B7-H3 Trike Enhances Killing of Myeloid Derived Suppressor Cells in Multiple Myeloma

Abstract

Multiple myeloma (MM) is sensitive to Natural Killer (NK) cell-mediated killing but develops methods to attenuate NK cell function. One method to enhance NK cell specificity against myeloma is antibody dependent cellular cytotoxicity through the CD16 receptor. We developed a tri-specific killer engager (TriKE®) with a camelid single-domain antibody (sdAb) fragment against CD16 (on NK cells) as well as a linker region containing IL-15, to enhance NK cell survival. Delivery of IL-15 by TriKE supports NK cell proliferation while avoiding off-target effects on T cells. The second sdAb targets B7-H3 (CD276), a tumor antigen that is expressed on myeloma and is associated with decreased progression free survival. B7-H3 is differentially expressed on myeloma tumor lines MM1S, RPMI-8226, U266, and H929 but all four MM lines are significantly killed by peripheral blood NK cells in the presence of 3nM B7-H3 TriKE compared to NK cells alone. Bone marrow aspirates from 5 MM patients (3 newly diagnosed and 2 relapsed) were analyzed by flow cytometry and showed 58-96% expression of B7-H3 on CD138+ plasma cells. The bone marrow biopsy aspirates also showed monocytic myeloid derived suppressor cells (Mo-MDSC, CD14+, CD11b+) that expressed B7-H3 (38-87%) as well as the damage associated molecular pattern (DAMP) S100A8/9. In comparison, CD14+ cells from the peripheral blood of healthy donors expressed very low levels of B7-H3 or S100A8/9 (<5%). Mo-MDSC are important to the pathogenesis of MM because they support MM growth and contribute to suppression of NK in the tumor microenvironment. B7-H3 is highly expressed on mature osteoclasts and MDSC can develop into osteoclasts in mouse models of MM and breast cancer. We saw that Mo-MDSC expressing B7-H3 also co-expressed RANK, a receptor necessary for signaling osteoclastic development. We developed Mo-MDSC from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF or co-culture with MM cell lines at 1:1000 ratio. Both types of MDSC suppressed NK proliferation. Peripheral blood derived MDSC expressed >80% B7-H3 and RANK. We separated B7-H3 positive and negative populations by flow cytometry and attempted to develop osteoclasts with M-CSF and RANK-L over 14 days. Tartrate resistant acid phosphatase (TRAP) staining was performed but both groups were negative and exhibited no phenotypic changes by light microscopy. We then isolated CD14+ cells from the bone marrow of an MM patient who was in complete remission (CR) after autologous stem cell transplant but had a distinct population of B7-H3+, RANK+, S100A8/9+ Mo-MDSC in her surveillance bone marrow aspirate. After fourteen days of culture with M-CSF and RANK-L, TRAP staining was positive, and cells had grown larger, consistent with osteoclastic differentiation (Figure 1A). We tested the ability of B7-H3 TriKE to eliminate MDSC by NK cells, therefore diminishing there immune suppression. Cytotoxicity assays were conducted over 48 hours with NK cells with or without B7-H3 TriKE. Killing was significantly enhanced by B7-H3 TriKE (Figure 1B). Since MDSC are known to suppress NK cell function, we assessed metabolic fitness in NK cells with MDSC at 1:1 ratio for 48 hours with or without 3nM B7-H3 TriKE and found that B7-H3 TriKE improved both the oxygen consumption and extracellular acidification rates. These data show that B7-H3 TriKE enhances NK cell killing of MM through direct targeting and eliminates suppressive Mo-MDSC. B7-H3+ Mo-MDSC can differentiate into osteoclasts and persist even in patients who are in CR, possibly contributing to relapse. B7-H3 TriKE may be particularly useful for patients with bone lesions or as consolidative therapy. Commercial manufacturing of B7-H3 TriKE (GTB-5550) has begun, and a phase I trial is expected in late 2023. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal