Functional Studies on the C-Type Lectin Receptor CD302 Present on Dendritic Cells and Macrophages

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2198-2198
Author(s):  
Derek NJ Hart ◽  
Pablo Silveira ◽  
Tsun Ho Lo ◽  
Nirupama Verma ◽  
Ai Vu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Immune Cell ◽  
Blood Monocytes ◽  
Lectin Receptors ◽  
Functional Studies ◽  
Cell Functions ◽  
Equity Ownership

Abstract Introduction: C-type lectin receptors (CLR) play an important role in the immune system by recognising molecular patterns expressed by exogenous and endogenous threats. They have been shown to capture and internalise antigens and to mediate other important immune cell functions. DEC205 and CLEC9A are being actively investigated as targets for clinical therapeutic cancer vaccines. We discovered CD302 as a new CLR expressed on human dendritic cells (DC), monocytes and macrophages (J Immunol 2007;179:6052). Our initial studies suggested the molecule could play a role in cell adhesion or migration due to its co-localisation with migratory structures on macrophages. Our study set out to investigate the potential immunological function of CD302 using mouse models and to define its wider tissue expression in man. Methods: We generated CD302 knockout (KO) mice lacking exon 1 of its gene, abrogating transcription, for functional studies. We characterised the transcriptional expression of CD302 in mouse immune cells using real-time PCR. We developed monoclonal mAb to mCD302. Human studies utilized the anti-CD302 mAbs, MMRI-20 & 21 in flow cytometry and confocal microscopy studies of human immune cell populations. Results: CD302 was primarily expressed in mouse liver, lungs, lymph nodes (LN) and spleen. In spleen, macrophages, granulocytes and dendritic cells (DC) expressed CD302. Analysis of LN DC subsets revealed 2.5-fold higher CD302 mRNA expression in migratory compared to resident DC populations. Enumeration of various immune populations in lymphoid organs by flow cytometry uncovered a modest deficiency in migratory DC number and proportion within LN of CD302 KO mice compared to wild-type (WT) mice. In vitro studies showed CD302 KO and WT DC had an equivalent capacity to be activated by various stimuli, prime T cells and migrate towards the lymphoid-homing chemokines CCL19/CCL21. CD302 KO migratory DC exhibited a reduced in vivo migratory capacity to LN after FITC skin-painting. However, CD302 KO macrophages migrated similarly to WT macrophages in vivo in response to thioglycollate. In man, CD302 was present in high density in liver and peripheral blood monocytes and myeloid but not plasmacytoid DC. Current studies are aimed at clarifying its distribution on tissue DC and macrophage subsets. Anti-CD302 coated microbeads were taken up by human monocyte derived macrophages and anti-CD302 mAb was also internalized by DC. Confocal studies showed that CD302 co-localized with F-actin structures at the near basal surface such as filopodia and lamellipodia and podosomes of human macrophages and EGFP tagged CD302 expressed in COS-1 cells associated with F-actin. Conclusion: Our data suggests that CD302 may play a specialist role in DC and macrophage membrane functions. This appears to relate to its ability to associate with F-actin and may contribute to the membrane interactions required for DC to migrate towards the draining LN. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership. Clark:DendroCyte BioTech Pty Ltd: Equity Ownership.

scholarly journals Resolvins as Regulators of the Immune System

2010 ◽  
Vol 10 ◽  
pp. 818-831 ◽  
Author(s):  
Hiroyuki Seki ◽  
Takaharu Sasaki ◽  
Tomomi Ueda ◽  
Makoto Arita
Keyword(s):  
Immune System ◽  
Acute Inflammation ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Bioactive Metabolites ◽  
Cell Functions ◽  
First Response ◽  
Beneficial Impact

Inflammation is the first response of the immune system to infection or injury, but excessive or inappropriate inflammatory responses contribute to a range of acute and chronic human diseases. Clinical assessment of dietary supplementation of ω-3 polyunsaturated fatty acids (i.e., eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) indicate that they have beneficial impact on these diseases, although the mechanisms are poorly understood at the molecular level. In this decade, it has been revealed that EPA and DHA are enzymatically converted to bioactive metabolites in the course of acute inflammation and resolution. These metabolites were shown to regulate immune cell functions and to display potent anti-inflammatory actions bothin vitroandin vivo. Because of their ability to resolve an acute inflammatory response, they are referred to as proresolving mediators, or resolvins. In this review, we provide an overview of the formation and actions of these lipid mediators.

