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The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibodydependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcgRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, cotargeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential. [ABSTRACT FROM AUTHOR]

Immune checkpoint blockade is a compelling approach in tumor immunotherapy. Blocking inhibitory pathways in T cells has demonstrated clinical efficacy in different types of cancer and may hold potential to also stimulate innate immune responses. A novel emerging potential target for immune checkpoint therapy is leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1). LILRB1 belongs to the superfamily of leukocyte immunoglobulin-like receptors and exerts inhibitory functions. The receptor is expressed by a variety of immune cells including macrophages as well as certain cytotoxic lymphocytes and contributes to the regulation of different immune responses by interaction with classical as well as non-classical human leukocyte antigen (HLA) class I molecules. LILRB1 has gained increasing attention as it has been demonstrated to function as a phagocytosis checkpoint on macrophages by recognizing HLA class I, which represents a 'Don't Eat Me!' signal that impairs phagocytic uptake of cancer cells, similar to CD47. The specific blockade of the HLA class I:LILRB1 axis may provide an option to promote phagocytosis by macrophages and also to enhance cytotoxic functions of T cells and natural killer (NK) cells. Currently, LILRB1 specific antibodies are in different stages of preclinical and clinical development. In this review, we introduce LILRB1 and highlight the features that make this immune checkpoint a promising target for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcg receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies' effector functions. [ABSTRACT FROM AUTHOR]

ULBP2:HER2-scFv simultaneously bound to HER2 and NKG2D and triggered NK cells to kill HER2-expressing tumor cells in the nanomolar concentration range. Keywords: ADCC; antibody therapy; EGFR; NK cells; NKG2D EN ADCC antibody therapy EGFR NK cells NKG2D 545 556 12 04/12/22 20220401 NES 220401 Introduction Exploiting natural killer (NK) cells is a promising approach in cancer immunotherapy. ULBP2:HER2-scFv induces NK cell cytotoxicity To analyze whether ULBP2:HER2-scFv was able to trigger NK cell cytotoxicity SP 51 sp Cr release experiments were performed (Figure 3A). [Extracted from the article]

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 glycoengineered, 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. [ABSTRACT FROM AUTHOR]

Abstract: In recent years, therapy with monoclonal antibodies has become standard of care in various clinical applications. Despite obvious clinical activity, not all patients respond and benefit from this generally well tolerated treatment option. Therefore, rational optimization of antibody therapy represents a major area of interest in translational research. Animal models and clinical data suggested important roles of Fc-mediated effector mechanisms such as antibody dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) in antibody therapy. These novel insights into the mechanisms of action mediated by monoclonal antibodies inspired the development of different engineering approaches to enhance/optimize antibodies’ effector functions. Fc-engineering approaches by altering the Fc-bound glycosylation profile or by exchanging amino acids in the protein backbone have been intensively studied. Here, advanced and emerging technologies in Fc-engineering resulting in altered ADCC and CDC activity are summarized and experimental strategies to evaluate antibodies’ effector functions are discussed. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]