resistance, several studies propose...

Abstract PR03: Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer

Abstract Approximately 25% of lung cancers are driven by mutationally-activated KRAS. A major obstacle in treating lung cancer is resistance to current therapeutic treatments. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, the twin problems of primary or acquired resistance to direct pharmacological inhibition of KRASG12C remain major obstacles in sustaining the deepest and most durable patient responses. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. Moreover, combined inhibition of RAS pathway signaling plus autophagy had potent anti-tumor effects in preclinical models of melanoma or pancreatic cancer. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagic flux in response to treatment with KRASG12C inhibitors. Moreover, combined treatment of such cells with a specific, selective and potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy, led to superior anti-tumor effects in human cell line xenograft models. Previous work to understand why inhibiting autophagy sensitizes KRAS mutant cancer cells to targeted therapy has been confounded by the use of non-specific lysosomal inhibitors such as hydroxychloroquine. Using selective autophagy inhibitors, such as ULK1/2i, provides a unique opportunity to investigate the precise mechanism as to why inhibition of autophagy sensitizes cells to KRASG12C inhibition. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in regulation of nutrient sensing and autophagy. Patients with KRAS-mutated lung cancer whose tumors also lack LKB1 expression are characterized by aggressive behavior and resistance to standard treatment. In other KRAS-driven cancers, the LKB1>AMPK>ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data suggests that LKB1 is dispensable for sotorasib-induced autophagy in KRASG12C-driven lung cancer cells. To this end, KRASG12C-driven lung cancer cell lines with loss of LKB1 expression significantly increased autophagic flux after treatment with either a KRASG12C inhibitor or inhibitors of RAF>MEK>ERK signaling suggesting that the mechanism(s) of autophagy induction in KRASG12C-driven may be different to those detected in other KRAS mutated cancers. Consequently, we have generated new genetically engineered mouse models of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. Citation Format: Phaedra C. Ghazi, Conan Kinsey, Madhumita Bogdan, Bryan D. Smith, Daniel L. Flynn, Martin McMahon. Cooperative anti-tumor effects of combined inhibition of KRASG12C plus autophagy in preclinical models of KRASG12C-driven lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR03.

Abstract LB199: A personalized neoantigen vaccine is well tolerated and induces specific T-cell immune response in patients with resected melanoma

Abstract Tumor mutations giving rise to neoantigens have recently emerged as promising targets for cancer immunotherapy. Vaccines delivering tumor-specific neoantigens have demonstrated favorable efficacy and safety results in numerous preclinical and early clinical studies. However, selecting therapeutically relevant neoantigens amongst the myriad of cancer mutations has proven challenging. To overcome this, we have developed a proprietary AI-based target discovery platform, PIONEER, with enhanced predictive performance and increased precision. The PIONEER top-ranked neoantigens were selected and included in the personalized DNA vaccine candidate EVX-02. To assess the ability of the PIONEER designed neoantigen vaccine candidate to induce neoantigen immunogenicity and anti-tumor effect in preclinical models, mice were immunized with a mouse surrogate EVX-02 molecule, mEVX-02. mEVX-02 vaccination completely prevented establishment of tumors and induced neoantigen-specific, polyfunctional CD4+ and CD8+ T cells in the blood and spleen of immunized mice. An enhanced antitumor effect was obtained when combining mEVX-02 with anti-PD-1 mAb treatment. Encouraged by the preclinical results, we conducted a first-in-human multicenter Phase I clinical study of EVX-02 in combination with nivolumab in patients with resected malignant melanoma. The objectives of the trial were to investigate safety and tolerability, operational and clinical feasibility, and immunogenicity of the applied personal neoantigens. Each patient received a fully personalized drug that was produced in a complex process, from biopsy, through genome sequencing, AI-profiling, vaccine design, manufacturing, quality testing, and drug product release. This was succeeded with every single step for each patient. In all patients, EVX-02 treatment was well tolerated, and only very mild adverse events (AEs) have been observed in relation to immunization with EVX-02. Interim data demonstrated neoantigen-specific T-cell immune responses upon EVX-02 treatment and that the responses were mediated by activated CD4+ and CD8+ T cells. The measured T-cell responses were robust and long lasting. Together, these findings validate the precision and predictive power of our proprietary AI platform, PIONEER, and provide proof of mechanism for the DNA-delivery technology in that the encoded neoantigens gave rise to significant immune reactions. Citation Format: Daniela Kleine-Kohlbrecher, Nadia Viborg Petersen, Michail Angelos Pavlidis, Thomas Trolle, Stine Friis, Jens Kringelum, Anders Bundgaard Soerensen, Thomas Strandet Jepsen, Camilla Højgaard, Anders Jespersen, Erik D. Heegaard, Britt Winding Lauenborg, Birgitte Rønø. A personalized neoantigen vaccine is well tolerated and induces specific T-cell immune response in patients with resected melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB199.

