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Cancer Technologies

Cancer Technologies - Poster Session A

Poster H6 - Characterizing Antitumor Activity of a Novel MUC1-C Targeting Antibody-Drug Conjugate for Treatment of Non-Small Cell Lung Cancer

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Over the past few decades, antibody-drug conjugates (ADCs) have revolutionized the cancer therapeutics space. By providing targeted delivery of cytotoxic payloads, ADCs can mitigate the systemic toxic effects of chemotherapy. However, differentially expressed target antigens and off-tumor toxicity present considerable challenges to ADC development.

Mucin 1 (MUC1) is a transmembrane protein typically found on the apical surface of healthy epithelial cells. Aberrantly overexpressed in over 80% of all solid tumors and 99% of non-small cell lung cancers (NSCLCs), MUC1 is linked to angiogenesis, epithelial-to-mesenchymal transition, chemoresistance, and immune evasion, making it a marker of the cancer stem cell state and aggressive, metastatic carcinomas. Recognizing the potential of MUC1 to target a wide range of tumors, the National Cancer Institute has ranked MUC1 amongst the top (second highest) antigens for therapeutic development. Interestingly, due to the loss of apical-basal polarity, cancer cells express MUC1 across the cell surface; therefore, the tumor-associated MUC1 antigen is selectively accessible to ADCs via the vasculature, while healthy cells are shielded.

MUC1 is composed of N- and C-termini subunits: MUC1-C is anchored in the cell membrane, while MUC1-N is bound extracellularly. Previous attempts at creating MUC1 ADCs have targeted MUC1-N. However, MUC1-N is shed into the serum, increasing “on-target, off-tumor” toxicities. Therefore, the development of a first-in-class MUC1-C-targeting ADC provides a unique opportunity for greater therapeutic window.

Here, we present the biophysical characterization, in-vitro studies of NSCLC MUC1-C expression and antitumor activity of our ADC. We also summarize preclinical efficacy and toxicity.

Materials and

Methods:  We raised a monoclonal antibody (mAb) against the α-3 helix of the MUC1-C extracellular domain. This mAb, 3D1, is covalently bound to monomethyl auristatin MMAE via a maleimide cysteine valine-citrulline dipeptide cleavable linker to form the complete ADC. To assess binding kinetics between 3D1 and MUC1-C, we utilized surface-plasmon resonance (SPR), producing the extracellular domain of MUC1-C with recombinant DNA technology.

The NSCLC cell lines we tested included MUC1-C positive H1975 and MUC1-C negative A549. We analyzed MUC1-C expression with the BD FACSCalibur flow cytometer, using FITC-conjugated anti-human IgG as a secondary antibody. To determine the EC50, the drug concentration needed for half-maximal cytotoxicity response, we conducted a five-day treatment assay in a 96-well plate. Plate reading was performed three hours after the addition of the Alamar blue viability stain.

In conjunction with the vitro analysis, a preliminary preclinical study was conducted in NSCLC xenografted mouse models. Six animals were treated with 7.5 mg/kg ADC delivered intravenously once per week, and five animals were given a phosphate-buffered saline control. Mean tumor volume and body weight changes were recorded three times per week.

The ADC was also assessed for gross toxicities in the lung, trachea, heart, thymus, thyroid, stomach, small intestine, colon, liver, pancreas, spleen, ovary, prostate, brain, spinal cord, and leg muscles through histological tissue analysis. Blood cell counts and troponin and myoglobin expression were additionally recorded to analyze hematological and cardiac toxicity.

Results, Conclusions, and Discussions: SPR analysis of 3D1-MUC1-C binding kinetics yielded a dissociation constant of KD = 17nM with a kon of 1.2*10E8 1/s and a koff of 2.1*10E-3 1/s. The numbers fall within the range of typical values for antigen-antibody bindings.

Flow cytometry analysis demonstrated that 32.5% of H1975 cells expressed MUC1-C, while A549 cells exhibited insignificant fluorescence in the FITC range. For H1975 cells, the EC-50 was found to be 52nM (R2 = 0.992) and maximal cytotoxicity around 75%. The cell viability assay for A549 cells was largely unaffected by the therapeutic doses, indicating that the ADC has appropriately minimal effects on cell lines that do not present MUC1-C. This would potentially limit off-target toxicities. Further research could be done to assess the bystander effect of the ADC through a co-culture of H1975 and A549. This would provide insight on whether ADC binding in the tumor microenvironment would be able to eliminate a tumor with heterogeneous MUC1 expression.

In the preclinical study, 3D1-MMAE exhibited strong antitumor activity in NSCLC xenografts. On day 26, mice in the control group displayed a mean tumor volume of 2578.3 mm3, while mice in the ADC-treated group had a mean tumor volume of 15.8 mm3, a statistically significant difference (p < 0.001). While the control group needed to be sacrificed at this point due to tumor burden, all six mice in the experimental group subsequently demonstrated total cure effects with complete tumor regression followed by no visible tumor for 4 weeks.

After treatment with 3D1-MMAE, histopathology analysis of the tissue samples did not demonstrate any gross toxicities. In addition, blood cell counts and troponin and myoglobin levels were not significantly altered from those of the control group, indicating a lack of hematotoxicities and cardiac toxicities.

Detailed studies of bioavailability, determination of the minimum effective dose needed for anti-tumor potential and the maximum tolerated dose that does not induce mortality, morbidity, or signs of significant toxicities are ongoing studies being conducted by colleagues in the principal investigator's laboratory.

Acknowledgements (Optional):  This 3D1-MMAE ADC NSCLC work is part of larger immuno-oncology studies being conducted on anti-MUC1-C monoclonal antibodies by XYone Therapeutics Inc. We would like to acknowledge Dr. Anshu Goyal for his contributions in supporting the work.