Abstract
Blinatumomab is an anti-CD19 bispecific T cell engager designed to bring together CD3+ T cells with CD19+ malignant B cell targets to enable synapse formation and disease eradication of B-ALL. We hypothesised that the proportion of T cells and their ability to bind to their targets may may be a biomarker of response. In this study, we assessed the effect of blinatumomab co-administered with reduced intensity chemotherapy in newly diagnosed B-ALL patients. Furthermore, we developed a method to detect T and B cell conjugates, as measured by conventional flow cytometry, and confirmed using imaging flow cytometry. The research is a cooperative trial group study lead by Australasian Leukemia and Lymphoma Group (ALLG), as ALLG ALL08.
Patients with untreated Ph-ve B-ALL aged 40-65 years were enrolled on the ALLG sponsored ALL08 study (ACTRN12617000084381) and treated with alternating cycles of blinatumomab and methotrexate/cytarabine. Peripheral blood mononuclear cells (PBMC) were collected from patients (n=30) at baseline and after C1D28, and healthy age matched donors (n=5). Immune phenotyping of PBMC was performed using spectral cytometry (31-plex, Cytek Aurora). Live video microscopy of purified T cells co-incubated with CD19+ Raji target cells +/- blinatumomab (10 ng/ml, Leica microscope), and whole PBMC synapse assays +/- blinatumomab (BD Fortessa and Cytek Amnis ImageStream flow cytometer) were performed. T:B cell synapse formation was determined by co-expression of T and tumour cell markers. Analysis was conducted using FlowJo, IMARIS, IDEAS and PRISM software.
After 1 cycle of blinatumomab treatment, CD19+ B cells decreased and naïve CD8+TIM3+ T cells significantly increased in the blood of B-ALL patients. Live video microscopy demonstrated the number of synapses formed after C1D28 halved compared to the screening sample. Furthermore, patients with a complete response had significantly higher synapse formation at screening, compared to patients with relapsed disease. Subsequent analysis using the whole PBMC synapse assay indicated fewer T cell multimers bound to endogenous B cells when the tumour burden exceeded 20% of circulating PBMC. After blinatumomab engagement multiple T cells were bound to each B cell in complexes comprising of CD4+CD8-, CD4+CD8+ and CD4-CD8+ T cells. Imaging flow cytometry confirmed that the number and size of multimers increased significantly in the presence of bispecific antibodies.
Blinatumomab-driven synapse formation was reduced in patients with high B-ALL burden and resulted in increased naive CD8+ T cell frequency in the blood following one cycle of therapy. Decreased synapse formation by T cells collected prior to blinatumomab therapy was associated with clinical relapse, indicating impaired blinatumomab-mediated T cell function. Despite evident ongoing T cell-B cell synapse formation there was a failure of clearance of residual B cells. Optimal blinatumomab therapeutic efficacy may require modifications of dosing schedule based on disease burden and demonstration of effective T cell engagement. Our novel whole PBMC synapse assay identified T cell multimers bound to target cells with different frequencies depending on the percentage of endogenous B cells present in the blood, which may be used as a biomarker to provide a real-world assessment of bispecific antibody efficacy.
No conflict of interest to disclose. Drug and funding support was provided by Amgen for the conduct of this study, however they had no role in the study design, conduct, analysis or interpretation of results
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