No abstract

Tumors exploit numerous immune checkpoints, including those deployed by myeloid cells to curtail antitumor immunity. Here, we show that the C-type lectin receptor CLEC-1 expressed by myeloid cells senses dead cells killed by programmed necrosis. Moreover, we identified Tripartite Motif Containing 21 (TRIM21) as an endogenous ligand overexpressed in various cancers. We observed that the combination of CLEC-1 blockade with chemotherapy prolonged mouse survival in tumor models. Loss of CLEC-1 reduced the accumulation of immunosuppressive myeloid cells in tumors and invigorated the activation state of dendritic cells (DCs), thereby increasing T cell responses. Mechanistically, we found that the absence of CLEC-1 increased the cross-presentation of dead cell–associated antigens by conventional type-1 DCs. We identified antihuman CLEC-1 antagonist antibodies able to enhance antitumor immunity in CLEC-1 humanized mice. Together, our results demonstrate that CLEC-1 acts as an immune checkpoint in myeloid cells and support CLEC-1 as a novel target for cancer immunotherapy.

Myeloid cells represent one of the most abundant immune cell types in solid tumors and are generally associated with poor clinical outcome. We previously identified the orphan C-type lectin receptors (CLRs) CLEC-1 as over-expressed in situation of established immune tolerance and reported that CLEC-1 expression by dendritic cells (DCs) and macrophages is enhanced by TGFβ and tempers downstream T cells responses. Recent literature demonstrates that tumors could hijack physiological mechanisms of CLRs that normally restrain immune cell-mediated tissue damage to suppress myeloid cell activation and promote tumor immune evasion. Here we report that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment (e.g. ovarian cancer) and significantly more expressed by in vitro generated M2 macrophages compared to monocytes or M1 macrophages. RNAseq public data indicates that Clec1a is highly expressed in XCR1-expressing DC including CD8α+ and CD103+ DCs from lymphoid and peripheral tissues respectively. As these DCs are specialized in cross-presentation of dying/dead cell associated antigens, we evaluated whether CLEC-1 could be a receptor of damaged cells. We found that both human and mouse CLEC-1 fusion protein, but not irrelevant CLRs or negative controls, binds specifically to late apoptotic and secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray) or culture stress conditions. No binding was observed with viable, primary necrotic or with early apoptotic cells suggesting that CLEC-1 ligand(s) (CLEC-1L) correspond(s) to DAMP modified during the cell death process. Using a CLEC-1 reporter NFAT-lacZ BWZ.36 cell line, we show that this interaction is functional. Importantly, we observed in vivo that dendritic cells from Clec1a deficient mice more efficiently cross-present antigen (OVA-loaded dead cells) to CD8+ T cells (OT-1) and that CLEC-1 deficient mice, but not wild-type, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy (eg. cyclophosphamide). Treatment of wild-type mice with a Fc-CLEC-1 fusion protein also synergized with chemotherapy and induced complete response in 37% of mice versus 0% in chemotherapy group. We then generated, screened and identified different anti-human Clec-1 antagonist monoclonal antibodies (mAbs) with the capacity to block the CLEC-1/CLEC-1L interaction. We discovered that antagonist CLEC-1 mAbs, but not non-antagonist CLEC-1 control mAbs, increase the phagocytosis of CLEC-1L-positive human tumor cells by human TGFβ-polarized DCs but also of CLEC-1L-positive opsonized tumor cells (eg: Raji cells opsonized with anti-CD20 Rituximab) by human macrophages. Altogether, these data indicate that tumor cells inhibit myeloid cells phagocytosis through CLEC-1 and that antagonists of the CLEC-1/CLEC-1L novel myeloid checkpoint pathway constitute a novel cancer immunotherapy approach synergistic with chemotherapy. Citation Format: Vanessa Gauttier, Marion Drouin, Javier Saenz, Bérangère Evrard, Caroline Mary, Géraldine Teppaz, Ariane Desalle, Virginie Thépénier, Emmanuelle Wilhelm, Nicolas Poirier, Elise Chiffoleau. CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy controlling damaged and tumor cells phagocytosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3259.

