Antibody-drug Conjugates For Treatment Research Articles

Preclinical Characterization of ARX517, a Site-specific Stable PSMA-Targeted Antibody Drug Conjugate for Treatment of Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an advanced disease in which patients ultimately fail standard of care androgen-deprivation therapies and exhibit poor survival rates. The prostate-specific membrane antigen (PSMA) has been validated as a mCRPC tumor antigen with over-expression in tumors and low expression in healthy tissues. Using our proprietary technology for incorporating synthetic amino acids (SAAs) into proteins at selected sites, we have developed ARX517, an antibody drug conjugate (ADC) which is composed of a humanized anti-PSMA antibody site-specifically conjugated to a tubulin inhibitor at a drug-to-antibody ratio of 2. After binding PSMA, ARX517 is internalized and catabolized, leading to cytotoxic payload delivery and apoptosis. To minimize premature payload release and maximize delivery to tumor cells, ARX517 employs a non-cleavable PEG linker and stable oxime conjugation enabled via SAA protein incorporation to ensure its overall stability. In vitro studies demonstrate that ARX517 selectively induces cytotoxicity of PSMA-expressing tumor cell lines. ARX517 exhibited a long terminal half-life and high serum exposure in mice, and dose-dependent anti-tumor activity in both enzalutamide-sensitive and -resistant CDX and PDX prostate cancer models. Repeat dose toxicokinetic studies in non-human primates demonstrated ARX517 was tolerated at exposures well above therapeutic exposures in mouse pharmacology studies, indicating a wide therapeutic index. In summary, ARX517 inhibited tumor growth in diverse mCRPC models, demonstrated a tolerable safety profile in monkeys, and had a wide therapeutic index based on preclinical exposure data. Based on the encouraging preclinical data, ARX517 is currently being evaluated in a Phase 1 clinical trial ([NCT04662580]).

Imaging and Monitoring HER2 Expression in Tumors during HER2 Antibody-Drug Conjugate Therapy Utilizing a Radiolabeled Site-Specific Single-Domain Antibody Probe: 68Ga-NODAGA-SNA004-GSC.

The overexpression of HER2 is pivotal in the initiation and progression of breast cancer. Developing HER2-targeted radiotracers is crucial for noninvasive assessment of HER2 expression, patient selection for HER2-targeted therapy, monitoring treatment response, and identifying resistance. Here, we reported a nonsite-specific coupled radiotracer, 68Ga-NOTA-SNA004-His6, and a site-specific coupled radiotracer, 68Ga-NODAGA-SNA004-GSC, based on a novel HER2 nanobody, SNA004. Both radiotracers exhibited high affinity, specific targeting, and rapid clearance in vitro and in vivo. Additionally, these tracers and trastuzumab showed noncompetitive binding to HER2. Compared to 68Ga-NOTA-SNA004-His6, 68Ga-NODAGA-SNA004-GSC demonstrated significantly reduced renal and liver uptake. PET/CT imaging with 68Ga-NODAGA-SNA004-GSC sensitively detected the responsiveness of various tumor models to trastuzumab and its antibody-drug conjugates (ADCs). Overall, the site-specific coupled radiotracer 68Ga-NODAGA-SNA004-GSC offered significant advantages in biodistribution and signal-to-noise ratio, making it a valuable tool for monitoring HER2 expression levels before, during, and after trastuzumab and ADC treatment.

Antibody-Drug Conjugates-Evolution and Perspectives.

Antineoplastic therapy is one of the main research themes of this century. Modern approaches have been implemented to target and heighten the effect of cytostatic drugs on tumors and diminish their general/unspecific toxicity. In this context, antibody-drug conjugates (ADCs) represent a promising and successful strategy. The aim of this review was to assess different aspects regarding ADCs. They were presented from a chemical and a pharmacological perspective and aspects like structure, conjugation and development particularities alongside effects, clinical trials, safety issues and perspectives and challenges for future use of these drugs were discussed. Representative examples include but are not limited to the following main structural components of ADCs: monoclonal antibodies (trastuzumab, brentuximab), linkers (pH-sensitive, reduction-sensitive, peptide-based, phosphate-based, and others), and payloads (doxorubicin, emtansine, ravtansine, calicheamicin). Regarding pharmacotherapy success, the high effectiveness expectation associated with ADC treatment is supported by the large number of ongoing clinical trials. Major aspects such as development strategies are first discussed, advantages and disadvantages, safety and efficacy, offering a retrospective insight on the subject. The second part of the review is prospective, focusing on various plans to overcome the previously identified difficulties.

