Antimitotic Research Articles

Control of cell proliferation by memories of mitosis.

Mitotic duration is tightly constrained, and extended mitosis is characteristic of problematic cells prone to chromosome missegregation and genomic instability. We show here that mitotic extension leads to the formation of p53-binding protein 1 (53BP1)-ubiquitin-specific protease 28 (USP28)-p53 protein complexes that are transmitted to, and stably retained by, daughter cells. Complexes assembled through a Polo-like kinase 1-dependent mechanism during extended mitosis and elicited a p53 response in G1 that prevented the proliferation of the progeny of cells that experienced an approximately threefold extended mitosis or successive less extended mitoses. The ability to monitor mitotic extension was lost in p53-mutant cancers and some p53-wild-type (p53-WT) cancers, consistent with classification of TP53BP1 and USP28 as tumor suppressors. Cancers retaining the ability to monitor mitotic extension exhibited sensitivity to antimitotic agents.

High OXPHOS efficiency in RA-FUdr-differentiated SH-SY5Y cells: involvement of cAMP signalling and respiratory supercomplexes

Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.

Exploring the potential of Ziziphus nummularia and luteolin-7-O-glucoside as tubulin inhibitors in cancer therapy and survival

Cancer is responsible for approximately 10 million deaths worldwide, with 70% of the deaths occurring in low- and middle-income countries; as such safer and more effective anti-cancer drugs are required. Therefore, the potential benefits of Ziziphus nummularia and Ziziphus spina-christi as sources of anti-cancer agents were investigated. Z. nummularia and Z. spina-christi extracts were prepared using chloroform, ethanol, ethyl acetate, and water. The extracts’ anti-cancer properties were determined using the MTT Cell Viability Assay in four cancer cell lines: breast (KAIMRC2 and MDA-MB-231), colorectal (HCT8), and liver (HepG2). The ApoTox-Glo Triplex Assay and high-content imaging (HCI)-Apoptosis Assay were used to assess KAIMRC2 and HCT8 cells further. In addition, KAIMRC2 cells were tested for microtubule staining, and AKT/mTOR protein expression was determined by western blot analysis. Liquid chromatography-mass spectrometry (LC–MS) was performed to identify the secondary metabolites in the ethanol and ethyl acetate extracts, followed by in silico techniques to predict molecular targets and interactions, safety, and pharmacokinetic profile for identified metabolites. Out of the eight extracts, the ethanolic extract of Z. nummularia, exhibited the most potent activity against KAIMRC2 cells with an IC50 value of 29.2 μg/ml. Cancer cell treatment with the ethanolic extract of Z. nummularia resulted in a dose-dependent decrease in cell viability with increased apoptosis and cytotoxic effects. Microtubule staining showed a disrupted microtubular network. The ethanolic extract treatment of KAIMRC2 cells led to upregulated expression of pAKT and pmTOR. In silico studies predicted luteolin-7-O-glucoside to be a ligand for tubulin with the highest docking score (− 7.686) and similar binding interactions relative to the native ligand. Further computational analysis of the metabolites showed acceptable pharmacokinetic and safety profiles, although ethanolic extract metabolites were predicted to have cardiotoxic effects. Ethanolic extraction is optimal for solubilizing active anticancer metabolites from Z. nummularia, which may act by causing M-phase arrest via inhibition of tubulin polymerization. Luteolin-7-O-glucoside is the lead candidate for further research and development as an anti-cancer agent. In addition, this study suggests that herbal treatment could switch on mechanisms of adaptation and survival in cancer cells.

Therapy-related chronic myelomonocytic leukemia does not have the high-risk features of a therapy-related neoplasm

AbstractTherapy-related myeloid neoplasms (t-MNs) arise after exposure to cytotoxic therapies and are associated with high-risk genetic features and poor outcomes. We analyzed a cohort of patients with therapy-related chronic myelomonocytic leukemia (tCMML; n = 71) and compared its features to that of de novo CMML (dnCMML; n = 461). Median time from cytotoxic therapy to tCMML diagnosis was 6.5 years. Compared with dnCMML, chromosome-7 abnormalities (4% vs 13%; P = .005) but not complex karyotype (3% vs 7%; P = .15), were more frequent in tCMML. tCMML was characterized by higher TP53 mutation frequency (4% vs 12%; P = .04) and lower NRAS (6% vs 22%, P = .007) and CBL (4% vs 12%, P = .04) mutation frequency. Prior therapy with antimetabolites (odd ratio [OR], 1.22; 95% confidence interval [CI], 1.05-1.42; P = .01) and mitotic inhibitors (OR, 1.24; 95% CI, 1.06-1.44; P = .009) was associated with NF1 and SETBP1 mutations whereas prior mitotic inhibitor therapy was associated with lower TET2 mutation frequency (OR, 0.71; 95% CI, 0.55-0.92; P = .01). Although no differences in median overall survival (OS) were observed among tCMML and dnCMML (34.7 months vs 35.9 months, P = .26), multivariate analysis for OS revealed that prior chemotherapy was associated with increased risk of death (hazard ratio, 1.76; 95% CI, 1.07-2.89; P = .026). Compared with a cohort of therapy-related myelodysplastic syndrome, tCMML had lower TP53 mutation frequency (12% vs 44.4%, P < .001) and less unfavorable outcomes. In summary, tCMML does not exhibit the high-risk features and poor outcomes of t-MNs.

