Activity Of Antibody-drug Conjugate Research Articles

CT109-SN-38, a Novel Antibody-drug Conjugate with Dual Specificity for CEACAM5 and 6, Elicits Potent Killing of Pancreatic Cancer Cells.

CEACAM5 and CEACAM6 are glycosylphosphatidylinositol (GPI)- linked members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which are frequently upregulated in epithelial cancers where they contribute to invasion, metastasis, immune evasion, and resistance to anoikis. CT109 is a novel antibody with dual specificity to both CEACAM5 and 6. In this study, we aimed to perform the preclinical characterization of CT109 and antibody- drug conjugate (ADCs) derivatives of CT109, focusing on CT109-SN-38. CT109's cognate epitope was characterized by scanning mutagenesis. CT109 specificity and internalization kinetics were assessed by immunoblot and flow cytometry, respectively. Cognate antigen expression prevalence in colorectal cancer and normal tissue arrays was determined by immunohistochemistry. CT109 conjugations were generated by the reaction of reduced CT109 cysteines with maleimide-functionalized payload linkers. In vitro cytotoxic activity of CT109 ADCs was characterized on antigen-positive and negative pancreatic ductal adenocarcinoma cell (PDAC) lines using a luminometric viability assay. In vivo efficacy of CT109-SN-38 was assessed on a PDAC tumor xenograft model at 10 and 25 mg/kg concentrations. CT109 showed to bind a glycoepitope centered on N309. CT109 is internalized in the CEACAM5+/CEACAM6+ double-positive PDAC line, BxPC-3, with a t1/2 of 2.3 hours. CT109 ADCs elicit a dose and antigen-dependent cytotoxic effect, with CT109-SN-38 exhibiting an IC50 value of 21 nM in BxPC-3 cells. In a BxPC-3 tumor xenograft model, CT109-SN-38 reduced tumor growth and induced regression in 3/10 mice at 25 mg/kg concentration. These data suggest that further preclinical and clinical development of CT109-SN-38 is warranted.

Unlocking the Potential: Biomarkers of Response to Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) have reshaped the cancer treatment landscape across a variety of different tumor types. ADCs' peculiar pharmacologic design combines the cytotoxic properties of chemotherapeutic agents with the selectivity of targeted therapies. At present, the approval of many ADCs used in clinical practice has not always been biomarker-driven. Indeed, predicting ADCs' activity and toxicity through the demonstration of specific biomarkers is still a great unmet need, and the identification of patients who can derive significant benefit from treatment with ADCs may often be uncertain. With the lack of robust predictive biomarkers to anticipate primary, intrinsic resistance to ADCs and no consolidated biomarkers to aid in the early identification of treatment resistance (ie, acquired resistance), the determination of precise biologic mechanisms of ADC activity and safety becomes priority in the quest for better patient-centric outcomes. Of great relevance, whether the target antigen expression is a determinant of ADCs' primary activity is still to be clarified, and available data remain quite controversial. Antigen expression assessment is typically performed on tissue biopsy, hence only providing information on a specific tumor site, therefore unable to capture heterogeneous patterns of tumor antigen expression. Quantifying the expression of the target antigen across all tumor sites would help better understand tumor heterogeneity, whereas molecularly characterizing tumor-intrinsic features over time might provide information on resistance mechanisms. In addition, toxicity can represent a critical concern, since most ADCs have a safety profile that resembles that of chemotherapies, with often unique adverse events requiring special management, possibly because of the differential in pharmacokinetics between the small-molecule agent versus payload of a similar class (eg, deruxtecan conjugate-related interstitial lung disease). As such, the identification of robust predictive biomarkers of safety and activity of ADCs has the potential to improve patient selection and enrich the population of patients most likely to derive a substantial clinical benefit, especially in those disease settings where different ADCs happen to be approved in competing clinical indications, with undefined biomarkers to make precise decision making and unclear data on how to sequence ADCs. At this point, the identification of clinically actionable biomarkers in the space of ADCs remains a top research priority.

