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.

scholarly journals 212 CLEC-1 is a novel myeloid immune checkpoint for cancer immunotherapy limiting tumor cells phagocytosis and synergizing with tumor-targeted antibodies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A231-A231
Author(s):  
Vanessa Gauttier ◽  
Marion Drouin ◽  
Sabrina Pengam ◽  
Javier Saenz ◽  
Bérangère Evrard ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cells ◽  
Cell Activation ◽  
Immune Cell ◽  
Myeloid Cells ◽  
Human Tumor ◽  
Carcinoma Cells ◽  
Dead Cell ◽  
Lectin Receptors

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.

732 Chemerin reactivates PTEN and suppresses PD-L1 in tumor cells via a novel CMKRL1-mediated pathway

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A776-A776
Author(s):  
Russell Pachynski ◽  
Keith Rennier ◽  
Woo Jae Shin ◽  
Ethan Krug ◽  
Gurpal Virdi ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Human Tumor ◽  
In Vivo Studies ◽  
Cell Phenotype ◽  
Immune Effector ◽  
First Time

BackgroundChemokines and chemoattractants play critical roles in trafficking that help regulate leukocyte infiltrates in the tumor microenvironment. Chemokines/chemoattractants can also modulate tumor cell phenotype and function, as tumor cells express functional receptors for these agents. Chemerin (retinoic acid receptor responder 2, RARRES2) is an endogenous leukocyte chemoattractant that recruits innate immune cells through its receptor, CMKLR1. RARRES2 is widely expressed in nonhematopoietic tissues and often downregulated across multiple tumor types compared with normal tissue. We and others have shown that augmenting chemerin in the tumor microenvironment significantly suppresses tumor growth, in part, by immune effector cell recruitment. As chemerin has various roles outside of leukocyte trafficking (eg adipocyte differentiation and metabolic processes), we hypothesized that it may have additional, tumor-intrinsic effects.MethodsWe investigated the effect of exogenous chemerin on human prostate and sarcoma tumor lines. Key signaling pathway components were elucidated using qPCR, Western blotting, siRNA knockdown, and specific inhibitors. Functional consequences of chemerin treatment were evaluated using in vitro and in vivo studies.ResultsWe show for the first time that human tumors exposed to exogenous chemerin significantly upregulate PTEN expression/activity, and concomitantly suppress programmed death ligand-1 (PD-L1) expression. CMKLR1 knockdown abrogated chemerin- induced PTEN and PD-L1 modulation, revealing a novel CMKLR1/PTEN/PD-L1 signaling cascade. Targeted inhibitors suggest that signaling occurs through the PI3K/AKT/mTOR pathway. We found that chemerin treatment significantly reduced tumor migration, while significantly increasing T-cell–mediated cytotoxicity. Chemerin treatment was as effective as both PD-L1 knockdown and the anti–PD-L1 antibody atezolizumab in augmenting T cell mediated tumor lysis. Forced expression of chemerin in human DU145 prostate tumors significantly suppressed in vivo tumor growth, significantly increasing PTEN and decreasing PD-L1 expression. Primary prostate tumor cultures that were treated with recombinant chemerin showed significant increases in PTEN and decreases in PD-L1 expression compared to controls. Lastly, analyses of clinical trial data from human metastatic prostate cancer patients receiving treatment with ipilimumab (NCT02113657) showed higher tumoral levels of RARRES2 expression correlated with higher levels of PTEN, higher effector immune cell (eg cytotoxic T cells, NK cells) signatures, and improved clinical outcomes, suggesting a strategy to augment chemerin/RARRES2 levels in tumors may improve responses to immunotherapy.ConclusionsCollectively, our data show for the first time a novel link between chemerin, PTEN, and PD-L1 in human tumor lines. These results show that chemerin – in addition to its ability to suppress tumor growth by recruitment of immune effector cells, may also have a role in improving T-cell–mediated immunotherapies through favorable modulation of PTEN and PD-L1.