Abstract 3127: Development of three-dimensional cancer cell line spheroid models for the in vitro functional characterization of cytotoxic antibody-drug conjugates

Abstract Background: Antibody-drug conjugates (ADCs) are an effective class of cancer therapeutics which have gained prominence for the treatment of several malignancies. A cytotoxic ADC consists of a linker-payload conjugated to a monoclonal antibody, which targets a distinct tumor-associated antigen (TAA) to enable the delivery of the cytotoxic payload to cancer cells. Presently, there is a need for in vitro models that better recapitulate in vivo tumor tissue complexity to aid in the screening and evaluation of ADCs during preclinical development. Hence, efforts have been made to develop in vitro three-dimensional (3D) models with improved translation to in vivo tumor models compared to traditional two-dimensional (2D) cancer cell line monolayer models. We aimed to generate monoculture cancer cell line spheroids in a high-throughput manner. Subsequently, we sought to evaluate the spheroid penetration capability of structurally distinct ADCs and to assess the 3D cytotoxic activity of a variety of microtubule inhibitor (MTI) and topoisomerase 1 inhibitor (TOPO1i)-bearing ADCs targeting multiple TAAs, comparing to activity in 2D models. Methods: Monoculture cancer cell spheroids were generated by seeding cells from a variety of tumor types, using an automated liquid-handling robot, into microtiter plates treated with ultra-low attachment coating, which allows for scaffold-free self-assembly of cancer cells into a 3D arrangement. Following spheroid formation by incubation for 2-3 days under standard culturing conditions, spheroids were treated with ADCs at a range of concentrations for functional evaluation: the spheroid penetration capability of ADCs of different antibody formats was assessed using high-content confocal imaging of spheroids treated with fluorescently labeled antibodies; the cytotoxic activity of ADCs was characterized using an ATP quantification luminescent reagent, live/dead cell fluorescent stains, and confocal imaging. Results: Monoculture spheroids of varying morphologies were successfully and reproducibly generated with a large panel of &amp;gt;50 cancer cell lines derived from &amp;gt;10 tumor types. Antibodies with different formats demonstrated target-mediated binding and a range of spheroid penetration depths. Additionally, MTI and TOPO1i ADCs evaluated in a panel of cancer cell spheroids demonstrated robust 3D cytotoxicity and differentiated activity when compared to the 2D monolayer assay. In a number of cases, the potency trends across multiple ADCs in 3D cytotoxicity assays were more predictive of in vivo efficacy compared to the 2D assay. Taken together, our high-throughput spheroid assays present important and straightforward in vitro tools to aid in the screening and selection of therapeutic cytotoxic ADC candidates for the treatment of solid tumors. Citation Format: Jodi Wong, Andrea Hernández Rojas, Allysha Bissessur, Lemlem T. Degefie, Araba P. Sagoe-Wagner, Samir Das, Vincent Fung, Kevin Yin, Renee Duan, Sam Lawn, Laurence Madera, Catrina Mi Jung Kim, Alex Wu, Mark E. Petersen, Raffaele Colombo, Jamie R. Rich, Stuart D. Barnscher. Development of three-dimensional cancer cell line spheroid models for the in vitro functional characterization of cytotoxic antibody-drug conjugates [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 3127.

Abstract 5269: Evaluation of mitotic spindle inhibitors as cell autonomous inflammatory stimulants in MYC overexpressing breast cancers

Abstract The oncogenic transcription factor MYC is overexpressed in most cancer types, including difficult to treat human malignancies such as receptor triple-negative breast cancers (TNBC). MYC overexpression induces aneuploidy and suppresses anticancer immunity. Elevated levels of MYC can induce mitotic spindle assembly defects and chromosomal instability (CIN) events, exemplified by an increase in cells with micronuclei. These defects are partially mitigated by concomitant upregulation of various mitotic spindle genes. Micronuclei result in cytosolic DNA which can activate cGAS-STING signaling, activating an interferon regulatory transcriptional program responsible for mounting an inflammatory immune response that potentially enhances immune cell recognition. We hypothesized that pharmacological inhibition of specific MYC deregulated mitotic spindle genes will further exacerbate mitotic defects in MYC high cells resulting in synthetic-lethal cell death and additional CIN to activate robust cGAS-STING activation. Interferon activation can increase surface expression of antigen presentation machinery, such as MHC-I, restoring the immunogenicity of MYC high cells. Here, we used mouse breast tumor cells in which MYC expression is conditionally expressed as well as human breast cancer cell lines with different levels of MYC expression. We assessed the anti-proliferative and cytotoxic effects of 9 mitotic small molecular inhibitors that affect spindle function. cGAS-STING activation in inhibitor treated and untreated samples was measured by immunoblotting for pathway proteins phosphorylated TBK1, total TBK1, and IFIT1. Differences in MHC-I cell surface expression was quantified using flow cytometry. Our findings revealed that inhibition of kinesin-like protein KIFC1 and transforming acidic coiled-coil-containing protein 3 (TACC3) induced greater levels of apoptosis in MYC high models. Moreover, inhibition of KIFC1 robustly triggered cGAS-STING activity in MYC high cells as evidenced by increased protein abundance of phospho-TBK1 and IFIT1 in a dose-dependent manner. In addition, sublethal KIFC1 inhibition resulted in increased levels of MHC-I surface expression in MYC high models. Our findings suggest that inhibition of MYC regulated mitotic spindle genes, specifically KIFC1, can serve to preferentially eliminate MYC overexpressing aggressive cancers. Furthermore, sublethal concentrations of a KIFC1 inhibitor can stimulate a tumor cell-intrinsic inflammatory-pathway. Future studies will determine if targeting KIFC1 can enhance the response of MYC high cancers to immunotherapy. Citation Format: Mauricio Jacobo, Andrei Goga. Evaluation of mitotic spindle inhibitors as cell autonomous inflammatory stimulants in MYC overexpressing breast cancers [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 5269.