Abstract 1841: TAK-500 is a clinical stage immune-cell directed antibody drug conjugate (iADC) inducing STING activation in CCR2-expressing intratumor myeloid cells and favorable immunomodulation

Abstract Introduction: Myeloid cells are present in the tumor microenvironment (TME) of most human solid cancers, playing an essential role to influence local immunomodulatory functions. STING signaling in intratumor myeloid cells can enhance interferon (IFN) production leading to enhanced local innate and adaptive immunity, and synergism with other anti-tumor mechanisms. Here, we used a novel iADC, TAK-500, to selectively activate STING in CCR2-expressing cells. This approach enables systemic delivery and favors intratumor accumulation of a STING agonist to potentially achieve anti-tumor activity. Methods: TAK-500 and mTAK-500, a murine surrogate, were employed to study the immunomodulatory role and anti-tumor effects in preclinical models. CCR2 protein expression was characterized in the TME of >1,000 primary human tumors including non-small cell lung cancer (NSCLC, 2 cohorts, N= 411), colorectal carcinomas (CRC, 2 cohorts, N=350) and pancreatic ductal adenocarcinomas (PDAC, 1 cohort, N=228) represented in tissue microarrays using multiplexed quantitative immunofluorescence (mQIF). Results: TAK-500 triggered dose-dependent monocyte activation in vitro. mTAK-500 induced robust activation of innate and adaptive immune responses both in vitro and in vivo. In syngeneic mouse models with CCR2 expressing intratumoral myeloid cells, mTAK-500 treatment caused accumulation and activation of CD8+ effector T-cells in the TME resulting in prominent anti-tumor activity and enhanced survival. The baseline levels of CCR2-expressing mMDSCs in mouse models positively associated with anti-tumor response to mTAK-500. mQIF analysis of human tumors confirmed high expression of CCR2 in myeloid cells, lower expression in tumor infiltrating lymphocytes (TILs) and absence in tumor epithelial cells. Using an mQIF panel for simultaneous measurement of DAPI, CK, CCR2, CD11b and CD68, we identified CCR2 protein expression in 94% of NSCLCs, 87% CRCs and 89% PDACs. The levels of CCR2 protein were significantly higher in NSCLC than in CRC and PDAC. In NSCLC, high CCR2 protein in myeloid cells was associated with the presence of activating EGFR/KRAS mutations and increased CD8+ TILs and local PD-L1 expression. In CRC, higher levels of CCR2 in CD68+ macrophages were seen in tumors harboring microsatellite instability than in microsatellite stable carcinomas. Conclusion: The iADC TAK-500 and mTAK-500 induces CCR2-dependent immune cell activation and anti-tumor effect. CCR2 is highly expressed in intratumor myeloid cells from NSCLC. High CCR2 is associated with enhanced local adaptive immune responses and specific clinical/molecular tumor subsets. TAK-500 is currently being evaluated clinically as a single agent and in combination with pembrolizumab in adults with select locally advanced or metastatic solid tumors (NCT05070247). Citation Format: Kurt A. Schalper, Atsushi Matsuda, Michelle Ganno-Sherwood, Angel E. Maldonado-Lopez, Emily Rosentrater, Angelo Porciuncula, Dong Mei Zhang, Camilla L. Christensen, Samantha A. Merrigan, Tiquella Hatten, Hong Myung Lee, Min Young Lee, Linlin Dong, Jian Huang, Natasha Iartchouk, Jianing Wang, He Xu, Tomoki Yoneyama, Konstantin I. Piatkov, Carole E. Harbison, Alex Parent, Neil Lineberry, Adnan O. Abu-Yousif, Vicky Appleman. TAK-500 is a clinical stage immune-cell directed antibody drug conjugate (iADC) inducing STING activation in CCR2-expressing intratumor myeloid cells and favorable immunomodulation [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 1841.

