InVivoMAb anti-human CD47

• Cancer Research
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Oncolytic adenovirus H101 enhances the anti-tumor effects of PD-1 blockade via CD47 downregulation in tumor cells.

In Oncology Research on 29 April 2025 by Qiao, C., Xu, Y., et al.

To investigate the anti-tumor effects of an E1B55KD-deleted oncolytic adenovirus, H101, in combination with a humanized anti-PD-1 (Programmed cell death protein 1) monoclonal antibody, Camrelizumab. Anti-tumor efficacy of intratumoral injection of H101 or/and intraperitoneal injection of Camrelizumab were evaluated in an immune system humanized NOD Prkdc scid Il2rg -/- mice subcutaneous (S.C.) tumor model, established with human glioblastoma of unknown origin cell line U87-MG, and human bladder cancer cell line T24 and YTS-1. The mechanism by which H101 induced anti-tumor immunity were also investigated. Combining H101 with Camrelizumab demonstrated more potent anti-tumor effects than monotherapy in mouse S.C. tumor model. Increased tumor-infiltrating T cells were observed in the combined treatment group. H101 infection decreased the expression of CD47 in cancer cells, thereby promoting macrophages to phagocytose cancer cells. Following the H101-mediated activation of macrophages, increased levels of cytokines, including TNF, IL-12 and IFN-γ were observed. Moreover, when induced THP-1 cells were co-cultured with H101-treated cancer cells, expression of IFN-γ was increased in T cells. Elimination of IL-12 using an anti-IL-12 antibody abolished IFN-γ production from T cells. In addition, infection with H101 increased PD-L1 expression in YTS-1 cells. These results suggested that H101 may act synergistically to enhance the therapeutic efficacy of PD-1 blockade in cancer via suppressing CD47 signaling, which may promote macrophages to phagocytose tumor cells and activate CD8+ T cells. The combination of H101 with PD-1 blockade exhibits potential as a novel strategy for the treatment of cancer. © 2025 The Authors.

• Cancer Research
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• Immunology and Microbiology
,
• Neuroscience

High-Throughput Screening on Primary Tumor-Associated Microglia and Macrophages Identifies HDAC Inhibitors as Enhancers of Phagocytosis and Potent Partners for Immunotherapy in Glioblastoma

Preprint on BioRxiv : the Preprint Server for Biology on 27 March 2025 by Khalaj, M., Gutierrez, M. L., et al.

Glioblastoma multiforme (GBM) is a lethal brain tumor with limited treatment options. Tumor-associated macrophages and microglia (TAMs) drive immune suppression and tumor progression, making them a key therapeutic target for GBM. Enhancing TAM phagocytosis in GBM has shown promise, particularly with innate checkpoint inhibitors, such as CD47-blocking antibodies. However, small molecule approaches, which offer tunable and potentially synergistic mechanisms, remain underexplored in this context. In this study, we conducted the first large-scale chemical screen on primary TAMs from patients with GBM, identifying histone deacetylase (HDAC) inhibitors as potent inducers of phagocytosis. These compounds demonstrated phagocytosis-inducing effects across multiple GBM patient samples, with further amplification when combined with CD47 blockade. In a xenograft GBM model, HDAC inhibitors enhanced phagocytosis and suppressed tumor growth, with even greater efficacy in combination with CD47 antibodies. Our findings highlight HDAC inhibitors as promising agents to reprogram TAMs and synergize with immune checkpoint therapies, offering a novel strategy to bolster anti-tumor immunity in GBM.

• Cancer Research
,
• Immunology and Microbiology

CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

Preprint on BioRxiv : the Preprint Server for Biology on 26 November 2024 by Allen, J., Meglan, A., et al.

