Identification of New Antibodies Targeting Malignant Plasma Cells for Immunotherapy by Next-Generation Sequencing-Assisted Phage Display

Abstract

To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies’ enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified.

Combining Cellular Immunization and Phage Display Screening Results in Novel, FcγRI-Specific Antibodies

“…Total RNA from the spleen was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. A murine scFv antibody library was generated from RNA as previously described [ 13 ]. Briefly, cDNA was synthesized from total RNA using oligo(dT) 15 primers and reverse transcriptase.…”

“…Phages were prepared from E. coli and titrated as described [ 13 ]. For the depletion of unspecific Ba/F3 binders, 2 × 10 7 Ba/F3 cells were incubated with 10 11 –10 12 phages in 2 mL PBS supplemented with 4% bovine serum albumin (BSA) for 1 h on a roller incubator at room temperature.…”

“…For elution, cells were incubated in 1.5 mL 50 mM HCl solution for 10 min at room temperature and neutralized with 0.5 mL 1 M TRIS/HCl buffer (pH 7.5). Cells were separated by centrifugation (18,000× g , 10 min) and E. coli were infected with eluted phages as described [ 13 ]. Pooled E. coli were used to prepare phages, and a second cellular panning step was performed as described above without a depletion step with Ba/F3 cells.…”

“…Monoclonal phage antibodies were prepared from individual E. coli colonies as described [ 13 ]. For whole-cell ELISA, 96-well plates were blocked with PBS supplemented with 4% BSA.…”

“…For NGS, phagemids from pooled bacterial colonies of the initial scFv antibody library the antibody library after the first panning and the antibody library after the second panning were prepared using the NucleoBond Xtra Maxi kit (Machery-Nagel, Düren, Germany). Preparation of samples and NGS was performed as previously described [ 13 ]. Briefly, VHs were amplified by PCR to add adapters and indexes.…”