Abstract 2605: STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells

Abstract Dipeptidyl Peptidase 4 (DPP4/CD26) is a membrane glycoprotein involved in particular chemotactic and inflammatory responses, via its N-terminal protein cleavage enzymatic activities. DPP4 expression is found in various cell types, in certain subsets of lymphocytes and frequently overexpressed in primary tumors and metastases where its role remains still poorly understood. Recently, DPP4 was identified as a novel biomarker of senescent cells. In cancers, the harmful effect of senescent tumor cells is well recognized and mediated by the local production of pro-inflammatory cytokines, soluble immunosuppressive factors and metalloproteinases, called ‘SASP’ phenotype which could be induced by many chemotherapy and radiation regimens. This deleterious microenvironment promotes tumor growth, invasion and neoangiogenesis which can lead to treatment resistance, relapses and poor prognosis in many cancers, especially at advanced stages. STX-1 is a leading first-in-class ADC platform based on a proprietary anti-DPP4 mAb generated after a screen between normal and senescence-induced cells identifying DPP4 as a candidate for cancer targeting. Other biomarkers of interest were also identified as being selectively expressed for further preclinical evaluation. This IgG1/k mAb has potent in vitro activities on DPP4+ cells: i) in a bleomycin-induced senescent fibroblasts model; ii) on DPP4-expressing and chemotherapy- or radiotherapy-induced senescent human tumor cell lines with overexpression of DPP4; iii) binding of the mAb to its target results in its internalization into the endolysosomal compartment of cells. The first formulations of STX-1 are undergoing preclinical evaluation in vitro and in vivo and consist of a classical maleimide-based conjugation, an enzymatically cleavable cathepsin-dipeptide linker comprising a self-immolative spacer and either inhibitor of tubulin (MMAE/DAR4) or topoisomerase I inhibitor (exatecan/DAR8) payloads. Plasma stability and affinity, dose-response potency and permeability bystander activity on human tumor cell lines were evaluated in vitro. Preliminary efficacy is demonstrated in vivo on tumor xenograft models with various DPP4 expressions (constitutive or chemo/radio-induced to senescence) based on a 21-28 days weekly infusion schedule (3 or 5 µg/kg). The indications selected for further therapeutic development are prostate, liver and colorectal carcinoma models. Data on antitumor efficacy, tolerability and pharmacokinetics will be presented. Further studies will include optimization of the best STX-1 formulation, NHP studies and GMP scale-up with planned IND submission expected in Q3-2024. Exploiting the targeting of senescent cells in cancer based on selected biomarkers such as DPP4 constitutes a new therapeutic avenue to counteract tumor dormancy, recurrences and resistance to conventional chemo/radiotherapies. Citation Format: Eric Angevin, Virginie Lelarge, Remi Capelle, Justine Choeur, Thierry Mathieu, Benjamin Le Calvé. STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells [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 2605.

Abstract 511: Cell cycle inhibitors exhibit anti-tumor immunomodulatory roles through the HIF-1-DDX41 cytosolic DNA sensing pathway in HCC

Abstract Cell cycle inhibitors have a long history as anti-cancer therapeutics in clinical applications. Uncontrolled and infinite cell cycles are hallmarks of cancer, which enable them to proliferate rapidly and survive. To target these key features of cancer cells, cell cycle inhibitors are used to arrest their active cell cycle and induce cell death. In this study, beyond the known primary effect on cell cycle control, we identified that an expected and a secondary effect in immune modulation by the cell cycle inhibitors. Anti-mitotic agents including Paclitaxel (microtubule stabilizer), Palbociclib (CDK4/6 inhibitor) and AZD1152 and GSK1070916 (Aurora Kinase B inhibitors) can also eliminate cancer cells via an alternative mechanism - the activation of STING signaling. Our results demonstrated that these anti-mitotic agents caused DNA damage and cytosolic DNA accumulation. The cytosolic DNA was captured by a cytosolic DNA sensor DDX41,which triggered STING-TBK1-IRF3/7 pathway to upregulate secretion of pro-inflammatory senescence-associated secretory phenotype (SASP) factors in cancer cells. In addition, we revealed that transcription of DDX41 was mediated by the transcription factor known ashypoxia-inducible factor (HIF). In HCC, hypoxia induced DDX41 expression through HIF-1,thus hypoxic HCC cells were more prone to STING activation and SASP production under mitotic stress induced by cell cycle inhibitors. In the tumor microenvironment, the SASP enhanced infiltration of immune cells into the tumor core to eliminate cancer cells. We also observed the additive effects of cell cycles inhibitors (Paclitaxel, Palbociclib, and AZD1152)with anti-PD-1 mAb to arrest growth of HCC in mouse models. In summary, this study exhibited the novel immune-mediated aspect of cell cycle inhibitors in suppressing tumor growth. Our data suggested the potential combination regimen of cell cycle inhibitors and currently available immunotherapy with promising result. Citation Format: Cerise Yuen Ki Chan, Po Yee Wong, Helen Do Gai Xue, Chi Ching Goh, Jacinth Wing Sum Cheu, Aki Pui Wah Tse, Misty Shuo Zhang, Carmen Chak Lui Wong. Cell cycle inhibitors exhibit anti-tumor immunomodulatory roles through the HIF-1-DDX41 cytosolic DNA sensing pathway in HCC [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 511.

