CCR2-expressing Cells Research Articles

Abstract 1841: TAK-500 is a clinical stage immune-cell directed antibody drug conjugate (iADC) inducing STING activation in CCR2-expressing intratumor myeloid cells and favorable immunomodulation

Abstract Introduction: Myeloid cells are present in the tumor microenvironment (TME) of most human solid cancers, playing an essential role to influence local immunomodulatory functions. STING signaling in intratumor myeloid cells can enhance interferon (IFN) production leading to enhanced local innate and adaptive immunity, and synergism with other anti-tumor mechanisms. Here, we used a novel iADC, TAK-500, to selectively activate STING in CCR2-expressing cells. This approach enables systemic delivery and favors intratumor accumulation of a STING agonist to potentially achieve anti-tumor activity. Methods: TAK-500 and mTAK-500, a murine surrogate, were employed to study the immunomodulatory role and anti-tumor effects in preclinical models. CCR2 protein expression was characterized in the TME of >1,000 primary human tumors including non-small cell lung cancer (NSCLC, 2 cohorts, N= 411), colorectal carcinomas (CRC, 2 cohorts, N=350) and pancreatic ductal adenocarcinomas (PDAC, 1 cohort, N=228) represented in tissue microarrays using multiplexed quantitative immunofluorescence (mQIF). Results: TAK-500 triggered dose-dependent monocyte activation in vitro. mTAK-500 induced robust activation of innate and adaptive immune responses both in vitro and in vivo. In syngeneic mouse models with CCR2 expressing intratumoral myeloid cells, mTAK-500 treatment caused accumulation and activation of CD8+ effector T-cells in the TME resulting in prominent anti-tumor activity and enhanced survival. The baseline levels of CCR2-expressing mMDSCs in mouse models positively associated with anti-tumor response to mTAK-500. mQIF analysis of human tumors confirmed high expression of CCR2 in myeloid cells, lower expression in tumor infiltrating lymphocytes (TILs) and absence in tumor epithelial cells. Using an mQIF panel for simultaneous measurement of DAPI, CK, CCR2, CD11b and CD68, we identified CCR2 protein expression in 94% of NSCLCs, 87% CRCs and 89% PDACs. The levels of CCR2 protein were significantly higher in NSCLC than in CRC and PDAC. In NSCLC, high CCR2 protein in myeloid cells was associated with the presence of activating EGFR/KRAS mutations and increased CD8+ TILs and local PD-L1 expression. In CRC, higher levels of CCR2 in CD68+ macrophages were seen in tumors harboring microsatellite instability than in microsatellite stable carcinomas. Conclusion: The iADC TAK-500 and mTAK-500 induces CCR2-dependent immune cell activation and anti-tumor effect. CCR2 is highly expressed in intratumor myeloid cells from NSCLC. High CCR2 is associated with enhanced local adaptive immune responses and specific clinical/molecular tumor subsets. TAK-500 is currently being evaluated clinically as a single agent and in combination with pembrolizumab in adults with select locally advanced or metastatic solid tumors (NCT05070247). Citation Format: Kurt A. Schalper, Atsushi Matsuda, Michelle Ganno-Sherwood, Angel E. Maldonado-Lopez, Emily Rosentrater, Angelo Porciuncula, Dong Mei Zhang, Camilla L. Christensen, Samantha A. Merrigan, Tiquella Hatten, Hong Myung Lee, Min Young Lee, Linlin Dong, Jian Huang, Natasha Iartchouk, Jianing Wang, He Xu, Tomoki Yoneyama, Konstantin I. Piatkov, Carole E. Harbison, Alex Parent, Neil Lineberry, Adnan O. Abu-Yousif, Vicky Appleman. TAK-500 is a clinical stage immune-cell directed antibody drug conjugate (iADC) inducing STING activation in CCR2-expressing intratumor myeloid cells and favorable immunomodulation [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 1841.

Tracking fibrosis in myeloproliferative neoplasms by CCR2 expression on CD34+ cells.