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.

ANTITUMOR EFFECT OF COLLOIDAL SILVER BISILICATE IN EXPERIMENTAL IN VITRO AND IN VIVO STUDIES

2019 ◽  
Vol 65 (5) ◽  
pp. 760-765
Author(s):  
Margarita Tyndyk ◽  
Irina Popovich ◽  
A. Malek ◽  
R. Samsonov ◽  
N. Germanov ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Tumor Growth ◽  
Tumor Cells ◽  
Human Tumor ◽  
Significant Inhibition ◽  
In Vivo Studies ◽  
Ehrlich Carcinoma ◽  
In Vivo Experiments

The paper presents the results of the research on the antitumor activity of a new drug - atomic clusters of silver (ACS), the colloidal solution of nanostructured silver bisilicate Ag6Si2O7 with particles size of 1-2 nm in deionized water. In vitro studies to evaluate the effect of various ACS concentrations in human tumor cells cultures (breast cancer, colon carcinoma and prostate cancer) were conducted. The highest antitumor activity of ACS was observed in dilutions from 2.7 mg/l to 5.1 mg/l, resulting in the death of tumor cells in all studied cell cultures. In vivo experiments on transplanted Ehrlich carcinoma model in mice consuming 0.75 mg/kg ACS with drinking water revealed significant inhibition of tumor growth since the 14th day of experiment (maximally by 52% on the 28th day, p < 0.05) in comparison with control. Subcutaneous injections of 2.5 mg/kg ACS inhibited Ehrlich's tumor growth on the 7th and 10th days of the experiment (p < 0.05) as compared to control.

scholarly journals Differentiated glioblastoma cells accelerate tumor progression by shaping the tumor microenvironment via CCN1-mediated macrophage infiltration

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Atsuhito Uneda ◽  
Kazuhiko Kurozumi ◽  
Atsushi Fujimura ◽  
Kentaro Fujii ◽  
Joji Ishida ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Progression ◽  
Tumor Cells ◽  
In Silico ◽  
Tumor Tissue ◽  
The Cancer Genome Atlas ◽  
Macrophage Infiltration ◽  
Macrophage Migration

AbstractGlioblastoma (GBM) is the most lethal primary brain tumor characterized by significant cellular heterogeneity, namely tumor cells, including GBM stem-like cells (GSCs) and differentiated GBM cells (DGCs), and non-tumor cells such as endothelial cells, vascular pericytes, macrophages, and other types of immune cells. GSCs are essential to drive tumor progression, whereas the biological roles of DGCs are largely unknown. In this study, we focused on the roles of DGCs in the tumor microenvironment. To this end, we extracted DGC-specific signature genes from transcriptomic profiles of matched pairs of in vitro GSC and DGC models. By evaluating the DGC signature using single cell data, we confirmed the presence of cell subpopulations emulated by in vitro culture models within a primary tumor. The DGC signature was correlated with the mesenchymal subtype and a poor prognosis in large GBM cohorts such as The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project. In silico signaling pathway analysis suggested a role of DGCs in macrophage infiltration. Consistent with in silico findings, in vitro DGC models promoted macrophage migration. In vivo, coimplantation of DGCs and GSCs reduced the survival of tumor xenograft-bearing mice and increased macrophage infiltration into tumor tissue compared with transplantation of GSCs alone. DGCs exhibited a significant increase in YAP/TAZ/TEAD activity compared with GSCs. CCN1, a transcriptional target of YAP/TAZ, was selected from the DGC signature as a candidate secreted protein involved in macrophage recruitment. In fact, CCN1 was secreted abundantly from DGCs, but not GSCs. DGCs promoted macrophage migration in vitro and macrophage infiltration into tumor tissue in vivo through secretion of CCN1. Collectively, these results demonstrate that DGCs contribute to GSC-dependent tumor progression by shaping a mesenchymal microenvironment via CCN1-mediated macrophage infiltration. This study provides new insight into the complex GBM microenvironment consisting of heterogeneous cells.