Abstract 3861: c-MYC mediated resistance to trametinib plus hydroxychloroquine in pancreatic cancer is overcome by CDK4/6 and lysosomal inhibition

Abstract Pharmacological inhibition of KRAS>RAF>MEK>ERK signaling has provided no clinical benefit to patients with pancreatic ductal adenocarcinoma (PDA). Interestingly, combined inhibition of MEK1/2 (with trametinib, T) plus autophagy (with chloroquine, CQ, or hydroxychloroquine, HCQ) demonstrated striking anti-tumor effects in preclinical models and in a patient (Patient 1). However, not all patients respond to the T/HCQ regimen, and Patient 1 eventually developed resistant disease. Here we report that primary or acquired resistance is associated with focal DNA copy number gains encompassing c-MYC. Furthermore, ectopic expression of c-MYC in PDA cell lines rendered them T/HCQ resistant. Interestingly, a CDK4/6 inhibitor, palbociclib (P), also induced autophagy and overrode c-MYC-mediated T/HCQ resistance, such that P/HCQ promoted regression of T/HCQ resistant PDA tumors with elevated c-MYC expression. Finally, P/HCQ treatment of Patient 1 resulted in a biochemical disease response. These data suggest that elevated c-MYC expression is both a marker and a mediator of T/HCQ resistance, which may be overcome by the use of palbociclib plus hydroxychloroquine. Citation Format: Mark Silvis, Dilru Silva, Rohweder Riley, Sophia Schuman, Swapna Gudipaty, Amanda Truong, Jeffery Yap, Kajsa Affolter, Martin McMahon, Conan Kinsey. c-MYC mediated resistance to trametinib plus hydroxychloroquine in pancreatic cancer is overcome by CDK4/6 and lysosomal inhibition. [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 3861.

Current comprehensive understanding of denosumab (the RANKL neutralizing antibody) in the treatment of bone metastasis of malignant tumors, including pharmacological mechanism and clinical trials.

Denosumab, a fully humanized monoclonal neutralizing antibody, inhibits activation of the RANK/RANKL/OPG signaling pathway through competitive binding with RANKL, thereby inhibiting osteoclast-mediated bone resorption. Denosumab inhibits bone loss; therefore, it is used to treat metabolic bone diseases (including postmenopausal osteoporosis, male osteoporosis, and glucocorticoid-induced osteoporosis), in clinical practice. Since then, multiple effects of denosumab have been discovered. A growing body of evidence suggests that denosumab has a variety of pharmacological activities and broad potential in clinical diseases such as osteoarthritis, bone tumors, and other autoimmune diseases. Currently, Denosumab is emerging as a treatment for patients with malignancy bone metastases, and it also shows direct or indirect anti-tumor effects in preclinical models and clinical applications. However, as an innovative drug, its clinical use for bone metastasis of malignant tumors is still insufficient, and its mechanism of action needs to be further investigated. This review systematically summarizes the pharmacological mechanism of action of denosumab and the current understanding and clinical practice of the use of denosumab for bone metastasis of malignant tumors to help clinicians and researchers deepen their understanding of denosumab.

MYC-mediated resistance to trametinib and HCQ in PDAC is overcome by CDK4/6 and lysosomal inhibition.

