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Defucosylation of Tumor-Specific Humanized Anti-MUC1 Monoclonal Antibody Enhances NK Cell-Mediated Anti-Tumor Cell Cytotoxicity.

ABSTRACT: Antibodies are commonly used in cancer immunotherapy because of their high specificity for tumor-associated antigens. The binding of antibodies can have direct effects on tumor cells but also engages natural killer (NK) cells via their Fc receptor. Mucin 1 (MUC1) is a highly glycosylated protein expressed in normal epithelial cells, while the under-glycosylated MUC1 epitope (MUC1-Tn/STn) is only expressed on malignant cells, making it an interesting diagnostic and therapeutic target. Several anti-MUC1 antibodies have been tested for therapeutic applications in solid tumors thus far without clinical success. Herein, we describe the generation of fully humanized antibodies based on the murine 5E5 antibody, targeting the tumor-specific MUC1-Tn/STn epitope. We confirmed that these antibodies specifically recognize tumor-associated MUC1 epitopes and can activate human NK cells in vitro. Defucosylation of these newly developed anti-MUC1 antibodies further enhanced antigen-dependent cellular cytotoxicity (ADCC) mediated by NK cells. We show that endocytosis inhibitors augment the availability of MUC1-Tn/STn epitopes on tumor cells but do not further enhance ADCC in NK cells. Collectively, this study describes novel fully humanized anti-MUC1 antibodies that, especially after defucosylation, are promising therapeutic candidates for cellular immunotherapy.

SUBMITTER: Gong Y

PROVIDER: S-EPMC8197514 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

Project description:BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties.MethodsTo further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions.ResultsIn Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions.ConclusionThis study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

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Defucosylation of Tumor-Specific Humanized Anti-MUC1 Monoclonal Antibody Enhances NK Cell-Mediated Anti-Tumor Cell Cytotoxicity.

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