scholarly journals MLN8054, an Inhibitor of Aurora A Kinase, Induces Senescence in Human Tumor Cells Both In vitro and In vivo

2010 ◽  
Vol 8 (3) ◽  
pp. 373-384 ◽  
Author(s):  
Jessica J. Huck ◽  
Mengkun Zhang ◽  
Alice McDonald ◽  
Doug Bowman ◽  
Kara M. Hoar ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Human Tumor ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Human Tumor Cells

scholarly journals In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1020
Author(s):  
Stefan Grote ◽  
Guillermo Ureña-Bailén ◽  
Kenneth Chun-Ho Chan ◽  
Caroline Baden ◽  
Markus Mezger ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Solid Tumors ◽  
Immune Cells ◽  
Immune Cell ◽  
Nk Cell ◽  
Migration And Invasion ◽  
Cellular Immunotherapy ◽  
Knock Out ◽  
Immunosuppressive Tumor Microenvironment

Background: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME). Methods: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity. Results: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity. Conclusions: The strong cytotoxic effect of a CD276-specific CAR in combination with an “off-the-shelf” NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.

CSIG-17. IMMUNE MODULATORY ROLE OF TYPE I INTERFERON IN SYNGENEIC MOUSE GLIOMA MODELS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi36-vi37
Author(s):  
Evelina Blomberg ◽  
Manuela Silginer ◽  
Michael Weller
Keyword(s):  
Tumor Cells ◽  
Immune Cell ◽  
Synergistic Effects ◽  
Xenograft Model ◽  
Prolonged Survival ◽  
Type I ◽  
Syngeneic Mouse ◽  
Glioma Initiating Cells ◽  
Glioma Growth

Abstract Glioblastoma is characterized by a poor prognosis and a challenging phenotype for drug development. Although multimodal treatment, including surgery, radio- and chemotherapy is applied, the overall survival remains just above one year. Numerous clinical trials have studied targeted therapies against commonly deregulated pathways, but an efficient targeted drug is yet to be discovered. Likewise, immunotherapy has not been shown to be active. A subset of glioma tumor cells demonstrates stem-like properties; these cells are commonly referred to as glioma initiating cells (GIC). These types of cells are pluripotent and can by definition initiate and recapitulate glioma growth in experimental animals in vivo. Furthermore, these cells are often resistant to conventional therapies. Interferon β (IFN-β) is an immunomodulatory molecule with anti-cancer properties. We have previously shown that IFN-β greatly reduces sphere-formation capability of GIC. It was also confirmed that IFN-β sensitized resistant GIC to irradiation or the chemotherapeutic agent, temozolomide (TMZ). IFN-β treatment significantly prolonged survival in a xenograft model with GIC cells. In the current project, we want to use syngeneic mouse models to study the immunomodulatory effects of type I IFNs. Preliminary results indicate that abrogation of IFN signalling in tumor cells by CRISPR/Cas9 technology prolonged survival in mice only in cell lines which have substantial baseline autocrine IFN signalling. On the contrary, we did not observe a difference in survival when wild-type tumor cells were implanted in either IFNAR1 deficient or proficient hosts. Flow cytometry analysis will elucidate changes in immune cell recruitment and infiltration upon IFN signalling disruption. Moreover, we explore different treatments in combination with IFN-β as there are indications that TMZ or radiotherapy can have synergistic effects with stimulation of interferon type I signalling.

scholarly journals Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Dalia Martinez-Marin ◽  
Courtney Jarvis ◽  
Thomas Nelius ◽  
Stéphanie Filleur
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Phagocytic Activity ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Functional Assay ◽  
Loss Of Function ◽  
Multiple Cancer

Abstract Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.

scholarly journals 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

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