Pharmacological inhibition of KRAS>RAF>MEK1/2>ERK1/2 signaling has provided no clinical benefit to patients with pancreatic ductal adenocarcinoma (PDAC). Interestingly, combined inhibition of MEK1/2 (with trametinib [T]) plus autophagy (with chloroquine [CQ] or hydroxychloroquine [HCQ]) demonstrated striking anti-tumor effects in preclinical models and in a patient (Patient 1). However, not all patients respond to the T/HCQ regimen, and Patient 1 eventually developed resistant disease. Here we report that primary or acquired resistance is associated with focal DNA copy number gains encompassing c-MYC. Furthermore, ectopic expression of c-MYC in PDAC cell lines rendered them T/HCQ resistant. Interestingly, a CDK4/6 inhibitor, palbociclib (P), also induced autophagy and overrode c-MYC-mediated T/HCQ resistance, such that P/HCQ promoted regression of T/HCQ-resistant PDAC tumors with elevated c-MYC expression. Finally, P/HCQ treatment of Patient 1 resulted in a biochemical disease response. These data suggest that elevated c-MYC expression is both a marker and a mediator of T/HCQ resistance, which may be overcome by the use of P/HCQ.

Matrix metalloproteinases as therapeutic targets in breast cancer.

Matrix metalloproteinases (MMPs) are the most prominent proteinases involved in tumorigenesis. They were initially recognized to promote tumor progression by remodeling the extracellular matrix through their proteolytic activity. However, accumulating evidence has revealed that some MMPs have protective roles in cancer progression, and the same MMP can exert opposing roles depending on the cell type in which it is expressed or the stage of cancer. Moreover, studies have shown that MMPs are involved in cancer progression through their roles in other biological processes such as cell signaling and immune regulation, independent of their catalytic activity. Despite the prognostic significance of tumoral or stromal expression of MMPs in breast cancer, their roles and molecular mechanisms in breast cancer progression remain unclear. As the failures of early clinical trials with broad-spectrum MMP inhibitors were mainly due to a lack of drug specificity, substantial efforts have been made to develop highly selective MMP inhibitors. Some recently developed MMP inhibitory monoclonal antibodies demonstrated promising anti-tumor effects in preclinical models of breast cancer. Importantly, anti-tumor effects of these antibodies were associated with the modulation of tumor immune microenvironment, suggesting that the use of MMP inhibitors in combination with immunotherapy can improve the efficacy of immunotherapy in HER2-positive or triple-negative breast cancer. In this review, the current understanding of the roles of tumoral or stromal MMPs in breast cancer is summarized, and recent advances in the development of highly selective MMP inhibitors are discussed.

Optimized Therapeutic 177Lu-Labeled PSMA-Targeted Ligands with Improved Pharmacokinetic Characteristics for Prostate Cancer.

Clinical trials have shown the significant efficacy of [177Lu]Lu-PSMA-617 for treating prostate cancer. However, the pharmacokinetic characteristics and therapeutic performance of [177Lu]Lu-PSMA-617 still need further improvement to meet clinical expectations. The aim of this study was to evaluate the feasibility and therapeutic potential of three novel 177Lu-labeled ligands for the treatment of prostate cancer. The novel ligands were efficiently synthesized and radiolabeled with non-carrier added 177Lu; the radiochemical purity of the final products was determined by Radio-HPLC. The specific cell-binding affinity to PSMA was evaluated in vitro using prostate cancer cell lines 22Rv1and PC-3. Blood pharmacokinetic analysis, biodistribution experiments, small animal SPCET imaging and treatment experiments were performed on normal and tumor-bearing mice. Among all the novel ligands developed in this study, [177Lu]Lu-PSMA-Q showed the highest uptake in 22Rv1 cells, while there was almost no uptake in PC-3 cells. As the SPECT imaging tracer, [177Lu]Lu-PSMA-Q is highly specific in delineating PSMA-positive tumors, with a shorter clearance half-life and higher tumor-to-background ratio than [177Lu]Lu-PSMA-617. Biodistribution studies verified the SPECT imaging results. Furthermore, [177Lu]Lu-PSMA-Q serves well as an effective therapeutic ligand to suppress tumor growth and improve the survival rate of tumor-bearing mice. All the results strongly demonstrate that [177Lu]Lu-PSMA-Q is a PSMA-specific ligand with significant anti-tumor effect in preclinical models, and further clinical evaluation is worth conducting.