Abstract 479: Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots

Abstract Acute Myeloid Leukemia (AML) treatment faces considerable challenges, in particular the delivery of therapeutics with sufficient potency and specificity. Traditional Antibody-Drug Conjugates (ADCs) offer a strategic approach to cancer therapy by combining the targeting capabilities of monoclonal antibodies with the cell-killing effect of cytotoxic drugs. However, the limitations in drug-to-antibody ratio (DAR) and the challenges in controlling the release kinetics have often hindered their therapeutic window suggesting novel approaches are necessary. Our approach integrates DNA nanobot technology to facilitate targeted delivery of anthracycline agents directly to CD37-expressing leukemic cells. CD37, a tetraspanin superfamily antigen, has shown promise as a target for AML due to its selective expression on immune cells and favorable internalization properties. By harnessing DNA origami assembly techniques, we developed a customizable platform capable of accommodating a significantly higher payload of chemotherapeutic agents than traditional ADCs. This increased payload potential, paired with the precision targeting of CD37, presents a potent therapeutic strategy with the possibility of reduced systemic toxicity. Here we present preclinical evidence demonstrating targeted delivery of our DNA nanobot drug delivery device and anthracycline payload to CD37+ AML target cells in vitro. In addition, we show targeted efficacy in vitro and in vivo of our anthracycline-loaded DNA nanobot-enabled delivery system, which significantly outperformed free anthracycline in AML target cells. Collectively, our findings suggest an improvement in the therapeutic index of anthracycline and antimitotic agents as well as the ability to combine multiple drug pathways in a single delivery device. The implications of this work extend beyond AML, offering a versatile platform that could revolutionize ADC chemistry and targeted drug delivery. We propose that our DNA nanobot delivery system signifies a pivotal advancement in the field of targeted cancer therapy, with the potential to overcome longstanding barriers in the treatment of AML and potentially other malignancies. Citation Format: Nicholas Vantangoli, Patrick D. Halley, Jeffrey R. Spitzner, John C. Byrd, Karilyn T. Larkin, Carlos E. Castro, Christopher R. Lucas. Enhanced payload delivery for acute myeloid leukemia treatment through CD37-targeting DNA nanobots [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 479.

Abstract 3131: Novel CDH17-targeting antibody-drug conjugate exhibits anti-tumor efficacy in preclinical models of gastrointestinal cancers

Abstract Antibody-drug conjugates (ADC) for oncology indications have been developed, however, only few of them are approved for the treatment of gastrointestinal (GI) cancers, which represent as a huge unmet medical need. As such, we developed a novel ADC of which monomethyl auristatin E (MMAE), an antimitotic agent, was conjugated to a cadherin-17 (CDH17)-targeting monoclonal antibody via protease-cleavable peptide valine-citrulline linker. CDH17 is highly restricted to the intestinal adherent junctions which is not accessible in healthy individuals. In contrast, it is overexpressed and becomes reachable in over half of the gastric and pancreatic cancer patients and in more than 95% of patients with colorectal cancer, making it a perfect target for ADC. Our ADC is designed to deliver MMAE specifically to CDH17-expressing tumors to exert the cytotoxic effect of MMAE on cancer cell, mitigating the undesired off-target cytotoxicity. LC/MS analysis revealed that MMAE was conjugated in a drug-to-antibody ratio of 4 with a yield of higher than 75%. ELISA showed that the binding of naked antibody and ADC to CDH17 was comparable (EC50, 0.17 nM &amp; 0.3 nM, respectively), indicating the conjugation process did not affect antigen binding. This ADC also bound to different CDH17-expressing cell lines, including pancreatic AsPC1 and gastric AGS (EC50, 2.1 nM &amp; 1.53 nM, respectively). By labelling the ADC with pH-sensitive fluorescent indicator, we illustrated that ADC internalization only occurred in CDH17+ but not CDH17- cells, suggesting the high specificity of the ADC. Similarly, the ADC showed selective killing on CDH17+ cells (EC50, 0.02-0.5 nM), and the data together proposed a positive correlation between ADC internalization level and its cytotoxicity (R2 = 0.4307). This supported the specificity and safety of the ADC in killing CDH17+ cancer cells only without affecting normal cells. Furthermore, a preliminary in-vivo study on subcutaneous AsPC1 xenograft model demonstrated that the ADC significantly suppressed tumor growth without causing any changes in body weight. Our ADC would potentially be a “First-in-class” ADC for the treatment of GI cancers with minimal adverse systemic toxicity. Further pharmacokinetics, safety, and other IND-enabling studies are underway. Citation Format: Po Yee Wong, Lin He, Kronos Chow, Kwan Wa Wong, Chui Yee O, Dennis Wong, John Moon Luk, Pui-Chi Lo, Kwong Fai Wong. Novel CDH17-targeting antibody-drug conjugate exhibits anti-tumor efficacy in preclinical models of gastrointestinal cancers [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 3131.

Abstract 507: HLS-22001 induces G2/M cell cycle arrest and apoptotic cell death via tubulin depolymerization in colon cancer

Abstract Colorectal cancer (CRC) is one of the main malignant tumors and among the leading cause of death in cancer patients worldwide considering that recurrence and chemotherapeutic drug resistance are highly common in the advanced stage of CRC. Mutations in KRAS and BRAF occur frequently in colorectal cancers, and abnormal cell cycles caused by these mutations affect tumor progression and metastasis. Accordingly, inhibition of cell cycle has been a potential target for colorectal cancers and therefore, we have developed a new lead compound, HLS-22001. Our studies confirmed that HLS-22001 inhibited the proliferation of several cancer cells and significantly induced G2/M arrest and apoptosis of colon cancer cells and chemotherapy resistant colon cancer cells. The docking model studies showed that HLS-22001 was expected to bind to the colchicine binding site of beta-tubulin, and in fact, as a tubulin inhibitor, it had the effect of inhibiting tubulin dimerization and polymerization in the microtubule dynamics. Consistently, in xenograft mouse model, oral administration of HLS-22001 significantly suppressed tumor weight and volume without acute toxicity in mice. Collectively, these results suggest further development of HLS-22001 as a novel and promising oral drug for colorectal cancer. Citation Format: Chi-hyun Ahn, Gyu-beom Jang, Chunhwa Kim, Junho Lee, Taewon Kim, Youngeun Yang, Eunji Cho, Minchae Kim, Yun-tai Kim, Yong-Hae Han, Heebum Kang. HLS-22001 induces G2/M cell cycle arrest and apoptotic cell death via tubulin depolymerization in colon 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 507.