Antibody-drug conjugates (ADC) in patients with advanced/metastatic HER2-low expressing breast cancer: A systematic review and meta-analysis.

e13174 Background: Historically, HER2-targeted therapies failed to improve survival of patients with HER2-low-expressing tumors. Recently, trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate (ADC), demonstrated superior outcomes compared with treatment of physician´s choice (TPC) in this population. Other ADCs have also shown encouraging results, including sacituzumab govitecan (SG), MRG002, and RC48-ADC. Hence, we performed a systematic review and meta-analysis to assess the effectiveness of ADCs in patients with HER2-low advanced/metastatic (a/m) breast cancer (BC). Methods: We searched PubMed, Embase, and Cochrane databases and ASCO, ESMO, and SABCS websites for studies evaluating ADCs on patients with HER2-low a/mBC. Outcomes of interest were objective response rate (ORR); disease control rate (DCR); clinical benefit rate (CBR); progression-free survival (PFS); and overall survival (OS). We used R software (v.4.2.2) and random effects models for all analyses. Heterogeneity was assessed using I2 test. Results: Overall, 12 studies were included (3 real-world studies and 9 clinical trials), with 1,292 HER2-low a/mBC patients, 378 received TPC, and 914 received ADCs. Most were treated with T-DXd (63%, n=572), SG (29%, n=268), MRG002 (6%, n=56), and RC48-ADC (2%, n=18). Median age ranged from 48.1 to 58 years, and follow-up from 9.5 to 18.4 months. Patients treated with T-DXd achieved a significantly higher ORR and DCR compared to those who received SG, RC48-ADC, and MRG002 (Table). CBR was numerically higher in patients on T-DXd than those on SG but did not reach a significant difference. Patients with HR-positive tumors treated with SG had an ORR of 45% (95% CI 23 – 67%), and patients with pure HER2-low treated with any ADC, had an ORR of 38% (95% CI 30 – 45%). In the pooled analysis of 3 RCTs, ADCs significantly prolonged PFS compared with TPC (n=963, HR: 0.44, 95% CI 0.32 – 0.61, I2 = 66%, p<0.001), and OS (n= 680, HR: 0.56, 95% CI 0.36 – 0.89, I2 = 73%, p=0.015). Patients on ADCs also achieved a greater anti-tumor response than TPC, including better ORR (OR: 4.18, 95% CI 2.40 - 7.28, I2 = 52%, p<0.001), DCR (OR: 2.82, 95% CI 2.06 - 3.85, I2 = 7%, p<0.001), and CBR (OR: 3.41, 95% CI 2.36 - 4.93, I2 = 25%, p<0.001). Conclusions: Our systematic review and meta-analysis supports the efficacy of ADCs in HER2-low a/mBC patients over TPC. Future studies should focus on bringing ADCs into earlier lines of therapy in this population.Anti-tumor activity of ADCs presented as overall % (95% CI). [Table: see text]

Bystander effects, pharmacokinetics, and linker-payload stability of EGFR-targeting antibody-drug conjugates Losatuxizumab vedotin and Depatux-M in glioblastoma models.

Antibody-drug conjugates (ADCs) are targeted therapies with robust efficacy in solid cancers, and there is intense interest in using EGFR-specific ADCs to target EGFR-amplified glioblastoma (GBM). Given the molecular heterogeneity of GBM, bystander activity of ADCs may be important for determining treatment efficacy. In this study, the activity and toxicity of two EGFR-targeted ADCs, Losatuxizumab vedotin (ABBV-221) and Depatuxizumab mafodotin (Depatux-M), with similar auristatin toxins, were compared in GBM patient-derived xenografts (PDXs) and normal murine brain following direct infusion by convection enhanced delivery (CED). EGFRviii-amplified and non-amplified GBM PDXs were used to determine in vitro cytotoxicity, in vivo efficacy, and bystander activities of ABBV-221 and Depatux-M. Non-tumor bearing mice were used to evaluate pharmacokinetics and toxicity of ADCs using LC-MS/MS and immunohistochemistry. CED improved intracranial efficacy of Depatux-M and ABBV-221 in three EGFRviii-amplified GBM PDX models (Median survival: 125 to >300 days vs 20-49 days with isotype-control AB095). Both ADCs had comparable in vitro and in vivo efficacy. However, neuronal toxicity and CD68+ microglia/macrophage infiltration were significantly higher in brains infused with ABBV-221, with the cell-permeable MMAE, as compared to Depatux-M, with the cell-impermeant MMAF. CED infusion of ABBV-221 into brain or incubation of ABBV-221 with normal brain homogenate resulted in significant release of MMAE, which is consistent with linker instability in the brain microenvironment. EGFR-targeting ADCs are promising therapeutic options for GBM when delivered intra-tumorally by CED. However, the linker and payload for the ADC must be carefully considered to maximize the therapeutic window.

