DuoBody®-CD3x5T4 Induces Efficient T-cell Activation, Tumor Cell Killing in HNSCC Cell Lines


The DuoBody®-CD3x5T4 was found to induce secretion of granzyme B and efficiently kill tumor cells in co-cultures of healthy donor T cells and patient-derived head and neck squamous cell carcinoma cell lines.

Rieneke van de Ven, PhD

Rieneke van de Ven, PhD

The DuoBody®-CD3x5T4 (GEN1044) was found to induce secretion of granzyme B and efficiently kill tumor cells in co-cultures of healthy donor T cells and patient-derived head and neck squamous cell carcinoma (HNSCC) cell lines, according to data presented during the 2021 Society for Immunotherapy of Cancer (SITC) Annual Meeting.1

The agent also activated autologous CD4- and CD8-positive tumor-infiltrating lymphocytes (TILs), despite abundant PD-L1 expression in freshly dissociated 5T4-positive HNSCC biopsies. Preliminary findings demonstrated specific eradication of 5T4-expressing HSNCC tumor cells.

“5T4 was broadly expressed in HNSCC cell lines, tumor biopsies, and primary tumor cell suspensions,” Rieneke van de Ven, PhD, lead study author and a research associate in otolaryngology/head & neck surgery, cancer immunology, and cancer biology and immunology, at Amsterdam Universitair Medische Centra, said in a presentation on the data. “This dataset contributes to the preclinical evidence for targeting 5T4-expressing solid cancers with DuoBody®-CD3x5T4.”

5T4, or trophoblast glycoprotein, is expressed in several solid cancers, including HNSCCs. The CD3 bispecific antibody, DuoBody®-CD3x5T4, has been found to efficiently induce T-cell–mediated cytotoxicity in 5T4-postive tumor cells.

For the study presented during the meeting, investigators sought to examine the preclinical mechanism of action of DuoBody®-CD3x5T4 both in vitro and ex vivo, specifically using HNSCC as a case study.

Investigators identified 5T4 protein expression in HNSCC tumor specimens by utilizing immunohistochemistry (IHC) and flow cytometry. T-cell–mediated cytotoxicity and T-cell activation induced by DuoBody®-CD3x5T4 were examined in co-cultures of healthy donor T cells and patient-derived HNSCC cell lines in vitro. The capacity of the agent to then activate TILs was evaluated in freshly dissociated 5T4-expressing HNSCC tumor specimens ex vivo.

Patient samples were stained for 5T4 (EPR5529), PanCK (AE1/AE3), and CD3 (2GV6). A pathologist then scored the samples for 5T4 H-score and tumor-proportion score (TPS). An image analysis was also used to evaluate CD3 density in the samples. Results showed that 5T4 is expressed in primary HNSCC, lymph node metastases, and recurrent disease. No significant difference in 5T4 expression was noted between primary and lymph node metastases.

Investigators also utilized quantitative flow cytometry to determine the level of 5T4 surface expression on a panel of HNSCC cell lines. In total, 6 HNSCC cell lines were co-cultured with purified healthy donor T cells using various effector:target ratios and increasing concentrations of DuoBody®-CD3x5T4, BsAb-CD3xctrl, or BsAb-ctrl5T4. Granzyme B was quantified in the supernatants use ELISA and related to the T-cell mediated cytotoxicity induced by DuoBody®-CD3x5T4. Neither T-cell mediated cytotoxicity or granzyme B was reported in the BsAb-CD3xctrl or BsAb-ctrl5T4 conditions. DuoBody®-CD3x5T4 was found to induce production of granzyme B and tumor cell kill in co-cultures of T cells and HNSCC cell lines.

Using freshly dissociated HNSCC biopsies, investigators looked at 5T4 expression on tumor cells collected from anatomical subsites that included lung squamous cell carcinoma, human papillomavirus (HPV)­–negative oral squamous cell carcinoma (OSCC), OSCC, HPV-positive oropharyngeal squamous cell carcinoma, and hypopharyngeal squamous cell carcinoma. Evidence of 5T4 expression was found on the tumor cells, independent of tumor site.

The functional activity of DuoBody®-CD3x5T4 was examined in freshly dissociated tumor samples by incubating with 5 mg/mL of the agent or BsAb-CD3xctrl for 24 to 72 hours. TIL activation was then determined by flow cytometry. Investigators found that DuoBody®-CD3x5T4 can activate autologous CD4- and CD8-positive TILs.


  1. van de Ven R, Ganzevles SH, Veth M, et al. DuoBody®-CD3x5T4 induces efficient T-cell activation and killing of patient-derived head and neck cancer cells in vitro and ex vivo. Present at: 2021 Society for Immunotherapy of Cancer (SITC) Annual Meeting; November 10-14, 2021; Washington, DC. Poster 889.
Related Videos
Shridar Ganesan, MD, PhD
Patrick Boland, MD
Brett L. Ecker, MD
Kevin Kalinsky, MD, MS
Reva K Basho, MD
Alison K. Conlin, MD
Terry P. Mamounas, MD
Aditya Bardia, MD, MPH