Development of Novel Human Anti-HLA-Monoclonal Antibodies for Clinical Applications Using Peripheral Blood B Cells Derived from Anti-HLA Antibody-Positive Donors

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. Potential efficacy of HLA allele-specific mAbs in treating malignant lymphoma has been shown by several studies. However, treatment of B-cell malignancies with humanized mAbs against HLA-DR alleles is associated with infusion-related toxicities (Lin et al., Leuk Lymphoma, 2009). In addition, previous attempts to develop mAbs specific to some HLA alleles (e.g., HLA-B61) by immunizing mice with recombinant HLA proteins have failed, likely because of a wide variety of polymorphisms of HLA molecules. To overcome these problems, 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 took approximately 1 month to produce human anti-HLA mAbs.

Methods: Approximately 20 ml of PB was collected from anti-HLA antibody-positive donors, and mononuclear cells (MNCs) isolated using the Ficoll-Hypaque method were cultured in RPMI-1640 + 10% FCS containing 5 μg/ml R-848, 1000 IU/ml hIL2, 2.5 μg/ml CpG2006, 2.5 μg/ml anti-CD40 antibody, 100 ng/ml hIL21, 2 ng/ml hIL17, 10 ng/ml hIL4, and 100 ng/ml hBAFF for 6 days. CD138⁺ cells were then isolated using anti-CD138-antibody-conjugated microbeads. A microwell array chip was manufactured using micromachining techniques, as previously described (Jin et al., Nature Medicine, 2009). Microwells (diameter, 10 μm; depth, 15 μm) were formed on a silicon surface. The chip was coated with a PBS-containing purified HLA antigen (10 μg/ml) and incubated overnight at 4°C. After removing the antigen solution, 50 μl of the CD138⁺ cell suspension was added to the chip, and the mixture was incubated for 4 h at 37°C. After gentle washing with PBS, 2 μg/ml of anti-human IgG Fc-Cy3 solution was added to the microwells, and the plate was left at room temperature for 15 min. Finally, the cells were stained with 1 μM Oregon Green for 2 min. The microwells were screened for the antigen-specific antibodies released from single cells under a fluorescence microscope. Antigen-specific antibody-secreting cells (ASCs) from individual wells were isolated using a micromanipulator fitted with capillaries under the fluorescence microscope and were then expelled to microtubes for reverse transcription. The antibody cDNA fragments for VH and VL fragments were amplified using the single-cell 5′-RACE method and inserted into expression vectors containing the complete constant region of cDNA for heavy or light chains. Thereafter, CHO cells were transfected with both the heavy and light chain expression vectors to obtain a supernatant containing complete antibody molecules. The antigen specificity of the recombinant antibodies was examined using ELISA and flow cytometry (FCM). The frequency of ASCs in the PB-MNCs of donors was quantified using allele-specific oligonucleotide PCR. Complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of the mAbs were assessed using conventional methods.

Results and Discussion: Two novel human mAbs specific to HLA-A24 and HLA-B61 were successfully generated. The antigen specificity of the mAbs was confirmed using ELISA and FCM. The mAbs bound to normal and malignant blood cells that expressed corresponding HLA alleles and showed CDC/ADCC activities against five B lymphoblastoid cell lines but not against lymphocytes/monocytes/granulocytes derived from five healthy donors, probably because of low expression levels of the target HLA alleles. The frequency of ASCs in PB-MNCs of donors was less than 0.001%.

Conclusions: The present method enabled the generation of mAbs specific to HLA alleles, which can be used for detecting minimal residual diseases of hematological malignancies as well as for treating B-cell lymphoma, within 1 month.