ABSTRACT Macrophages hold tremendous promise as effectors of cancer immunotherapy, but the best strategies to provoke these cells to attack tumors remain unknown. Here, we evaluated the therapeutic potential of targeting two distinct macrophage immune checkpoints: CD47 and CD24. We found that antibodies targeting these antigens could elicit maximal levels of phagocytosis when combined together in vitro. However, to our surprise, via unbiased genome-wide CRISPR screens, we found that CD24 primarily acts as a target of opsonization rather than an immune checkpoint. In a series of in vitro and in vivo genetic validation studies, we found that CD24 was neither necessary nor sufficient to protect cancer cells from macrophage phagocytosis in most mouse and human tumor models. Instead, anti-CD24 antibodies exhibit robust Fc-dependent activity, and as a consequence, they cause significant on-target hematologic toxicity in mice. To overcome these challenges and leverage our findings for therapeutic purposes, we engineered a collection of 77 novel bispecific antibodies that bind to a tumor antigen with one arm and engage macrophages with the second arm. We discovered multiple novel bispecifics that maximally activate macrophage-mediated cytotoxicity and reduce binding to healthy blood cells, including bispecifics targeting macrophage immune checkpoint molecules in combination with EGFR, TROP2, and CD71. Overall, our findings indicate that CD47 predominates over CD24 as a macrophage immune checkpoint in cancer, and that the novel bispecifics we created may be optimal immunotherapies to direct myeloid cells to eradicate solid tumors.

• Endocrinology and Physiology
,
• Immunology and Microbiology

Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker.

In Nature Communications on 9 November 2024 by Martins, T. A., Kaymak, D., et al.

A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive microenvironment, which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Myeloid immune checkpoint therapy targeting the CD47-signal regulatory protein alpha (SIRPα) axis induces GAM phagocytic function, but CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. In this work, we engineer a CAR T cell against epidermal growth factor receptor variant III (EGFRvIII), constitutively secreting a signal regulatory protein gamma-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eradicate orthotopic EGFRvIII-mosaic GBM in vivo, promoting GAM-mediated tumor cell phagocytosis. In a subcutaneous CD19+ lymphoma mouse model, anti-CD19-SGRP CAR T cell therapy is superior to conventional anti-CD19 CAR T. Thus, combination of CAR and SGRP eliminates bystander tumor cells in a manner that could overcome main mechanisms of CAR T cell therapy resistance, including immune suppression and antigen escape. © 2024. The Author(s).

• Cancer Research

In vivo perturb-seq of cancer and microenvironment cells dissects oncologic drivers and radiotherapy responses in glioblastoma.

In Genome Biology on 7 October 2024 by Liu, S. J., Zou, C., et al.

Genetic perturbation screens with single-cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking. Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single-cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery of sgRNA libraries into mouse models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo-dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune and stromal cells following in vivo CRISPRi perturbations that can affect tumor cell phagocytosis. In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single-cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application. © 2024. The Author(s).

• Cancer Research
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• Cardiovascular biology
,
• Immunology and Microbiology

Blockade of the CD47/SIRPα checkpoint axis potentiates the macrophage-mediated anti-tumor efficacy of tafasitamab.

In Haematologica on 27 June 2024 by Biedermann, A., Patra-Kneuer, M., et al.

Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.

In Nature Communications on 14 September 2023 by Zhang, X., Wang, Y., et al.

PubMed

GPR84 is a unique orphan G protein-coupled receptor (GPCR) that can be activated by endogenous medium-chain fatty acids (MCFAs). The signaling of GPR84 is largely pro-inflammatory, which can augment inflammatory response, and GPR84 also functions as a pro-phagocytic receptor to enhance phagocytic activities of macrophages. In this study, we show that the activation of GPR84 by the synthetic agonist 6-OAU can synergize with the blockade of CD47 on cancer cells to induce phagocytosis of cancer cells by macrophages. We also determine a high-resolution structure of the GPR84-Gi signaling complex with 6-OAU. This structure reveals an occluded binding pocket for 6-OAU, the molecular basis of receptor activation involving non-conserved structural motifs of GPR84, and an unusual Gi-coupling interface. Together with computational docking and simulations studies, this structure also suggests a mechanism for the high selectivity of GPR84 for MCFAs and a potential routes of ligand binding and dissociation. These results provide a framework for understanding GPR84 signaling and developing new drugs targeting GPR84. © 2023. Springer Nature Limited.

• Endocrinology and Physiology
,
• Immunology and Microbiology

Enhancing anti-EGFRvIII CAR T cell therapy against glioblastoma with a paracrine SIRPγ-derived CD47 blocker

Preprint on BioRxiv : the Preprint Server for Biology on 3 September 2023 by Martins, T. A., Tatari, N., et al.

