Background: Engineering of the antibody's fragment crystallizable (Fc) by modifying the amino acid sequence (Fc protein engineering) or the glycosylation pattern (Fc glyco-engineering) allows enhancing effector functions of tumor targeting antibodies. Here, we investigated whether complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) of CD20 antibodies could be improved simultaneously by combining Fc protein engineering and glyco-engineering technologies. Methods and Results: Four variants of the CD20 antibody rituximab were generated: a native IgG1, a variant carrying the EFTAE modification (S267E/H268F/S324T/G236A/I332E) for enhanced CDC as well as glyco-engineered, non-fucosylated derivatives of both to boost ADCC. The antibodies bound CD20 specifically with similar affinity. Antibodies with EFTAE modification were more efficacious in mediating CDC, irrespective of fucosylation, than antibodies with wild-type sequences due to enhanced C1q binding. In contrast, non-fucosylated variants had an enhanced affinity to FcγRIIIA and improved ADCC activity. Importantly, the double-engineered antibody lacking fucose and carrying the EFTAE modification mediated both CDC and ADCC with higher efficacy than the native CD20 IgG1 antibody. Conclusion: Combining glyco-engineering and protein engineering technologies offers the opportunity to simultaneously enhance ADCC and CDC activities of therapeutic antibodies. This approach may represent an attractive strategy to further improve antibody therapy of cancer and deserves further evaluation.

Antibody-dependent cellular phagocytosis (ADCP) by macrophages, an important effector function of tumor targeting antibodies, is hampered by ‘Don´t Eat Me!’ signals such as CD47 expressed by cancer cells. Yet, human leukocyte antigen (HLA) class I expression may also impair ADCP by engaging leukocyte immunoglobulin-like receptor subfamily B (LILRB) member 1 (LILRB1) or LILRB2. Analysis of different lymphoma cell lines revealed that the ratio of CD20 to HLA class I cell surface molecules determined the sensitivity to ADCP by the combination of rituximab and an Fc-silent variant of the CD47 antibody magrolimab (CD47-IgGσ). To boost ADCP, Fc-silent antibodies against LILRB1 and LILRB2 were generated (LILRB1-IgGσ and LILRB2-IgGσ, respectively). While LILRB2-IgGσ was not effective, LILRB1-IgGσ significantly enhanced ADCP of lymphoma cell lines when combined with both rituximab and CD47-IgGσ. LILRB1-IgGσ promoted serial engulfment of lymphoma cells and potentiated ADCP by non-polarized M0 as well as polarized M1 and M2 macrophages, but required CD47 co-blockade and the presence of the CD20 antibody. Importantly, complementing rituximab and CD47-IgGσ, LILRB1-IgGσ increased ADCP of chronic lymphocytic leukemia (CLL) or lymphoma cells isolated from patients. Thus, dual checkpoint blockade of CD47 and LILRB1 may be promising to improve antibody therapy of CLL and lymphomas through enhancing ADCP by macrophages.

Background: Native cluster of differentiation (CD) 19 targeting antibodies are poorly effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are crucial effector functions of therapeutic antibodies in cancer immunotherapy. Both functions can be enhanced by engineering the antibody’s Fc region by altering the amino acid sequence (Fc protein-engineering) or the Fc-linked glycan (Fc glyco-engineering). We hypothesized that combining Fc glyco-engineering with Fc protein-engineering will rescue ADCC and CDC in CD19 antibodies. Results: Four versions of a CD19 antibody based on tafasitamab’s V-regions were generated: a native IgG1, an Fc protein-engineered version with amino acid exchanges S267E/H268F/S324T/G236A/I332E (EFTAE modification) to enhance CDC, and afucosylated, Fc glyco-engineered versions of both to promote ADCC. Irrespective of fucosylation, antibodies carrying the EFTAE modification had enhanced C1q binding and were superior in inducing CDC. In contrast, afucosylated versions exerted an enhanced affinity to Fcγ receptor IIIA and had increased ADCC activity. Of note, the double-engineered antibody harboring the EFTAE modification and lacking fucose triggered both CDC and ADCC more efficiently. Conclusions: Fc glyco-engineering and protein-engineering could be combined to enhance ADCC and CDC in CD19 antibodies and may allow the generation of antibodies with higher therapeutic efficacy by promoting two key functions simultaneously.

Human γδ T cells are innate-like T cells which are able to kill a broad range of tumour cells and thus may have potential for cancer immunotherapy. The activating receptor natural killer group 2 member D (NKG2D) plays a key role in regulating immune responses driven by γδ T cells. Here, we explored whether recombinant immunoligands consisting of a CD20 single-chain fragment variable (scFv) linked to a NKG2D ligand, either MHC class I chain-related protein A (MICA) or UL16 binding protein 2 (ULBP2), could be employed to engage γδ T cells for tumour cell killing. The two immunoligands, designated MICA:7D8 and ULBP2:7D8, respectively, enhanced cytotoxicity of ex vivo-expanded γδ T cells against CD20-positive lymphoma cells. Both Vδ1 and Vδ2 γδ T cells were triggered by MICA:7D8 or ULBP2:7D8. Killing of CD20-negative tumour cells was not induced by the immunoligands, indicating their antigen specificity. MICA:7D8 and ULBP2:7D8 acted in a dose-dependent manner and induced cytotoxicity at nanomolar concentrations. Importantly, chronic lymphocytic leukaemia (CLL) cells isolated from patients were sensitized by the two immunoligands for γδ T cell cytotoxicity. In a combination approach, the immunoligands were combined with bromohydrin pyrophosphate (BrHPP), an agonist for Vδ2 γδ T cells, which further enhanced the efficacy in target cell killing. Thus, employing tumour-directed recombinant immunoligands which engage NKG2D may represent an attractive strategy to enhance antitumour cytotoxicity of γδ T cells.

To identify new antibodies for the treatment of plasma cell disorders including multiple myeloma (MM), a single-chain Fragment variable (scFv) antibody library was generated by immunizing mice with patient-derived malignant plasma cells. To enrich antibodies binding myeloma antigens, phage display with cellular panning was performed. After depleting the immune library with leukocytes of healthy donors, selection of antibodies was done with L-363 plasma cell line in two consecutive panning rounds. Monitoring the antibodies’ enrichment throughout the panning by next-generation sequencing (NGS) identified several promising candidates. Initially, 41 unique scFv antibodies evolving from different B cell clones were selected. Nine of these antibodies strongly binding to myeloma cells and weakly binding to peripheral blood mononuclear cells (PBMC) were characterized. Using stably transfected Chinese hamster ovary cells expressing individual myeloma-associated antigens revealed that two antibodies bind CD38 and intercellular adhesion molecule-1 (ICAM-1), respectively, and 7 antibodies target yet unknown antigens. To evaluate the therapeutic potential of our new antibodies, in a first proof-of-concept study the CD38 binding scFv phage antibody was converted into a chimeric IgG1. Further analyses revealed that #5-CD38-IgG1 shared an overlapping epitope with daratumumab and isatuximab and had potent anti-myeloma activity comparable to the two clinically approved CD38 antibodies. These results indicate that by phage display and deep sequencing, new antibodies with therapeutic potential for MM immunotherapy can be identified.