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K. Klausz et al.
growth and the survival benefit compared to control mice, but significance was not reached (P=0.11). Of note, treat- ment with 0.1 mg/kg MSH-TP15 significantly retarded tumor growth and prolonged survival (27 days; P=0.03 vs. control group; Figure 5C and D). Treatment with 1 mg/kg and 10 mg/kg further improved tumor control and result- ed in a median survival of 33 days and 36 days, respective- ly (P<0.01 for both doses vs. control group). Importantly, with the Fc-optimized MSH-TP15 Fc-eng. survival was not only significantly prolonged compared to PBS-treated animals at doses of 0.1 mg/kg (29 days, P=0.02), 1 mg/kg (41 days, P<0.01) and 10 mg/kg (50 days, P<0.01), but also when compared to 10 mg/kg MSH-TP15 treated mice (P=0.03). This tendency of being more effective than the wt IgG1 mAb was also seen at 1 mg/kg doses, although differences did not reach statistical significance (P=0.12; Figure 5C). These results point to the importance of effi- cient effector cell recruitment for the anti-myeloma activ- ity of MSH-TP15 in vivo.
Discussion
In this study, we characterized the ICAM-1 binding epi- tope and modes of action of the novel human IgG1 anti- body MSH-TP15 originally isolated as scFv antibody by phage display and myeloma cell screening.22 Detailed binding analyses revealed that the MSH-TP15 epitope is located at the N-terminal D1-2 of human ICAM-1, which is a trans-membrane glycoprotein that consists of five, heavily glycosylated extracellular Ig domains.32 The major- ity of the ICAM-1 ligands bind to D1, including the β2-integrin LFA-1 (CD11a/CD18) expressed on all lym- phocytes.32 ICAM-1 and β2-integrin interactions play a pivotal role in lymphocyte activation and adhesion, leuko- cyte trafficking, and cellular immune responses. In gener- al, ICAM-1 and its ligands are expressed on lymphocytes, leukocytes and vascular endothelium, are increased upon stimulation with inflammatory cytokines like IFN-γ and mediate adhesion of immune cells to allow migration into tissue.9 Many anti-ICAM-1 antibodies, like RR1/1 and 84H10, bind to D1 or D2 and inhibit LFA-1-mediated adhesion of lymphocytes to ICAM-1 although they recog- nize different epitopes.30 Our cross-blocking experiments confirmed a unique ICAM-1 epitope recognized by RR1/1 distinct from that of 84H10, MSH-TP15 and BI-505. Potent blockade and thus overlapping epitopes were observed for MSH-TP15 and BI-505, while only partly or no overlapping epitopes were seen for the two human mAb with 84H10 and RR1/1. Accordingly, MSH-TP15 was incapable of inhibiting LFA-1 and ICAM-1 interac- tion. Of note, MSH-TP15 and BI-505 were both identified by phage display and are derived from a fully human scFv library based on the same VH (VH-DP47), but different LC (VL-DPL3 vs. DPK9) framework.22,33 This resulted in 89% VH sequence identity between the human IgG1l mAb BI- 505 and the human IgG1κ mAb MSH-TP15. Interestingly, in contrast to MSH-TP15, BI-505 was found by screening for apoptosis-inducing antibodies against Ramos Burkitt’s lymphoma cells.34 Its exact epitope was not determined, but our studies indicate that BI-505 and MSH-TP15 bind close epitopes of human ICAM-1 D1-2.
Despite its important role in immunological processes, ICAM-1 was found to be highly expressed on malignant plasma cells.35,36 There ICAM-1 seems to play a pivotal role
in myeloma cell adhesion to BMSC, and it is, thus consid- ered to be an important molecule in the tumor-microenvi- ronment and to be essential for immune escape of myelo- ma cells. In addition, ICAM-1 is thought to be involved in macrophage-induced drug resistance and was shown to be overexpressed on chemo-resistant residual myeloma cells. Furthermore, ICAM-1 expression correlates with tumor progression and metastasis in melanoma and renal cell car- cinoma patients,38,39 and was recently identified as poten- tial cancer stem cell marker in esophageal squamous cell carcinoma.40
Targeting ICAM-1 for therapy was initially evaluated with mouse antibodies that were clinically tested in rheumatoid arthritis, acute stroke and renal transplanta- tion.11-13 Production of human anti-mouse Ab caused seri- ous problems after repeated treatment.41,42 To date, BI-505 is the only clinically tested human anti-ICAM-1 Ab tested. Of note, repeated administration of 10 mg/kg BI-505 already saturated ICAM-1 on patient’s BM myeloma cells and up to 43 mg/kg were well tolerated.10,43
Many ICAM-1 targeting mAb, including BI-505 and MSH-TP15, exert only little Fab-mediated anti-tumor effects. MSH-TP15 also did not directly affect myeloma cell proliferation, but was capable of inhibiting IL-6- dependent INA-6 growth in the presence of BMSC from myeloma patients. This might be an interesting function regarding the supportive role of the BM microenviron- ment for survival and drug resistance of myeloma cells. Additionally, MSH-TP15 induced apoptosis of ICAM-1 expressing lymphoma cells after cross-linking on the cell surface – a mechanisms which was also described for BI- 505.6 No CDC but moderate ADCP activity and activa- tion of human NK cells for ADCC of myeloma cells was observed for MSH-TP15. Together these data suggest engagement of NK cells and phagocytes as a predominant effector function of MSH-TP15, a fact already evident for scFv-Fc antibody TP15-Fc.22 In an attempt to improve the Fc-mediated effector functions of MSH-TP15, we applied Fc protein-engineering. As intended, the DE mutations introduced into the Fc domain of MSH-TP15 Fc-eng. sub- stantially improved the antibody’s affinity for FcγRIIIa and FcγRIIa,15,44 resulting in enhanced ADCC and ADCP activity of MSH-TP15 Fc-eng. against myeloma cells in vitro. Of note, significant NK cell-mediated tumor cell lysis was also achieved for patient-derived myeloma cells. In vivo, wt and Fc-optimized MSH-TP15 controlled tumor growth in two myeloma xenograft models, while with the k.o. Ab variant no significant survival benefit com- pared to control mice was achieved. Myeloma cell growth was completely prevented or dose-dependently inhibited by MSH-TP15 and MSH-TP15 Fc-eng. and both antibody variants significantly prolonged survival of the mice. Importantly, improvement in tumor control and survival was also seen in vivo with the Fc-optimized MSH- TP15 Fc-eng. compared to MSH-TP15 pointing to an important role of immune cells in mice as well. Due to the fact that in SCDI/bg mice NK cell functions are impaired, most likely mouse myeloid effector cells (monocytes/macrophages, granulocytes) account for the observed differences. This is in line with previous find- ings that human IgG1 antibodies efficiently bind to mouse FcγRIV, which is the orthologue of human FcγRIIIa, but which is, in contrast to the human system, restricted to mouse myeloid cells and is not expressed on mouse NK cells.45,46 Since our novel anti-ICAM-1 antibod-
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haematologica | 2021; 106(7)