Disulfiram-loaded hollow copper sulfide nanoparticles show anti-tumor effects in preclinical models of colorectal cancer

Colorectal cancer is one of the most common malignancies causing the majority of cancer-related deaths. There is an urgent need to develop new anticancer modalities. Recently, efforts have been made to turn clinically approved drugs into anticancer agents in specific tumor microenvironments via NPs. Disulfiram (DSF) as an effective copper (Cu2+)-dependent anti-tumour drug, which has been more widely used in antitumor research. Here, we constructed a novel therapeutic nanoplatforms, DSF@CuS, by encapsulating DSF in hollow CuS NPs to enable in situ chemoselective activation of DSF and hyperthermal amplified chemotherapy. The anticancer effect of DSF was enhanced by the thermal energy generated under NIR irradiation through the intrinsic photothermal conversion of CuS. As a result, significant apoptosis was induced in vitro, and tumor elimination was achieved in vivo. Collectively, DSF@CuS combined with photothermal therapy can significantly promote the apoptosis of CT26 colorectal cancer cells both in vitro and in vivo, providing a potential theoretical agent for the treatment of colorectal cancer.

Abstract 2576: ATG-037, a highly potent small molecule CD73 inhibitor has superior activity of reversing immunosuppression in higher-AMP environments compared with anti-CD73 antibodies

Abstract Background: CD73 is a cell surface enzyme which is highly expressed in the tumor microenvironment (TME). Degradation of adenosine monophosphate (AMP) into adenosine by CD73 results in the generation of an immunosuppressed and pro-angiogenic niche within the TME that promotes the onset and progression of cancer. Targeting CD73 has resulted in favourable anti-tumor effects in preclinical models and in clinic. One of the key mechanisms of therapeutic activity by CD73 inhibition is restoring the normal function of effector T cells from adenosine inhibition. ATG-037 is a highly potent and selective oral small molecule inhibitor of CD73. This study compared the ability of ATG-037 and clinical CD73 antibodies in reverting immunosuppression in higher-AMP environments in vitro. Methods: The activity of ATG-037 and CD73 antibodies (Hu101-28, MEDI9447) in inhibiting enzyme function of cell surface CD73 was evaluated by measuring the ATP-dependent oxidation of luciferin, which is inhibited by AMP, using Cell Titer Glo assay. Reversal of AMP/adenosine-mediated immune suppression of human T cells by CD73 inhibitors was determined by measuring T cell function in the presence of different concentrations of exogenous AMP. Markers for T cell proliferation, activation and cytotoxicity were assessed by flow cytometry, and the cytokine levels were measured by ELISA. Results: ATG-037, Hu101-28 and MEDI9447 inhibited the enzyme activity of cell surface-expressed CD73 on human A375 cells under 100 µM AMP with a half-maximal inhibitory concentration (IC50) of 0.36 nM,20.94 nM and 3.46 nM, respectively. ATG-037 demonstrated complete inhibition of CD73 activity, whereas MEDI9447 did not reach complete CD73 inhibition. T cell proliferation and activation induced by CD3/CD28 dynabeads were suppressed by 100 µM of extracellular AMP and the suppression was relieved by the addition of ATG-037 with half-maximal effective concentration (EC50) of 21.6 nM for CD8+ T cell proliferation, 27 nM for T cell activation, 2 nM for granzyme B expression, and 6.5 nM for T cell IFN-γ production. While Hu101-28 and MEDI9447 fail to restore the T cell function. In a mixed lymphocyte reaction assay, ATG-037, but not Hu101-28 or MEDI9447 reversed AMP-dependent T cell suppression. In addition, ATG-037 rescued immune cell activation in high AMP (500 µM, 1000 µM) environments. Conclusion: ATG-037 demonstrated potent and complete CD73 enzyme inhibition and stronger ability in restoring T cell function from high-level AMP mediated suppression, compared with clinical anti-CD73 antibodies. These data implicate the potential therapeutic advantages of small molecule CD73 inhibitors over blocking antibodies. Citation Format: Gang Bian, Jiahui Hua, Linjie Tian, Kevin Lynch, Jay Mei, Bo Shan, Bing Hou. ATG-037, a highly potent small molecule CD73 inhibitor has superior activity of reversing immunosuppression in higher-AMP environments compared with anti-CD73 antibodies [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 2576.

