Poster Presentation Melbourne Immunotherapy Spring Symposium 2025

Identification of T:B cell multimers after bispecific T cell engagement, using both conventional and imaging flow cytometry (#132)

Mandy Ludford-Menting 1 2 , Joanne E Davis 1 2 , Darryl Johnson 3 , David S Ritchie 1 2 4 , Rachel Koldej 1 2
  1. ACRF Translational Research Laboratory, The Royal Melbourne Hospital, Melbourne, Victoria, Australia
  2. Department of Medicine, University of Melbourne, Melbourne, Victoria, Australia
  3. Materials Characterisation & Fabrication Platform, University of Melbourne, Melbourne, Victoria, Australia
  4. Clinical Haematology, Peter MacCallum Cancer Centre and Royal Melbourne Hospital, Melbourne, Victoria, Australia

Aim:
Bispecific T cell engagers bring together T cells with malignant B cell targets to enable synapse formation and disease eradication of haematological malignancies. The proportion of T cells and their ability to bind to their targets varies between patients and may be a biomarker of response. In this study, we developed a method to detect T and B cell conjugates, as measured by conventional flow cytometry, and confirmed using imaging flow cytometry.

 

Method:

Purified T cells from healthy donors were co-incubated with one of CD19+CD20+ tumour cell lines (Raji, Mino, JeKo-1) at a ratio of 1:1 in the presence of one of the bispecific antibodies blinatumomab, epcoritamab or glofitamab (10 ng/ml) for 1 hour at 37°C. Samples were acquired using a BD Fortessa and Cytek Amnis ImageStream flow cytometer, and synapse formation was determined by co-expression of T and tumour cell line markers. Analysis was performed using FlowJo, IDEAS, and PRISM software.

 

Results:

After bispecific antibody engagement multiple T cells were bound to each B cell (8.5-fold increase in Raji + blinatumomab), in complexes comprising of CD4+CD8-, CD4+CD8+ and CD4-CD8+ T cells. Imaging cytometry confirmed that the number and size of multimers increased significantly in the presence of bispecific antibodies. CD45RA and CCR7 cell surface memory markers on individual T cells within cell complexes were analysed and compared to conventional cytometry results.  

 

Conclusion:

Imaging flow cytometry confirmed conventional flow cytometry data showing increased T:B synapse and multimer formation after incubation with bispecific T cell engagers. Our novel gating strategy identified T cell multimers bound to target cells with different frequencies depending on the bispecific antibody used. This assay has been applied to blood samples from patients on clinical trials, providing a real-world assessment of bispecific antibody efficacy.