A major challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive tumor microenvironment (TME), which is densely populated and supported by protumoral glioma-associated microglia and macrophages (GAMs). Targeting of CD47, a “don’t-eat-me” signal overexpressed by tumor cells, disrupts the CD47-SIRPα axis and induces GAM phagocytic function. However, antibody-mediated CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. To overcome these limitations, we combined local CAR T cell therapy with paracrine GAM modulation for more effective elimination of GBM. To this end, we engineered a new CAR T cell against epidermal growth factor receptor variant III (EGFRvIII) that constitutively secretes a SIRPγ-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII + GBM in a dose-dependent manner in vitro and eradicated orthotopically xenografted EGFRvIII-mosaic GBM by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon tumor re-challenge. The combination of anti-CD47 antibodies with anti-EGFRvIII CAR T cells failed to achieve a similar therapeutic effect, underscoring the importance of sustained paracrine GAM modulation. Multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry on GBM-xenografted brains showed that anti-EGFRvIII-SGRP CAR T cells accelerated GBM clearance, increased CD68 + cell trafficking to tumor scar sites, and induced myeloid-mediated tumor cell uptake. Additionally, in a peripheral lymphoma mouse xenograft model, anti-CD19-SGRP CAR T cells had superior efficacy compared to conventional anti-CD19 CAR T cells. Validation on human GBM explants revealed that anti-EGFRvIII-SGRP CAR T cells had similar tumor-killing capacity to anti-EGFRvIII CAR monotherapy, but showed a slight improvement in maintenance of tumor-infiltrated CD14 + myeloid cells. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate immune TME, resulting in the additive elimination of bystander EGFRvIII - tumor cells in a manner that overcomes major mechanisms of CAR T cell therapy resistance, including tumor innate immune suppression and antigen escape.

• Cancer Research

Oncolytic adenovirus H101 enhanced antitumor effects of PD-1 blockade by downregulating CD47 on tumor cells

Preprint on Research Square on 19 April 2023 by Qiao, C., Wang, S., et al.

Programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) blockade are standard of care for many patients with advanced or metastatic cancer. However, a majority of patients remain resistant to these treatments. It has been reported that local oncolytic viral infection of tumors is capable of overcoming systemic resistance to PD-1 blockade, and strongly suggest the combination therapy of virotherapy with PD-1 blockade to improve therapeutic efficacy in tumors that are refractory to checkpoint blockade. We investigate the antitumor effects of an E1B55KD deleted oncolytic adenovirus H101, in combination with a humanized anti-PD-1 monoclonal antibody Camrelizumab on cancer. Combination of H101 with Camrelizumab demonstrated more potent antitumor effects than monotherapy in immune system humanized NSG mice subcutaneous (S.C.) tumor model. Increased tumor infiltrating T cells including the total and IFN-γ-expressing CD8 + T cells in the combination treatment group were observed. H101 infection induced decreased expression of CD47 on cancer cells, thereby promoting macrophage to phagocytose cancer cells. With the activation of macrophage by H101, increased levels of cytokines including TNF, IL-12 and IFN-γ were observed when induced THP-1 cells were co-cultured with H101-treated cancer cells, which further induced increased expressions of IFN-γ in T cells. Eliminating the IL-12 by anti-IL-12 neutralizing antibodies abolished IFN-γ production from T cells, showing activation of macrophages by H101 induced oncolysis to promote IFN-γ secretion of T cells via IL-12. Meanwhile, infection with H101 induced upregulation of PD-L1 on YTS-1 cells. These results suggested that H101 works synergistically to enhance therapeutic efficacy of PD-1 blockade on cancer by suppressing CD47 signaling, which may promote phagocytose of macrophages to tumor cells and activate CD8 + T cells. Combination of H101 with PD-1 blockade would be a novel strategy for treating cancer.

• Biochemistry and Molecular biology

In The Journal of Biological Chemistry on 1 April 2023 by Govatati, S., Pichavaram, P., et al.

PubMed

Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)-phospholipase Cβ3-nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin β3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase-interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia. Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

• Cancer Research
,
• Immunology and Microbiology

In Journal for Immunotherapy of Cancer on 1 September 2022 by Mandel, I., Haves Ziv, D., et al.

PubMed

Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. NCT04717375. © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

• Homo sapiens (Human)
,
• Cancer Research
,
• Immunology and Microbiology

Immunotherapy of glioblastoma explants induces interferon-γ responses and spatial immune cell rearrangements in tumor center, but not periphery.

