Mechanism Of Action Research Articles

Integrating metabolomics and network pharmacology to study the mechanism of Er-Xian decoction in improving intervertebral disc degeneration.

Integrating metabolomics and network pharmacology to study the mechanism of Er-Xian decoction in improving intervertebral disc degeneration.

Abstract 6755: Discovery of the synergistic dual-payload antibody-drug conjugate (CTPH-02) by combination of MMAE and novel payloads

Abstract Many ADC drugs in clinical trials are showing poor performance without providing sufficient dose due to dose-limiting toxicity, which is rooted from on-target toxicity related to payload itself. There are many efforts to increase the therapeutic index (TI) of ADC, but research on discovering novel payloads based on unique anticancer mechanisms of action is relatively limited. We have developed unique ADC platform technology by adopting multi-drug payloads concept, where we aim to achieve three different goals, (1) increasing MTD and lowering MED (2) showing cytotoxic activity in antigen low-expressed cell lines (3) overcoming resistance cell lines with better cytotoxic effects. This study began with the search for a drug combination that could maintain or increase efficacy of MMAE even with lower dose of MMAE. Drugs with various mechanisms of action were tested to select a combination that showed a high synergy effect with MMAE, and a cell cycle regulating drug was found to offer the most synergistic combination efficacy with MMAE. By conjugating a dual-payload with two-different mechanisms of action to HER2 antibody(trastuzumab), the resulting ADC(CTPH-02) was confirmed to show synergistic effect in terms of cytotoxicity. Very excitingly, CTPH-02 shows significantly high synergistic efficacy in the case of cell lines with low HER2 antigen expression. These findings based on the right combination of two-different payloads open a new paradigm for ADC drug design strategies in cancer therapy in order to provide treatment options to wider range of cancer patients such as low TPS % with low IHC score. Citation Format: Chang-Sun Lee, Myeong Joo Kim, Da hyun Kim, Soyeon Lim, Jiwoo Lee, Eunseok Choi, Wonbin Ha, Han ah Kim, Jaebeom Park, Seung Chan Kim. Discovery of the synergistic dual-payload antibody-drug conjugate (CTPH-02) by combination of MMAE and novel payloads [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6755.

Abstract 5477: Cell avidity - the next-gen cell binding to understand the mechanism of action through cell-cell and cell-protein interaction

Abstract Traditional assays such as affinity measurements, cytokine secretion, and cytotoxicity provide informative insights but are insufficient for comprehensively characterizing and selecting the most effective cell therapy candidates. The complexity of cell-cell interactions that drive functional outcomes remains inadequately understood. Cell avidity, defined as the aggregate strength of interactions between effector and target cells, serves as a novel pivotal metric for uncovering the mechanism of action behind therapeutic candidates. The z-Movi Cell Avidity Analyzer, part of our versatile Cell Avidity platform, leverages the integration of fluorescence microscopy with precise acoustic force application to measure cell binding under physiologically relevant conditions. This approach allows for a detailed analysis of binding profiles, encompassing cell surface receptors and ligands, cell phenotype, cell status, and more to elucidate potency, safety, sensitivity, and binding kinetics essential for robust therapeutic evaluation. Our platform is designed to support the characterization and screening of a wide array of therapeutic modalities, including molecular binders like antibodies and small molecules, as well as cell-based therapies. By providing extensive insights into the mechanism of action and functional performance, Cell Avidity enables the development of next-generation therapeutics with enhanced safety and efficacy profiles. Citation Format: Zhou Zhong, Keith Bailey, Justin Moser, Bella Perez, Brian Kierce, Michael Savage. Cell avidity - the next-gen cell binding to understand the mechanism of action through cell-cell and cell-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5477.

Abstract 1276: UNLOCK, a preclinical platform of PDX models resistant to innovative therapies

Abstract Introduction: Over the past 20 years, advances in precision oncology and cancer genetics have identified numerous actionable oncogenic drivers, leading to new drug development and clinical application. However, tumor cells can adapt and develop resistance mechanisms (MoR), and the mechanisms of action (MoA) of these drugs are only partially understood, highlighting the need for research focused on both MoR and MoA. Following the success of the MATCH-R clinical trial (NCT02517892, 2015-2022), the UNLOCK program was launched in 2022 to better understand the MoA and MoR of innovative drugs (phase 1 to approval) through integrated clinical and translational research. As part of this initiative, we developed preclinical models, including PDX models from patients who developed resistance, to support the development of new therapeutic strategies. Methods: Fresh tumor biopsy specimens were prospectively collected from patients through the MATCH-R clinical trial (NCT02517892) or the UNLOCK program. PDX models were established in NOD Scid Gamma (NSG) mice, which were then developed and characterized. These models underwent immunohistochemistry, comprehensive molecular profiling (including whole exome sequencing [WES] and bulk RNA sequencing [RNA-seq]), and pharmacological validation. Results: As of November 2024, 197 PDX models have been successfully generated from 519 biopsies, with a global take rate of 38%. These models are developed across several cohorts, including castration-resistant prostate cancer (22 PDX: 18 AR pathway inhibitors, 1 post-PARPi, and 3 Lu-PSMA), lung cancer (40 PDX with EGFR inhibitors, including 28 post-osimertinib, and 22 PDX with ALK inhibitors, including 2 post-brigatinib, 9 post-lorlatinib, and 6 post-alectinib). Additionally, 34 PDX models have been developed for the FGFR cohorts with FGFR2/FGFR3 alterations across various cancers, including cholangiocarcinomas, bladder carcinomas, gynecological carcinomas, and pancreatic cancers. We also developed 32 PDX models with KRAS mutations (G12C, n=23; G12D, n=9) from various tumors, including lung, pancreatic, gynecological, and colon cancers. Recently, we launched a new UNLOCK cohort focused on antibody-drug conjugate therapy, developing 12 additional PDX models. These PDX models faithfully replicate the genetic, transcriptional, phenotypic, and pharmacologic features of the original biopsies, providing a unique preclinical platform for testing novel drugs and combination therapies. Adaptive treatments with new or combinatorial strategies are being explored to restore drug sensitivity. Conclusion: The development of 197 PDX models under the UNLOCK program represents a significant resource, enabling the evaluation of novel drugs, adaptive treatment strategies, and combinatorial approaches. This unique collection of PDX models paves the way for advancing precision oncology and overcoming therapeutic resistance. Citation Format: Ludovic Bigot, Nobre Nobre, Alice Da silva, Melissandre Meteau, Mathis Delavigne, Miguel Soares, Francesco Facchinetti, Loic Poiraudeau, Floriane Braye, Inmaculada Inmaculada Alonso Garcia, Natalie Ngoi, Alexandre Halimi, Emlie Natali, Damien Vasseur, Kristi Beshiri, Claudio Claudio Nicotra, Maud Ngo-Camus, Lambros Tselikas, Beatriz Beatriz Alonso de Castro, Julieta Rodriguez, Maria Fernanda Mosele, Maud Kamal, Gérôme Jules-Clement, Karim Fizazi, Christophe Massard, Benjamin BESSE, Luc Friboulet, Yohann Loriot. UNLOCK, a preclinical platform of PDX models resistant to innovative therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1276.