Abstract 4708: Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro

Abstract Previously we have reported on the relevance of the joint ProLiFiler and Cancer DataMiner platforms when studying the antitumor efficacy of novel antibody-drug conjugates (ADCs), such as Sacituzumab govitecan. In this study, we use our platforms to investigate and compare the selectivity of two FDA-approved Trastuzumab-based ADCs targeting ERBB2: Kadcyla, the first-in-class ADC employing a microtubule inhibitor bound via a non-cleavable linker, and Enhertu, which combines a topoisomerase inhibitor with a protease-cleavable linker. We performed a cell proliferation and survival assay using the ProLiFilerTM (Reaction Biology) to characterize the in vitro antitumor effects of Enhertu, Kadcyla, and their individual cytotoxins, deruxtecan &amp; mertansine on 160 human cancer cell lines (CLs). The resulting data were uploaded to 4HF´s Cancer DataMiner platform for data analysis. First, we confirmed high potency and limited selectivity of mertansine, as it displayed an IC50 &amp;lt; 250 nM (mean: 27.3 nM) in 95% of cell lines (CLs). A COMPARE analysis confirmed that mertansine data correlated best with those of other microtubule inhibitors in our drug databases (e.g., MMAE, Spearman rho = 0.79, p=9.6E-27). Conversely, a paired analysis showed that CLs were less sensitive to Kadcyla than to mertansine except for CLs overexpressing ERBB2. The 160-CL panel included a total of nine CLs (six mammary, two gastric, one ovarian) with strong ERBB2 overexpression (Affymetrix data &amp;gt;10), mostly associated with ERBB2 gene amplification; seven of them were the most sensitive CLs to Kadcyla (IC50 &amp;lt; 6 nM). Focusing solely on breast cancer cell lines (breast cancer is the approved indication for Kadcyla), we observed a strong correlation between sensitivity to Kadcyla and ERBB2 expression (Spearman r = -0.78, p = 0.003). Interestingly, in line with published data, the ERBB2-overexpressing cell lines SNU-216 (stomach cancer) and JIM-T1 (breast cancer) were less sensitive to Kadcyla. In non-ERBB2-amplified CLs, Kadcyla sensitivity did not correlate with ERBB2 expression levels. Focusing on ERBB2-negative CLs such as hematological CLs, we unexpectedly observed consistent cell inhibition (mean IC50 =33 nM) with Kadcyla, suggesting that the ADC was cleaved, and the payload released into the medium. Overall, this study has demonstrated the relevance of our platforms to study and compare newly developed ADCs. Cytotoxic activities recorded for conjugates and free payloads, along with molecular data for the test cell lines, provides insight in the mode of action of conjugates and their target cell populations and hence can support the development of ADCs. Investigations are underway with Enhertu and its payload deruxtecan, and results will be compared with those obtained for Kadcyla and mertansine. Citation Format: Anne-Lise Peille, Kaja Holtorf, Pablo Anton Garcia, Nadine Obier, Daniel Feger, Thomas Metz, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Ehlert, Vincent Vuaroqueaux. Potency and selectivity of ERBB2-targeting antibody drug conjugates in vitro [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 4708.

Abstract 4701: A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile

Abstract Initial evaluation of the prenylated hydroxy-stilbene isolated from bee propolis, AUS_001, in the NCI 60 human cell line anticancer screen showed significant growth inhibitory effects with the most robust being observed in leukemia, central nervous system and breast cancer cell lines. The aim of the current study was to further assess the potency of AUS_001 in vitro and investigate its safety profile and mechanism of action. A commercially available cell-based profiling screen (OncoPanel®) identified significant AUS_001-induced growth inhibition in 273 out of 280 cancer cell lines with a concentration causing 50% cell growth inhibition in the range of 0.021-0.94 µM, while non-neoplastic cells required an average of 20x higher concentration to generate similar efficacy. These findings indicate that AUS_001 exerts a high degree of potency across 30 cancer types with a selectivity for cancer versus normal cells. Maximum tolerated dose studies in mice illustrated that orally administered AUS_001 is well tolerated at doses 4 times greater than those that conferred antitumor efficacy, with no myelosuppression or other overt toxicities observed. Predictive toxicology and safety screening indicated that AUS_001 exerts a strong safety profile as evidenced by absence of mutagenic potential in the Ames test using up to 100 µM drug doses and low hERG-blocking liability with a half-maximal inhibitory concentration of 65 µM. Notably, AUS_001 is a poor substrate of the drug efflux transporter, P-glycoprotein 1 (Pgp), as it does not inhibit drug-stimulated Pgp ATPase activity and retains efficacy in high Pgp-expressing models in vitro. Subsequently, the effects of AUS_001 were explored in the ToxTracker® assay, consisting of a panel of mammalian stem cell lines that contain fluorescent reporters for the exploration of DNA damage, oxidative stress, and protein damage using flow cytometry. Robust activation of the Rtkn genotoxicity reporter under low cytotoxicity conditions, in combination with accumulation of cells in G2/M phases of the cell cycle within 4 h and an increase in aneuploid cells after 24 h of drug treatment, suggested an aneugenic mode of action, typically caused by tubulin-targeting agents or mitotic kinase inhibitors. Biochemical tubulin polymerization assays then revealed that AUS_001 acts as a direct tubulin destabilizing agent and monitoring microtubule dynamics in GFP-tubulin reporter cells provided evidence that AUS_001 blocks microtubule assembly. Our studies have elaborated the mechanism of AUS_001 as an inhibitor of tubulin polymerization. The favorable safety profile of AUS_001, along with its ability to circumvent Pgp-mediated multidrug resistance, provides potential for efficacy against multiple cancers where microtubule destabilization is proven to be an effective target. Citation Format: Herman Lelie, Inger Brandsma, Giel Hendriks, Lee R. Cavedine, Brogan A. Epkins, Steven M. Garner, Andrew J. Cook, Muthukrishnan Renganathan, Yong Zhao, Alastair J. King, Peter Y.W. Chan, April Risinger, Marina Koutsioumpa. A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile [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 4701.