In Science Advances on 1 July 2022 by Shekarian, T., Zinner, C. P., et al.

A patient-tailored, ex vivo drug response platform for glioblastoma (GBM) would facilitate therapy planning, provide insights into treatment-induced mechanisms in the immune tumor microenvironment (iTME), and enable the discovery of biomarkers of response. We cultured regionally annotated GBM explants in perfusion bioreactors to assess iTME responses to immunotherapy. Explants were treated with anti-CD47, anti-PD-1, or their combination, and analyzed by multiplexed microscopy [CO-Detection by indEXing (CODEX)], enabling the spatially resolved identification of >850,000 single cells, accompanied by explant secretome interrogation. Center and periphery explants differed in their cell type and soluble factor composition, and responses to immunotherapy. A subset of explants displayed increased interferon-γ levels, which correlated with shifts in immune cell composition within specified tissue compartments. Our study demonstrates that ex vivo immunotherapy of GBM explants enables an active antitumoral immune response within the tumor center and provides a framework for multidimensional personalized assessment of tumor response to immunotherapy.

• In Vitro
,
• Homo sapiens (Human)
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• Cancer Research
,
• Immunology and Microbiology

In Immunity on 14 June 2022 by MacNabb, B. W., Tumuluru, S., et al.

PubMed

Antigen cross-presentation, wherein dendritic cells (DCs) present exogenous antigen on major histocompatibility class I (MHC-I) molecules, is considered the primary mechanism by which DCs initiate tumor-specific CD8+ T cell responses. Here, we demonstrate that MHC-I cross-dressing, an antigen presentation pathway in which DCs acquire and display intact tumor-derived peptide:MHC-I molecules, is also important in orchestrating anti-tumor immunity. Cancer cell MHC-I expression was required for optimal CD8+ T cell activation in two subcutaneous tumor models. In vivo acquisition of tumor-derived peptide:MHC-I molecules by DCs was sufficient to induce antigen-specific CD8+ T cell priming. Transfer of tumor-derived human leukocyte antigen (HLA) molecules to myeloid cells was detected in vitro and in human tumor xenografts. In conclusion, MHC-I cross-dressing is crucial for anti-tumor CD8+ T cell priming by DCs. In addition to quantitatively enhancing tumor antigen presentation, MHC cross-dressing might also enable DCs to more faithfully and efficiently mirror the cancer cell peptidome. Copyright © 2022 Elsevier Inc. All rights reserved.

• Cancer Research
,
• Pharmacology
,
• IHC

In Frontiers in Pharmacology on 14 June 2022 by Shi, C., Li, J., et al.

PubMed

Background: Bevacizumab (Avastin®), a humanized antiangiogenic monoclonal antibody, is widely used in the clinical treatment of tumour diseases. However, recent research has shown that the beneficial antiangiogenic effects of these agents have been limited in a number of patients due to complex immunosuppressive mechanisms. Here, we report a synergistic antitumour strategy through simultaneous blockade of VEGF and CD47 signalling to enhance the curative effect of advanced gastric cancer. Method: A BGC-823 gastric tumour model was chosen to evaluate antitumour efficacy. Macrophage migration and phagocytosis were evaluated to determine immune-related resistance to bevacizumab therapy. Synergistic antitumour activity was observed on the basis of tumour volume, tumour weight, tumour inhibition rate, tumour angiogenesis and tumour metastasis when bevacizumab was combined with an anti-CD47 monoclonal antibody. Results: Our study demonstrated that synergistic therapy targeting CD47 and VEGF reversed macrophage migration and phagocytosis, which were inhibited by antiangiogenic therapy and enhanced antitumour effects. Moreover, blockade of CD47 induced by antiangiogenic therapy inhibited tumour metastasis. Conclusion: Our data provide an effective strategy to attenuate resistance to bevacizumab therapy, promoting clinical cancer treatment with antiangiogenic drugs in combination with CD47-targeting inhibitors. Copyright © 2022 Shi, Li, Fan and Liu.

• Immunology and Microbiology

In Journal for Immunotherapy of Cancer on 1 March 2022 by Wu, Z. H., Li, N., et al.