Abstract 2169: TTK/PLK1 dual antagonist BAL0891 synergizes with pembrolizumab in a vascularized 3D tumor microenvironment model

Abstract Introduction: BAL0891, a first-in-class dual TTK/PLK1 inhibitor, activates the cGAS/STING pathway and induces a senescence-associated secretory phenotype (SASP), resulting in increased PD-L1 expression and pro-inflammatory cytokine production in cancer cells. These changes enhance tumor susceptibility to immunotherapies. However, evaluating the synergy between BAL0891 and immune checkpoint inhibitors like pembrolizumab (PD-1 inhibitor) is challenging in traditional preclinical models, which lack human immune cells and key features of the tumor microenvironment. To address these limitations, Qureator’s advanced 3D tumor microenvironment (TME) model, incorporating perfusable vessels, human primary cells, and patient-derived organoids (PDOs) was utilized. This physiologically relevant system enabled a detailed investigation of BAL0891's mechanism of action (MOA) and its combination with pembrolizumab across multiple cancer types, aiming to guide clinical trial design and patient stratification. Methods: Vascularized TME models were developed by co-culturing endothelial cells, fibroblasts, and PDOs derived from renal cell carcinoma, triple-negative breast cancer, gastric cancer, and colorectal cancer within Qureator’s Microphysiological System (MPS). Following vasculature formation, models were primed with BAL0891 for two days and then treated with PBMCs and pembrolizumab or isotype control antibodies. Tumor growth, immune infiltration, and tumor cell killing were assessed via live cell imaging and immunofluorescence staining. Post-treatment immuno-profiling included flow cytometry to analyze macrophage polarization and T cell activation/exhaustion. Multiplex secretome analysis was performed to characterize chemokine and cytokine profiles associated with BAL0891 and the combination therapy. Results: BAL0891 priming sensitized a subset of PDOs to pembrolizumab, demonstrating synergistic anti-tumor activity in the vascularized TME model. Responsive PDOs exhibited significant organoid shrinkage, increased immune cell infiltration, enhanced T cell activation, and macrophage polarization towards an anti-tumoral phenotype. Cytokine and chemokine analysis revealed a shift to a pro-inflammatory microenvironment. Conversely, non-responders showed limited immune infiltration and minimal activation. Conclusion: Qureator’s vascularized TME model provided a robust platform to evaluate the synergy between BAL0891 and pembrolizumab. The study demonstrated BAL0891's ability to reshape the tumor immune landscape and enhance the efficacy of the immune checkpoint inhibitor. These findings offer valuable insights into the MOA of BAL0891 within this highly representative tumor model, supporting its further clinical development in combination with checkpoint inhibitors. Citation Format: Junfeng Wang, MinKyung Kang, Byungjun Lee, Delaney Donnelly, Jihye Baek, Bushra Rajput, Tsung-Li Liu, Hyeon-geun Park, Yunha Shin, Aneesh Sathe, Yangji Kim, Kyusuk Baek, Sanghee Yoo, Salman Ahmed, Jae Jung Lee, Seunghyun Ma. TTK/PLK1 dual antagonist BAL0891 synergizes with pembrolizumab in a vascularized 3D tumor microenvironment model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2169.

Abstract 5208: The use of human small intestinal organoids as a preclinical screening tool to assess gastrointestinal toxicity induced by CDK inhibition

Dysregulated cell division is one of the most important hallmarks of cancer. Cyclindependent kinases (CDKs), involved in cell cycle regulation, have been identified to be overexpressed in many cancers. The development of CDK inhibitors has emerged as a promising and novel approach for cancer treatment in both solid and hematological malignancies. However, first generation pan-CDKIs, which initially showed reliable results in vitro and in Phase I trials, lacked selectivity. Many were discontinued due to adverse effects, including Gastrointestinal (GI) toxicity, and reduced efficacy in vivo. The development of specific CDKIs, which can provide optimum therapeutic outcomes with good efficacy and reduced off-target effects are therefore needed. In this study, we have used a human small intestinal organoid model (adapted from Sato et al. 2009), as a screening tool to predict GI toxicity and subsequent mucosal regeneration. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids and resembled the frequency of linage observed in vivo. The effect of several clinically available CDKIs, such as Dinaciclib, Palbociclib and Abemaciclib, some of them known to induce GI toxicity were assessed. Treatment was applied on Day 3 after plating of fragments, when the organoid structures had formed. On Day 5, the number of dead, viable unbranched and branched organoids were counted and IC50 values were calculated. Furthermore, morphometric analysis of acquired images were performed. To compare the toxicity profiles on a molecular level, organoids were harvested for RNA-seq. The second generation pan-CDK inhibitor Dinaciclib was the most toxic. Whereas Palbociclib was the least toxic compound within the human organoid assay, reflecting clinical data that shows the lowest incidence of severe diarrhea. We conclude that organoids are a predictive preclinical model that can be used to identify potential on-target, off-tissue GI toxicities induced by novel therapeutics such as CDKIs. Toxicity and mechanism of action (MOA) can all be addressed in vitro to potentially reduce in vivo experimentation. Citation Format: Frida Ponthan, Thomas Kedward, Valentina Ubertini, Gary Beale, Cath Booth. The use of human small intestinal organoids as a preclinical screening tool to assess gastrointestinal toxicity induced by CDK inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5208.

