Introduction
Multiple Myeloma (MM) is a malignant plasma cell disorder with high incidence and mortality. Monoclonal antibodies represent promising new treatment options and are widely applied in cancer therapy, but none have yet been approved for the treatment of MM. Daratumumab and other antibodies have recently shown encouraging results in clinical trials, but there is still a need to identify plasma cell specific target structures suitable for this approach.
Methods
To identify novel plasma cell surface antigens or epitopes, synthetic human single-chain fragment variable (scFv) phage display libraries were screened by using a newly established subtractive, cellular screening approach. Pre-absorption steps with granulocytes and T lymphocytes were applied with subsequent positive selection for phages that show binding to plasma cell lines INA-6 and JK-6L. The binding patterns of the resulting phage antibodies on various cell types were evaluated by cellular ELISA and flow cytometry. Based on its favorable binding profile, MSH-TP15 was selected and the coding sequence was used to generate a fully human scFv-Fc fusion protein (mini-antibody) for functional assays.
Results
Screening of phage display libraries by using our novel strategy resulted in a number of phage clones with preferential binding to MM cells. MSH-TP15 was selected based upon its strong binding to five MM cell lines (INA-6, JK-6L, L363, RPMI-8226, U266), while not recognizing a B cell precursor leukemia cell line (SEM), an immature acute myeloid leukemia cell line (KG-1a), and T cell derived acute lymphoblastic leukemia cell lines (CEM, Jurkat). In addition to MM cells, two Burkitt lymphoma cell lines (Daudi, Ramos) were also positive. Importantly, patient-derived CD138-positive malignant plasma cells were recognized while no reactivity was seen on other mononuclear cells of the same patient. No significant binding was seen on peripheral blood T lymphocytes, NK cells, monocytes, and granulocytes from healthy donors. Additional screening on a panel of stably transfected cell lines that express antigens already considered for antibody-based MM therapy (CD38, CD40, CD56, CD70, CD138, CS1, FGFR3, IGF-1R, IL-6R) revealed no reactivity. To subsequently evaluate direct (e.g. growth inhibition) and indirect (e.g. antibody-dependent cell-mediated cytotoxicity; ADCC) antibody effector functions, the coding sequence was used to generate a scFv-Fc fusion protein. This mini-antibody successfully retained its prior established binding properties. The antibody by itself was not capable to induce significant growth inhibition of INA-6, L363, and RPMI-8226 plasma cell lines. In contrast, MSH-TP15 was able to trigger ADCC, an important killing mechanism of therapeutic antibodies, against these myeloma cells with peripheral blood mononuclear cells as effector cells.
Conclusions
Phage display technology together with a cell-based screening approach led to the generation of an antibody that preferentially binds to MM cells, and is effective in antibody-mediated killing mechanisms. The recognized antigen is distinct from any of the MM antigens currently explored as targets for antibody therapy, and therefore represents a promising target structure suitable for an antibody-based therapeutic approach in myeloma.
Disclosures:
No relevant conflicts of interest to declare.
Development of Anti-Infectives Using Phage Display: Biological Agents against Bacteria, Viruses, and Parasites