Abstract A013: NF1 loss is syntetic lethal with Trastuzumab emtansine

Abstract There is great interest in the identification of biomarkers to guide development of antibody-drug conjugates (ADC). Most research has focused on target expression, but key predictors of payload efficacy have not been indeitifed. NF1 is a tumor suppressor classically considered as an inhibitor of RAS signaling, and often mutated in metastatic HER2+ breast cancer (BC). We screened multiple approved drugs for differential sensitivityin CRISPR-engineeredf NF1 KO cells. HER2-targeted agents (small molecules or antibodies) were found to be less effective upon NF1 loss; surprisingly, we identified increased sensitivity to the approved ADC T-DM1, but not to Trastuzumab Deruxtecan (T-Dxd). We then elucidated the underlying molecular cause employing in vivo, in vitro and in vitro reductionist systems. To measure in vivo MT dynamics, we transiently transfected the MT end-binding protein EB3- GFP and reconstructed MT trajectories by live-cell imaging. Upon DM1 treatment, KO cells showed a highly significant reduction in MT speed, demonstrating a direct role for NF1 on MT dynamics in cells. In tubulin polymerization assays, recombinant NF1 greatly accelerated polymerization and completely rescued DM1-induced inhibition. NF1 induced significant MT bundling, a defining feature of many MT-associated proteins, which generates signal indistinguishable from true MT polymerization in turbidity assays. To follow the dynamics of individual microtubules, we applied Total Internal Reflection (TIRF) microscopy on glass-immobilized MTs. As expected, polymerization in the presence of NF1 led to a dose-dependent significant increase in MT dynamics (fraction of elongating MTs, elongation speed, catastrophe rate). Expectedly, DM1 led to significant reduction in the fraction of elongating MTs and speed, but these defects were completely or partially rescued by NF1. Importantly, DM1 did not only lead to MT shortening (as proposed by the current model), but also to clear and frequent MT fracturing, indicating that the drug is not only engaging MT ends but also intra-tubular binding sites. This is consistent with recent models of MT formation which incorporate the frequent presence of areas of discontinuity or damage induced by mechanical stress, exposing intratubular DM1 binding sites. Interestingly, adding NF1 to DM1-treated MTs generated areas of de novo intra-tubular tubulin insertion, coincident with damaged sites, suggesting an entirely novel role for NF1 in MT repair. In conclusion, we provide evidence for a model in which maytansinoids bind not only to soluble tubulin dimers and MT ends, but also to intratubular damaged sites. Thus, the number of binding sites in cells would be proportional to MT damage, suggesting a mechanism for differential efficacy across tumor types and a potential avenue for combinatorial drug development. These results prompt the use of NF1 as a biomarker to select patients for ADC treatment. Funding: FIEO fellowship 2023, AIRC (n25791), Italian MoH-Ricerca Corrente di Rete (ACCORD) 2022, Next Generation EU – PNRR M6C2 – PNRR-MAD-2022-12376934 Citation Format: Eleonora Messuti, Bruno Achutti Duso, Alessia Castiglioni, Giulia Tini, Emanuele Bonetti, Giuseppe Ciossani, Silvia Monzani, Daria Khuntsariya, Marcus Braun, Zdenek Lansky, Luigi Scietti, Luca Mazzarella. NF1 loss is syntetic lethal with Trastuzumab emtansine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A013.

Surface-Available HER2 Levels Alone Are Not Indicative of Cell Response to HER2-Targeted Antibody-Drug Conjugate Therapies.

HER2-targeting therapies have advanced breast cancer treatment over the past decade. Clinically, eligibility for HER2 therapies is determined by assessing HER2 levels on tumor cell surfaces through immunohistochemistry or by gene regulation through fluorescence in situ hybridization. HER2 therapies are not always effective in patients with elevated levels of HER2, questioning whether the amount of HER2 is sufficiently predictive of patient outcomes. Additionally, the HER2-targeting antibody-drug conjugate (ADC) Enhertu® was recently approved for metastasized HER2-low cancers, confirming the benefits of HER2 treatment for patients with low HER2 levels. To evaluate the correlation between HER2 levels and treatment efficacy, we quantified HER2 on eight cell lines using flow cytometry while simultaneously determining the toxicity of two HER2-targeting ADCs. Both HER2-high cell lines and HER2-low cell lines had significant toxicity responses to ADCs. We quantified HER2 internalization and found no correlation between HER2 levels and the percentage of internalization. We found a useful metric suggesting that a minimum number of HER2 receptors trafficked to lysosomes is sufficient to provide effective treatment. Our results indicate that the current standards of determining eligibility for HER2 therapy could limit patients' access to effective treatment. In conclusion, HER2 levels are not wholly adequate to determine the response to ADC treatment.

Identifying mutations in ctDNA to predict antibody-drug conjugate response in breast cancer.

e13074 Background: Metastatic breast cancer (mBC) is a complex disease, and outcomes remain poor because new therapies are needed. A new class of drugs, antibody-drug conjugate (ADC), has shown effectiveness across all breast cancer subtypes. Yet FDA- approval for these therapies is restricted only to HER2 and triple-negative subtypes. Therefore, we urgently need to identify which patients, regardless of subtype, will respond to ADCs. The advent of circulating tumor DNA (ctDNA) offers a unique noninvasive method to capture breast cancer (BC) heterogeneity. Recently, ctDNA testing has been employed in clinical practice to guide targeted therapies for mBC, particularly in cases of hormone receptor positive (HR+) disease with mutations in PIK3CA and ESR1. The clinical implications to predict response to ADC therapy in BC remains unclear. Here we test the hypothesis that ctDNA can detect mutations that predict response to novel ADCs. Methods: We analyzed a subset of patients from the Dallas Metastatic Breast Cancer Study comprised of patients with mBC who underwent ctDNA testing using the Tempus xF liquid biopsy ctDNA sequencing panel. The data was analyzed from a single academic medical center between the initial year of ctDNA collection in 2019 to 2023. We identified detectable mutations from patients and addressed a relationship by using Metascape. Relevant signaling pathways were identified: APOBEC, ErbB signaling, p53 and Ras-MAPK pathway. Patients with triple negative, HER2, and HR BC were examined for ADC response and associated mutations. Survival analyses were estimated using Kaplan-Meier and Gehan Breslow Wilcoxon test for statistical analysis. Results: We identified 43 patients with HR+ mBC who had at least one mutation within the Ras-MAPK pathway and 10 of 43 received ADC therapy. Median survival of patients who received ADC therapy 58 months (n=10) vs not receiving ADC therapy 33 months (n=33) (p < 0.05). Of the ten patients who received ADC treatment, 60% had 1+ HER2 staining on tissue biopsy. 70% of patients received Trastuzumab deruxtecan (T-DXd) and 30% received Sacituzumab govitecan (SG). SG was given to patients with negative HER2 staining on biopsy. Patients with at least one mutation within ABOBEC, ErbB signaling, and p53 pathways did not have statistically significant differences in mOS. Conclusions: In HR+ mBC, detection of mutated genes associated with the Ras-MAPK pathway by ctDNA was associated with a more favorable response when treated with ADCs. These findings are exciting because HR+ BCs are being targeted with ADCs regardless of HER2 or TROP2 expression, highlighting the need for new biomarkers to distinguish which HR+ patients to treat. Our retrospective analysis findings are limited by the number of patients with ctDNA testing and also timing of testing. Further studies are needed to solidify the observed correlation between Ras-MAPK pathway mutations and favorable response to ADC treatment.