Abstract 3133: PF-08046032: a CD25-directed vedotin ADC leveraging a well characterized safety and pharmacokinetic profile for enhanced regulatory T cell depletion in the tumor microenvironment

Abstract PF-08046032 is a novel vedotin antibody-drug conjugate (ADC) engineered to target and deplete immunosuppressive regulatory T cells (Tregs) within the tumor microenvironment (TME) through direct cytotoxicity. PF-08046032 directs delivery of the cytotoxic MMAE (monomethyl auristatin E) vedotin payload to cells expressing CD25, the alpha chain of the IL-2 receptor, which is highly expressed on intratumoral Tregs and lymphoma cells. The MMAE-based vedotin drug linker platform, utilized in four approved ADCs for hematologic and solid tumors, has a well-characterized mechanism of action (MoA), exposure, and target-independent toxicity profile. PF-08046032 leverages this platform, exerting anti-tumor activity through the multifaceted MMAE-driven MoA, including direct cytotoxicity, bystander killing, and immunogenic cell death. Additionally, PF-08046032 is differentiated from other vedotin ADCs and anti-CD25 depleting agents through affinity-tuned binding, which enables selective targeting of CD25hi intratumoral Tregs while sparing peripheral Tregs. Here, we present preclinical data demonstrating safety, pharmacokinetics, and anti-tumor activity of PF-08046032 which is highly consistent with the vedotin ADC platform, while additionally harnessing a Treg-directed anti-tumor approach. PF-08046032 selectively kills Tregs compared to CD8 T cells, with preferential activity on Tregs with the highest CD25 expression and shows dose-dependent antitumor activity in CD25-expressing lymphoma xenograft models. In human CD25-expressing mice bearing MC38 colorectal carcinoma tumors, PF-08046032 significantly suppressed intratumoral Treg levels with minimal activity on peripheral Tregs and no effect on intratumoral CD8+ T cells. A mouse surrogate of PF-08046032 demonstrated that targeted depletion of immunosuppressive tumoral Tregs resulted in robust antitumor activity as both a single agent and in combination with anti-PD1 checkpoint inhibitor blockade. Pharmacokinetic and toxicity studies in non-human primates demonstrated safety and PK profiles consistent with the vedotin platform despite targeting CD25. Leveraging the cumulative knowledge of the vedotin platform, PF-08046032 enables a novel additional mechanism of action for vedotin ADCs at potentially efficacious and safe doses. These data support further investigation of PF-08046032 in clinical trials for advanced malignancies. Citation Format: Christopher Carosino, Sherif Abdelhamed, Priyanka Gupta, Xinqun Zhang, Bryan Grogan, Weiping Zeng, Lauren Bou, Devra Olson, Paromita Raha, Andrea R. Lim, Brian P. O'Connor, Ryan A. Heiser, Matthew R. Levengood. PF-08046032: a CD25-directed vedotin ADC leveraging a well characterized safety and pharmacokinetic profile for enhanced regulatory T cell depletion in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3133.

Abstract 2122: FS-8002, a bifunctional antibody fusion protein of anti-VEGF and anti-TGF-β, exhibits significant anti-tumor activity in preclinical studies

Abstract Background: FS-8002 is a bifunctional antibody fusion protein targeting vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β), which is generated by fusing a humanized IgG1 antibody against VEGF and the extracellular domain of TGF-β receptor II (TGF-βRII). FS-8002 can simultaneously bind to and neutralize VEGF and TGF-β, thus achieving anti-tumor effects through the following proposed mechanisms: 1) inhibiting tumor neovascularization and normalizing tumor blood vessels; 2) relieving the suppression of anti-tumor immune cells in the tumor microenvironment (TME); 3) inhibiting the formation of the immune barrier or extracellular matrix (ECM) and promoting cytotoxic T cell infiltration into tumors, and 4) blocking the invasion and metastasis of tumor cells. Methods: The mechanism of action (MOA) and anti-tumor effects of FS-8002 were evaluated in a series of studies including in vitro target binding, biological activity assays, and in vivo efficacy study in xenograft tumor models. Results: in vitro, FS-8002 binds to VEGF and TGF-β simultaneously. FS-8002 bound to VEGF165 and VEGF121 and thus inhibited VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation. FS-8002 also specifically bound to TGF-β1 and TGF-β3, but only had weak affinity to TGF-β2. By neutralizing TGF-β, FS-8002 could block TGF-β/Smad signaling pathway, inhibit TGF-β1-induced tumor cell metastasis, and relieve immunosuppression in the TME by activating NK cells and inhibiting Treg proliferation. FS-8002 exhibited significant anti-tumor effect in two "cold tumor" xenograft models (human PBMC reconstituted MDA-MB-231 model and HT-29 cells model) as single agent or in combination with anti-PD-1 antibody (Keytruda®). IHC analysis of the tumors from the two mouse xenograft models revealed that FS-8002 treatment could inhibit the tumor neovascularization, inhibit the formation of the immune barrier by reducing fibrosis formation in tumor tissues, and promote T cells infiltration into tumor tissue to kill tumor cells, therefore exhibit anti-tumor effect. The results of GLP repeat-dose toxicity study in cynomolgus monkeys combined with safety pharmacology assessment indicated that FS-8002 was well tolerated under the doses ranged from 15 to 135 mg/kg/dose (HNSTD) after four weeks of weekly intravenous infusion administration, and the adverse effects were within expectations and can be managed in clinical trials. FS-8002 is currently under clinical evaluation for safety and activity in both US (IND approved) and China. Conclusion: All the preclinical data supported FS-8002 to be studied in clinical trials. Citation Format: Libo Zhou, Qiran Guo, Ruxia Zhou, Hua Jiang, Chaoyi Xu, Xinzhong Jon Wang. FS-8002, a bifunctional antibody fusion protein of anti-VEGF and anti-TGF-β, exhibits significant anti-tumor activity in preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2122.

