Abstract
Introduction: The development of monoclonal antibodies (mAbs), such as anti-CD20 mAb (rituximab) and anti-CD38 mAb (daratumumab), has revolutionized the treatment of lymphoid malignancies. The potential efficacy of anti-HLA mAbs in treating hematological malignancies has been reported in several studies. Previous attempts to develop mAbs specific for some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of HLA molecule polymorphisms. To overcome this problem, we recently created a novel method to develop mAbs using microarray technology and human peripheral blood (PB) B lymphocytes derived from donors who are positive for anti-HLA antibodies. The process takes approximately 1 month to produce human anti-HLA mAbs to treat myeloma and lymphoma.
Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells were cultured in RPMI-1640 + 10% FCS containing a cytokine cocktail. CD138+ cells were then isolated using anti-CD138-antibody-conjugated microbeads. Microarray chips were coated with a PBS-containing purified HLA antigen (10 μg/ml). After removing the antigen solution, 100 μl of the CD138+ cell suspension was added to the chip. After washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells. Finally, antigen-specific antibody-secreting cells (ASCs) from individual microwells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA was amplified and transfected into HEK293 cells to obtain a supernatant containing complete antibody molecules (Zaimoku et al., Blood 2017). The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry. Next-generation sequencing (Adaptive Biotechnologies Corp) was used to quantify the ASC frequency in PB of donors. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of mAbs were assessed using conventional methods. The following cell lines and PB mononuclear cells (PBMCs) from 5 healthy individuals who possessed Bw4 and/or B61 were used for CDC/ADCC assays: 5 B-lymphoblastoid cell lines (B-LCLs), 11 myeloma cell lines (AM01, IM-9, KMS-12BM, KMS-18, KMS-28PE, KMS-34, LP-1, NCI929, OPM-2, RPMI8226, and U266), 4 mantle cell lymphoma cell lines (JVM, Jeko, Mino, and Z138), and 2 diffuse-large B cell lymphoma cell lines (KPUM-UH1 and KPUM-MS3). Daratumumab, elotuzumab, and anti-influenza virus human mAbs were used as control mAbs.
Results and Discussion: Two novel human mAbs specific for HLA-Bw4 or HLA-B61 were successfully generated. The ASC frequency that produced anti-Bw4 and B61 mAbs in the cytokine-stimulated PBMCs derived from the donors were 9.0 × 10−5 and 1.4 × 10−5, respectively. Flow cytometry showed that mAbs bound to all normal PB cells and malignant lymphoid cells with corresponding alleles, but the expression levels of the HLA alleles were lower in normal PBMCs than in malignant cells (HLA-Bw4-mean florescence intensity (MFI), 1098 ± 246 vs. 6556 ± 4045, P =0.02). mAbs showed CDC /ADCC activities against all 5 B-LCLs, 6 of 9 myeloma, and 2 of 2 lymphoma cell lines with corresponding alleles but not against 2 myeloma and 4 lymphoma cell lines without corresponding alleles; they showed very weak CDC activities against PBMCs derived from 5 healthy donors who have corresponding alleles (CDC, 39 ± 16 vs. 4 ± 3%, P =0.001). Four myeloma cell lines (IM-9, KMS-28PE, KMS-34, and U266) that had low CD38/high CD59 expression and were resistant to daratumumab were killed by anti-Bw4 and/or HLA-B61 mAbs. In keeping with a current report showing low CDC activities of daratumumab on myeloma cells that highly express CD55/CD59 (Nijhof et al., Blood 2016), some myeloma cell lines highly expressing CD59 were resistant to CDC induced by anti-Bw4/HLA-B61 mAbs despite their high expression levels of HLA alleles (Figure 1AB). Elotuzumab showed obvious CDC and ADCC activities against 0 of 11 and 2 (KMS-34 and U266) of 11 myeloma cell lines, respectively.
Conclusions: NovelmAbs specific for HLA-Bw4 or B61 showed in vitro preferential killing of B-cell malignancies while sparing normal PBMCs, suggesting their potential usefulness in treating malignant lymphoma and myeloma.
Tanaka: HLA Laboratory: Employment. Kawai: Wakunaga Pharmaceutical Co., Ltd.: Employment.