Abstract 1902: ADRX-0706 Nectin-4 antibody-drug conjugate PK/PD characterization elucidates its widened therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers, while expression in normal tissues is limited. A first-generation antibody-drug conjugate targeting Nectin-4, called enfortumab vedotin-ejfv (EV), was granted accelerated FDA approval in 2019 for urothelial cancers, and is currently approved for frontline therapy in cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma, providing groundbreaking improvement in patient survival. However, clinical use of EV has also revealed safety-driven limitations, which result in reduced and/or interrupted drug exposures impacting efficacy. To overcome these limitations in therapeutic window, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio (DAR) of 8. ADRX-0706 utilizes a novel tubulin inhibitor payload AP052 with a proprietary cleavable linker utilizing Adcentrx’ proprietary i-ConjugationTM technology, which results in excellent biophysical features. Most noteworthy, ADRX-0706 displays a very stable PK profile with overlapping curves for total antibody and ADC as well as full DAR retention in circulation as determined by mass spectrometry. This translates to superior efficacy of ADRX-0706 over EV in multiple in vivo cancer cell line- and patient-derived xenograft mouse models across indications. Detailed PK/PD characterization of ADRX-0706 validates the mechanism of action as an anti-mitotic agent inducing cell cycle arrest and apoptosis. In addition, ADRX-0706 treatment leads to increased PD-L1 expression in mouse pharmacology models, which is a consequence of immunogenic cell death as described for this payload class. These findings are in line with emerging data on EV and provide rationale for combination regimens with checkpoint inhibitors. Besides the improvement in efficacy, ADRX-0706 was also designed for superior safety. A payload distribution study in tumor-bearing mice reveals a preferential and increased delivery of payload to tumors and lower payload delivery to normal tissues as well as lower free payload levels in plasma for ADRX-0706 compared to EV. Taken together, these data provide association of the improved drug-linker stability of ADRX-0706 to an improved Nectin-4 directed delivery of AP052 payload to the tumor and lower exposure in normal tissues, which may translate to an improved therapeutic window. In summary, the preclinical efficacy and safety properties of ADRX-0706 support clinical evaluation of this next generation anti-Nectin-4 ADC. ADRX-0706 is currently in a phase 1a/b study (NCT06036121). Citation Format: Maria Shahmoradgoli, Andrew Hau, Dong Jun Lee, Anna Wang, Peter P. Challita, Oscar Betancourt, Wes Sisson, Mario M. Kuo, Kris Zhang, Albert Goldson, Sam Janssen, Paul Datta, Felipe Acosta, Alexander Chu-Kung, Hui Li, Pia M. Challita-Eid, Sabine Rottmann. ADRX-0706 Nectin-4 antibody-drug conjugate PK/PD characterization elucidates its widened therapeutic window [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 1902.

Abstract 1885: Developing a biparatopic anti-ROR1 antibody drug conjugate BR111 for hematological and solid tumor treatment

Abstract ROR1 is a type I transmembrane protein that belongs to the ROR family. It is a receptor for Wnt family signaling molecules Wnt5a, and plays a critical role in various cellular processes, such as cell proliferation, survival, and migration. Being an oncofetal protein, ROR1 exhibits limited expression in most normal tissues. However, it is abnormally expressed in various hematological and solid cancers, contributing to the development and progression of many types of cancer. Due to its overexpression in cancer, ROR1 presents as a highly attractive target for antibody-drug conjugate (ADC) therapy. The current clinical results of ROR1 ADCs have been promising in treating patients with relapsed and/or refractory (R/R) hematologic malignancies. BR111 is anti-ROR1 ADC that consists of a biparatopic antibody, stably conjugated to an antimitotic agent. The biparatopic antibodies demonstrate superior binding affinity to ROR1-expressing cancer cells compared to single epitope antibodies. Notably, our unique and innovative conjugation platform CysX™, not only prevents payload detachment during circulation and reducing the off-target toxicity, but also enables a uniformed drug-to-antibody ratio (DAR) of 4. Both in vitro and In vivo studies have highlighted the remarkable antitumor activity of BR111. Preclinical data have shown great safety profile in cynomolgus monkeys. BR111 demonstrated a wider therapeutic window for cancer treatment, outperforming the lead anti-ROR1 ADC currently in phase II/IIl trial. The promising outcome holds great potential as a therapeutic treatment for hematological and solid cancers, offering a better safety profile. BR111 has great potential in further clinical evaluations. Citation Format: Xiaobei Zhao, Jie Zhu, zhenhua Wu, Yiqun Li, Jing Li, Yaqiong Zhou, Lei Nie, Haibin Wang, Gang Chen. Developing a biparatopic anti-ROR1 antibody drug conjugate BR111 for hematological and solid tumor treatment [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 1885.