Abstract 6378: Brain-penetrant molecular glue degraders targeting ALK via a novel degron: a potential therapeutic approach for ALK-positive NSCLC

Abstract Aberrant expression of anaplastic lymphoma kinase (ALK) is the oncogenic driver of 3-7% of non-small cell lung cancer (NSCLC) cases. Six ATP-competitive tyrosine kinase inhibitors (TKIs) of ALK have been approved by the FDA to treat patients with ALK-positive NSCLC, but mutations conferring drug resistance often occur in the ATP-binding pocket of ALK signifying a need for alternative modalities to inhibit ALK function. Here we report that ALK-driven tumor growth can be suppressed by novel molecular glue cereblon E3 ligase modulatory drugs (CELMoDs) that trigger ubiquitylation and subsequent proteasomal degradation of ALK. A cryo-EM structure of the ALK-CELMoD-cereblon ternary complex revealed that ALK interacts with cereblon and drug through a site distal to its ATP-binding pocket and features a surprising degron structure distinct from the canonical G-loop degron established for other cereblon neosubstrates. Subsequent lead optimization identified a series of ALK CELMoDs with improved brain penetration. Our in vitro and in vivo studies support the hypothesis that a brain penetrant ALK CELMoD would be an effective therapy for ALK-positive NSCLC with similar or better efficacy and safety profile compared to TKIs. Furthermore, combination treatment with TKIs might enhance ALK CELMoD efficacy due to distinct binding sites and mechanisms of action. Finally, the novel degron suggests that the degradome of CELMoDs goes beyond G-loop-containing proteins, thus emphasizing the importance and potential of this modality to target a wide spectrum of previously un-druggable proteins in human diseases. Citation Format: Zheng Wang, Massimo Ammirante, Martina Malatesta, Alex Cortez, Young Chen, Jinyi Zhu, Dahlia Weiss, Vijaya Lakshmi Dommeti, Geraldine Hernandez, Joshua M. Baughman, Adwait Sathe, Matthew Groza, Maria Donoso, Roxxana Beltran, Regina Paramitha, Ryan Davison, Atefeh Garzan, Barbra Pagarigan, Michelle Slade, Jacobo Fuentes, Jennifer Buenviaje, Shan Yu, Xinde Zheng, Andres H de la Peña, Mariko Riley, Gabe Mintier, Surendra Nayak, Shuichan Xu, Rama Narla, Ellen Filvaroff, Deborah Mortensen, Lihong Shi, Celia Fontanillo Fontanillo, Christoph Zapf, Neil Bence, Mark Rolfe. Brain-penetrant molecular glue degraders targeting ALK via a novel degron: a potential therapeutic approach for ALK-positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6378.

Abstract 5647: Targeting HAUS7/gamma-tubulin interaction with HTIP1 suppresses tumorigenesis in esophageal squamous cell carcinoma

Abstract Background: Unlimited cell division resulting from cell cycle deregulation is a cancer hallmark. We hypothesized targeting deregulated cell cycle molecule with prognostic significance could be viable to counteract tumorigenesis. Our on-going effort showed HAUS7 was such a molecule highly relevant to esophageal squamous cell carcinoma (ESCC). This research targeted HAUS7/gamma-tubulin interaction using a novel peptide HTIP1, which was strategically designed based on our new understanding on the therapeutic mechanism associated with HAUS7 suppression. Methods: Clinical relevance of HAUS7 upregulation was studied in local and public ESCC cohorts. Physiological microtubule-regulating and tumorigenic functions of HAUS7 relative to other HAUS members were examined in vitro and/or in vivo. A multidisciplinary approach, which included using RNA-seq, mass spectrometry, electron microscopy, in silico predictions, bioinformatics analysis, biochemical methods, cellular assays, immunoassays and animal models, was employed to decode the mechanism underlying HAUS7 suppression. HTIP1 was designed by structure modelling and docking simulation. Its properties, cancer selectivity, anti-tumor efficacy, off-target effect and mechanism of action were comprehensively evaluated. Patient-derived tumor xenograft models were used to provide preclinical evidence. Results: Among HAUSs, HAUS7 was most essential for regulating microtubule dynamics in ESCC. Its overexpression was correlated with aggressive tumor phenotypes and poor prognosis. Mechanistically, HAUS7 suppression released its binding partner g-tubulin. The free g-tubulin upon phosphorylation seized LMAN1 and together both of them relocalized to the nucleus. Given the protein-transporting function of LMAN1 at endoplasmic reticulum (ER), its removal from the innate location triggered ER stress and apoptosis. Capable to perturb HAUS7/gamma-tubulin interaction, HTIP1 treatment manifested similar anti-tumor effects like HAUS7 suppression, while demonstrating high tumor specificity, negligible off-target effects and more potent than relevant compounds. Conclusions: We first discovered a unique physiological function and pathological relevance of HAUS7 among other HAUSs in ESCC. A new therapeutic mechanism was dissected under HAUS7 suppression. To target cancer with HAUS7 dependency, a novel peptide HTIP1 was designed to interrupt HAUS7/gamma-tubulin interaction. This invention paved a new strategy to counter tumorigenesis. By translating the scientific evidence to support further R&D of HTIP1, our ultimate goal is to produce a brand new drug. Citation Format: Ziqing CAI, Yu Hong Ian WONG, Ying Kit Simon LAW. Targeting HAUS7/gamma-tubulin interaction with HTIP1 suppresses tumorigenesis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5647.