Abstract PS08-01: Antibody-Drug Conjugates (ADCs) in Breast Cancer: Real World Analysis of Outcomes

Abstract Methods: We estimated the distribution of progression-free survival (PFS), overall survival (OS), and time to treatment failure (TTF) using the Kaplan-Meier method. Differences in survival curves between groups categorized by initial ADC treatment and subsequent ADC treatments were assessed using the log-rank test. Cox proportional hazards regression models were employed to evaluate the association between each survival outcome and various measures of interest, such as age, gender, race, number of prior lines of treatment, hormone receptor status, and HER2 status. Results: The analysis included a total of 469 breast cancer patients, with the distribution of clinical characteristics summarized in Table 1. The median age at the start of ADC treatment was 50 years (range: 20-85). Among the patients, 263 patients (56%) received SC as their initial ADC treatment, while 44% received T-DXd. Additionally, 29 patients received both ADCs during metastatic treatment. The median follow-up time for all patients was 7.9 months (range: 0.1- 39.1). Out of the total patient population, 29% died, while 71% were still alive at the last follow-up. The median OS for all patients was 21.6 months. Median OS of patients receiving SC or T-DXd as their initial ADC treatment, was 14 and 37.1 months, respectively. Patients who received both SC and T-DXd had a median OS of 26.1 months. The median PFS for all patients was 6.0 months. Patients initially treated with SC had a median PFS of 4.7 months, whereas those treated with T-DXd had a median PFS of 9.0 months. In patients who received both, median of PFS was 4.9 months for T-DXd after SC, and 5.0 months for SC after T-DXd. The median TTF for all patients was 6.1 months. Patients initially treated with SC had a median TTF of 4.7 months, while those treated with T-DXd had a median TTF of 9.0 months. In patients who received T-DXd after SC, TTF was 4.9 months and SC after T-DXd was 5.0 months. Clinician-judged responses were seen in 180 (74%)of patients with SC only, 80 (41%) with T-DXd only, 9 (100%) with SC as 2nd ADC and 20 (100%) with T-DXd as 2nd ADC. Furthermore, the analysis explored the association of OS, PFS, and TTF with various measures of interest, including age, gender, race, number of prior lines of treatment, type of treatment, hormone receptor status, HER2 status, Ki67, and best response to ADC. Some notable associations were observed, such as ER-positive status being associated with marginally longer OS compared to ER-negative status (median 24.7 vs. 16.4 months; p=0.06), and HER2-positive status being associated with longer PFS compared to HER2-negative status (median 14.2 vs. 5.4 months; p< 0.001). However, these associations should be interpreted cautiously as they were obtained from univariate analysis. Conclusions: Overall, these results highlight the significant differences in survival outcomes between different ADC treatments in breast cancer patients, likely due to differing patient characteristics. Distribution of clinical characteristics across ADCs Citation Format: Akshara Singareeka Raghavendra, Zhongya Wang, Roland Bassett, Debu Tripathy. Antibody-Drug Conjugates (ADCs) in Breast Cancer: Real World Analysis of Outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS08-01.

Abstract PO5-16-10: The efficacy of novel antibody-drug-conjugates on brain metastases in metastatic breast cancer patients – a multicenter real-world analysis

Abstract 15 – 30% of all patients with metastatic breast cancer (MBC) develop brain metastases (BMs). Treatment of BMs include radiation therapy and surgical resection while systemic treatment is challenging and prospective evidence for the treatment of active brain metastases is restricted to the small molecule Tucatinib. Recently, the antibody-drug-conjugates (ADCs) Sacituzumab-Govitecan (SG) and Trastuzumab-Deruxtecan (T-DXd) have shown to be highly effective in the treatment of triple-negative (SG), HER2-positive (T-DXd) and HR+/HER2- (SG and, in case of HER2-low-expression, T-DxD) MBC. However, there is only limited data, whether these macromolecules are also effective in patients with BMs. We therefore aimed to examine the efficacy of SG and T-DXd in patients with stable and active BMs in a multicenter real-world analysis. Female patients with stable or active BMs who were treated with either SG or T-DXd at the breast centers of Tuebingen University, Ulm University or Freiburg University in Germany before June 30, 2023 were included into this analysis. Data cut-off for this analysis was July 10, 2023. BMs were classified as active if they were newly diagnosed and did not require local treatment or if they were progressing and did not require local treatment. Preexisting BMs without intracranial disease progression or preexisting/newly diagnosed BMs that had been treated by surgery and/or radiotherapy directly prior to SG/T-DXd therapy were considered as stable. All patients underwent baseline magnetic resonance imaging (MRI) of the brain and received follow-up MRIs at least every three months. Growth dynamics of BMs were assessed by board certified neuroradiologists. Intracranial disease control rate (icDCR) was defined as the percentage of patients with at least intracranial stable disease at the first follow-up MRI. Intracranial progression-free survival (icPFS) was determined as the period between the first application of SG or T-DXd and intracranial disease progression or treatment cessation due to the patients’ will or the onset of intolerable toxicity. If ADC treatment was discontinued due to extracranial disease progression albeit at least stable intracranial disease patients were censored. Overall survival (OS) was defined as the period between the first application of SG or T-DXd and death. In total, 26 patients were included with a median of two prior therapy lines in the metastatic setting (range 2 – 15). 10 (38%) and 14 (54%) patients received SG and T-DXd, respectively. 2/26 (8%) patients receiving SG were consecutively subjected to T-DXd treatment. SG was applied to 12/12 patients (100%) due to triple-negative MBC and 12/16 patients (75%) treated with T-DXd were HER2-positive. The remaining 4/16 patients (25%) treated with T-DXd showed HER2 low tumor biology. 5/12 (42%) and 8/16 (50%) patients treated with SG or T-DXd had active BMs at treatment initiation. The icDCR was 33% (95% CI: 5% - 61%)/81% (95% CI: 62% - 100%) for all patients treated with SG/T-DXd and 40% (95% CI: 0% - 88%)/75% (95% CI: 43% - 100%) in case of active BMs. After a median follow-up of 7.2 months, 10/12 (83.3%) patients treated with SG and 4/16 (25%) patients subjected to T-DXd had discontinued treatment. Median icPFS was 3.1 months (95% CI: 0.4 – 5.8 months) for SG and not reached for T-DXd. 7/12 (58%) patients treated with SG and 4/16 patients (25%) treated with T-DXd died until data cut-off. Median OS was 7.2 months (95% CI: 3.0 – 11.4 months) for patients treated with SG and not reached for patients treated with T-DXd. SG and T-DXd showed promising clinical activity in both, stable and active cerebral breast cancer metastasis. Updated PFS and OS data will be reported at the meeting. Citation Format: Dominik Dannehl, Henning Schäffler, Tobias Engler, Lea Volmer, Arne Estler, Ilinca Popp, Stefanie Lorenz, Eva-Maria Grischke, Markus Hahn, Ghazaleh Tabatabai, Ingolf Juhasz-Böss, Wolfgang Janni, Sara Brucker, Florin-Andrei Taran, Andreas Hartkopf. The efficacy of novel antibody-drug-conjugates on brain metastases in metastatic breast cancer patients – a multicenter real-world analysis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-10.