PubMed

The CD47-SIRPα pathway acts as an important myeloid cell immune checkpoint and targeting the CD47/SIRPα axis represents a promising strategy to promote antitumor immunity. Several CD47-targeting agents show encouraging early activity in clinical trials. However, due to ubiquitous expression of CD47, the antigen sink and hematologic toxicity, such as anemia and thrombocytopenia, are main problems for developing CD47-targeting therapies. Considering the limited expression of SIRPα, targeting SIRPα is an alternative approach to block the CD47-SIRPα pathway, which may result in differential efficacy and safety profiles. SIRPα-targeting antibody BR105 was generated by hybridoma fusion and following humanization. BR105 was characterized for binding to human SIRPα alleles and blockade of the interaction with CD47. The functional activity was determined in in vitro phagocytosis assays by using human macrophages. The effect of BR105 on human T cell activation was studied using an OKT3-induced T-cell proliferation assay and an allogeneic mixed lymphocyte reaction. Human SIRPα-humanized immunodeficient mice were used in cancer models for evaluating the in vivo antitumor efficacy of BR105. Safety was addressed in a repeat-dose toxicity study in cynomolgus monkeys, and toxicokinetic analysis was further evaluated. BR105 shows broad binding activity across various SIRPα variants, and potently blocks the interaction of SIRPα and CD47. In vitro functional assays demonstrated that BR105 synergizes with therapeutic antibodies to promote phagocytosis of tumor cells. Moreover, the combination of BR105 and therapeutic antibody significantly inhibits tumor growth in a xenograft tumor model. Although BR105 may slightly bind to SIRPγ, it does not inhibit T cell activation, unlike other non-selective SIRPα-targeting antibody and CD47-targeting agents. Toxicity studies in non-human primates show that BR105 is well tolerated with no treatment-related adverse effects noted. The novel and differentiated SIRPα-targeting antibody, BR105, was discovered and displays promising antitumor efficacy in vitro and in vivo. BR105 has a favorable safety profile and shows no adverse effects on T cell functionality. These data support further clinical development of BR105, especially as a therapeutic agent to enhance efficacy when used in combination with tumor-targeting antibodies or antibodies that target other immune checkpoints. © Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.

• Homo sapiens (Human)
,
• Cancer Research
,
• Immunology and Microbiology

Preprint on BioRxiv : the Preprint Server for Biology on 21 January 2022 by Shekarian, T., Zinner, C. P., et al.

PubMed

Recent therapeutic strategies for glioblastoma (GBM) aim at targeting immune tumor microenvironment (iTME) components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform for GBM would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of therapy response and resistance. We cultured 47 GBM explants from tumor center and periphery from 7 patients in perfusion bioreactors to assess iTME responses to immunotherapy. Explants were exposed to antibodies blocking the immune checkpoints CD47, PD-1 or or their combination, and were analyzed by highly multiplexed microscopy (CODEX, co -detection by in dex ing) using an immune-focused 55-marker panel. Culture media were examined for changes of soluble factors including cytokines, chemokines and metabolites. CODEX enabled the spatially resolved identification and quantification of >850,000 single cells in explants, which were classified into 10 cell types by clustering. Explants from center and periphery differed significantly in their cell type composition, their levels of soluble factors, and their responses to immunotherapy. In a subset of explants, culture media displayed increased interferon-γ levels, which correlated with shifts in immune cell composition within specific tissue compartments, including the enrichment of CD4 + and CD8 + T cells within an adaptive immune compartment. Furthermore, significant differences in the expression levels of functional molecules in innate and adaptive immune cell types were found between explants responding or not to immunotherapy. In non-responder explants, T cells showed higher expression of PD-1, LAG-3, TIM-3 and VISTA, whereas in responders, macrophages and microglia showed higher cathepsin D levels. Our study demonstrates that ex vivo immunotherapy of GBM explants enables an active antitumoral immune response within the tumor center in a subset of patients, and provides a framework for multidimensional personalized assessment of tumor response to immunotherapy.

• WB
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• Homo sapiens (Human)
,
• Immunology and Microbiology

In PLoS Pathogens on 1 June 2021 by Sarute, N., Cheng, H., et al.