Abstract 1579: PA289, a prodrug linker-payload with a novel mechanism of action for the development of antibody drug conjugates

Abstract Background: PA289, is a novel seco-CBI-based prodrug payload which appears to exert cell-killing activity through the alkylation of one or more cytosolic proteins (Tietze et al, 2010). PA289, is the lead linker-payload from our ProAlk payload technology platform and is designed with tumor-selective glucuronide triggers that attenuate the potency of the payload until uptake into target cancer cells and selective release in the lysosome. The glucuronide linker and PermaLink (PL) conjugation chemistry ensure ADC stability. The unique mechanism of action of PA289 compared to currently used chemotherapies and ADC payloads, led us to generate proof-of-concept data for this novel ADC payload. Methods: The free payload and ADCs were tested for cell-killing activity in vitro by CellTiter-Glo assay. The prodrug linker-payload PA289, was conjugated using PermaLink technology to generate ADCs with a drug to antibody ratio (DAR) of approximately 4. Stability was determined in mouse and human plasma and in vivo efficacy was performed with subcutaneous human tumor xenografts in nude mice. Bystander effect was analyzed in vitro and compared to ADCs composed of MMAE and MMAF payloads. An exploratory toxicology study was conducted in cynomolgus monkeys. Results: The free payload showed highly potent in vitro activity against 248 cancer cell lines representing 15 solid cancer indications with IC50 values in the fM to pM range. Isumab01-PL-PA289, a model ADC targeting FRA, displayed excellent plasma stability and was highly potent against the JEG-3 cell line in vitro, with IC50 values in the pM range. A single administration of Isumab01-PL-PA289 in the FRA-positive JEG-3 xenograft model induced complete tumor regression by day 30 when dosed at 2.2 mg/kg, while the Isotype-PL-PA289 non-binding control resulted in weak tumor growth inhibition. Evaluation of bystander killing in vitro with a model ADC targeting CA242, Isumab04-PL-PA289, demonstrated stronger bystander cell-killing than benchmark ADCs containing MMAE and MMAF. A pilot toxicity study in cynomolgus monkeys treated with Isumab04-PL-PA289 showed the ADC was well-tolerated with minimal to no test-article related findings at the highest dose tested (7 mg/kg single dose). Conclusions: ADCs comprised of PA289, a novel protein alkylator prodrug linker-payload, demonstrate 1) strong in vitro potency and bystander effect, 2) durable tumor regressions in xenograft models, and 3) excellent tolerability in monkeys. The free payload is highly active against a broad array of cancer cell lines representing a variety of solid tumors suggesting that PA289 is a promising candidate for the development of ADCs with a novel MOA for the treatment of solid tumors. Citation Format: Justyna H. Mysliwy, Jutta Deckert, Adam Lodge, Jenny Thirlway, James Stephenson, Patrick Higgs, Daniel Williamson, Robert J. Lutz. PA289, a prodrug linker-payload with a novel mechanism of action for the development of antibody drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1579.

Abstract 5731: BGB-C354, a novel B7H3 ADC with high DAR stability and strong bystander effect, demonstrates robust antitumor activity in preclinical models

Abstract B7H3 is a type I transmembrane protein belonging to the B7 immunoregulatory family. B7H3 is overexpressed on a wide range of solid tumors such as lung cancers, gastrointestinal cancers and gynecological cancers, but its expression is absent or low in normal tissues, making it an attractive target for anticancer therapies. BGB-C354 is a B7-H3-targeting antibody-drug conjugate (ADC) composed of a humanized anti-B7-H3 monoclonal antibody conjugated via a cleavable linker to a novel TOP1i payload, and the drug to antibody ratio (DAR) is approximately 8. Utilizing a stable ring-open conjugator, BGB-C354 demonstrated improved ADC stability and sustained high DAR in vivo, which may enable more efficient payload delivery to B7H3-expressing tumors. In nonclinical pharmacological studies, BGB-C354 exhibited strong target binding activity, high target-dependent internalization, and potent cytotoxicity toward B7H3-expressing tumor cells. In addition, BGB-C354 demonstrated strong bystander killing effect in the presence of B7H3-expressing cell lines to potentially overcome the tumor heterogeneity. Notably, robust anti-tumor activity was observed in a panel of cell-derived xenograft (CDX) models with varying levels of B7-H3 expression, as well as in patient-derived xenograft (PDX) models. Consistent with its mechanism of action, DNA damage and apoptosis biomarker changes were induced by BGB-C354 in a dose-dependent manner after single dosing in preclinical models. Recently, a phase I study of BGB-C354 has been initiated to investigate its potential safety and preliminary efficacy in patients with B7H3-expressing advanced solid tumors (NCT06422520). Citation Format: Xiao Ding, Charng-Sheng Tsai, Mei-Hsuan Tsai, Maggie Tang, Liu Xue, Chi Guan, Meiling Tan, Yiren Xiao, Yuanyuan Xie, Kunpeng Lyu, Xiaotong Chen, Wenhao Li, Qi Liu, Yun Chen, Jie Pan, Wenjin Yao, Yang Lyu, Kaiying Jia, Chang Song, Weiwei Song, Ce Wang, Fan Wang, Zhitao Wan, Mingming Guo, Beibei Jiang, Xiaomin Song, Wei Jin, Qiansheng Ren, Yu Shen, Zhirong Shen. BGB-C354, a novel B7H3 ADC with high DAR stability and strong bystander effect, demonstrates robust antitumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5731.

Abstract 1702: OBI-992, a novel TROP2 antibody-drug conjugate, exhibits distinct resistance profiles compared to other TROP2 targeting ADCs

