UNDERSTANDING THE T-CELL AND B-CELL RECEPTOR REPERTOIRE IN CHILDREN AND YOUNG ADULTS WITH ANTI-DRUG ANTIBODIES AGAINST PEG ASPARAGINASE

The aim of this sub study is to identify biomarkers in children and young adults with Acute Lymphoblastic Leukemia with hypersensitivity to PEG-asparaginase.

Evidence strongly implicates humoral immunity in Asparaginase immunogenicity with neutralizing antibodies18. Asparaginase is a bacterial enzyme not naturally present in the human body, and therefore highly immunogenic. Upon administration, antigen-presenting cells internalize and present fragments of the enzyme to naïve T cells, driving their differentiation into T-helper cells signaling differentiation of B-cells. A subset of activated B-cells matures into long-lived plasma cells that home to the bone marrow, where they continuously secrete anti-asparaginase antibodies. Others differentiate into Memory-B-cells that enable rapid antibody responses. This immunological memory means that once sensitization occurs, re-exposure to asparaginase triggers strong hypersensitivity reactions that make further treatment unsafe. Yet, despite the clinical importance, only one study exists on TCR repertoire in response to anti-asparaginase antibodies, but no studies to date have directly profiled the B-cell receptor (BCR) repertoire in children with anti-asparaginase antibodies. The BCR sequencing has recently been developed. By integrating BCR-seq from blood and BM with serum proteomics (Ig-Seq), we can directly link specific plasma cell clones to circulating anti-asparaginase antibodies. This approach will provide mechanistic insight into drug immunogenicity and enable the identification of molecular biomarkers of hypersensitivity risk in children and young adults with ALL.

Methods:

The investigators will perform a longitudinal Danish cohort study in pediatric and young adult ALL patients receiving PEG-asparaginase as part of induction and post-consolidation therapy treated on the A2G-1. The investigators will include 20 patients with hypersensitivity and 20 without. Biological samples will be collected at baseline, during early post-exposure with PEG-asparaginase, at clinical hypersensitivity, and at follow-up timepoints according to the A2G-1. The investigators will pair these samples with EDTA-plasma samples from the same timepoints. The investigators will perform enrichment of plasma cells together with bulk TCR and BCR repertoire and clonotype sequencing. From plasma the investigators will perform serum proteomics to identify peptide fragments of circulating anti-asparaginase antibodies. Ig-Seq peptides will be aligned with BCR sequences from PBMC and BM to map specific antibodies to their clonal plasma cell origin. This approach yields a direct functional link between repertoire data and pathogenic antibodies.

The investigators will compare bone marrow repertoires with blood samples to determine whether circulating clones reflect the dominant marrow-resident plasma cells. If anti-asparaginase antibodies-specific BCRs can be reliably detected in blood, this may serve as a non-invasive biomarker, reducing the need for bone marrow sampling.