A radioenhancing nanoparticle mediated immunoradiation improves survival and generates long-term antitumor immune memory in an anti-PD1-resistant murine lung cancer model

BackgroundCombining radiotherapy with PD1 blockade has had impressive antitumor effects in preclinical models of metastatic lung cancer, although anti-PD1 resistance remains problematic. Here, we report results from a triple-combination therapy in which NBTXR3, a clinically approved nanoparticle radioenhancer, is combined with high-dose radiation (HDXRT) to a primary tumor plus low-dose radiation (LDXRT) to a secondary tumor along with checkpoint blockade in a mouse model of anti-PD1-resistant metastatic lung cancer.MethodsMice were inoculated with 344SQR cells in the right legs on day 0 (primary tumor) and the left legs on day 3 (secondary tumor). Immune checkpoint inhibitors (ICIs), including anti-PD1 (200 μg) and anti-CTLA4 (100 μg) were given intraperitoneally. Primary tumors were injected with NBTXR3 on day 6 and irradiated with 12-Gy (HDXRT) on days 7, 8, and 9; secondary tumors were irradiated with 1-Gy (LDXRT) on days 12 and 13. The survivor mice at day 178 were rechallenged with 344SQR cells and tumor growth monitored thereafter.ResultsNBTXR3 + HDXRT + LDXRT + ICIs had significant antitumor effects against both primary and secondary tumors, improving the survival rate from 0 to 50%. Immune profiling of the secondary tumors revealed that NBTXR3 + HDXRT + LDXRT increased CD8 T-cell infiltration and decreased the number of regulatory T (Treg) cells. Finally, none of the re-challenged mice developed tumors, and they had higher percentages of CD4 memory T cells and CD4 and CD8 T cells in both blood and spleen relative to untreated mice.ConclusionsNBTXR3 nanoparticle in combination with radioimmunotherapy significantly improves anti-PD1 resistant lung tumor control via promoting antitumor immune response.Graphical

Effect of Celecoxib vs Placebo as Adjuvant Therapy on Disease-Free Survival Among Patients With Breast Cancer

Patients with breast cancer remain at risk of relapse after adjuvant therapy. Celecoxib has shown antitumor effects in preclinical models of human breast cancer, but clinical evidence is lacking. To evaluate the role of celecoxib as an addition to conventional therapy for women with ERBB2 (formerly HER2)-negative primary breast cancer. The Randomized European Celecoxib Trial (REACT) was a phase 3, randomized, double-blind study conducted in 160 centers across the UK and Germany testing 2 years of adjuvant celecoxib vs placebo among 2639 patients recruited between January 19, 2007, and November 1, 2012, with follow-up 10 years after treatment completion. Eligible patients had completely resected breast cancer with local and systemic therapy according to local practice. Patients with ERBB2-positive or node-negative and T1, grade 1 tumors were not eligible. Randomization was in a 2:1 ratio between celecoxib or placebo. Statistical analysis was performed from May 5, 2019, to March 5, 2020. Patients received celecoxib, 400 mg, or placebo once daily for 2 years. The primary end point was disease-free survival (DFS), analyzed in the intention-to-treat population using Cox proportional hazards regression and log-rank analysis. Follow-up is complete. A total of 2639 patients (median age, 55.2 years [range, 26.8-86.0 years]) were recruited; 1763 received celecoxib, and 876 received placebo. Most patients' tumors (1930 [73%]) were estrogen receptor positive or progesterone receptor positive and ERBB2 negative. A total of 1265 patients (48%) had node-positive disease, and 1111 (42%) had grade 3 tumors. At a median follow-up of 74.3 months (interquartile range, 61.4-93.6 years), DFS events had been reported for 487 patients (19%): 18% for those who received celecoxib (n = 323; 5-year DFS rate = 84%) vs 19% for those who received placebo (n = 164; 5-year DFS rate = 83%); the unadjusted hazard ratio was 0.97 (95% CI, 0.80-1.17; log-rank P = .75). Rates of toxic effects were low across both treatment groups, with no evidence of a difference. In this randomized clinical trial, patients showed no evidence of a DFS benefit for 2 years' treatment with celecoxib compared with placebo as adjuvant treatment of ERBB2-negative breast cancer. Longer-term treatment or use of a higher dose of celecoxib may lead to a DFS benefit, but further studies would be required to test this possibility. ClinicalTrials.gov Identifier: NCT02429427 and isrctn.org Identifier: ISRCTN48254013.