In myeloproliferative neoplasm (MPNs), bone marrow fibrosis - mainly driven by the neoplastic megakaryocytic clone - dictates a more severe disease stage with dismal prognosis and higher risk of leukemic evolution. Therefore, accurate patient allocation into different disease categories and timely identification of fibrotic transformation are mandatory for adequate treatment planning. Diagnostic strategy still mainly relies on clinical/laboratory assessment and bone marrow histopathology, which, however, requires an invasive procedure and frequently poses challenges also to expert hemopathologists. Here we tested the diagnostic accuracy of the detection, by flow cytometry, of CCR2+CD34+ cells to discriminate among MPN subtypes with different degrees of bone marrow fibrosis. We found that the detection of CCR2 on MPN CD34+ cells has a very good diagnostic accuracy for the differential diagnosis between “true” ET and prePMF (AUC 0.892, P<0.0001), and a good diagnostic accuracy for the differential diagnosis between prePMF and overtPMF (AUC 0.817, P=0.0089). Remarkably, in MPN population, the percentage of CCR2-expressing cells parallels the degree of bone marrow fibrosis. In ET/PV patients with a clinical picture suggestive for transition into spent phase, we demonstrated that only patients with confirmed secondary MF showed significantly higher levels of CCR2+CD34+ cells. Overall, flow cytometric CCR2+CD34+ cell detection can be envisioned in support of conventional bone marrow histopathology in compelling clinical scenarios, with the great advantage of being extremely rapid. For patients in follow-up, its role can be conceived as an initial patient screening for subsequent bone marrow biopsy when disease evolution is suspected.

CCR2-Mediated Uptake of Constitutively Produced CCL2: A Mechanism for Regulating Chemokine Levels in the Blood.

C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium.

Frontline Science: Blood-circulating leukocytes fail to infiltrate the spinal cord parenchyma after spared nerve injury

The development of neuropathic pain after peripheral nerve injury involves neuroimmune-glial interactions in the spinal cord. However, whether the development of neuropathic pain depends on the infiltration of peripheral immune cells, such as monocytes, into the spinal cord parenchyma after peripheral nerve damage remains unclear. Here, we used a combination of different techniques such as transgenic reporter mouse (Cx3cr1GFP/+ and Ccr2RFP/+ mice), bone marrow chimeric mice, and parabiosis to investigate this issue in spared nerve injury (SNI) model. Herein, we provided robust evidence that, although microglial cells are activated/proliferate at the dorsal horn of the spinal cord after SNI, peripheral hematopoietic cells (including monocytes) are not able to infiltrate into the spinal cord parenchyma. Furthermore, there was no evidence of CCR2 expression in intrinsic cells of the spinal cord. However, microglial cells activation/proliferation in the spinal cord and mechanical allodynia after SNI were reduced in Ccr2-deficient mice. These results suggest that blood-circulating leukocytes cells are not able to infiltrate the spinal cord parenchyma after distal peripheral nerve injury. Nevertheless, they indicate that CCR2-expressing cells might be indirectly regulating microglia activation/proliferation in the spinal cord after SNI. In conclusion, our study supports that CCR2 inhibition could be explored as an interventional approach to reduce microglia activation and consequently neuropathic pain development after peripheral nerve injury.

Effect of CCR2 inhibitor-loaded lipid micelles on inflammatory cell migration and cardiac function after myocardial infarction.