Abstract PO4-26-01: Computational pathology: Revolutionizing diagnostics and clearing the way for precision medicine

Abstract While targeted cancer therapies often rely on subjective and semi-quantitative visual assessment of protein biomarkers by pathologists through immunohistochemically stained tissue, the transformative force of computational pathology is reshaping healthcare by unleashing unprecedented diagnostic accuracy and unlocking personalized treatments. In recent years, we have established a large integrated computational pathology unit to foster collaboration between interdisciplinary teams of computer scientists, pathologists, molecular biologists, and data scientists. This integration of cutting-edge technologies enabled us to develop a computational pathology approach called Quantitative Continuous Scoring (QCS). QCS deploys the power of Deep Learning (DL) to provide objective and continuous expression data of biomarkers in digitized IHC whole slide images (WSI), particularly of proteins expressed at low levels. While manual scoring of IHC WSIs is limited by subjectivity and semi-quantitative assessment of protein expression, QCS overcomes these limitations with an unprecedented accuracy. QCS utilizes two DL-based algorithms, which we developed fully supervised by using pathologist input as the reference standard. These algorithms identify invasive tumour areas and segment each tumour cell across the WSI into cell nuclei, cytoplasm and membrane. Based on an accurate subcellular segmentation, we can compute biomarker expression, on a continuous scale, as mean Optical Density (OD) in each subcellular compartment based on the Hue-Saturation-Density (HSD) model. Therefore, this approach enables precise detection of the low biomarker expression range with single-cell resolution. Importantly, it also allows the computation of the spatial distribution of tumour cells across the WSI. We have successfully used QCS to drive the selection of antibody clones for IHC assays and to delineate the mode of action and PK/PD mechanisms. Of note, the combination of assessing continuous target expression and capturing the spatial distribution of tumor cells has provided surrogate markers to predict potential bystander activity of antibody drug conjugates (ADCs). This approach outperformed traditional pathologist scoring in identifying patient populations having maximum treatment benefit through retrospective analysis of multiple clinical trials. At present, all computational pathology approaches are developed based on conventional IHC assays that have been optimized for manual scoring. Importantly, we draw a vision in which the assay serves as a critical catalyst for unleashing the full potential of computational pathology by providing high-quality, standardized data inputs. We suggest an approach utilizing orthogonal methods as a reference standard to develop highly sensitive IHC assays, capable of detecting even subtle molecular and cellular changes with precision and exhibiting exceptional specificity for accurately identifying and distinguishing target biomarkers from background noise. In summary, we here describe and discuss a computational pathology-based approach for precise biomarker quantification and superior patient selection with broad applicability and the potential to transform the very fabric of how we diagnose and treat cancer. Citation Format: Mark Gustavson, Markus Schick, Ansh Kapil, Anatoliy Shumilov, Carl Barrett, Hadassah Sade. Computational pathology: Revolutionizing diagnostics and clearing the way for precision medicine [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 PO4-26-01.

Abstract PO2-04-05: FINAL OUTCOME ANALYSIS FROM THE PHASE II TUXEDO-1 TRIAL OF TRASTUZUMAB-DERUXTECAN IN HER2-POSITIVE BREAST CANCER PATIENTS WITH ACTIVE BRAIN METASTASES