Abstract 1802: CD39 inhibition shapes the transcriptional landscape of myeloid cells and induces proinflammatory states in the CT26 syngeneic tumor model

Abstract Extracellular adenosine triphosphate (ATP) generated by tissue damage or immunogenic cell death initiates proinflammatory responses that are potently restricted by adenosine produced through ATP hydrolysis. The ectonucleotidases CD39 and CD73 limit immune responses by sequentially converting ATP to adenosine monophosphate (AMP) and adenosine, respectively. Increased activity of CD39, the rate-limiting enzyme in ATP hydrolysis in tumor microenvironments (TME), results in significant reductions in extracellular ATP. The subsequent accumulation of adenosine contributes to tumor immune escape, induction of angiogenesis, and metastatic progression. Although pharmacological targeting of CD39 has antitumor effects in preclinical models, the immunological mechanisms of therapeutic CD39 blockade have not been finely parsed. In this study, the immunological effects of CD39 inhibition on tumor infiltrating lymphocytes (TILs) were examined by single-cell transcriptome analysis. Mice bearing syngeneic CT26 tumors were treated with an anti-murine CD39 antibody that blocks the conversion of ATP to AMP. After 3 doses, tumors were harvested on Day 15 post-implant, dissociated, and enriched for CD45+ cells. Isolated TILs were captured in droplets, and single-cell sequencing libraries were generated through standard 10× Genomics protocols. Raw reads were pre-processed through the CellRanger pipeline. Data quality, normalization, integration, and clustering were performed with Seurat. Cell type annotation and differential expression was performed with SingleR and EdgeR, respectively. Several defined immunocyte populations identified in the CT26 TME were conserved across all conditions tested. These cell clusters segregated along the lymphoid/myeloid axis, with many sub-clusters identified for each major lineage. Data for each cell were aggregated in a pseudo-bulk RNA-seq analysis to define broad effects of CD39 blockade, which revealed major changes to immunocyte transcriptional landscapes. Notably, CD39 blockade upregulated several proinflammatory genes, including Gzmf, Gzmg, Cxcl9, and Csf3. Mapping of these changes to single-cell clusters revealed that CD39 blockade predominantly altered the transcriptional profiles of myeloid cell subsets, generally inducing proinflammatory gene modules. This analysis also demonstrated a significant effect of CD39 inhibition on plasmacytoid dendritic cells, inducing Cxcl2, Il1b, Gadd45g, and Fabp4 expression. Interestingly, CD39 blockade repressed Klk1, a kallikrein previously implicated in tumorigenesis. In summary, single-cell RNA-seq of the CT26 syngeneic tumor model suggests that CD39 inhibition predominantly shaped the transcriptional landscape of myeloid cells and generally induced proinflammatory conditions. Citation Format: Devapregasan Moodley, Mayra Carneiro, Sonia D. Gas, Austin Dulak, Ricard Masia, Secil Koseoglu, Matthew Rausch, Marisella Panduro, Michael C. Warren, John Stagg, Benjamin Lee, Pamela M. Holland, Vito J. Palombella, Andrew C. Lake. CD39 inhibition shapes the transcriptional landscape of myeloid cells and induces proinflammatory states in the CT26 syngeneic tumor model [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 1802.