Abstract 7144: BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death

Abstract BPR0L075 (also known as SCB01A in phase II clinical trial) is a novel anti-microtubule drug with potent anti-tumor and anti-angiogenic activity in vitro and in vivo. BPR0L075 inhibits tubulin polymerization (microtubule de-stabilizer) by binding to the colchicine-binding site of tubulin. We previously reported that BPR0L075 overcomes paclitaxel (microtubule stabilizer) resistance and induces vascular-disruption in vitro and in vivo. We observed that breast and ovarian cancer cells treated with BPR0L075 in vitro induced key hallmarks of immunogenic cell death (ICD). In both human and murine, ovarian (OVCAR3, ID8) and breast cancer (MDA-MB-231, 4T1) cell lines, treatment with low nanomolar concentration of BPR0L075 significantly decreased the viability of cells evidenced by SRB assay. Using a known ICD inducer mitoxantrone as a positive control, both flow cytometric analysis and immunofluorescence staining assays demonstrated that BPR0L075 treatment led to a time- and dose-dependent translocation of calreticulin, an endoplasmic reticulum chaperone protein, on the plasma membrane of dying cells which sends an “eat me” signal. The BPR0L75 treatment also led to secretion of High Mobility Group Box 1 (HMGB1), a non-histone chromatin-binding nuclear protein, as well as adenosine triphosphate (ATP) from the cell into the extracellular space using ELISA assay. These damage-associated molecular patterns (DAMPs) are known to lead to antigen-presenting cell recruitment, phagocytosis, maturation of dendritic cells, and chemotaxis. The appearance of the ICD biomarkers (calreticulin exposure, HMGB1 and ATP release) supports BPR0L075 as an ICD-inducing antimitotic agent. Further in vivo studies are warranted to delineate the effect of BPR0L075 on immune activation and recruitment of immune cells to the tumor microenvironment. Citation Format: Ayesha Bano, Xinli Liu. BR0L075 (SCB01A), a microtubule inhibitor targeting the colchicine binding site, induces immunogenic cell death [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 7144.

Abstract 6355: Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC)

Abstract Background: Glioblastoma Multiforme (GBM), primarily arising in the brain's frontal and temporal lobes, is a devastating malignancy originating from astrocytes. Despite treatment advancements, the prognosis remains bleak, with a median survival of 15-18 months and a 10% five-year survival rate.Our study explored a novel targeted approach to combat this challenge. uPARAP is an endocytic receptor expressed at low levels on selected mesenchymal cell types but highly overexpressed on the majority of Soft-Tissue- and Bone Sarcomas. It internalizes fragments of collagen and transfers them to the lysosome for degradation. Methods &amp; Results: First, we analyzed formalin-fixed paraffin-embedded (FFPE) tumors from GBM patients through immunohistochemistry (IHC) staining. Interestingly, a majority of the samples showed strong but varying uPARAP expression. In parallel, we detected and quantified uPARAP receptor expression in GBM cell lines by flow cytometry, underscoring its potential as a therapeutic target.Next, we generated an antibody-drug conjugate (ADC) linked to monomethyl auristatin E (MMAE), a potent antimitotic agent. Confocal microscopy showed rapid internalization of the ADC in several GBM cell lines in vitro. Cellular cytotoxicity was demonstrated in Incucyte and Alamar Blue in vitro assays. Finally, anti-tumor activity of the uPARAP-ADC was studied in a GBM patient-derived xenograft (PDX) model in vivo, where it resulted in significant tumor regression with a tumor growth inhibition (TGI) of 92 and 97% at 3 and 6 mg/kg, respectively. Conclusions: Our findings show that uPARAP is overexpressed on GBM tumors and that a uPARAP-targeted ADC has anti-tumor activity in a PDX model of GBM. This research provides initial evidence that uPARAP-ADCs could become a novel treatment option for this highly aggressive disease. Citation Format: Ida Gregersen, Cecillie R. Løkke, Jette B. Lange, Pernille Barkholt, Christophé Côme, Tina Broberg, Marianne M. Petersen, Carmel M. Lynch, Dominik Mumberg, Niels Behrendt, Lars H. Engelholm. Urokinase plasminogen activator receptor-associated protein (uPARAP) is overexpressed in human glioblastomas and is a therapeutic target for antibody-drug conjugates (ADC) [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 6355.

Abstract 5804: Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload

Abstract LCB84 is an antibody-drug conjugate (ADC) that consists of an antibody targeting human trophoblast cell-surface antigen-2 (TROP2) linked to monomethyl auristatin E (MMAE) via a beta-glucuronidase cleavable linker with a drug-to-antibody ratio (DAR) of 4. LCB84 demonstrates tumor selective killing by multiple mechanisms, increasing both safety and efficacy in parallel. Firstly, the over-expression of TROP2 in many solid tumors allows for targeted delivery of MMAE by LCB84 to Trop2-positive tumor tissues. Secondly, the overexpression of beta-glucuronidase in the lysosome of most tumors facilitates the selective release of active payload within tumor cells compared to normal healthy tissues. Thirdly, the intrinsic selectivity of tubulin inhibitor payloads, such as MMAE, for proliferating cells provides additional protection for non-proliferating normal cells. MMAE is also cell-permeable in its free drug form and can lead to increased efficacy of LCB84 via a bystander effect, enabling activity in heterogenous tumors. LCB84 demonstrates superior efficacy in mouse tumor xenograft studies when compared to benchmark TROP2-targeting ADCs, including sacituzumab govitecan and datopotamab deruxtecan. LCB84 also shows an improved safety profile compared to these clinical benchmarks, as well as compared to traditional MMAE-bearing ADCs, in both in vitro and in vivo toxicity assessments. Additionally, LCB84 shows a stable preclinical pharmacokinetic profile with minimal loss of payload over time. The utilization of an ADC for target-mediated delivery, in combination with a stable beta-glucuronide trigger for selective payload release in tumors, allows for a superior preclinical therapeutic index which is expected to translate to a best-in-class ADC in the clinic. Citation Format: Stephen Slocum, Hyejung Kim, Chul-Woong Chung, Changsik Park. Preclinical evaluation of LCB84, a novel next-generation TROP2 directed ADC utilizing a cancer selective linker and an MMAE payload [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 5804.

