Study on the Mechanism of ADC Drug Evaluation Based on Immune Co-culture of Lung Cancer Organoids

A case-control study was conducted to evaluate the efficacy and mechanism of action of antibody-drug conjugates (ADCs) in lung cancer, utilizing patient-derived organoid (PDO)-immune co-cultures. Focusing on HER2-positive and TROP2-positive non-small cell lung cancer (NSCLC) cases, ADC candidates were screened for in vitro activity based on organoid-immune interaction models.

Key assessments included:

Tumor killing efficiency, assessed by dose-response relationships; Drug internalization (cellular uptake), as a measure of penetration into cancer cells; Antibody-dependent cellular cytotoxicity (ADCC) and bystander effect, with negative control targets employed to delineate specificity; Single-cell RNA sequencing, to profile transcriptional alterations at single-cell resolution.

Data demonstrated distinct ADC responses correlating with target expression and immune microenvironment features. The integrated approach provided cell-based evidence of ADC potency and revealed mechanistic insights-including immune-mediated cytotoxicity pathways and intracellular trafficking-supporting the rational design of clinical trials. These findings established a foundation for precision immunotherapy strategies and offered a mechanistic rationale for patient selection in HER2/TROP2-positive lung cancer.

Study Overview

• Status: Recruiting
• Conditions: HER2 Positive OR TROP2 Positive Non-Small Cell Lung Cancer
• Intervention / Treatment: Drug: Antibody-drug conjugate (ADC) combination therapy

Detailed Description

A case-control study was conducted to systematically evaluate the therapeutic efficacy and underlying mechanisms of antibody-drug conjugates (ADCs) in non-small cell lung cancer (NSCLC), utilizing an integrated patient-derived organoid (PDO)-immune cell co-culture platform. Focusing on HER2-positive and TROP2-positive NSCLC cases, a comprehensive research pipeline was established, comprising three core components: the construction of a PDO-immune co-culture model, multidimensional tumor killing assessment, and mechanistic dissection of cellular internalization.

Clinically resected tumor tissues and malignant pleural effusion specimens were harvested to generate PDOs, which were rigorously validated for histological fidelity and phenotypic stability via H&E staining and TTF-1 immunohistochemistry; cases were subsequently stratified based on HER2/TROP2 expression intensity. The functional integrity of the co-culture system was confirmed through flow cytometric analysis of immune cell purity and activation status, coupled with ELISA quantification of cytokines to verify effective immune-tumor crosstalk.

Pharmacodynamic evaluations were performed using ATP-based viability assays, PDO viability imaging, and Caspase-3/7 apoptosis detection. These assays simulated clinically relevant peak plasma concentrations (C max) to directly reflect in vivo drug exposure, while also assessing the synergistic potential of "ADC + Immuno-oncology" combination strategies to optimize clinical dosing regimens. Mechanistically, pHrodo dye tracking was employed to visualize and quantify cellular internalization and phagocytosis, complemented by single-cell RNA sequencing to delineate transcriptional profiles and identify specific subpopulations sensitive to ADC therapy. Furthermore, high-sensitivity Olink proteomics and multiplex fluorescence immunohistochemistry provided "cellular-molecular-spatial" evidence of immune activation and intracellular trafficking dynamics.

Collectively, the data revealed that distinct ADC responses correlated with target expression and the immune microenvironment, precisely characterizing the molecular signatures of sensitive cell subpopulations and their enhanced endocytic activity. These findings provide critical molecular targets and a theoretical basis for patient selection and the rational design of next-generation ADC therapies in HER2/TROP2-positive lung cancer.

• Study Type: Observational
• Enrollment (Estimated): 10

Contacts and Locations

• Study Contact: Name: chengzhi Professor Zhou, PhD; Phone Number: 13560351186; Email: 13560351186@163.com
• Study Contact Backup: Name: Xinqing Lin, PhD; Phone Number: 18819281507; Email: linxinging81@163.com

Study Locations

• China
  • Guangdong
    • Guangzhou, Guangdong, China
      • Recruiting
      • The First Affiliated Hospital of Guangzhou Medical University
      • Contact: chengzhi Professor Zhou, PhD; Phone Number: 13560351186; Email: 13560351186@163.com
      • Contact: Xinqing Lin, PhD; Phone Number: 18819281507; Email: linxinging81@163.com
      • Principal Investigator: Chengzhi Zhou, PhD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Patients with a pathological diagnosis of non-small cell lung cancer (NSCLC), whose tumor patient-derived organoids (PDOs) exhibited strong or weak positive expression of the HER2 or TROP2 targets as determined by immunohistochemistry.