Abstract Background Brain metastases (BM) are a severe complication of HER2-positive metastatic breast cancer. In the absence of any immediate indication for local therapy, the tyrosine-kinase inhibitor tucatinib combined with trastuzumab and capecitabine (TTC) is regarded as the preferred systemic treatment option for active BM, while data on the activity of antibody-drug conjugates (ADC) is limited. In the primary outcome analysis of TUXEDO-1, an intracranial response rate (RR) of 73.3% was reported with the ADC trastuzumab-deruxtecan (T-DXd). Here, we report final progression-free survival (PFS) and overall survival (OS) results of this study. Patients and Methods: TUXEDO-1 is a prospective, single-centre, single-arm phase 2 trial. Adult patients with HER2-positive BC and active BM (newly diagnosed untreated or progressing after prior local therapy), prior treatment with trastuzumab and pertuzumab, ECOG performance status 0 or 1 without indication for immediate local therapy were accrued and received T-DXd until progression, inacceptable toxicity, or withdrawal for any other reason. The primary endpoint was intracranial RR centrally assessed by Response Assessment in Neuro-Oncology (RANO)-BM criteria in the intention-to-treat population; secondary endpoints included PFS, OS, safety, quality-of-life (QoL), and neurocognitive function. PFS and OS were estimated with the Kaplan-Meier method and analysed in the per-protocol population (PPP). Results A total number of 15 patients were accrued; one patient was found to have dural metastases only upon restaging, resulting in a PPP of 14 patients. Patients had received a median number of two prior treatment lines (range, 1-5), 60% had progressive brain metastases and 60% had received prior T-DM1. At 26.5 months median follow-up, median PFS was 21 months (95% CI 13.3-n.r.) and median OS was not reached (95% CI 22.2-n.r.). A total of 238 cycles of T-DXd were administered (median 11.5 cycles; range 4-42). With longer follow-up, no new safety signals were observed. The most common grade 3 adverse event (AE) was fatigue (n=3; 20%). A total of 8 serious AEs were reported in 8 patients. Grade 2 interstitial lung disease and a grade 3 symptomatic drop of left-ventricular ejection fraction were observed in one patient each. QoL and neurocognitive functioning were maintained over the entire treatment duration and a significant deterioration of global QoL was observed upon progression (p=0.036). Most patients received TTC (n=5) or local therapy (n=4) as next subsequent treatment line. Ancillary biomarker studies are ongoing, and results will be presented at the meeting. Discussion In TUXEDO-1, T-DXd yielded prolonged intra- and extracranial disease control in patients with active HER2-positive breast cancer BM. The safety profile was consistent with previous reports. T-DXd did not impair QoL and neurocognitive functioning was maintained. Results therefore support the concept of ADCs as systemic therapy for active BM. Citation Format: Rupert Bartsch, Anna Sophie Berghoff, Julia Furtner, Maximilian Marhold, Elisabeth Sophie Bergen, Sophie Roider-Schur, Angelika Maria Starzer, Maximilian Mair, Heidrun Forstner, Beate Rottenmanner, Marie-Bernadette Aretin, Karin Dieckmann, Zsuzsanna Bago-Horvath, Helmuth Haslacher, Georg Widhalm, Aysegül Ilhan-Mutlu, Christoph Minichsdorfer, Thorsten Fuereder, Thomas Szekeres, Leopold Oehler, Birgit Gruenberger, Georg Pfeiler, Christian F. Singer, Ansgar Weltermann, Luzia Berchtold, Matthias Preusser. FINAL OUTCOME ANALYSIS FROM THE PHASE II TUXEDO-1 TRIAL OF TRASTUZUMAB-DERUXTECAN IN HER2-POSITIVE BREAST CANCER PATIENTS WITH ACTIVE BRAIN METASTASES [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 PO2-04-05.

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.

Preparation and evaluation of the PD0721‑DOX antibody‑drug conjugate targeting EGFRvIII to inhibit glioblastoma

Preparation and evaluation of the PD0721‑DOX antibody‑drug conjugate targeting EGFRvIII to inhibit glioblastoma