BackgroundMyeloid cells represent one of the most abundant immune cell types in solid tumors that impede myeloid phagocytosis by triggering ‘don’t eat me’ and ‘don’t find me’ signals. Recent literature demonstrates that C-type lectin receptors (CLRs) normally constrain immune cell–mediated tissue damage by suppressing myeloid cell activation and then promote tumor immune evasion. We previously identified the orphan (CLRs) CLEC-1 as over-expressed in situation of established immune tolerance and reported that CLEC-1 expression by dendritic cells (DCs) and macrophages is enhanced by TGFβ and tempers downstream T cells responses. Furthermore, we reported that CLEC-1 is highly expressed by myeloid cells purified from human tumor micro-environment significantly more expressed by suppressive macrophages.MethodsAs DCs and macrophages are professional phagocytes of dying/dead cell, we evaluated whether CLEC-1 could be a receptor of damaged cells in the phagocytosis.ResultsWe found that CLEC-1 fusion protein, binds specifically to late apoptotic and secondary necrotic healthy or tumor cells induced by chemotherapy, radiation (UV, X-ray) or culture stress conditions. Importantly, we observed in vivo that CLEC-1 deficient mice, but not wild-type, eradicate MC38 colorectal tumors in combination with cytotoxic and immunogenic chemotherapy (eg. Cyclophosphamide. We then generated, screened and identified different anti-human Clec-1 antagonist monoclonal antibodies (mAbs) with the capacity to block the CLEC-1/CLEC-1L interaction. We discovered that various antagonist CLEC-1 mAbs, but not non-antagonist CLEC-1 control mAbs, increase the phagocytosis of CLEC-1L-positive human tumor cells by human CLEC-1 expressing TGFβ-polarized DCs or macrophages. Indeed, TGFβ-polarized DCs phagocytosed more efficiently Rituximab (anti-CD20 mAb)-opsonized Burkitt lymphoma cells (Raji) as well as bare NSCLC cells (A549) when CLEC-1 is antagonized by antibodies. Furthermore, macrophages more productively engulfed Rituximab-opsonized Raji cells as well in the context of CLEC-1 blockade (2–3 fold increase). Moreover, Cetuximab opsonized colon carcinoma cells (DLD-1; EGFR+) and Trastuzumab opsonized mammary carcinoma cells (SK-BR-3; Her2+) were likewise more phagocytosed by CLEC-1 blocked macrophages.ConclusionsAltogether, these data indicate illustrate that CLEC-1 broadly inhibits tumor-cell phagocytosis and synergized with tumor-targeted cytotoxic monoclonal antibodies in both solid and hematological tumors.

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.

No abstract

Myeloid cells represent the most abundant immune component of the tumor microenvironment, where they often assume immunosuppressive roles. Therefore, developing therapeutic strategies reinvigorating immunosuppressive myeloid cell subsets is of raising interest in oncology. We have identified CLEC-1, a member of the C-type lectin receptor (CLR) family as being expressed by myeloid cells, especially by conventional type one dendritic cells (cDC1), and by tumor-associated macrophages (TAM). However, the role of CLEC-1 in myeloid function and its mechanism of action remained to be fully elucidated. Here, we investigated the effect of CLEC-1 blockade, either by genetic deletion or by antibody targeting, on myeloid function and anti-tumor response. First, we observed that CLEC-1 genetic deletion significantly increases the survival of syngeneic tumor-bearing mice in the Hepa1.6 orthotopic model of hepatocarcinoma, as well as in the AK-7 orthotopic model of mesothelioma. Moreover, the synergy with chemotherapy treatment (cyclophosphamide, gemcitabine) in the MC38 model of colon adenocarcinoma in Clec1a deficient animals significantly enhanced complete responses. Eventually, cured mice developed a robust memory antitumor immune response against the tumor. Next, we generated anti-human CLEC-1 monoclonal antibodies (mAbs) as well as CLEC-1 humanized mice. We found that CLEC-1 targeting through mAb treatment was able to prolong mouse survival as efficiently as by CLEC-1 genetic deletion in MC38 and Hepa1.6 models. CLEC-1 blockade by genetic deletion or by different mAbs treatment profoundly impacted the tumor microenvironment: we observed an increase in the frequency of invigorated dendritic cells (DCs) and macrophages, activated and memory T cells, while frequencies of immunosuppressive myeloid cells and PD1-expressing T cells largely decreased. Mechanistically, the in vivo phagocytosis of tumor cells (Hepa1.6 and MCA101) by macrophages was enhanced in CLEC-1 deficient animals compared to WT animals. Altogether, our results demonstrate that CLEC-1 acts as a novel immune checkpoint in myeloid cells and highlight its high potential as a target for innovative immunotherapy in oncology. Citation Format: Vanessa Gauttier, Irène Baccelli, Marion Drouin, Isabelle Girault, Sabrina Pengam, Emmanuelle Wilhelm, Javier Saenz, Julien Taurelle, Emmanuel Mérieau, Bérangère Evrard, Caroline Mary, Géraldine Teppaz, Ariane Desselle, Virginie Thépénier, Nicolas Poirier, Elise Chiffoleau. CLEC-1 inhibitory myeloid checkpoint blockade enhances antitumor responses and tumor phagocytosis by macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6358.