Description

Inclusion Criteria:

• Age ≥ 18 years.
• Availability of patient-derived organoids (PDOs) with matched autologous tumor-infiltrating lymphocytes (TILs) or peripheral blood mononuclear cells (PBMCs) from non-small cell lung cancer (NSCLC) cases.
• Patients currently undergoing or scheduled to receive Trastuzumab deruxtecan (T-DXd) therapy who meet clinical eligibility criteria.
• Provision of written informed consent.
• PDOs exhibiting strong positive HER2 and TROP2 expression by immunohistochemistry (IHC) assigned to the experimental group.
• PDOs exhibiting weak positive HER2 and TROP2 expression by IHC assigned to the negative control group.

Exclusion Criteria:

• PDOs derived from patients with pathologically confirmed small cell lung cancer (SCLC).
• Unavailability of matched autologous PDOs, TILs, or PBMCs.
• Presence of any contraindications to T-DXd treatment.
• Presence of other serious comorbidities resulting in an estimated survival of <3 months.
• Pregnant or breastfeeding women.

Study Plan

How is the study designed?

Number of groups / cohorts

1

Cohorts and Interventions

Group / Cohort

Intervention / Treatment

HER2 Positive OrTROP2 Positive non-small cell lung cancer; The abundance of HER2 or TROP2 target expression was determined by immunohistochemistry and categorized into strongly positive and weakly positive groups, representing the experimental group and negative control group, respectively. Using an immunococulture system based on patient-derived organoids, the in vitro activity of antibody-drug conjugates (ADCs)-including trastuzumab emtansine (an approved ADC administered intravenously)-that are either approved or currently in clinical trials was evaluated. In parallel, clinical patients provided ex vivo cytological assay results indicating their sensitivity to ADC therapy.

Drug: Antibody-drug conjugate (ADC) combination therapy; In a co-culture system of tumor patient-derived organoids (PDOs) and autologous immune cells, at least ten drug combinations comprising antibody-drug conjugates (ADCs) plus tyrosine kinase inhibitors (TKIs) or immune checkpoint blockers (ICBs) were employed to screen for ADCs with sensitivity against the tumor PDOs. The ADCs selected for evaluation included: Trastuzumab deruxtecan for injection (intravenous) Trastuzumab emtansine for injection (intravenous) Sacituzumab govitecan for injection (intravenous); Other Names: ADC + TKI/ICB + Cellular Therapy DS-8201; ADC + TKI/ICB + Cellular Therapy T-DM1; ADC + TKI/ICB + Cellular Therapy SG

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Changes in ATP assay of tumor patient-derived organoids (PDOs) under treatment with ADCs and various drug combinations	The single high-concentration ADC design was formulated to mimic the peak plasma concentration achieved after clinical administration, thereby directly reflecting the cytotoxic potency of the drug at effective concentrations. The combination therapy assessment focuses on the current trend of "ADC + immuno" strategies in oncology, providing ex vivo data to support optimization of clinical dosing regimens.	Tumor viability assessed by luminescence measurement at 72 hours after co-culture with immune cells
Real-time high-content imaging reveals efficient internalization of pHrodo-conjugated ADCs	Kinetic analysis of ADC internalization was performed using a high-content imaging system. Briefly, cells were incubated with pHrodo-conjugated ADCs at 37°C. Real-time fluorescence imaging was conducted at 15-minute intervals for 24 hours to track the internalization efficiency. Quantitative data regarding cellular uptake were analyzed using the integrated high-content analysis software."	24hours
Integrated analysis of ADC IC50 determination and scRNA-seq data post co-culture	We observed a wide range of sensitivities to ADC treatment across different PDO models, as reflected by varying IC50 values (ranging from X nM to Y nM). To uncover the mechanistic basis for this heterogeneity, we performed scRNA-seq on tumor cells following the co-culture and ADC treatment. Unsupervised clustering revealed distinct tumor cell subpopulations, including a cluster characterized by high proliferative signature and another marked by stress-response pathways.	96hours
Multiplex immunofluorescence (mIF) and high-sensitivity Olink proteomics for inflammatory cytokines.	To characterize the immune landscape, we performed multiplex immunofluorescence (mIF) staining on tissue sections to analyze the spatial distribution of immune cells. Additionally, high-sensitivity Olink proteomics was utilized to quantify a panel of inflammatory cytokines in the culture supernatant.	96hours

