Enhancement of antibody-dependent cellular phagocytosis of lymphoma cells by the blockade of HLA class I receptors

Zeller, Tobias Sebastian (2025): Enhancement of antibody-dependent cellular phagocytosis of lymphoma cells by the blockade of HLA class I receptors. Dissertation, LMU München: Medizinische Fakultät

Abstract

Antibody-dependent cellular phagocytosis (ADCP) of cancer cells by macrophages is a major mechanism of action of therapeutic antibodies. However, ADCP is hampered by the expression of ‘Don’t Eat Me!’ signals such as CD47 on tumor cells. In clinical studies in lymphoma patients, encouraging outcomes were obtained by combining the CD20 antibody rituximab (RTX) with the CD47 blocking antibody magrolimab. Recently, in solid tumor models also class I human leukocyte antigens (HLA), which ligate the macrophage receptors leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) and LILRB2, were found to protect cells from phagocytosis. Whether ADCP of lymphoma cells can be augmented by antibody blockade of LILRB1 or LILRB2 is currently not known and was analyzed in this thesis. Along this line, investigations in a set of lymphoma cell lines showed a positive correlation between the CD20-to-HLA class I expression ratio and the extent of ADCP induced by co-treatment with RTX and a CD47 antibody. This indicates that the expression of HLA class I hampered ADCP under CD47 blockade. To specifically block HLA class I receptors, anti-LILRB1 and anti-LILRB2 antibodies were generated as Fc-silent versions (LILRB1-IgGσ and LILRB2-IgGσ, respectively) with abrogated Fcγ receptor (FcγR) binding. Both were produced by expression in human cells and purification via affinity chromatography. Antigen-specificity was demonstrated using flow cytometry. ADCP was analyzed by fluorescence microscopy or live cell imaging. To this, monocytes were enriched from peripheral blood and differentiated to macrophages with M0, M1 or M2 polarization status ex vivo. As a result, LILRB1-IgGσ significantly improved ADCP of different lymphoma cell lines by M0, M1 or M2 macrophages when the antibody was combined with RTX and an Fc-silent magrolimab variant (CD47-IgGσ). LILRB1-IgGσ was even effective with DG-75 Burkitt lymphoma cells, which were barely phagocytozed upon treatment with RTX and CD47-IgGσ only. LILRB1-IgGσ facilitated the uptake of multiple target cells by individual macrophages, but constantly required the simultaneous blockade of CD47 and the combination with a CD20 antibody for FcγR activation to unfold its effect. Remarkably, LILRB1-IgGσ notably increased the phagocytosis of freshly isolated cancer cells from patients with chronic lymphocytic leukemia or mantle cell lymphoma. LILRB2-IgGσ, on the other hand, was ineffective for yet unknown reasons, even though the antibody effectively impeded receptor ligation by HLA class I. Consequently, the expression of HLA class I by lymphoma cells limits the potential of the combination of CD20 and CD47 antibodies to initiate phagocytosis. This hinderance can be overcome by masking LILRB1, but not LILRB2. Thus, dual LILRB1/CD47 checkpoint blockade is an encouraging new approach to further improve CD20 antibody therapy of lymphomas.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: antibody-based immunotherapy, macrophages, phagocytosis, ADCP, CD20, CD47, LILRB1, LILRB2, innate immune checkpoint blockade, lymphoma
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 16. Oktober 2025
• 1. Bericht­erstatter:in: Kellner, Christian