After myocardial infarction (MI), inflammatory cells infiltrate the infarcted heart in response to secreted stimuli. Monocytes are recruited to the infarct via CCR2 chemokine receptors along a CCL2 concentration gradient. While infiltration of injured tissue with monocytes is an important component of the reparatory response, excessive or prolonged inflammation can adversely affect left ventricular remodeling and worsen clinical outcomes. Here, we developed poly(ethylene glycol) (PEG)-distearoylphos-phatidylethanolamine (PEG-DSPE) micelles loaded with a small molecule CCR2 antagonist to inhibit monocyte recruitment to the infarcted myocardium. To specifically target CCR2-expressing cells, PEG-DSPE micelles were further surface decorated with an anti-CCR2 antibody. Targeted PEG-DSPE micelles showed eight-fold greater binding to CCR2-expressing RAW 264.7 monocytes than plain, non-targeted PEG-DSPE micelles. In a mouse model of MI, CCR2-targeting PEG-DSPE micelles loaded with a CCR2 small molecule antagonist significantly decreased the number of Ly6Chigh inflammatory cells to 3% of total compared with PBS-treated controls. Furthermore, CCR2-targeting PEG-DSPE micelles significantly reduced the infarct size based on epicardial and endocardial infarct arc lengths. Both non-targeted and CCR2-targeting PEG-DSPE micelles showed a trend toward improving cardiac function. As such, PEG-DSPE micelles represent a promising cardiac therapeutic platform.

A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo

AbstractExtravascular fibrin deposition accompanies many human diseases and causes chronic inflammation and organ damage, unless removed in a timely manner. Here, we used intravital microscopy to investigate how fibrin is removed from extravascular space. Fibrin placed into the dermis of mice underwent cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin(ogen) receptors, αMβ2 and ICAM-1, the myeloid cell integrin-binding site on fibrin or the endocytic collagen receptor, the mannose receptor. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance and shows that interstitial fibrin and collagen are cleared by different subsets of macrophages employing distinct molecular pathways.

Localized CCR2 Activation in the Bone Marrow Niche Mobilizes Monocytes by Desensitizing CXCR4

Inflammatory (classical) monocytes residing in the bone marrow must enter the bloodstream in order to combat microbe infection. These monocytes express high levels of CCR2, a chemokine receptor whose activation is required for them to exit the bone marrow. How CCR2 is locally activated in the bone marrow and how their activation promotes monocyte egress is not understood. Here, we have used double transgenic lines that can visualize CCR2 activation in vivo and show that its chemokine ligand CCL2 is acutely released by stromal cells in the bone marrow, which make direct contact with CCR2-expressing monocytes. These monocytes also express CXCR4, whose activation immobilizes cells in the bone marrow, and are in contact with stromal cells expressing CXCL12, the CXCR4 ligand. During the inflammatory response, CCL2 is released and activates the CCR2 on neighboring monocytes. We demonstrate that acutely isolated bone marrow cells co-express CCR2 and CXCR4, and CCR2 activation desensitizes CXCR4. Inhibiting CXCR4 by a specific receptor antagonist in mice causes CCR2-expressing cells to exit the bone marrow in absence of inflammatory insults. Taken together, these results suggest a novel mechanism whereby the local activation of CCR2 on monocytes in the bone marrow attenuates an anchoring signalling provided by CXCR4 expressed by the same cell and mobilizes the bone marrow monocyte to the blood stream. Our results also provide a generalizable model that cross-desensitization of chemokine receptors fine-tunes cell mobility by integrating multiple chemokine signals.

Inflammatory Monocytes Mediate Early and Organ-Specific Innate Defense During Systemic Candidiasis

Candida albicans is a commensal fungus that can cause systemic disease in patients with breaches in mucosal integrity, indwelling catheters, and defects in phagocyte function. Although circulating human and murine monocytes bind C. albicans and promote inflammation, it remains unclear whether C-C chemokine receptor 2 (CCR2)- and Ly6C-expressing inflammatory monocytes exert a protective or a deleterious function during systemic infection. During murine systemic candidiasis, interruption of CCR2-dependent inflammatory monocyte trafficking into infected kidneys impaired fungal clearance and decreased murine survival. Depletion of CCR2-expressing cells led to uncontrolled fungal growth in the kidneys and brain and demonstrated an essential antifungal role for inflammatory monocytes and their tissue-resident derivatives in the first 48 hours postinfection. Adoptive transfer of purified inflammatory monocytes in depleted hosts reversed the defect in fungal clearance to a substantial extent, indicating a compartmentally and temporally restricted protective function that can be transferred to enhance systemic innate antifungal immunity.