Abstract PO3-05-14: Practice patterns for sequential use of antibody-drug conjugate after antibody-drug conjugate in metastatic breast cancer: results from a physician survey

Abstract Introduction: Antibody-drug conjugates (ADCs) have become a key part of metastatic breast cancer (MBC) treatment. Trastuzumab deruxtecan (T-DXd), and Sacituzumab Govitecan (SG) are approved ADCs available for the treatment of MBC. Completed trials that led to the approval of these ADCs did not include patients who had received the other ADC as prior therapy. Little is known about the optimal sequencing of these agents and how clinicians are incorporating ADC sequencing decisions at bedside. Methods: An online survey regarding the use of sequential ADCs was distributed electronically from April 2023 to July 2023 among US clinicians who treat patients with breast cancer. The survey included eight questions assessing clinician practice settings and the use of ADC sequencing and five case-based scenarios (table 1). Descriptive statistics were used to assess survey responses. Results: 107 survey responses were received, out of which 72% were female responders and 93% were medical oncologists. The majority of responders practiced in an academic setting (83%) and reported being involved in clinical research (82%). 58% of responders have been in practice for >10 years and 68% treat >20 breast cancer patients per week. 87% had prescribed an ADC post-ADC for MBC. 46% of responders thought that the degree of benefit for an ADC post-ADC would be similar to the benefit noted in the pivotal ADC trials, and 54% thought that the degree of benefit with sequencing would be lower than in the pivotal trials. Regarding the case-based questions for patients with triple-negative breast cancer and HER2 low disease who had disease progression on first-line metastatic therapy, 64-71% of clinicians opted to give SG as the first ADC followed by T-DXd as the second ADC in sequence, regardless of age, PDL1 status and prior metastatic therapies. For a patient with hormone-positive (HR+), HER2 low MBC, who had received capecitabine as the first metastatic chemotherapy, 50% opted to give T-DXd as the first ADC followed by SG as the second ADC, and 40% opted to give weekly paclitaxel followed by SG as the first ADC. For a patient with HR+, HER2 low MBC who had received two prior chemotherapies for metastatic disease, 77% opted for T-DXd as the first ADC, followed by SG as the second sequential ADC. When grade 1 pneumonitis was added as a comorbidity to the case, only 16% opted to give T-DXd as the first ADC choice. Conclusion: Almost 90% of clinicians report prescribing sequential ADCs for MBC, yet there is little uniformity in how these ADCs are sequenced. More than 50% felt that the benefit of the second ADC in the sequence will be lower than the registration trials that led to the approval of the ADC in a setting of no prior ADC treatment. The optimal sequencing of ADCs remains an area of unmet need and further studies are needed to guide optimal medical decisions regarding sequential ADC-based treatment algorithms for patients with MBC. Table 1. Case-based scenarios for ADC sequencing Citation Format: Yara Abdou, Prarthna Bhardwaj, Rachel Abelman, Laura Spring, Aditya Bardia, Lisa Carey, Priyanka Sharma. Practice patterns for sequential use of antibody-drug conjugate after antibody-drug conjugate in metastatic breast cancer: results from a physician survey [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-05-14.

Abstract PO1-14-04: A multivariate biomarker to guide antibody-drug conjugate selection and provide insight on response differences across breast cancer subtypes