Abstract 5089: Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates

Abstract Background: Endometrial cancer is the most frequently occurring gynecologic cancer in developed countries and the third leading cause of cancer deaths among women. It remains one of the few malignant tumors with rapidly increasing mortality and morbidity. Patients with recurrent or metastatic endometrial cancer continue to experience poor outcomes. Antibody-drug conjugates (ADCs) represent a class of targeted therapeutic modalities that offer biomarker-based therapeutic options for patients with endometrial cancer. In this study, we proposed to identify a novel molecular target for the preclinical development of an ADC for endometrial cancer-targeted therapy. Methods: Firstly, to determine the level of target expression within endometrial cancer tissue components as well as differences, we performed immunohistochemical staining of human endometrial cancer and paracancerous tissue. Secondly, the expression of the target on the cell membrane surface was quantitated and qualitatively verified using flow cytometry and single-photon confocal microscopy. In the third step, an endocytosis assay was used to verify the endocytosis of the target antibody by EC cells. Confocal imaging can directly observe the endocytosis of the target antibody, and flow cytometry can be analyzed and quantified. Co-localization experiments with antibody fluorescence can verify the specific site where the target antibody is endocytosed into cancer cells. We then designed and characterized two targeted ADCs. Next, the in vitro inhibitory activities of these ADCs against endometrial cancer cell lines and normal cells were evaluated using the CCK8 assay. Finally, we established a cell line-derived xenograft (CDX) model of endometrial cancer to validate the in vivo efficacy and bio-safety of the ADCs. Results: We have determined a correlation between ICAM1 overexpression and EC, and IHC staining of patient tissues showed significantly higher levels of ICAM1 expression in endometrial cancer tissues than in paracancerous tissues. The high cell surface expression of ICAM1 was verified in a panel of various human endometrial cancer cell lines. Moreover, two ICAM1 ADCs were established using clinically-approved ADC linker and payload combinations. It was visually confirmed that ICAM1 ADCs selectively recognize endometrial tumors and efficiently enter endometrial cells via antigen-mediated endocytosis. We compared the anti-tumor activity of ICAM1 ADCs with standard-of-care chemodrugs, and ICAM1 ADCs outperformed chemodrugs in the CDX model of endometrial cancer without toxicity. Conclusion: We identified that ICAM1 is a promising ADC target for endometrial cancer and confirmed the potent anti-tumor activity and biosafety of ICAM1 ADCs in vitro and in vivo, warranting further pre-clinical and clinical investigations. Citation Format: Hanfei Xie, Lu Sun, ShiLi Yao, Peng Guo, YingLi Zhang. Therapeutically targeting endometrial cancer by ICAM1 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 5089.

Abstract 1084: Preclinical testing of a cell adhesion molecule 1 (CADM1) targeting antibody-drug conjugate in osteosarcoma and other pediatric solid tumor models

Abstract Introduction: Survival outcomes of osteosarcoma have remained stagnant for decades, emphasizing the urgent need to identify new cell surface targets and develop novel therapies. Antibody-drug conjugates (ADCs), known for their targeted delivery of cytotoxic agents to specific targets, present an attractive therapeutic class for potential use in osteosarcoma. Methods: We previously identified Cell Adhesion Molecule 1 (CADM1) is highly expressed on the cell surface in osteosarcoma using an integrated proteomic and transcriptomic surfaceome profiling approach. As a proof of concept, a humanized CADM1 antibody (clone PTA021-A1) was conjugated with Tesirine (SG3249), a pyrrolobenzodiazepine (PBD) dimer payload. The antitumor activity of this CADM1 ADC was validated in vivo in 8 osteosarcoma patient-derived xenografts (PDXs). CADM1 is also highly expressed in other pediatric solid malignancies such as neuroblastoma and Wilms tumor. The CADM1 ADC was tested in vitro in neuroblastoma and Wilms tumor models. Negative controls included a CD19 targeting ADC with the same payloads and CADM1 negative Ewing sarcoma models. Results: The expression of the CADM1 was validated by flow cytometry in osteosarcoma, neuroblastoma, and Wilms tumor cell lines, and by immunohistochemistry (IHC) in osteosarcoma patient samples and PDX models. In vivo testing of CADM1 ADC in 8 osteosarcoma PDX models showed that the ADC was well-tolerated and significantly prolonged progressive-free survival in all osteosarcoma models. Complete response (CR) and maintained CR was observed in 3 models. In vitro testing for neuroblastoma and Wilms tumor is ongoing, and preliminary data suggest promising antitumor activities. Conclusions: CADM1 is highly expressed in osteosarcoma and other pediatric solid tumors. CADM1 ADC demonstrated promising antitumor in vitro and in vivo activity in osteosarcoma, and initial in vitro studies in neuroblastoma and Wilms tumor show similarly promising responses. These data support the need for further studies assessing the potential of a CADM1 ADC as a novel agent for the treatment of pediatric solid cancers. Citation Format: Zhongting Zhang, Yifei Wang, Wendong Zhang, Xiangjun Tian, Qi Wang, Rossana N Lazcano Segura, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Yianhua Yi, Sylvester Jusu, Giuseppe Longo, Xin Zhou, Jing Wang, Richard Gorlick. Preclinical testing of a cell adhesion molecule 1 (CADM1) targeting antibody-drug conjugate in osteosarcoma and other pediatric solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1084.