Abstract BACKGROUND/AIM: Trophoblast cell surface antigen-2 (TROP2), a transmembrane glycoprotein often overexpressed in a variety of cancers, has been proposed as a potential cancer driver in colorectal cancer (CRC) cells. As a result, several antibody-drug conjugates (ADC) have been developed targeting TROP2, such as Sacituzumab Govitecan (SG) and Datopotamab Deruxtecan (Dato-Dxd). One of the major challenges in the development of ADC is the emergence of resistance via different mechanism of actions. This study aims to characterize the potential resistant mechanisms of TROP2 ADCs, including a novel TROP2 ADC OBI-992 in CRC cells. METHODS: DLD1 CRC cells resistant to OBI-992, SG, and Dato-Dxd analogs were established with sequential increase of respective ADC concentrations. Cytotoxicity of three ADCs and their payloads, including exatecan, SN-38, and Dxd, was evaluated to assess the fold of resistance. Cell surface and total TROP2 protein were measured by flow cytometry and western blotting. DNA mutation and gene expression profiling were conducted by whole exome sequencing (WES) and RNA sequencing (RNA-seq). RESULTS: TROP2 reduction and multidrug resistance proteins (MRPs) enhancement were not detected in OBI-992-induced resistant cells. Notable different DNA mutations in ATRX, BRCA2, and PALB2 of OBI-992 resistant cells were detected compared to those found with Dato-Dxd and SG-selected resistant cells. A TOP1 inhibitor-resistant mutation, TOP1 R364H, was identified as 100% variant allele frequency (VAF) in Dato-Dxd and SG resistant cells, whereas lower percentage of VAF was observed in OBI-992 resistant cell lines (29 % and 48.6 % in 0.5 uM and 2uM of OBI-992, respectively). OBI-992 resistant cells showed a clear divergence in differential expression gene (DEG) profiles, whereas Dato-Dxd and SG resistant cells were similar. c-MET and REG4 levels were greatly increased in cells with Dato-Dxd and SG resistance but not with OBI-992. Extracellular matrix (ECM) receptor interaction pathway was enriched in OBI-992 resistant cells. CONCLUSION: OBI-992, a novel TROP2 targeting ADC, emerges as a potential promising therapeutic option for patients who are resistant to other TROP2 targeting ADCs such as SG and Dato-Dxd. OBI-992 exhibits distinct resistance profiles compared to other TROP2 targeting ADCs. Understanding of potential mechanisms of resistance can provide potential combination options for TROP2-targeting ADCs to combat the drug resistance. Citation Format: Lifen Shen, Tzer-Min Kuo, Yu-Hsuan Tsao, Ya-Chi Chen, Ming-Tain Lai. OBI-992, a novel TROP2 antibody-drug conjugate, exhibits distinct resistance profiles compared to other TROP2 targeting ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1702.

Infectivity and structure of SARS-CoV-2 after hydrogen peroxide treatment.

Hydrogen peroxide (H2O2) exhibits broad-spectrum antiviral activity and is commonly used as an over-the-counter disinfecting agent. However, its potential activities against SARS-CoV-2 have not been systematically evaluated, and mechanisms of action are not well understood. In this study, we investigate H2O2's antiviral activity against SARS-CoV-2 infection and its impact on the virion's structural integrity as compared to the commonly used fixative agent paraformaldehyde (PFA). We show that H2O2 rapidly and directly inactivates SARS-CoV-2 with a half-maximal inhibitory concentration (IC50) of 0.0015%. Cryogenic electron tomography (cryo-ET) with subtomogram averaging reveals that treatment with PFA induced the viral trimeric spike protein (S) to adopt a post-fusion conformation, and treatment of viral particles with H2O2 locked S in its pre-fusion conformation. Therefore, H2O2 treatment likely has induced modifications, such as oxidation of cysteine residues within the S subunits of the spike trimer that locked them in their pre-fusion conformation. Locking of the meta-stable pre-fusion trimer prevents its transition to the post-fusion conformation, a process essential for viral fusion with host cells and entry into host cells. Together, our cellular, biochemical, and structural studies established that hydrogen peroxide can inactivate SARS-CoV-2 in tissue culture and uncovered its underlying molecular mechanism.IMPORTANCEHydrogen peroxide (H2O2) is the commonly used, over-the-counter antiseptic solution available in pharmacies, but its effect against the SARS-CoV-2 virus has not been evaluated systematically. In this study, we show that H2O2 inactivates the SARS-CoV-2 infectivity and establish the effective concentration of this activity. Cryogenic electron tomography and sub-tomogram averaging reveal a detailed structural understanding of how H2O2 affects the SARS-CoV-2 spike in comparison with that of the commonly used fixative PFA under identical conditions. We found that PFA promoted a post-fusion conformation of the viral spike protein, while H2O2 could potentially lock the spike in its pre-fusion state. Our findings not only substantiate the disinfectant efficacy of H2O2 as a potent agent against SARS-CoV-2 but also lay the groundwork for future investigations into targeted antiviral therapies that may leverage the virus' structural susceptibilities. In addition, this study may have significant implications for developing new antiviral strategies and improving existing disinfection protocols.

Abstract 3111: PI3K-dependent GAB1/Erk phosphorylation renders head and neck squamous cell carcinoma sensitive to PI3Kα inhibitors

Head and neck cancer is the seventh most common cancer in the world, of which about 90% are head and neck squamous cell carcinoma (HNSCC). Frequent alterations of PIK3CA in HNSCC suggest that PI3Kα is a promising target for HNSCC treatment. CYH33 is a novel PI3Kα-selective inhibitor discovered by our group, which is currently undergoing a phase I clinical trial (NCT03544905) for the treatment of advanced solid tumors, including HNSCC. However, there is an urgent need to elucidate its mechanism of action and improve its efficacy against HNSCC. In this study, we found CYH33 displayed promising but variable therapeutic activity against HNSCC. Inhibition of PI3K/Akt pathway by CYH33 was not sufficient for its activity against HNSCC. Tandem-Mass-Tag (TMT) proteomics and phosphoproteomics were performed to reveal comprehensive regulation of kinome by CYH33. Among them, phosphorylation of MAPK1/Erk at activation sites (T185/Y187) were significantly attenuated by CYH33 in sensitive HNSCC cells. CYH33 significantly suppressed phosphorylation of Erk in sensitive but not resistant cells. Further inhibition of Erk phosphorylation enhanced the activity of CYH33 against HNSCC. Signaling network of differentially phosphorylated proteins illustrated that phosphorylation at multiple sites (Y659, Y406, S651, S648) of the adaptor protein GAB1, which integrates the signaling from PI3K and receptor tyrosine kinases, were significantly downregulated upon CYH33 treatment. CYH33 dose-dependently suppressed the phosphorylation of GAB1 at Y659 in sensitive HNSCC cells, whereas it had little effect in resistant cells. We found that CYH33 attenuated the membrane localization and phosphorylation of GAB1, resulting in reduced Erk phosphorylation and ultimately inhibiting the proliferation of sensitive cells. Meanwhile, activation of EGFR induced GAB1 phosphorylation independent of PI3K in HNSCC. Concurrent inhibition of EGFR synergistically potentiated the activity of CYH33 against HNSCC.In conclusion, we found that PI3Kα-selective inhibitor CYH33 displayed potent activity against HNSCC, which was associated with inhibition of Akt signaling as well as attenuation of PI3K-dependent GAB1/Erk phosphorylation. Simultaneous inhibition of GAB1 phosphorylation independent of PI3K potentiated the anti-HNSCC activity of CYH33. These findings revealed the insight mechanism of CYH33 against HNSCC and provided rational combination regimen for HNSCC. Citation Format: Xu Zhang, Jiao Xu, Xuan Wang, Lan Xu, Xi Zhang, Yi Wang, Shujuan Jiang, Yixiang Zhang, Jian Ding, Chen Qing, Linghua Meng. PI3K-dependent GAB1/Erk phosphorylation renders head and neck squamous cell carcinoma sensitive to PI3Kα inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3111.