Genetic and Pharmacological Targeting of CSF-1/CSF-1R Inhibits Tumor-Associated Macrophages and Impairs BRAF-Induced Thyroid Cancer Progression

Advanced human thyroid cancers are densely infiltrated with tumor-associated macrophages (TAMs) and this correlates with a poor prognosis. We used BRAF-induced papillary thyroid cancer (PTC) mouse models to examine the role of TAMs in PTC progression. Following conditional activation of BRAFV600E in murine thyroids there is an increased expression of the TAM chemoattractants Csf-1 and Ccl-2. This is followed by the development of PTCs that are densely infiltrated with TAMs that express Csf-1r and Ccr2. Targeting CCR2-expressing cells during BRAF-induction reduced TAM density and impaired PTC development. This strategy also induced smaller tumors, decreased proliferation and restored a thyroid follicular architecture in established PTCs. In PTCs from mice that lacked CSF-1 or that received a c-FMS/CSF-1R kinase inhibitor, TAM recruitment and PTC progression was impaired, recapitulating the effects of targeting CCR2-expressing cells. Our data demonstrate that TAMs are pro-tumorigenic in advanced PTCs and that they can be targeted pharmacologically, which may be potentially useful for patients with advanced thyroid cancers.

The Expression of Tumor-Associated Macrophages in Papillary Thyroid Carcinoma

Solid tumors contain not only malignant cells, but also extracellular matrix and many other nonmalignant cells including fibroblasts, endothelial cells, and inflammatory cells such as macrophages, neutrophils, and lymphocytes. The presence of inflammatory cells within solid tumors suggests that the inflammatory microenvironment could play a major role in promoting tumorigenesis and progression. Macrophage infiltrates in the context of or surrounding a variety of malignancies represent the host's immune response to the tumor [1,2]. It was recently reported that tumor-associated macrophages (TAMs) had important roles in the tumor progression and metastasis of various cancers, including advanced thyroid cancer [3-9]. However, the role of TAMs in papillary thyroid carcinoma (PTC) has not been fully elucidated. Fiumara et al. [10] studied the tissue distribution and prognostic significance of TAMs in a retrospective series of 121 PTCs. They found tumor-infiltrating macrophages in approximately 70% of cases. Phagocytosis of neoplastic cells by macrophages was observed in approximately 15% of tumors, and none of these tumors developed distant metastases. These data suggest that neoplastic cell phagocytosis by macrophages and lymphocytic infiltration play a protective role in the development of distant metastases in patients with PTC. Conversely, emerging data suggest that TAMs promote tumor progression and metastasis in PTC. Ryder et al. [8] reported that an increased density of TAMs was associated with tumor progression in advanced thyroid cancers and that there was a significant correlation between increased TAMs and histological grade, tumor invasiveness, and decreased cancerrelated survival in PTC. Very recently, they also reported that TAMs promoted PTC progression in BRAF-induced PTC mouse models, and that targeting CCR2-expressing cells during BRAF induction reduced TAM density and impaired PTC development [11]. These results suggested that therapeutic strategies targeting TAMs may be beneficial in the treatment of advanced PTC. Qing et al. [9] investigated TAMs density in both benign thyroid lesions and PTC tumors by CD68 immunostaining. They found that the overall density of TAMs was significantly higher in PTC tumors compared with thyroid goiter and follicular adenoma. In addition, the density of TAMs was positively associated with lymph node (LN) metastasis in TNM stages III/VI compared with stages I/II. However, no association was observed with other common tumor features, including BRAF mutation. In this issue, Kim et al. [12] investigated the expression of TAMs in 36 PTC patients with LN metastasis using immunohistochemical staining with anti-CD68 antibody. They reported that a higher density of TAMs was correlated with larger tumor size, suggesting a protumorigenic role of TAMs in PTCs. Comparing clinicopathologic characteristics among low (<25%) and high (25% to 70%) TAM density groups, primary tumor size was larger in the high density group compared to the low density group (2.0±0.1 vs. 1.5±0.1; P=0.009). However, there was no significant association between high TAM density and poorer clinicopathologic characteristics including multifocality, LN metastasis, and extrathyroidal extension. These data were not in agreement with those of previous studies, which reported that high TAM density was correlated with tumor invasiveness and poor clinicopathologic characteristics, excepting tumor size [8,9,11]. Kim et al. [12] also analyzed the morphological characteristics of TAMs in PTC. They found that TAMs had thin, elongated cytoplasmic extensions, forming a canopy structure over tumor cells, and that the morphological characteristics of thyroid cancer tissue were well maintained irrespective of the presence of TAMs. In view of this, they inferred that TAMs do not play key role in tumor development in PTC. However, Caillou et al. [13] reported that anaplastic thyroid cancer displayed a very dense network of interconnected ramified TAMs in direct contact with intermingled cancer cells, and that this TAM network was directly related to the aggressiveness of thyroid cancer. Unfortunately, Kim et al. [12] conducted this study using anti-CD68 antibody on only a small sample of PTCs with LN metastasis. A major limitation of this study was the lack of follow-up data with which to evaluate long-term outcomes. Therefore, further studies using a more specific antibody and larger sample sizes of various stages of PTC are warranted to investigate the role of TAMs in tumor development, tumor invasiveness, and metastasis of PTC. Finally, TAM-targeted pharmacologic therapy in patients with advanced thyroid cancer should also be investigated.