Abstract 7142: Optimizing estradiol dimers through linker design: Enhancing anticancer efficacy by targeting microtubule dynamics

Abstract We investigated the development and anticancer efficacy of novel estradiol dimers (ED), targeting the dynamics of microtubules—an essential factor in effective cancer therapy. Employing copper-catalyzed azide-alkyne cycloaddition (CuAAC), two distinct series of ED variants were synthesized. The initial series included thirteen dimers, derived from 17α-ethinyl estradiol, incorporating varied five-atom linkers with central carbon, nitrogen, or oxygen atoms, and, in some instances, modified with benzyl groups containing hydroxy or methoxy substituents. These dimers were evaluated for their cytotoxic properties and impact on the cell cycle, revealing certain structures with substantial efficacy, compared with the original ED. The subsequent series, encompassing twelve variants, concentrated on structural alterations within the aromatic bridges linking the estradiol units. In this series, linkers of simpler composition and fewer substituents demonstrated increased selectivity and potency against cancer cell lines, particularly ED3 and ED5, which exhibited cytotoxicity levels comparable to established tubulin inhibitors. Both dimers series showed the ability to disrupt microtubule assembly and mitotic processes, confirmed through cell-based evaluations and in vitro tubulin assembly assays. Complementing these results, in silico modelling and binding free energy calculations exhibited a strong correlation with the demonstrated biological activities, especially in terms of binding affinity to the colchicine site. Collectively, these findings highlight the promise of estradiol dimers as potential anticancer agents and emphasize the critical role of linker structures in enhancing their biological effectiveness. Work was supported by the Ministry of Education, Youth and Sports of the Czech Republic through infrastructural projects (CZ-OPENSCREEN - LM2018130; EATRIS-CZ - LM2018133) and National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Petr Dzubak, Michal Jurášek, Jiří Řehulka, Soňa Gurská, Pavel Polishchuk, Pavel Dráber, Lukáš Huml, Pavel B. Drašar, Alexandra Ivanova, Olena Mokshyna, Lenka Hrubá, Marián Hajdúch. Optimizing estradiol dimers through linker design: Enhancing anticancer efficacy by targeting microtubule dynamics [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 7142.

Abstract 1891: Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window

Abstract Nectin-4 is a well validated tumor target, which is highly expressed in several solid cancers such as urothelial, head-and-neck, breast, lung, pancreatic, ovarian, and cervical cancer, while expression in normal tissues is limited. A first-generation antibody-drug conjugate targeting Nectin-4, called enfortumab vedotin-ejfv (EV), was granted accelerated FDA approval in 2019 for urothelial cancers, and is currently approved for frontline therapy in cisplatin-ineligible patients with locally advanced or metastatic urothelial carcinoma, providing groundbreaking improvement in patient survival. However, clinical use of EV has also revealed safety-driven limitations, which result in reduced and/or interrupted drug exposures impacting efficacy. To overcome these limitations in therapeutic window and to maximize on the potential of Nectin-4 as a tumor target beyond urothelial cancers, Adcentrx has developed ADRX-0706, a Nectin-4-targeting next-generation ADC with a drug-antibody ratio of 8. ADRX-0706 is comprised of a novel fully human IgG1k monoclonal antibody linked to an optimized tubulin inhibitor payload AP052 through a proprietary cleavable linker. ADRX-0706 has strong binding affinity and selectively induces cytotoxicity in Nectin-4-expressing tumor cells. Importantly, ADRX-0706 is designed to have improved bystander effect, which allows tumor regression even in cases of heterogeneous Nectin-4 expression. This translates to superior in vivo efficacy of ADRX-0706 over EV in multiple mouse xenograft models of other indications beyond bladder cancer. In a mouse clinical trial of patient-derived cervical cancers with a range of Nectin-4 expression levels, ADRX-0706 reaches a 73% ORR. Since peripheral neuropathy is a main platform toxicity for this payload class, ADRX-0706 is assessed in an in vitro co-culture model to evaluate its neurotoxic potential. While EV displays a 75% decrease in neurite outgrowth, ADRX-0706 strikingly has no impact at concentrations up to 1mg/ml, suggesting a lower risk of peripheral neuropathy in humans for ADRX-0706. In preclinical repeat dose toxicology study, ADRX-0706 reaches an HNSTD of 18mg/kg in NHPs well above therapeutic exposures in mouse pharmacology studies, indicating a tremendous increase in the therapeutic index compared to EV. In summary, the highly selective and potent anti-tumor activity in multiple tumor types and wide preclinical therapeutic index of ADRX-0706 support clinical evaluation of this next generation anti-Nectin-4 ADC. ADRX-0706 is currently in a phase 1a/b study (NCT06036121). Citation Format: Andrew M. Hau, Maria Shahmoradgoli, Dong Jun Lee, Wes Sisson, Anna Wang, Peter P. Challita, Erin Nye, Mario M. Kuo, Alexis Mahloch, Monica Leung, Oscar Betancourt, Alexander Chu-Kung, Maojun Guo, Hui Li, Sabine Rottmann, Pia M. Challita-Eid. Preclinical characterization of ADRX-0706: A next-generation anti-Nectin-4 antibody-drug conjugate with improved therapeutic window [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 1891.