Abstract 6993: Development of synthetic lethal compounds targeting RB1-SUCLA2 deletion in advanced prostate cancer

Abstract The deletion of the tumor suppressor gene RB1 frequently occurs upon metastatic and/or castration-resistant conversion in prostate cancer. This extensive chromosomal loss associated with RB1 deletion also results in the loss of additional neighboring genes. Inhibition of gene products that compensate for these defective deletions can induce tumor-specific synthetic lethality. Our previous research demonstrated that SUCLA2 is collaterally deleted with RB1 often in advanced prostate cancer and that 5-Isopropyl-2-methyl-1, 4-benzoquinone (TQ) selectively kills SUCLA2-deficient tumors. In this study, we explored the target of TQ and the mechanism of action. To determine the binding targets of TQ, we synthesized a biotinylated alkyl TQ enabling target identification via mass spectrometry analysis. Four mitochondrial enzymes were identified (W, X, Y, and Z). Enzymatic assays revealed that only enzyme X was inhibited by TQ in a dose-dependent manner. Enzyme X composes qn enzymatic complex consisting of six alpha and six beta subunits, which TQ dissociated. Notably, TQ irreversibly depleted 2-oxoglutarate and glycine in SUCLA2 knockout (KO) cells, while TQ did not affect these metabolites in SUCLA2-reconstituted cells. Adding cell-permeable 2-oxoglutarate prevented TQ-induced apoptosis in SUCLA2 KO cells, suggesting that TQ induces apoptosis by depleting tricarboxylic acid (TCA) cycle intermediates, likely through the inhibition of enzyme X. We are currently developing new TQ derivatives to improve IC50 and pharmacokinetics. This research opens avenues for novel therapeutic strategies targeting metabolic pathways through this distinctive mechanism. Citation Format: Susumu Kohno, Chiaki Takahashi. Development of synthetic lethal compounds targeting RB1-SUCLA2 deletion in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6993.

Abstract 838: The novel eIF4A inhibitor synergizes potently with the XPO-1 inhibitor in T-cell lymphoma

Abstract Malignant lymphomas are categorized into B-cell and T-cell lymphomas. While treatment outcomes for B-cell lymphoma have significantly improved with targeted therapies and cellular treatments, T-cell lymphoma remains a therapeutic challenge due to the lack of standardized first-line treatments. Consequently, T-cell lymphoma exhibits lower remission rates, higher relapse rates, and poor overall prognosis. Although T-cell lymphomas account for only 15-20% of all malignant lymphomas, their incidence is notably higher in Asia compared to Western countries, making Korea an ideal location for research on this disease. Also, T-cell lymphoma (TCL) is an aggressive malignancy with poor prognosis, characterized by high relapse rates and limited therapeutic options. Novel therapeutic approaches are urgently needed to improve outcomes for patients with TCL. Previous studies have demonstrated the efficacy of eIF4A inhibitors in downregulating oncogenic protein synthesis and inducing cell death in various lymphoma models. We hypothesized that the combination of eIF4A inhibitor (CR-1-31-B) and XPO-1 inhibitor (Selinexor) would exhibit synergistic anti-lymphoma activity in TCL and have potential clinical applications. The anti-lymphoma effects of CR-1-31-B in combination with a XPO-1 inhibitor were evaluated in TCL cell lines using flow cytometry. Cell apoptosis assays were performed at increasing concentrations of CR-1-31-B and the XPO-1 inhibitor alone, as well as in combination, over 48 hours. To investigate the molecular mechanisms underlying the observed synergistic effects in TCL, RNA-seq profiling was conducted on cells treated with single agents and the combination therapy. Gene set enrichment analysis (GSEA) was performed to identify pathways involved in the combination's mechanism of action. CR-1-31-B and the XPO-1 inhibitor demonstrated concentration- and time-dependent cytotoxicity in TCL cell lines at low nanomolar concentrations. The combination of the eIF4A inhibitor and the XPO-1 inhibitor showed strong synergistic effects in all tested TCL cell lines (p<0.0001). RNA-seq profiling revealed that the transcriptional signature of the combination therapy was distinct from that of single-agent treatments. The combination of eIF4A inhibitor and XPO-1 inhibitor demonstrates potent anti-lymphoma activity in T-cell lymphoma. This novel combination represents a promising therapeutic strategy for patients with TCL, warranting further preclinical investigations and potential clinical translation. Citation Format: So-Young Seol, Chung Hyun Park, Soo Jeong Kim, Hyunsoo Cho, Jin Seok Kim, Yu Ri Kim. The novel eIF4A inhibitor synergizes potently with the XPO-1 inhibitor in T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 838.

The Endowment Effect in Second-hand Market and the "Double Eleven Shopping Festival"

The endowment effect is common in behavioral economics and consumer psychology, which has a wide range of applications in the consumer and trading market. At the same time, the endowment effect has different positive and negative impacts on various occasions. In life, as consumers, people are often affected by the endowment effect, thus carrying out some irrational consumption. In the market transaction, merchants will also be affected by the endowment effect, thus affecting the market. This paper mainly studies the different effects of endowment effect in other economic behaviors through two examples, it analyzes the case of experiments and the Double Eleven shopping festivals and conclude that the endowment effect has an impact on the trading market. Therefore, understanding the endowment effect and its mechanism of action can help people to identify life. At the same time, by analyzing the impact of the endowment effect, it is conducive to a more targeted response strategy, which is beneficial to both businesses and consumers.