Monocytic CCR2+ Myeloid-Derived Suppressor Cells Promote Immune Escape by Limiting Activated CD8 T-cell Infiltration into the Tumor Microenvironment

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate during tumor formation, facilitate immune escape, and enable tumor progression. MDSCs are important contributors to the development of an immunosuppressive tumor microenvironment that blocks the action of cytotoxic antitumor T effector cells. Heterogeneity in these cells poses a significant barrier to studying the in vivo contributions of individual MDSC subtypes. Herein, we show that granulocyte-macrophage colony stimulating factor, a cytokine critical for the numeric and functional development of MDSC populations, promotes expansion of a monocyte-derived MDSC population characterized by expression of CD11b and the chemokine receptor CCR2. Using a toxin-mediated ablation strategy to target CCR2-expressing cells, we show that these monocytic MDSCs regulate entry of activated CD8 T cells into the tumor site, thereby limiting the efficacy of immunotherapy. Our results argue that therapeutic targeting of monocytic MDSCs would enhance outcomes in immunotherapy.

Monocytic CCR2+ Myeloid Derived Suppressor Cells Promote Immune Escape by Limiting Activated CD8 T Cell Infiltration Into the Tumor Microenvironment

Abstract 2171Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate during tumor progression in a process driven by soluble factors such as granulocyte-macrophage colony stimulating factor (GM-CSF). These cells contribute to the suppressive nature of the tumor microenvironment and interfere with the functions of cytotoxic anti-tumor T effector cells. To date, MDSC heterogeneity has presented a barrier to studying the properties of individual MDSC constituents in vivo.Herein, we find that GM-CSF, a cytokine that promotes the numeric and functional development of monocytes, granulocytes and dendritic cells, and is frequently used as a vaccine adjuvant, is also critical for the expansion of a monocyte-derived MDSC population characterized by the expression of CD11b and the chemokine receptor CCR2. We demonstrate that these cells mediate T cell suppression in a contact dependent fashion and via the function of Arginase and inducible nitric oxide synthase, consistent with known MDSC functions. CD11b+CCR2 negative cells do not have suppressive capability despite also being expanded numerically by the actions of GM-CSF. Utilizing a toxin-mediated ablation strategy that targets CCR2-expressing cells, we demonstrate that monocytic MDSCs regulate activated CD8 T cell entry into the tumor site in vivo, thereby limiting the efficacy of immunotherapy.Our results extend observations on the dual role of GM-CSF in both stimulation and suppression of tumor immunity and suggest therapeutic targeting of monocytic MDSC could enhance the outcomes of immunotherapy. Disclosures:No relevant conflicts of interest to declare.