scholarly journals The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation

2014 ◽  
Vol 211 (9) ◽  
pp. 1741-1758 ◽  
Author(s):  
Sachin Kumar ◽  
Juying Xu ◽  
Rupali Sani Kumar ◽  
Sribalaji Lakshmikanthan ◽  
Reuben Kapur ◽  
...  
Keyword(s):  
Immune Cell ◽  
Neutrophil Migration ◽  
Clinical Importance ◽  
Endotoxin Shock ◽  
Small Gtpase ◽  
Akt Activation ◽  
Cellular Defense ◽  
Cell Functions

Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation.

scholarly journals Neem Leaf Glycoprotein Partially Rectifies Suppressed Dendritic Cell Functions and Associated T Cell Efficacy in Patients with Stage IIIB Cervical Cancer

2011 ◽  
Vol 18 (4) ◽  
pp. 571-579 ◽  
Author(s):  
Soumyabrata Roy ◽  
Shyamal Goswami ◽  
Anamika Bose ◽  
Krishnendu Chakraborty ◽  
Smarajit Pal ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Dendritic Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Stage Iiib ◽  
Immune Functions ◽  
Cell Functions

ABSTRACTMyeloid-derived dendritic cells (DCs) generated from monocytes obtained from stage IIIB cervical cancer (CaCx IIIB) patients show dysfunctional maturation; thus, antitumor T cell functions are dysregulated. In an objective to optimize these dysregulated immune functions, the present study is focused on the ability of neem leaf glycoprotein (NLGP), a nontoxic preparation of the neem leaf, to induce optimum maturation of dendritic cells from CaCx IIIB patients.In vitroNLGP treatment of immature DCs (iDCs) obtained from CaCx IIIB patients results in upregulated expression of various cell surface markers (CD40, CD83, CD80, CD86, and HLA-ABC), which indicates DC maturation. Consequently, NLGP-matured DCs displayed balanced cytokine secretions, with type 1 bias and noteworthy functional properties. These DCs displayed substantial T cell allostimulatory capacity and promoted the generation of cytotoxic T lymphocytes (CTLs). Although NLGP-matured DCs derived from CaCx monocytes are generally subdued compared to those with a healthy monocyte origin, considerable revival of the suppressed DC-based immune functions is notedin vitroat a fairly advanced stage of CaCx, and thus, further exploration ofex vivoandin vivoDC-based vaccines is proposed. Moreover, the DC maturating efficacy of NLGP might be much more effective in the earlier stages of CaCx, where the extent of immune dysregulation is less and, thus, the scope of further investigation may be explored.

scholarly journals Dendritic Cells under Hypoxia: How Oxygen Shortage Affects the Linkage between Innate and Adaptive Immunity

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Sandra Winning ◽  
Joachim Fandrey
Keyword(s):  
Dendritic Cells ◽  
Immune Responses ◽  
Adaptive Immune Response ◽  
Mature Dendritic Cell ◽  
Innate And Adaptive Immunity ◽  
Cell Functions ◽  
Adaptive Immune ◽  
Oxygen Shortage

Dendritic cells (DCs) are considered as one of the main regulators of immune responses. They collect antigens, process them, and present typical antigenic structures to lymphocytes, thereby inducing an adaptive immune response. All these processes take place under conditions of oxygen shortage (hypoxia) which is often not considered in experimental settings. This review highlights how deeply hypoxia modulates human as well as mouse immature and mature dendritic cell functions. It tries to linkin vitroresults to actualin vivostudies and outlines how hypoxia-mediated shaping of dendritic cells affects the activation of (innate) immunity.

scholarly journals Dickkopf-1: A Promising Target for Cancer Immunotherapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Hang Yin Chu ◽  
Zihao Chen ◽  
Luyao Wang ◽  
Zong-Kang Zhang ◽  
Xinhuan Tan ◽  
...  
Keyword(s):  
Cancer Immunotherapy ◽  
Immune Cell ◽  
Immune Surveillance ◽  
Suppressor Cells ◽  
Functional Studies ◽  
Promising Target ◽  
Using Data ◽  
Dickkopf 1

Clinical studies in a range of cancers have detected elevated levels of the Wnt antagonist Dickkopf-1 (DKK1) in the serum or tumors of patients, and this was frequently associated with a poor prognosis. Our analysis of DKK1 gene profile using data from TCGA also proves the high expression of DKK1 in 14 types of cancers. Numerous preclinical studies have demonstrated the cancer-promoting effects of DKK1 in both in vitro cell models and in vivo animal models. Furthermore, DKK1 showed the ability to modulate immune cell activities as well as the immunosuppressive cancer microenvironment. Expression level of DKK1 is positively correlated with infiltrating levels of myeloid-derived suppressor cells (MDSCs) in 20 types of cancers, while negatively associated with CD8+ T cells in 4 of these 20 cancer types. Emerging experimental evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. This review introduces the rapidly growing body of literature revealing the cancer-promoting and immune regulatory activities of DKK1. In addition, this review also predicts that by understanding the interaction between different domains of DKK1 through computational modeling and functional studies, the underlying functional mechanism of DKK1 could be further elucidated, thus facilitating the development of anti-DKK1 drugs with more promising efficacy in cancer immunotherapy.

