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K. Klausz et al.
malignant plasma cells from MM patients treated with chemotherapy and especially in tumors with a multi-drug resistance phenotype.7 This is in line with the observation that adhesion molecules such as ICAM-1 are involved in macrophage-induced drug resistance and tumor escape in myeloma.8 In contrast, expression of ICAM-1 in healthy tissue, e.g., on endothelial cells and leukocytes, is constitu- tively low and increases only after stimulation with cytokines, i.e., interferon (IFN)-γ or interleukin (IL)-1β, which are up-regulated and released during inflammation and infection.9 Clinical trials with anti-ICAM-1 antibodies in patients with rheumatoid arthritis, renal transplants, stroke and myeloma proved safety and tolerability, but often lacked significant activity.10-13
Most therapeutic antibodies used for cancer immunotherapy are of immunoglobulin (Ig) G1 isotype and exert their anti-tumor activity via antigen-binding fragment (Fab)- and/or fragment crystallizable (Fc)-medi- ated effector functions directly through antigen binding and/or interactions with the immune system.14 The Fc-mediated effector functions encompass antibody- dependent cell-mediated cytotoxicity (ADCC), antibody- dependent cellular phagocytosis (ADCP) and comple- ment-dependent cytotoxicity (CDC). Over the years many efforts have been made to improve Fc-mediated effector functions of mAb to increase their cytotoxic and therapeutic activity by Fc glyco- and Fc protein-engineer- ing techniques. Fc protein-engineering approaches make use of specific amino acid substitutions in the Fcγ recep- tors (FcγR) and C1q binding interfaces of a therapeutic antibody to specifically improve ADCC, ADCP and/or CDC activity.15-19 Lazar and colleagues identified the S239D and I332E (DE) mutations in the CH2 domain of IgG1 antibodies, which markedly enhanced binding to the activating FcγRIIIa/CD16a and ADCC activity of a variety of therapeutic IgG1 antibodies.15 The DE mutations have been applied to generate an Fc-engineered (Fc-eng.) CD19 antibody that demonstrates promising therapeutic activity in clinical trials for relapsed/refractory B-cell non-Hodgkin lymphoma.20 Margetuximab, an Fc-optimized anti-HER2 antibody with reduced binding to FcγRIIb/CD32b and enhanced affinity for FcγRIIIa/CD16a,21 is currently evalu- ated in a phase III study for breast cancer and also high- lights the potential of Fc-engineering as a strategy to improve antibodies for cancer immunotherapy.
Here, we applied Fc-engineering to MSH-TP15, a novel, fully human anti-ICAM-1 IgG1 antibody whose variable regions are derived from TP15-Fc originally isolated by phage display and screening of myeloma cells.22 Fab- and Fc-mediated effector functions of MSH-TP15 were ana- lyzed by comparing three antibody variants that differed in their affinity for FcγR’s on immune cells. Antigen bind- ing, direct anti-tumor effects and immune cell recruitment for tumor cell lysis by the MSH-TP15 antibody variants were tested in vitro and in vivo against myeloma cells.
Methods
Cell separation
Mononuclear cells from peripheral blood (PBMC), pleural effu- sion (PE) and BM of myeloma patients and healthy donors were isolated as previously described.23 Samples were taken after receiv- ing donors’ written informed consents. Experiments were in accordance with the Declaration of Helsinki and approved by the
Ethics Committee of the Christian-Albrechts-University, Kiel, Germany (D442/10).
Cell lines
Ramos, CHO-K1, L363 and U266 were obtained from DSMZ (Braunschweig, Germany), Lenti-X 293T were purchased from Takara Bio (Göteborg, Sweden), MM1.S, Raji CS and CHO- FcγRIIa-131H were gifts from YT Tai (Boston, MA, USA), MJ Glennie (Southampton, UK), and F Nimmerjahn (Erlangen, Germany),24 respectively, and cultured as recommended. INA-6, INA-6.Tu1 and BHK-FcγRIIIa-158V were established in our labo- ratory and cultured as previously described.25,26
Production of MSH-TP15 antibody variants
Variable light (VL) and heavy chain (VH) sequences derived from phage PIII-1522 and BI-5056,27 were cloned into modified pSecTag2/HygroC vectors harboring sequences for the constant region of the human kappa light chain (LC) or either wild-type (wt) or mutated human IgG1 heavy chain (HC).28 Antibodies were termed MSH-TP15 (wt IgG1), MSH-TP15 Fc-engineered (Fc-eng.) and MSH-TP15 Fc knockout (Fc k.o.) and produced as described in the Online Supplementary Appendix.
Flow cytometric analyses
Immunofluorescence analyses to investigate mAb binding, induction of programmed cell death (PCD) and ADCP were per- formed on a Navios flow cytometer and analyzed with Kaluza software (Beckman Coulter). For details please refer to the Online Supplementary Appendix.
Co-culture experiments
BM stromal cells (BMSC) were isolated from MM patient BM aspirates and used for co-culture experiments with INA-6 cells as previously described.29
Cytotoxicity assays
ADCC activity was analyzed in 3 hour (h) 51Cr release assays using PBMC and natural killer (NK) cells at E:T ratios of 80:1 and 10:1, respectively, as previously described.23
Live cell imaging
INA-6 killing was analyzed over 24 h with IncuCyte caspase- 3/7 reagent (250 nM) in the presence or absence of NK cells (E:T ratio 10:1) and 20 mg/mL of the indicated antibodies with IncuCyte live cell imaging (Essen Bioscience). Green cell counts per image were analyzed with Zoom2016A software.
Animal experiments
All animal experiments were performed according to the guide- lines of the Christian-Albrechts-University Kiel along with the German Animal Protection Law. For both models 7-8 week-old female SCID/beige mice (Charles River, Sulzfeld, Germany) and red fluorescent protein-expressing INA-6.Tu1 cells (INA- 6.Tu1_red; unpublished) were used. In the intraperitoneal (i.p.) model, ten mice/group were injected with 20x106 INA-6.Tu1_red cells 48 h prior start of twice weekly i.p. treatment with mAb (ini- tial doses 10 mg/kg followed by six doses of 5 mg/kg mAb/ani- mal) or vehicle control. In the established tumor model 5x106 INA- 6.Tu1_red cells were subcutaneously (s.c.) injected into the right hind flank of five mice/group. At day 10, i.p. treatment with five doses of 10, 1 or 0.1 mg/kg mAb/mouse twice weekly was started. Tumor growth was measured by caliper and volume was calculat- ed: (length x width2)/2. Human IL-6 receptor was analyzed with CD126 enzyme-linked immunosorbent assay (ELISA) kit (Diaclone, Besançon, France).
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haematologica | 2021; 106(7)