Abstract Background: Antibody-drug conjugates (ADCs), including trastuzumab deruxtecan (T-DXd; targeting HER2) and sacituzumab govitecan (SG; targeting TROP2), have transformed the management of metastatic breast cancer, with additional ADCs approved in other solid tumors or in late-stage development. To date, expression of ADC target alone has been poorly predictive of objective response rates (ORRs) in both breast cancer and other tumors. Hence, there is an urgent need to develop better predictive biomarkers to guide ADC vs. other treatments, T-DXd vs. SG treatment, and optimized predictive medicine opportunities involving other ADCs. Additionally, whether ADCs are similarly effective in less common breast cancer subtypes—such as invasive lobular carcinoma (ILC) vs. invasive ductal carcinoma (IDC), is unclear. Given the limited availability of trial tissue samples and cohorts with long-term follow-up, herein we sought to determine whether a tissue based, pan-solid tumor, multivariate biomarker algorithm could predict observed ORRs across ADCs and tumor types, as has been used previously to associate tumor mutation burden with immunotherapy ORRs. Methods: From 15,032 FFPE tumor tissue samples (from 21 tumor types) tested by clinical comprehensive genomic profiling plus RNA based quantitative transcriptional profiling (qTP) as part of the observational Strata Trial (NCT03061305), the ADC treatment response score (TRS) was discovered and validated to predict published ORRs across tumor types and approved/late-stage ADCs (n=16 observed ORRs [from 7 tumor types and 8 ADCs; SG not included]). The best performing 3-factor algorithm (by Pearson correlation coefficient of observed ORRs vs. tumor type and ADC specific predicted biomarker positivity rates) included only qTP components and combined ADC target expression, cell proliferation, and extracellular matrix adhesion (the latter being negatively associated with ORRs). Importantly, predictive biomarker positivity rates of TRS was more correlated vs. observed ORRs (n=16, r=0.81, p=0.0001) than target expression alone (n=16, r=0.54, p=0.03). TRS was then validated using held out SG ORRs from nine tumor types in the IMMU-132-01 basket trial and two ADC ORRs from ASCO 2023 abstracts (enfortumab vedotin [EV; targeting NECTIN-4] in head and neck cancer and patritumab vedotin [targeting HER3] in breast cancer), with TRS predictive biomarker positivity rates again being more significantly correlated vs. observed ORRs than target expression alone (n=11, TRS r=0.91, p=0.0001; target expression alone r=44, p=0.17). Lastly, the locked TRS model was then applied to the DESTINY-PanTumor02 T-DXd dataset presented at ASCO 2023 (n=21 tumor type/HER2 expression groups matched to the trial groups), with TRS predictive biomarker positivity rates again being highly correlated vs. observed ORRs (n=21, TRS r=0.80, p< 0.0001). Across the 21 tumor types in the Strata Trial dataset, breast cancer had the highest percentage (76%) of patients predicted positive for at least one ADC, with 23% and 55% positive for the approved ADCs mirvetuximab soravtansine (targeting FOLR1) and EV, respectively. Lastly, patients with ILC vs. IDC had significantly greater TRS positivity rates for T-DXd (61% vs. 47%) and SG (57% vs. 48%) due to significantly decreased extracellular matrix adhesion in ILC vs. IDC. Clinical outcomes data for patients treated with ADCs and available TRS enrolled in the Strata Trial are maturing and will be presented at the meeting. Conclusion: We have developed and validated TRS, a multivariate RNA based tumor tissue algorithm that predicts observed ORRs across tumor types and approved/late-stage ADCs. More than 75% of all patients with metastatic breast cancer are predicted to be responsive to one or more ADCs, including those approved in other tumor types. Citation Format: Laura Lamb, Jonathan Mowers, Azadeh Nasrazadani, Mark Burkard, Nickolay Khazanov, Daniel Hovelson, Kat Kwiatkowski, Scott Tomlins, D. Bryan Johnson, Daniel Rhodes. A multivariate biomarker to guide antibody-drug conjugate selection and provide insight on response differences across breast cancer subtypes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-14-04.

Antibody-Drug Conjugates in the Treatment of Genitourinary Cancers: An Updated Review of Data.

The treatment landscape of genitourinary cancers has significantly evolved over the past few years. Renal cell carcinoma, bladder cancer, and prostate cancer are the most common genitourinary malignancies. Recent advancements have produced new targeted therapies, particularly antibody-drug conjugates (ADCs), due to a better understanding of the underlying oncogenic factors and molecular mechanisms involved. ADCs function as a 'drug delivery into the tumor' system. They are composed of an antigen-directed antibody linked to a cytotoxic drug that releases cytotoxic components after binding to the tumor cell's surface antigen. ADCs have been proven to be extremely promising in the treatment of several cancer types. For GU cancers, this novel treatment has only benefited patients with metastatic urothelial cancer (mUC). The rest of the GU cancer paradigm does not have any FDA-approved ADC treatment options available yet. In this study, we have thoroughly completed a narrative review of the current literature and summarized preclinical studies and clinical trials that evaluated the utility, activity, and toxicity of ADCs in GU cancers, the prospects of ADC development, and the ongoing clinical trials. Prospective clinical trials, retrospective studies, case reports, and scoping reviews were included.

Abstract LB279: The combination therapy with B7-H3 antibody drug conjugates for effective radiation treatment

Abstract Purpose: Antibody-drug conjugate (ADC) is a promising treatment modality because it enables effective tumor-specific delivery of anticancer drugs by targeting cancer antigens with antibodies. By using ADC, each treatment can be used at an optimal dose, thereby minimizing side effects, and maximizing treatment effects. The ADC used in this experiment targets B7-H3, and the B7-H3 protein is highly expressed in various cancer types. Recent studies have shown that B7-H3 is increased by radiation in various cancer cells. This study aims to demonstrate the efficacy of the B7-H3 ADC and confirm its combined effect with radiotherapy. Method: In vitro, colony forming assay, CCK-8 assay, FACs and western blotting were performed in JIMT-1 and HCT116 cell lines. In vivo, JIMT-1, HCT116 and Colon PDX models were developed to monitor tumor growth and survival rate. Biodistribution was performed through the IVIS (In Vivo Imaging System) spectrum. Result: The B7-H3 ADC used in the experiment was developed by combining DUBA as a toxin with anti-B7-H3 antibody In in vitro experiments, the combined treatment of radiation and B7-H3 ADC reduced cell viability via apoptosis more effectively than treatment alone. In the in vivo mouse models, we observed that the combined treatment of radiation and B7-H3 ADC was more effective in inhibiting tumor growth than the single treatment groups. The survival rate of the mice was also improved when the radiation was combined with B7-H3 ADC treatment compared to the other treatment groups alone. In the biodistribution results, the accumulation of B7-H3 ADC-Cy7 was further enhanced when irradiated. Conclusion: The results show that B7-H3 ADC can be used as a promising therapeutic agent and the combined treatment with radiation can obtain an improved anticancer effect than the single treatment, suggesting another cancer treatment method. Citation Format: Gawon Son, Eunjin Ju, Jin Park, Seolhwa Shin, Eunjeong Ko, Miri Kwon, Hyewon Lee, Beomseok Seo, Sangkwang Lee, Seoksoon Park, Seongyun Jeong, Siyeol Song, Eunkyung Choi. The combination therapy with B7-H3 antibody drug conjugates for effective radiation treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB279.

Abstract 6352: Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types

Abstract Traditional antibody-drug conjugates leverage the specificity of antibodies to deliver cytotoxic payloads to tumor microenvironments (TMEs), thereby reducing systemic toxicities associated with non-targeted chemotherapeutics. Analogously, a novel class of immune-stimulating antibody conjugates (ISACs) has recently emerged to selectively deliver synthetic immune agonists to the TME without systemic immune activation. The first generation of ISACs and the majority of approved and investigational ADCs contain payloads linked to native lysine or cysteine residues, which can lead to heterogenous drug-antibody ratios (DARs), reduced antibody stability, and labile conjugation, all of which limit therapeutic index. Here we describe the generation and preclinical characterization of ARX622, a site-specific TLR7-selective agonist ISAC targeting HER2. To avoid the instability issues of previous generation ADCs and ISACs, ARX622 has a homogenous and stable DAR via oxime bond-mediated payload conjugation to a synthetic amino acid, para-acetyl phenylalanine (pAF), which is recombinantly incorporated at defined heavy chain positions. Similar to cytotoxic ADCs built on this site-specific conjugation platform, ARX622 ISAC and total mAb pharmacokinetic (PK) profiles in non-human primates (NHPs) are indistinguishable, demonstrating in vivo stability and minimal payload deconjugation. Accordingly, ARX622 displays an extended PK profile in mice compared to a DAR-matched random cysteine ISAC containing the same antibody and payload. In the presence of HER2-expressing cells, ARX622 selectively induces proinflammatory cytokine production, type I and III IFN secretion, and APC maturation. In a syngeneic tumor model, ARX622 promotes complete tumor regressions, immunologic memory, and epitope spreading. In xenograft systems, single doses of ARX622 induce complete regression of large, established tumors (>1000mm3), and robustly inhibit tumor growth in mice that have progressed through prior treatment with HER2-targeted mAbs or ADCs. In HER2-low xenografts, ARX622 monotherapy inhibits tumor growth, and promotes complete regression of late-stage (800mm3) tumors when sequentially combined with a HER2-ADC. Based on PK data at efficacious doses in mice and tolerated exposures in NHP toxicity studies, ARX622 has an estimated therapeutic index of ~60x. Taken together, ARX622’s mAb-like properties, high stability, broad immune mechanism induction, strong anti-tumor activity alone or in combination with ADCs, and wide therapeutic index make it a strong candidate for further development toward treating patients with a variety of HER2-expressing solid tumors. Citation Format: David Mills, Ji Young Kim, Erica Wood, Keith Tatsukawa, Nick Knudsen, Jay Nelson, Lillian Skidmore, Kedar GC, Hon Tran, Manoj Pal, Hamidreza Hashemi, Ying Buechler, Shawn Zhang, Dan O'Connor. Preclinical discovery of ARX622: A site-specific, TLR7 agonist, HER2-targeted immune-stimulatory antibody drug conjugate for treatment of multiple solid tumor types [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 6352.

Abstract 5080: Antibody drug conjugate trastuzumab deruxtecan suppresses leptomeningeal brain metastasis cells and is potentiated by addition of immune check point inhibitor

Abstract Leptomeningeal disease (LMD) is defined by dissemination of metastatic cancer cells into the subarachnoid space with subsequent involvement of the cerebrospinal fluid and leptomeninges. LMD is an aggressive, late-stage development of cancer, which is increasing in prevalence as improved treatments for systemic malignancies are discovered. It leads to significant neurological morbidity and mortality, and outcomes remain poor despite improving cancer treatment strategies. There is a large population of patients with human epidermal growth factor receptor 2 (HER2) expressing cancers, such as breast and lung cancers, who suffer from leptomeningeal metastasis. There has been evolving research looking at antibody drug conjugates for treatment of these primary pathologies, such as Trastuzumab Deruxtecan (TDxd), which shows improved progression free survival and overall survival in those with HER2 expressing primary cancers, as well as in intraparenchymal brain metastasis. However, as of yet there has been no study of its utility in LMD. Additionally, use of immune check point inhibitors (ICI) for treatment of LMD has been studied, showing some efficacy, but less robust response than that in intraparenchymal brain metastasis. Given the significant population of patients who continue to be affected by leptomeningeal metastasis, there is significant need for an improved treatment regimen. We hypothesize that TDxd will have a killing effect on HER2 expressing LMD cells, which can be further potentiated by the addition of ICIs. To test this hypothesis, we developed breast and lung cancer cell lines (SKBrM-LMD, and H2030BrM-LMD) that express HER2 and preferentially metastasize to leptomeningeal space. These cell lines were also labeled with luciferase. We then treated these cell lines with TDxd and found that TDxd had significant growth inhibitory effect on these cells in vitro when compared to the control. Furthermore, we educated human T Cells against the SkBrM-LMD cells and treated these cells with the aforementioned T cells plus the ICI Anti-PD1. Our results showed that ICI had significant killing effect when compared to the control. We have also created an in vivo LMD model by performing intraventricular injection of HER2 expressing tumor cells into a mouse model, which can be used for testing the efficacy of TDxd and ICI by intrathecal injection. In conclusion, our results indicate that TDxd and ICI have significant killing activity against HER2 expressing LMD cells, and therefore intrathecal administration of these drugs is a promising treatment modality that will be demonstrated using a mouse LMD model. Citation Format: Eleanor C. Smith, Shih-Ying Wu, Ravindra P. Deshpande, Abhishek Tyagi, Kounosuke Watabe. Antibody drug conjugate trastuzumab deruxtecan suppresses leptomeningeal brain metastasis cells and is potentiated by addition of immune check point inhibitor [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 5080.