scholarly journals Glioblastoma epigenome profiling identifies SOX10 as a master regulator of molecular tumour subtype

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yonghe Wu ◽  
Michael Fletcher ◽  
Zuguang Gu ◽  
Qi Wang ◽  
Barbara Costa ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Immune Cell ◽  
Sequencing Data ◽  
Master Regulator ◽  
Mesenchymal Transition ◽  
Functional Studies ◽  
Bona Fide ◽  
Oligodendrocyte Precursor

AbstractGlioblastoma frequently exhibits therapy-associated subtype transitions to mesenchymal phenotypes with adverse prognosis. Here, we perform multi-omic profiling of 60 glioblastoma primary tumours and use orthogonal analysis of chromatin and RNA-derived gene regulatory networks to identify 38 subtype master regulators, whose cell population-specific activities we further map in published single-cell RNA sequencing data. These analyses identify the oligodendrocyte precursor marker and chromatin modifier SOX10 as a master regulator in RTK I-subtype tumours. In vitro functional studies demonstrate that SOX10 loss causes a subtype switch analogous to the proneural–mesenchymal transition observed in patients at the transcriptomic, epigenetic and phenotypic levels. SOX10 repression in an in vivo syngeneic graft glioblastoma mouse model results in increased tumour invasion, immune cell infiltration and significantly reduced survival, reminiscent of progressive human glioblastoma. These results identify SOX10 as a bona fide master regulator of the RTK I subtype, with both tumour cell-intrinsic and microenvironmental effects.

TNB-486, a Novel Fully Human Bispecific CD19 x CD3 Antibody That Kills CD19-Positive Tumor Cells with Minimal Cytokine Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4070-4070
Author(s):  
Harbani Malik ◽  
Ben Buelow ◽  
Udaya Rangaswamy ◽  
Aarti Balasubramani ◽  
Andrew Boudreau ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cytokine Secretion ◽  
Employment Equity ◽  
Equity Ownership

Introduction The restricted expression of CD19 in the B-cell lineage makes it an attractive target for the therapeutic treatment of B-cell malignancies. Many monoclonal antibodies and antibody drug conjugates targeting CD19 have been developed, including bispecific T-cell redirecting antibodies (T-BsAbs). In addition, anti-CD19 chimeric antigen receptor T-cells (CAR-T) have been approved to treat leukemia and lymphoma. However, despite the impressive depth of responses achieved by T-cell redirecting approaches such as T-BsAbs and CAR-T cells, toxicity from over-activation of T-cells remains a substantial limitation for this type of therapy, in particular neurotoxicity. In designing TNB-486, a novel CD19 x CD3 T-BsAb, we endeavored to retain activity against CD19-positive tumor cells while limiting the cytokine secretion thought to underlie toxicity from T-cell redirecting therapies. Utilizing TeneoSeek, a next generation sequencing (NGS)-based discovery pipeline that leverages in silico analysis of heavy chain only/fixed light chain antibody (HCA/Flic, respectively) sequences to enrich for antigen specific antibodies, we made a high affinity αCD19 HCA and a library of αCD3 Flic antibodies that showed a >2 log range of EC50s for T cell activation in vitro. Of note, the library contained a low-activating αCD3 that induced minimal cytokine secretion even at concentrations that mediated saturating T-cell dependent lysis of lymphoma cells (when paired with an αCD19 HCA). We characterized the relative efficacy and potential therapeutic window of this unique molecule, TNB-486, in vitro and in vivo and compared it to two strongly activating bispecific CD19 x CD3 antibodies similar to those currently available and in clinical development. Methods Affinity measurements of the αCD19 moiety were made via Biacore (protein) and flow cytometry (cell surface). Stability measurements were made by subjecting the molecule to thermal stress and the %aggregation was measured by Size Exclusion Chromatography. T-cell activation was measured via flow cytometry (CD69 and CD25 expression) and cytokine was measured by ELISA (IL-2, IL-6, IL-10, INF-ɣ, and TNFα) in vitro. Lysis of B-cell tumor cell lines (Raji, RI-1, and Nalm6) was measured via flow cytometry in vitro. In vivo, NOG mice were engrafted subcutaneously with NALM-6 or SUDHL-10 cells and intravenously with human peripheral blood mononuclear cells (huPBMC), and the mice treated with multiple doses of TNB-486 or negative or positive control antibody. Tumor burden was evaluated via caliper measurement. Pharmacodynamic/Pharmacokinetic (PK/PD) studies were performed in NOG mice. A pharmacokinetic (PK) study was performed in BALB/c mice, and a tolerability and PK study are ongoing in cynomolgus monkeys. Results TNB-486 bound to cell surface CD19 with single digit nanomolar affinity (~3nM). EC50s for cytotoxicity were in the single-digit nanomolar range for TNB-486, and sub-nanomolar for the strongly activating controls; TNB-486 maximum achievable lysis was identical to the positive controls. TNB-486 induced significantly less cytokine release for all cytokines tested compared to the positive controls even at doses saturating for tumor lysis. No off-target activation was observed in the absence of CD19 expressing target cells. In vivo, TNB-486 eradicated all CD19-positive tumors tested (NALM-6 and SUDHL10) at doses as little as 1µg administered every four days after tumors had reached ~200mm3. TNB-486 showed a PK profile consistent with other IgG molecules in mice (T1/2 ~6 days in mice). Conclusions TNB-486 induced comparable lysis of CD19-positive tumor cells as the strongly activating control bispecific antibodies while inducing significantly reduced cytokine secretion, even at doses saturating for tumor lysis in vitro. In vivo TNB-486 eradicated all tested CD19 positive tumor cell lines in established tumor models. No off-target binding was observed. In summary, TNB-486 shows promise as a lymphoma therapeutic differentiated from T-cell targeted therapies currently in the clinic and in clinical trials. Disclosures Malik: Teneobio, Inc.: Employment, Equity Ownership. Buelow:Teneobio, Inc.: Employment, Equity Ownership. Rangaswamy:Teneobio, Inc.: Employment, Equity Ownership. Balasubramani:Teneobio, Inc.: Employment, Equity Ownership. Boudreau:Teneobio, Inc.: Employment, Equity Ownership. Dang:Teneobio, Inc.: Employment, Equity Ownership. Davison:Teneobio, Inc.: Employment, Equity Ownership. Force Aldred:Teneobio, Inc.: Equity Ownership. Iyer:Teneobio, Inc.: Employment, Equity Ownership. Jorgensen:Teneobio, Inc.: Employment, Equity Ownership. Pham:Teneobio, Inc.: Employment, Equity Ownership. Prabhakar:Teneobio, Inc.: Employment, Equity Ownership. Schellenberger:Teneobio, Inc.: Employment, Equity Ownership. Ugamraj:Teneobio, Inc.: Employment, Equity Ownership. Trinklein:Teneobio, Inc.: Employment, Equity Ownership. Van Schooten:Teneobio, Inc.: Employment, Equity Ownership.

Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 237-237 ◽  
Author(s):  
Michael P. Rettig ◽  
Matthew Holt ◽  
Julie Prior ◽  
Sharon Shacham ◽  
Michael Kauffman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Cell Lines ◽  
Nuclear Export ◽  
Employment Equity ◽  
Advisory Committees ◽  
Ic50 Values ◽  
Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

Microvesiculation and Disease

2013 ◽  
Vol 41 (1) ◽  
pp. 237-240 ◽  
Author(s):  
Jameel M. Inal ◽  
Una Fairbrother ◽  
Sheelagh Heugh
Keyword(s):  
Infectious Disease ◽  
Extracellular Vesicles ◽  
Immune Cell ◽  
In Vivo Models ◽  
Cell Functions ◽  
Models Of Disease ◽  
New Treatments ◽  
Acute Myeloid

The important roles of extracellular vesicles in the pathogenesis of various diseases are rapidly being elucidated. As important vehicles of intercellular communication, extracellular vesicles, which comprise microvesicles and exosomes, are revealing important roles in cancer tumorigenesis and metastases and in the spread of infectious disease. The September 2012 Focused Meeting ‘Microvesiculation and Disease’ brought together researchers working on extracellular vesicles. The papers in this issue of Biochemical Society Transactions review work in areas including HIV infection, kidney disease, hypoxia-mediated tumorigenesis and down-regulation of immune cell functions in acute myeloid leukaemia by tumour-derived exosomes. In all cases, microvesicles and exosomes have been demonstrated to be important factors leading to the pathophysiology of disease or indeed as therapeutic vehicles in possible new treatments. The aim was, having enhanced our molecular understanding of the contribution of microvesicles and exosomes to disease in vitro, to begin to apply this knowledge to in vivo models of disease.

scholarly journals 230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A245-A245
Author(s):  
Vanessa Gauttier ◽  
Marion Drouin ◽  
Sabrina Pengam ◽  
Javier Saenz ◽  
Bérangère Evrard ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Immune Cell ◽  
Human Tumor ◽  
Myeloid Cell ◽  
Lectin Receptors ◽  
Knock Out ◽  
Cell Functions ◽  
Preclinical Efficacy

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.

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