HMBD004, a Novel Anti-CD47xCD33 Bispecific Antibody Displays Potent Anti-Tumor Effects in Pre-Clinical Models of AML

CD47 is the “don't-eat-me” signal and is ubiquitously expressed on normal cells where binding to SIRPalpha on macrophages inhibits phagocytosis. CD47 is commonly over-expressed in tumors where it correlates with immune evasion and poor prognosis. Blocking CD47-SIRPalpha interaction restores macrophage phagocytosis of tumor cells and anti-CD47 mAbs have shown anti-tumor efficacy in mouse models of solid tumors and hematological malignancies. Nevertheless, the widespread expression of CD47 decreases the bioavailability of anti-CD47 mAbs at the tumor due to antigen sink effects, and also risks off-target toxicities, most significantly from cross-linking of erythrocytes that show high expression of CD47. Therefore, increasing the specificity for binding CD47, and inhibiting the interaction with SIRPalpha, to tumor cells alone should increase the efficacy and safety of anti-CD47 therapy.CD33 is expressed on myeloid cells, but is highly over-expressed in 90% of AML patients, where it marks leukemic blast cells. The safety of CD33 as an antibody target for directing effector molecules to tumor cells has been convincingly demonstrated by the efficacy of gemtuzumab ozogamicin (Mylotarg), an antibody-drug conjugate (ADC) that binds CD33 with high affinity, in the treatment of relapsed AML. However, ADCs are notable for their significant risk of toxicities, and gemtuzumab ozogmicin was withdrawn for this reason in 2010. Still, other companies continue development on next generation anti-CD33 ADCs, e.g. vadastuximab talirine.Hummingbird Bioscience has used its proprietary Rational Antibody Development Platform for the design and development of novel therapeutic antibodies against CD47. Computational sequence and structural analyses predicted a highly specific surface epitope, with strong antigenicity and good safety profile, where antibody binding would inhibit binding to SIRPalpha. Subsequently, an in-house mouse immunization and screening strategy efficiently yielded a monoclonal antibody (mAb, 11a1) that bound with low nM (KD 5 nM) affinity and high specificity to the desired epitope on human CD47. In vitro, 11a1 demonstrated 100% inhibition of SIRPalpha binding at 1 nM, and potently induced phagocytosis of Raji cells by isolated macrophages.11a1 was subsequently humanized by CDR grafting and back-mutation, and the resulting humanized mAb maintained high affinity binding to CD47. Hummingbird Bioscience then constructed a bispecific anti-CD47xCD33 antibody (bsAb), based on a standard 1+1 IgG format, with a humanized anti-CD47 variable domain as an effector arm and the gemtuzumab variable domain as an anti-CD33 specificity arm. This bsAb (HMBD004) maintained the potent inhibition of SIRPalpha binding and induction of phagocytosis but, unlike many CD47 mAbs, caused negligible hemagglutination (i.e. cross-linking) of erythrocytes in vitro . Furthermore, HMBD004 showed preferential sorting of CD47+ CD33+ cells during FACS of mixed populations of CD47+ cells. Finally, in vivo CDX mouse models of AML were established by engrafting NCr nude mice with the CD47+ CD33+ AML cell line, HL-60, via subcutaneous implantation. Treatment of these mice with HMBD004 showed a significant decrease in tumor burden and increased PFS.Together, these data highlight the benefit of using CD33 binding to enhance the specificity, efficacy and safety of anti-CD47 antibody therapy in AML. Hummingbird Bioscience is currently advancing HMBD004 towards the clinic in 2018. DisclosuresNo relevant conflicts of interest to declare.