Abstract 1911: Dato-DXd mediates anti-tumor activity in preclinical TROP2-expressing intracranial tumor model

Abstract Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 monoclonal antibody linked to a potent DNA topoisomerase I (TOP1) inhibitor payload via a plasma-stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd is currently under clinical investigation for the treatment of patients with solid tumors including non-small cell lung cancer (NSCLC), hormone receptor positive (HR+ BC) and triple negative breast cancer (TNBC). Pharmacotherapy of brain tumors can be limiting due to restricted drug delivery across blood brain and blood tumor barrier. Enhertu®, that uses the same DXd ADC technology, has reported clinical activity in patients with brain metastases from HER2+ breast cancer, but very little information is available on what drives ADC biodistribution and activity in CNS-involved cancers. Here, we investigated whether systemically administered ADCs can penetrate the brain microenvironment and mediate anti-tumor activity in a preclinical model. Luciferase-tagged H1373 (TROP2-expressing NSCLC) tumor cells were intracranially implanted into NSG mice. Tumor-bearing mice were dosed with Dato-DXd or matched isotype Control IgG-ADC (DAR4) at 10mpk 7 days or 14 days post intracranial tumor implant. Dato-DXd inhibited intracranial tumor growth better than Control ADC (Day 7: 105% TGI vs 38% TGI; Day 14: 65% TGI vs <10% TGI, respectively, compared to Vehicle). Immunohistochemistry analysis of brain tissue validated localization of Dato-DXd in the tumor, suggesting Dato-DXd can distribute into the local tumor microenvironment in this preclinical tumor model. Additionally, treatment with Dato-DXd provided a significant survival benefit over Control ADC (Median survival of 63 days vs 43 days, P=0.0002). This preclinical study supports the inclusion of Dato-DXd in treatment of patients with CNS-involved tumors. Understanding the pharmacological determinants of Dato-DXd activity in the CNS will help outline strategies to implement Dato-DXd-based treatment of patients with CNS-involved tumors. Citation Format: Kristen Jones Jones, Montira Suksomboon, SaraAnn rosenthal, Jacob Gordon, Corinne Reimer, Matthew Sung, Chetan Rane. Dato-DXd mediates anti-tumor activity in preclinical TROP2-expressing intracranial tumor model [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 1911.

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 >50 cancer cell lines derived from >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.

Activity of antibody-drug conjugates (ADCs) with radiation in preclinical bladder cancer models.

664 Background: Antibody-drug conjugates (ADCs) are a novel class of therapeutics that combine a tumor cell targeting antibody with a cytotoxic payload. Two ADCs are currently approved in the US for treatment of advanced bladder cancer: enfortumab vedotin (EV) and sacituzumab govitecan (SG). Radiation therapy (RT) plays a central role in trimodality therapy (TMT), a curative treatment approach for muscle-invasive bladder cancer (MIBC). However, the biological activity of ADCs combined with RT in preclinical MIBC models has not been reported. Methods: We use a molecularly annotated panel of human MIBC preclinical models to test the impact of RT on expression of ADC targets and define the combined activity of EV and SG with RT. We also test the in vivo activity of combining ADCs with image-guided, fractionated RT in bladder cancer flank xenograft mouse models. Results: Radiation has variable impact on expression levels of nectin-4 and trop-2, the targets of EV and SG, respectively, as determined by immunoblot, immunofluorescence microscopy, and immunohistochemistry (IHC). Whereas modest dose-dependent increases in nectin-4 and/or trop-2 levels were observed in some models, no significant changes were observed in other models. Importantly, RT did not lead to a significant decrease in nectin-4 or trop-2 expression in any of the models. EV and SG showed additive or synergistic cell killing when combined with RT across preclinical models in vitro. Combining EV or SG with fractionated RT in vivo was well-tolerated, showed improved tumor control, and prolonged survival in bladder cancer flank xenograft mouse models compared to either ADC or RT alone. Conclusions: ADCs demonstrate combination activity with RT across a panel of molecularly diverse bladder cancer preclinical models. These studies provide preclinical data supporting clinical trials to investigate the safety and efficacy of combining ADCs with RT as a bladder-preserving therapy for MIBC.

Spatiotemporal Quantification of HER2-targeting Antibody-Drug Conjugate Bystander Activity and Enhancement of Solid Tumor Penetration.