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Fluorescence imaging of PDO viability and apoptosis assessment via caspase-3/7 activity in pharmaco-sensitivity and PDO-immune co-culture assays.	We utilized fluorescent viability dyes to visualize the cytotoxic effects of ADCs in co-culture. Concurrently, caspase-3/7 cleavage was detected as a marker of intrinsic apoptosis induced by the drug payload.	96hours
Cytokine secretion profiles (IL-6, TNF-α, IFN-γ) in co-culture supernatants measured by ELISA	The levels of key effector cytokines, including IL-6, TNF-α, and IFN-γ, were quantified in the supernatant of the PDO-immune co-culture system using ELISA kits。	96hours
Flow cytometric analysis of T cell activation and exhaustion markers in co-culture assays	Cells harvested from co-cultures were stained with fluorophore-conjugated antibodies against surface markers. T cell activation was defined by the upregulation of CD69 and CD25, while T cell dysfunction was characterized by the expression of inhibitory receptors such as PD-1 and TIM-3.	96hours

Collaborators and Investigators

• Sponsor: Guangzhou Institute of Respiratory Disease
• Investigators: Principal Investigator: Chengzhi Zhou, PhD, The First Affiliated Hospital of Guangzhou Medical University

Publications and helpful links

General Publications

• Eltayeb E. Nance, Michael J. D. Gooden, Francesca M. Marcon; Antibody Fragments as Agonists and Antagonists in Cancer Therapy; Antibodies; 2023 Apr; 12; 72.
• Hammood M, Craig AW, Leyton JV. Impact of Endocytosis Mechanisms for the Receptors Targeted by the Currently Approved Antibody-Drug Conjugates (ADCs)-A Necessity for Future ADC Research and Development. Pharmaceuticals (Basel). 2021 Jul 15;14(7):674. doi: 10.3390/ph14070674.
• Fu Z, Li S, Han S, Shi C, Zhang Y. Antibody drug conjugate: the "biological missile" for targeted cancer therapy. Signal Transduct Target Ther. 2022 Mar 22;7(1):93. doi: 10.1038/s41392-022-00947-7.
• Birrer MJ, Moore KN, Betella I, Bates RC. Antibody-Drug Conjugate-Based Therapeutics: State of the Science. J Natl Cancer Inst. 2019 Jun 1;111(6):538-549. doi: 10.1093/jnci/djz035.

Helpful Links

• Antibody Fragments as Agonists and Antagonists in Cancer Therapy
• Antibody drug conjugate: the "biological missile" for targeted cancer therapy
• Synthesis and evaluation of highly releasable and structurally stable antibody-SN-38-conjugates

Study record dates

Study Major Dates

• Study Start (Actual): December 28, 2025
• Primary Completion (Estimated): October 1, 2027
• Study Completion (Estimated): October 1, 2027

Study Registration Dates

• First Submitted: May 12, 2026
• First Submitted That Met QC Criteria: May 25, 2026
• First Posted (Actual): May 28, 2026

Study Record Updates

• Last Update Posted (Actual): May 28, 2026
• Last Update Submitted That Met QC Criteria: May 25, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: Non-Small Cell Lung Cancer; HER2 Positive Non-Small Cell Lung Cancer; TROP2 Positive Non-Small Cell Lung Cancer
• Additional Relevant MeSH Terms: Neoplasms by Site; Neoplasms; Respiratory Tract Diseases; Lung Diseases; Respiratory Tract Neoplasms; Thoracic Neoplasms; Lung Neoplasms; Carcinoma, Bronchogenic; Bronchial Neoplasms; Carcinoma, Non-Small-Cell Lung; Immunologic Factors; Physiological Effects of Drugs; Amino Acids, Peptides, and Proteins; Proteins; Therapeutics; Pharmacologic Actions; Chemical Actions and Uses; Antibodies; Immunoglobulins; Blood Proteins; Serum Globulins; Globulins; Immunoconjugates; Combined Modality Therapy; arginine decarboxylase
• Other Study ID Numbers: ADC-2025-10-1

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: Yes