Direct Visualization of PR1/HLA-A2 on the Membrane of HLAA2+ CD13+CD33+ Myeloid Leukemia Blasts by a Novel Monoclonal Antibody (2008)

Abstract PR1 (VLQELNVTV), the HLA-A2-restricted leukemia-associated antigen (LAA) derived from proteinase 3 (P3) and neutrophil elastase (NE), is targeted by CD8+ T lymphocytes (PR1-CTL) that lyse leukemia but not healthy hematopoietic cells. It is thought that aberrant P3 and NE expression in leukemia causes differential susceptibility to PR1-CTL lysis, perhaps by overexpression of PR1/HLA-A2 on leukemia. Importantly, while PR1-CTL with high affinity T cell receptor (TCR)-αβ are more effective killers of leukemia than low affinity PR1-CTL, they are also deleted by target cells, inducing tolerance and outgrowth of leukemia. Until now, it has not been possible to measure the surface density of PR1/HLA-A2 so critical issues of target specificity and tolerance could be addressed. If PR1 could be directly detected on leukemia, patients might be identified that could benefit from PR1-directed immunotherapy. We sought to develop a monoclonal antibody with specificity for PR1/HLA-A2 to provide direct visualization of the target molecules on leukemia and normal cells. We used two strategies to immunize BALB/c mice: first, PR1-pulsed TAP-deficient T2 cells were injected into the footpads; in the second approach, PR1 refolded with recombinant HLA-A*0201 + β2-microglobulin was injected through intraperitoneal and subcutaneous routes. Draining lymph nodes and spleens was collected and the B cells were fused with myeloma cells to create hybridomas. In the first approach, 1600 hybridoma-derived clones were screened by ELISA with PR1/HLA-A2 monomers, but no specific clones were identified. However, hybridoma clones derived using the second approach resulted in one positive clone from among 950 (designated clone 8F4). Using PR1/HLA-A2 monomers in an ELISA, we confirmed PR1 specificity and showed absence of binding to control peptides including the HLA-A2-restricted peptides pp65 from CMV, FLU peptide from Influenza, and WT1 from Wilm’s tumor antigen. Peptides with amino acid substitutions created at each of the 9 positions in PR1 were used to confirm that modification at P1 from V to A completely abrogated binding by ELISA. 8F4 was identified as IgG2a by standard ELISA using isotype-specific antibodies. Binding affinity of 8F4 was determined to be KD=10 nM by BIACore, compared to KD=162 nM for the common anti-HLA-A*0201 monoclonal antibody BB7.2. The high binding of 8F4 to PR1/HLA-A2 was due largely to its relatively slow off-rate compared to BB7.2. Next, binding specificity was confirmed by FACS using T2 cells pulsed with PR1 and the same control peptides used in the ELISA. To determine whether PR1 could be identified on leukemia, circulating blasts from HLA-A2+ and A2− patients were analyzed with 8F4 directly conjugated to Alexa647 in combination with other surface makers to distinguish blasts. While only 31% of normal monocytes showed minimal 8F4 binding, 73% and 79% of FAB-M1 and FAB-M5 blasts (live CD13+CD33+) bound 8F4 with &amp;gt; 60% staining intensity (MFI) compared to normal monocytes. While PR1 appears to be present on normal cells, AML blasts are preferentially lysed by PR1-CTL but normal monocytes are not. Therefore, PR1-specific TCR-αβ can distinguish small differences in peptide density on normal cells and leukemia. While monoclonal antibodies have been used successfully to treat hematological malignancies, their lack of specificity for tumor results in significant toxicity. It is likely that an antibody against a known LAA+MHC that is expressed only on the leukemia cell membrane would be therapeutically useful without off-target toxicity. Thus, anti-PR1/HLA-A2 antibodies can quantify PR1/HLA-A2 molecules on the leukemia cell surface and can distinguish leukemia from healthy APC.