Antibody-drug conjugate (ADC) has had a transformative effect on the treatment of many solid tumors, yet it remains unclear how ADCs exert bystander activity in the tumor microenvironment. Here, we directly visualized and spatiotemporally quantified the intratumor biodistribution and pharmacokinetics of different ADC components by developing dual-labeled fluorescent probes. Mechanistically, we found that tumor penetration of ADCs is distinctly affected by their ability to breach the binding site barrier (BSB) in perivascular regions of tumor vasculature, and bystander activity of ADC can only partially breach BSB. Furthermore, bystander activity of ADCs can work in synergy with coadministration of their parental antibodies, leading to fully bypassing BSBs and enhancing tumor penetration via a two-step process. These promising preclinical data allowed us to initiate a phase I/II clinical study of coadministration of RC48 and trastuzumab in patients with malignant stomach cancer to further evaluate this treatment strategy in humans.

Prevalence and Therapeutic Targeting of High-Level ERBB2 Amplification in NSCLC

IntroductionERBB2 amplification in lung cancer remains poorly characterized. HER2 (encoded by ERBB2) is a transmembrane tyrosine kinase capable of ligand-independent dimerization and signaling when overexpressed, and a common cause of HER2 overexpression is ERBB2 amplification. Here, we evaluated the clinicopathologic and genomic characteristics of ERBB2-amplified NSCLC and explored a HER2 antibody–drug conjugate (ADC) therapeutic strategy. MethodsOur institutional next-generation DNA sequencing data (OncoPanel) from 5769 NSCLC samples (5075 patients) were queried for cases having high-level ERBB2 amplification (≥6 copies). Clinical and demographic characteristics were extracted from the electronic medical records. Efficacy of the pan-ERBB inhibitor afatinib or HER2 ADCs (trastuzumab deruxtecan and trastuzumab emtansine) was evaluated in NSCLC preclinical models and patients with ERBB2 amplification. ResultsHigh-level ERBB2 amplification was identified in 0.9% of lung adenocarcinomas and reliably predicted overexpression of HER2. ERBB2 amplification events are detected in two distinct clinicopathologic and genomic subsets of NSCLC: as the sole mitogenic driver in tumors arising in patients with a smoking history or as a concomitant alteration with other mitogenic drivers in patients with a light or never smoking history. We further reveal that trastuzumab deruxtecan is effective therapy in in vitro and in vivo preclinical models of NSCLC harboring ERBB2 amplification and report two cases of clinical activity of an anti-HER2 ADC in patients who acquired ERBB2 amplification after previous targeted therapy. ConclusionsHigh-level ERBB2 amplification reliably predicts HER2 overexpression in patients with NSCLC, and HER2 ADC is effective therapy in this population.

Antibody-Drug Conjugates for the Treatment of Renal Cancer: A Scoping Review on Current Evidence and Clinical Perspectives.

Antibody-drug conjugates (ADCs) are complex chemical structures composed of a monoclonal antibody, serving as a link to target cells, which is conjugated with a potent cytotoxic drug (i.e., payload) through a chemical linker. Inspired by Paul Ehrlich's concept of the ideal anticancer drug as a "magic bullet", ADCs are also highly specific anticancer agents, as they have been demonstrated to recognize, bind, and neutralize cancer cells, limiting injuries to normal cells. ADCs are among the newest pharmacologic breakthroughs in treating solid and hematologic malignancies. Indeed, in recent years, various ADCs have been approved by the Food and Drug Administration and European Medicines Agency for the treatment of several cancers, resulting in a "practice-changing" approach. However, despite these successes, no ADC is approved for treating patients affected by renal cell carcinoma (RCC). In the present paper, we thoroughly reviewed the current literature and summarized preclinical studies and clinical trials that evaluated the activity and toxicity profile of ADCs in RCC patients. Moreover, we scrutinized the potential causes that, until now, hampered the therapeutical success of ADCs in those patients. Finally, we discussed novel strategies that would improve the development of ADCs and their efficacy in treating RCC patients.

Epigenetically upregulating TROP2 and SLFN11 enhances therapeutic efficacy of TROP2 antibody drug conjugate sacitizumab govitecan

TROP2 antibody drug conjugates (ADCs) are under active development. We seek to determine whether we can enhance activity of TROP2 ADCs by increasing TROP2 expression. In metaplastic breast cancers (MpBC), there is limited expression of TROP2, and downregulating transcription factor ZEB1 upregulates E-cad and TROP2, thus sensitizing cancers to TROP2 ADC sacituzumab govitecan (SG). Demethylating agent decitabine decreases DNA methyltransferase expression and TROP2 promoter methylation and subsequently increases TROP2 expression. Decitabine treatment as well as overexpression of TROP2 significantly enhance SG antitumor activity. Decitabine also increases SLFN11, a biomarker of topoisomerase 1 inhibitor (TOP1) sensitivity and is synergistic with SG which has a TOP1 payload, in TROP2-expressing SLFN11-low BC cells. In conclusion, TROP2 and SLFN11 expression can be epigenetically modulated and the combination of demethylating agent decitabine with TROP2 ADCs may represent a novel therapeutic approach for tumors with low TROP2 or SLFN11 expression.