Myeloid cells represent the most abundant immune component of the tumor microenvironment, where they often assume immunosuppressive roles. Therefore, understanding the mechanisms regulating the immunosuppressive activity of myeloid cells is of major interest in oncology. We have identified CLEC-1, a member of the C-type lectin receptor (CLR) family, as a myeloid cell-driven brake for anti-tumor T cell response in different murine models, including mesothelioma, hepatocellular and colorectal carcinomas. However, the molecular mechanism underlying the immunomodulatory role of CLEC-1 remained to be fully elucidated. Here, using both murine and human cells, we identified and characterized a novel endogenous ligand for CLEC-1, shedding new light on CLEC-1 driven inhibitory myeloid checkpoint activity. First, using single cell RNA sequencing, we confirmed that CLEC-1 is expressed by human myeloid cells, especially by conventional type-one dendritic cells and by tumor-associated macrophages. Using an Fc-fusion human CLEC-1 protein (huCLEC-1) as a bait for potential endogenous ligands, we observed both in human and murine cells that huCLEC-1 binds specifically to stressed or dying cells following programmed necrosis, for instance after X-ray or chemotherapy treatments. CLEC-1 interaction with its ligand expressed by necrotic cells was able to activate the NFAT promoter in a CLEC-1-CD3zeta NFAT reporter cell line in vitro; furthermore, CLEC-1 ligand targeting via Fc-CLEC-1 treatment in vivo extended the overall survival of chemotherapy-treated mc38-colorectal cancer bearing mice, altogether demonstrating the functional relevance of CLEC-1 interaction with its ligand. Immunoprecipitation of proteins bound to huCLEC-1 subsequently enabled the identification of TRIM21, an intra-cellular Fc receptor and E3 ubiquitin ligase as a novel interaction partner for CLEC-1. Direct interaction between recombinant TRIM21 and CLEC-1 human proteins was subsequently confirmed by ELISA and Biacore affinity analysis. TRIM21 was indeed detected at the cell surface of necrotic cells following genotoxic treatments in vitro in several human cancer cell lines and, interestingly, high TRIM21 expression was predictive of worse overall survival in patients with hepatocellular carcinoma, pancreatic cancer, or glioma. Mechanistically, we demonstrated that CLEC-1 genetic deletion in mice enhances the capacity of dendritic cells to cross-present tumor antigens, a process known to be regulated by TRIM21 through its E3 ubiquitin ligase activity. Antagonist anti-CLEC-1 or anti-TRIM21 antibodies are therefore being evaluated to further confirm the involvement of the newly identified CLEC-1 interaction with TRIM21 in the regulation of CLEC-1’s function as an inhibitory myeloid checkpoint. Altogether our data highlight the CLEC-1/TRIM21 axis as a new target for cancer immunotherapy. Citation Format: Irene Baccelli, Vanessa Gauttier, Marion Drouin, Caroline Mary, Isabelle Girault, Emmanuelle Wilhelm, Sabrina Pengam, Géraldine Teppaz, Julien Taurelle, Ariane Desselle, Virginie Thepenier, Nicolas Poirier, Elise Chiffoleau. TRIM21 is a novel endogenous partner of the inhibitory myeloid checkpoint CLEC-1 involved in tumor antigen cross-presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6365.