Abstract 6249: Probabilistic causal network models from real-world prostate cancer data identify potential synergistic combinations with CBP/P300 inhibitors

Abstract The current standard of care for metastatic castration resistant prostate cancer (mCRPC) includes androgen-receptor signaling inhibitors (ARSIs), chemotherapy, PARP inhibitors (PARPi), or PSMA-radioligand therapies. Although these novel therapies have yielded substantial benefits for patients, many fail to respond or quickly develop resistance. Previous work identified key regulatory programs driving AR-altered, ARSI-resistant, late-stage prostate cancer, and further identified and validated that treatment with the CBP/p300 inhibitor pocenbrodib inhibits cancer cell growth both in vitro and in vivo. Here, we focus on identifying targeted therapies where combination with pocenbrodib could improve clinical response in late-stage prostate cancer. Previous analyses identified that treatment of prostate cancer cell lines with pocenbrodib downregulates a group of genes, termed a subnetwork, capturing G2M, E2F, and DNA repair mechanisms, suggesting that combination with a therapy that induces DNA damage or inhibits DNA repair could be additive or synergistic. Cell cycle profiling following treatment of cell lines with pocenbrodib confirmed induction of G2M cell cycle arrest. Next, we systematically ranked current targeted therapies based on publicly available RNA-seq signatures and signatures derived from RWD (e.g. BRCA2 alterations) to identify therapies that target our subnetwork of genes enriched for G2M, E2F, and DNA repair mechanisms. Given that both PSMA-radioligand therapies and PARP inhibitors induce DNA damage or inhibit DNA repair, we hypothesize that these drugs in combination with pocenbrodib would lead to additive or synergistic effects by enhancing the PARPi and PSMA-radioligand efficacy via their known mechanisms of action. To validate these hypotheses, we treated a panel of 5 genetically-diverse prostate cancer PDX-derived organoids with pocenbrodib, the PARPi olaparib, and in combination. The combination of pocenbrodib with olaparib led to additive or synergistic effects in 3 out of the 5 organoids, consistent with those showing some level of response to olaparib as a single agent. To evaluate the potential for combination benefit with PSMA-radioligand therapy, an in vivo efficacy study was performed using the PSMA+ 22Rv1 prostate cancer xenograft model. Addition of a low dose of pocenbrodib to a single dose of Lu177-PSMA617 (60 MBq) led to better tumor growth inhibition control than either single agent on its own. Mechanistic analyses from RNAseq are ongoing. Together, our integrated omics and experimental analyses support the hypothesis that combination of pocenbrodib with DNA damage-inducing or -inhibiting targeted therapies may provide further therapeutic benefit to prostate cancer patients than single agent therapies alone. Citation Format: Veronica Calvo, Bonnie Dougherty, Ananya Dutta, Duo Xu, Qi Pan, Andrew Armstrong, Jens Renstrup, Jason Somarelli, Eric Schadt. Probabilistic causal network models from real-world prostate cancer data identify potential synergistic combinations with CBP/P300 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6249.

Abstract 2887: Next generation antibody drug conjugates targeting HER2 and TROP2: Multi-Payload ConjugatesTM targeting orthogonal mechanisms of cell killing

Abstract Introduction: Antibody-Drug Conjugates (ADCs) have had tremendous impact on patient outcomes in breast and other cancers. T-DXd, for example, is second-line therapy for stage IV, HER2 positive metastatic breast cancer as well as HER2 low expressing tumors. Sacituzumab govitecan, a TROP2 targeted ADC, has been approved as a 3rd line therapy in locally advanced or metastatic HER2 negative breast cancer. However, many patients fail to respond or relapse after treatment with ADC therapies due to tumor heterogeneity and eventual resistance to the ADC payload. We are developing next generation Multi-Payload Conjugates™ (MPCs™) that deliver targeted combination chemotherapies within a single molecule to address shortcomings in current ADCs. Method: CatenaBio has developed novel, highly stable, single-molecule targeted combination therapies, MPCs™, with tunable payload ratios. Our selective conjugation platform allows the attachment of distinct payloads targeting different mechanisms of action at three unique sites on antibody scaffolds. Results: Catena’s MPCs show superior inhibition and excellent tolerability in mouse models of tumor growth in HER2 high and HER2 low/TROP2 expressing xenograft models including models of TNBC (triple negative breast cancer). In head-to-head comparisons MPCs out-perform current standards of care T-DXd and sacituzumab govitecan, demonstrating tumor elimination at low mg/kg doses. To produce the MPCs, combinations of different payloads targeting orthogonal mechanisms of cell division attached to trastuzumab as well as sacituzumab were screened at different DARs and payload ratios to optimize tumor cell killing. These targeted combination ADCs demonstrated robust killing in multiple cell lines containing high and low expression of HER2 and TROP2, as well as a T-DXd resistant cell line. Conclusion: While advances have been made in the design of ADCs to expand to previously unaddressed populations, high patient relapse and the failure of recent mono-payload ADCs in late-stage trials indicate a need for novel conjugate modalities. Multi-payload Conjugates™ offer the next step in ADC design and allow for the targeted combination of multiple mechanisms of action with a single MPC™ that are highly efficacious at eliminating tumors across multiple cell line derived xenograft models with a range of target expression. Successfully constructed with a variety of targeting antibodies, these molecules offer the potential to circumvent tumor resistance pathways to deliver deeper and more durable patient responses. Citation Format: Richard L. Kendall, Devin Trinter, Maxwell Nguyen, Daniel Gutierrez, Samantha Brady, Chanez Symister, Andrew Lau, Derek Garcia-Almedina, Saurabh Johri, Matthew Francis, Marco Lobba. Next generation antibody drug conjugates targeting HER and TROP: Multi-Payload ConjugatesTM targeting orthogonal mechanisms of cell killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2887.