Therapeutic efficacy of a MMAE-based anti-DR5 drug conjugate Oba01 in preclinical models of pancreatic cancer

Pancreatic cancer (PC) is among the most aggressive malignancies associated with a 5-year survival rate of <9%, and the treatment options remain limited. Antibody–drug conjugates (ADCs) are a new class of anticancer agents with superior efficacy and safety profiles. We studied the antitumor activity of Oba01 ADC and the mechanism underlying the targeting of death receptor 5 (DR5) in preclinical PC models. Our data revealed that DR5 was highly expressed on the plasma membrane of PC cells and Oba01 showed potent in vitro antitumor activity in a panel of human DR5-positive PC cell lines. DR5 was readily cleaved by lysosomal proteases after receptor-mediated internalization. Monomethyl auristatin E (MMAE) was then released into the cytosol to induce G2/M-phase growth arrest, cell death via apoptosis induction, and the bystander effect. Furthermore, Oba01 mediated cell death via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. For improved potency, we investigated the synergetic effect of Oba01 in combination with approved drugs. Oba01 combined with gemcitabine showed better antiproliferative activity than either standalone treatment. In cell- and patient-derived xenografts, Oba01 showed excellent tumoricidal activity in mono- or combinational therapy. Thus, Oba01 may provide a novel biotherapeutic approach and a scientific basis for clinical trials in DR5-expressing patients with PC.

Understanding the activity of antibody–drug conjugates in primary and secondary brain tumours

Antibody-drug conjugates (ADCs), a class of targeted cancer therapeutics combining monoclonal antibodies with a cytotoxic payload via a chemical linker, have already been approved for the treatment of several cancer types, with extensive clinical development of novel constructs ongoing. Primary and secondary brain tumours are associated with high mortality and morbidity, necessitating novel treatment approaches. Pharmacotherapy of brain tumours can be limited by restricted drug delivery across the blood-brain or blood-tumour barrier, although data from phase II studies of the HER2-targeted ADC trastuzumab deruxtecan indicate clinically relevant intracranial activity in patients with brain metastases from HER2+ breast cancer. However, depatuxizumab mafodotin, an ADC targeting wild-type EGFR and EGFR variant III, did not provide a definitive overall survival benefit in patients with newly diagnosed or recurrent EGFR-amplified glioblastoma in phase II and III trials, despite objective radiological responses in some patients. In this Review, we summarize the available data on the central nervous system activity of ADCs from trials involving patients with primary and secondary brain tumours and discuss their clinical implications. Furthermore, we explore pharmacological determinants of intracranial activity and discuss the optimal design of clinical trials to facilitate development of ADCs for the treatment of gliomas and brain metastases.

Antibody drug conjugates as targeted cancer therapy: past development, present challenges and future opportunities.

Antibody drug conjugates (ADCs) are promising cancer therapeutics with minimal toxicity as compared to small cytotoxic molecules alone and have shown the evidence to overcome resistance against tumor and prevent relapse of cancer. The ADC has a potential to change the paradigm of cancer chemotherapeutic treatment. At present, 13 ADCs have been approved by USFDA for the treatment of various types of solid tumor and haematological malignancies. This review covers the three structural components of an ADC-antibody, linker, and cytotoxic payload-along with their respective structure, chemistry, mechanism of action, and influence on the activity of ADCs. It covers comprehensive insight on structural role of linker towards efficacy, stability & toxicity of ADCs, different types of linkers & various conjugation techniques. A brief overview of various analytical techniques used for the qualitative and quantitative analysis of ADC is summarized. The current challenges of ADCs, such as heterogeneity, bystander effect, protein aggregation, inefficient internalization or poor penetration into tumor cells, narrow therapeutic index, emergence of resistance, etc., are outlined along with recent advances and future opportunities for the development of more promising next-generation ADCs.