Targeting Human B-Precursor Acute Lymphoblastic Leukemia Cells with Recombinant Human CD19 Ligand

Abstract 599

CD19 is a 95-kDa B-lineage restricted receptor molecule that functions as a key regulator of transmembrane signals in both B-cells and B-cell precursors. Here we report the cloning and characterization of a novel high-mobility group (HMG)-box protein as the membrane-associated natural ligand of human CD19 receptor (CD19-L) on human immature as well as mature lymphoid cells. We cloned the gene encoding CD19-L from a human thymus cDNA library by yeast two-hybrid screening using the cDNA encoding human CD19 extracellular domain (AA 1 to 273) (CD19^ECD) fused to the GAL4 DNA binding domain as the bait plasmid. The cDNA for the surface membrane-associated CD19-L protein is 2290-bp in length encoding a 487-aa protein with a predicted molecular mass of 54-kDa. The comparison of the amino acid sequence of CD19-L protein with reported sequences in Genebank Database revealed that CD19-L is a new member of the HMG-box protein family. CD19-L contains two leucine-rich hydrophobic nuclear export signal (NES) motifs associated with unconventional ER- and Golgi-independent transport of nuclear/cytoplasmic secretory proteins to the surface membrane. Expression of the CD19-L gene expression is limited to the lymphocyte compartment within the human lymphohematopoietic system and particularly abundant in T-lineage cells. CD19-L displays abundant expression on immature double-positive (DP) thymocytes as well as leukemic T-cell precursors from T-lineage ALL patients corresponding to immature double-negative (DN) pro-thymocyte and DP cortico-thymocyte stages of human T-cell ontogeny. CD19-L is also expressed on B-lineage lymphoid cells at all stages of human B-cell ontogeny, including fetal liver derived biphenotypic CD2⁺CD19⁺ pro-B/T cells, pro-B cells, pre-B cells and mature B-cells. Soluble recombinant human CD19-L protein produced in a baculovirus expression system exhibited exquisite specificity for the extracellular domain of CD19 and had profound effects on apoptosis-related signaling and gene expression in CD19⁺ human leukemia cells. Engagement of CD19 co-receptor on B-lineage ALL cells with soluble CD19-L protein perturbed CD19-associated signaling network and triggered tyrosine phosphorylation of CD19 in a time-dependent fashion with peak phosphorylation occurring within 1–5 min. CD19-phosphorylation was associated with rapid and transient activation of SYK tyrosine kinase. Treatment of B-lineage ALL cells with 100 ng/mL CD19-L for 24 h corrupted the regulation of gene expression and altered the expression levels of 13 genes directly involved in regulation of apoptosis. We next examined the ability of CD19-L to induce apoptosis in leukemic B-cell precursors from chemotherapy-resistant CD19-positive human B-lineage ALL cell lines NALM-6 (pre-B ALL), RS4;11 (MLL-AF4⁺ Pro-B ALL), and ALL-1 (BCR-ABL⁺ Pre-pre-B ALL). CD19-L (but not control proteins CD19^ECD or CD19^ICD) caused apoptosis in each of these 3 ALL cell lines. As CD19-L specifically targets CD19^ECD, excess CD19^ECD protein was able to compete with surface CD19 receptor for CD19-L binding and thereby prevent CD19-L induced apoptosis. Excess CD19 intracellular domain protein (CD19^ICD) that was included as a negative control did not affect CD19-L induced apoptosis. CD19-L was also capable of causing apoptosis in chemotherapy-resistant primary leukemic cells from relapsed CD19⁺ B-lineage ALL patients. This collection of experimental data provides compelling evidence that CD19-L is a potent biotherapeutic new agent candidate against CD19⁺ lymphoid malignancies. The identification of human CD19-L may lead to therapeutic innovation for B-lineage ALL as well as other B-lineage lymphoid malignancies by providing an effective alternative to CD19-directed monoclonal antibody-based biotherapeutic agents that have encountered several limitations in clinical settings.