Abstract 3184: Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action

Abstract Background: Tumor immunotherapy, especially the combination of PD-1/PD-L1 inhibitors and chemotherapy, has developed rapidly. However, the systemic side effects induced by chemotherapy remain a crucial problem that needs to be addressed. Antibody-drug conjugates (ADCs) are exceptional as a target-specific prodrugs that greatly improve the therapeutic window of chemotherapy drugs. Therefore, we designed the ADC based on a bispecific anti-PD-L1/anti-AXL antibody with toxic MMAE (Monomethyl auristatin E) for precise targeting of cancer cells and reduction of the side effects. Materials and Methods: The CPL976-MMAE conjugate was prepared with site-specific conjugation technology of Fc-glycan remodeling and click-chemistry. The binding to the extracellular domain of AXL and PD-L1 was confirmed with use of the Surface Plasmon Resonance (SPR). After the conjugation with MMAE, cytotoxicity and selectivity of the ADC were evaluated on the human cancer cell lines with different expression levels of AXL and PD-L1. The ADC's ability to force internalization of targets was shown on a panel of cell lines. The effectiveness of ADC was evaluated based on changes in the cell cycle and of treated cells. The high efficacy of the conjugates was confirmed in a mouse model of MDA-MB-231 xenografts in bi-weekly intravenous injection of 6 doses. Results: CPL976-MMAE showed improved cellular uptake, selectivity, and anticancer activity, compared to unconjugated MMAE in the cancer cell lines with different expression of the targets AXL/PD-L1. We confirmed ADCs efficacy using cells expressing high levels of AXL and PDL-1 shown as arrest in G2-M phase of the cell cycle and dose-depended apoptosis. The in vivo experiment on a mouse xenograft model confirmed complete tumor reduction (8/8) after ADC treatment, despite the application of an 11 times lower dose in comparison to the free-MMAE control group and was well-tolerated by mice. Importantly, even after the withdrawal of 976-MMAE for 3 weeks, no tumor regrowth was observed. Conclusions: We presented a new, ADC based on bispecific anti-PD-L1/anti-AXL antibody conjugated with MMAE, that combines the effects of cancer growth inhibition by interaction with AXL and immunotherapy by disruption of the PD-1/PD-L1 axis, with selective delivery of MMAE to cancer cells. The simultaneous action of delivering the toxin and acting on both therapeutic targets provides a potent synergistic anti-tumor effect, that can lead to complete tumor eradication and potentially overcome the primary and secondary resistance to PD-1/PD-L1 targeted therapies. Citation Format: Krysztof Lacek, Anna Jablonska, Olga Abramczyk, Martyna Janowska, Filip Mitula, Aleksandra Sowinska, Agnieszka Bojko-Matuszek, Tomasz Kornatowski, Damian Kolakowski, Magdalena Bojko, Monika Skupinska, Wiktor Pyra, Michal Gorka, Joanna Hucz-Kalitowska, Emilia Mroz, Malgorzata Choros, Jerzy Pieczykoland, Maciej Wieczorek, Delfina Popiel. Development of an innovative bifunctional CPL976-MMAE conjugate based on a bispecific anti-PD-L1/ant-AXL antibody with a dual mechanism of action [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 3184.

Abstract 2610: Discovery of AM E3: A novel α-TLSPR monoclonal antibody for the ADC treatment of CRLF2-rearranged Ph-like B-ALL

Abstract Background: Patients affected by Ph-like B-cell acute lymphoblastic leukemia (B-ALL) have a high mortality rate and elevated levels of disease relapse upon chemotherapy treatment. More than 60% of Ph-like B-ALL is characterized by genomic rearrangements resulting in high expression level of the CRLF2/TLSPR receptor on the cell surface. While significant advances have been made in developing targeted agents for B-ALL treatment, the CRLF2-rearranged subpopulation is in complete absence of selective and effective therapies. Here, we report the identification of α-TLSPR monoclonal antibodies with therapeutic potential and the preclinical validation of AM E3 as an Antibody-Drug Conjugate (ADC) for the treatment of CRLF2-rearranged Ph-like B-ALL. Material and Methods: Leveraging a high-complex phage display platform we have first identified several fully human α-TLSPR monoclonal antibodies with single-digit nanomolar affinity for the target. Among them, AM E3 emerged as suitable for ADC development due to its rapid internalization by CRLF2-expressing cells. As a proof of concept, upon conjugation with the cytotoxic payload tesirine (SG3249) AM E3 was validated for anti-tumor response in both CRLF2-rearranged in vitro cell lines and in vivo xenograft models. Further, since human/mouse CRLF2 homology is low (˂35%), we have also developed an ADC surrogate against murine TLSPR to establish a therapeutic window for the ADC therapy in CRLF2-rearranged preclinical models. Results: ADC AM E3 demonstrated strong cytotoxicity (EC50=40pM) on cell lines derived from hCRLF2-rearranged B-ALL patients. In xenograft models a single injection of ADC AM E3 (0.7mg/Kg) showed antitumoral activity proportional to hCRLF2/TSLPR receptor density at the cell surface. Moreover, ADC AM E3 treatment reduces tumor growth and disease burden of hCRLF2-rearranged B-ALL orthotopic xenografts and extends survival over standard induction therapy. ADC surrogate mB4 AM23, which retains similar properties as the human ADC AM E3, reduces tumor growth and is well tolerated in xenograft models engineered to express mCRLF2/TSLPR. Conclusions: AM E3 is a novel α-TLSPR mAb with ideal features for the development of a first-in-class ADC therapy. α-TLSPR ADC demonstrated in vitro and in vivo efficacy as a monotherapy for the treatment of CRLF2/TLSPR rearranged Ph-like B-ALLs and proven to be well-tolerated in surrogate animal studies. Citation Format: Claudia Dall'Armi, Valentina Di Biasio, Chantal Paolini, Daniela Natale, Francesco Scalabrì, Antonino Missineo, Nadine Alaimo, Maurizio Nuzzo, Anna Demartis, Pamela Di Pasquale, Roberto Benoni, Fulvia Caretti, Elisa Beghetto, Fabrizio Colaceci, Romina Sasso, Vincenzo Pucci, Alessandro Carugo, Licia Tomei, Carlo Toniatti. Discovery of AM E3: A novel α-TLSPR monoclonal antibody for the ADC treatment of CRLF2-rearranged Ph-like B-ALL [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 2610.

Associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 with molecular subtypes, PD-L1 expression, and FGFR3 mutational status in two advanced urothelial bladder cancer cohorts.

Treatment options for advanced urothelial carcinoma (aUC) rapidly evolved: besides immunomodulative therapeutic options and inhibitors targeting Fibroblast growth factor receptor (FGFR) alterations, two new antibody-drug conjugates (ADC), sacituzumab govitecan (SG) and enfortumab vedotin (EV), have been approved. However, little is known about the associations of specific aUC properties and the surface target expression of TROP2 and NECTIN-4. Our aim was to characterize associations of TACSTD2/TROP2 and NECTIN-4/NECTIN-4 protein and gene expression with morphomolecular and clinicopathological characteristics of aUC in two large independent cohorts. The TCGA BLCA (n = 405) and the CCC-EMN (n = 247) cohorts were retrospectively analysed. TROP2/TACSTD2 and NECTIN-4/NECTIN-4 are highly expressed at the protein and transcript level in aUC, and their expression status did not correlate with patient survival in both cohorts. NECTIN-4/NECTIN-4 expression was higher in luminal tumours and reduced in squamous aUCs. NECTIN-4 was negative in 10.6% of samples, and 18.4% of samples had low expression (H-score <15). The TROP2 negativity rate amounted to 6.5%. TACSTD2 and NECTIN-4 expression was reduced in neuroendocrine-like and/or protein-based double-negative tumours. TROP2- and NECTIN-4-negative tumours included one sarcomatoid and four neuroendocrine aUC. FGFR3 alterations and PD-L1 expression on tumour and immune cells did not associate with TROP2 or NECTIN-4 expression. TACSTD2/TROP2 and NECTIN-4/NECTIN-4 are widely expressed in aUC, independent of FGFR3 alterations or PD-L1 expression, thus representing a suitable target for ADC treatment in the majority of aUC. The expression loss was associated with aggressive morphomolecular aUC subtypes, i.e. neuroendocrine(-like) and sarcomatoid aUC.

CD46-ADC Reduces the Engraftment of Multiple Myeloma Patient-Derived Xenografts.

An antibody-drug conjugate (ADC) targeting CD46 conjugated to monomethyl auristatin has a potent anti-myeloma effect in cell lines in vitro and in vivo, and patient samples treated ex vivo. Here, we tested if CD46-ADC may have the potential to target MM-initiating cells (MM-ICs). CD46 expression was measured on primary MM cells with a stem-like phenotype. A patient-derived xenograft (PDX) model was implemented utilizing implanted fetal bone fragments to provide a humanized microenvironment. Engraftment was monitored via serum human light chain ELISA, and at sacrifice via bone marrow and bone fragment flow cytometry. We then tested MM regeneration in PDX by treating mice with CD46-ADC or the nonbinding control-ADC. MM progenitor cells from patients that exhibit high aldehyde dehydrogenase activity also have a high expression of CD46. In PDX, newly diagnosed MM patient samples engrafted significantly more compared to relapsed/refractory samples. In mice transplanted with newly diagnosed samples, CD46-ADC treatment showed significantly decreased engraftment compared to control-ADC treatment. Our data further support the targeting of CD46 in MM. To our knowledge, this is the first study to show preclinical drug efficacy in a PDX model of MM. This is an important area for future study, as patient samples but not cell lines accurately represent intratumoral heterogeneity.

Fertility-preserving myeloablative conditioning using single-dose CD117 antibody-drug conjugate in a rhesus gene therapy model

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.

Exposure-safety and exposure-efficacy analyses for tisotumab vedotin for patients with locally advanced or metastatic solid tumors.

The antibody-drug conjugate (ADC) tisotumab vedotin (TV) received accelerated approval from the US Food and Drug Administration for treatment of adults with recurrent or metastatic cervical cancer (r/mCC) with disease progression on or after chemotherapy. A population pharmacokinetic (PK) model, developed using dosing data from four clinical TV studies, was used to estimate individual exposure and explore safety and efficacy exposure-response (ER) relationships. Because PK analysis showed no appreciable accumulation of TV and monomethyl auristatin E (MMAE) with repeated dosing, cycle 1 exposure metrics and predicted average concentrations from time zero until end of the cycle in which an event occurred (CavgLast ) were used for ER analyses. The probability of achieving objective response increased significantly as the ADC cycle 1 maximum serum concentration (Cmax ) increased. The probability of treatment-related adverse events (AEs) leading to dose modification increased significantly as ADC cycle 1 area under the concentration-time curve (AUC) increased. Number of grade 2+ ocular AEs increased significantly as ADC cycle 1 AUC, Cmax , and ADC CavgLast increased. MMAE cycle 1 AUC predicted risk of serious treatment-related AEs. The relationship between ADC exposure and efficacy end points suggests ADC treatment was associated with clinically meaningful response across the observed exposures; greater exposure was associated with increased efficacy. The relationship between ADC and MMAE exposure and safety end points suggests increased exposure was associated with increased AE risk. These results align with clinical findings showing TV 2 mg/kg (≤200 mg for patients ≥100 kg) every 3 weeks is efficacious and tolerable for patients with r/mCC.