PubMed

Signal-regulatory protein alpha (SIRPA) is a well-known inhibitor of phagocytosis when it complexes with CD47 expressed on target cells. Here we show that SIRPA decreased in vitro infection by a number of pathogenic viruses, including New World and Old World arenaviruses, Zika virus, vesicular stomatitis virus and pseudoviruses bearing the Machupo virus, Ebola virus and SARS-CoV-2 glycoproteins, but not HSV-1, MLV or mNoV. Moreover, mice with targeted mutation of the Sirpa gene that renders it non-functional were more susceptible to infection with the New World arenaviruses Junín virus vaccine strain Candid 1 and Tacaribe virus, but not MLV or mNoV. All SIRPA-inhibited viruses have in common the requirement for trafficking to a low pH endosomal compartment. This was clearly demonstrated with SARS-CoV-2 pseudovirus, which was only inhibited by SIRPA in cells in which it required trafficking to the endosome. Similar to its role in phagocytosis inhibition, SIRPA decreased virus internalization but not binding to cell surface receptors. We also found that increasing SIRPA levels via treatment with IL-4 led to even greater anti-viral activity. These data suggest that enhancing SIRPA's activity could be a target for anti-viral therapies.

• Cancer Research

In Cancer Medicine on 1 March 2021 by Wang, X., Wang, Y., et al.

PubMed

CD47 is a widely expressed transmembrane protein located on the surface of somatic cells. It mediates a variety of cellular processes including apoptosis, proliferation, adhesion, and migration. An important role for CD47 is the transmission of a "Don't eat me" signal by interacting with SIRPα on the macrophage surface membrane, thereby preventing the phagocytosis of normal cells. However, cancer cells can take advantage of this autogenous signal to protect themselves from phagocytosis, thus enabling immune escape. Blocking the interaction between CD47 and SIRPα has proven to be effective in removing cancer cells. The treatment of various cancers with CD47 monoclonal antibodies has also been validated. We designed and synthesized a peptide (RS17), which can specifically bind to CD47 and block CD47-SIRPα signaling. The affinity of RS17 for CD47-expressing tumor cells was determined, while the inhibition of CD47-SIRPα signaling was evaluated in vitro and in vivo. The results indicated that RS17 significantly promotes the phagocytosis of tumor cells by macrophages and had a similar therapeutic effect compared with a positive control (CD47 monoclonal antibodies). In addition, a cancer xenograft mouse model was established using CD47-expressing HepG2 cells to evaluate the effect of RS17 on tumor growth in vivo. Using ex vivo and in vivo mouse models, RS17 demonstrated a high inhibitory effect on tumor growth. Based on our results, RS17 may represent a novel therapeutic peptide for cancer therapy. © 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

• Cancer Research
,
• Immunology and Microbiology

In Journal for Immunotherapy of Cancer on 1 March 2021 by Cao, X., Li, B., et al.

PubMed

Limited therapeutic options are available for triple-negative breast cancer (TNBC), emphasizing an urgent need for more effective treatment approaches. The development of strategies by targeting tumor-associated macrophages (TAMs) to stimulate their ability of Programmed Cell Removal (PrCR) provides a promising new immunotherapy for TNBC treatment. CD47 is a critical self-protective "don't eat me" signal on multiple human cancers against macrophage immunosurveillance. Using human and mouse TNBC preclinical models, we evaluated the efficacy of PrCR-based immunotherapy by blocking CD47. We performed high-throughput screens on FDA-approved anti-cancer small molecule compounds for agents potentiating PrCR and enhancing the efficacy of CD47-targeted therapy for TNBC treatment. We showed that CD47 was widely expressed on TNBC cells and TAMs represented the most abundant immune cell population in TNBC tumors. Blockade of CD47 enabled PrCR of TNBC cells, but the efficacy was not satisfactory. Our high-throughput screens identified cabazitaxel in enhancing PrCR-based immunotherapy. A combination of CD47 blockade and cabazitaxel treatment yielded a highly effective treatment strategy, promoting PrCR of TNBC cells and inhibiting tumor development and metastasis in preclinical models. We demonstrated that cabazitaxel potentiated PrCR by activating macrophages, independent of its cytotoxicity toward cancer cells. When treated with cabazitaxel, the molecular and phenotypic signatures of macrophages were polarized toward M1 state, and the NF-kB signaling pathway became activated. The combination of CD47 blockade and macrophage activation by cabazitaxel synergizes to vastly enhance the elimination of TNBC cells. Our results show that targeting macrophages is a promising and effective strategy for TNBC treatment. © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

• Cancer Research
,
• Immunology and Microbiology
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• FC/FACS

In Frontiers in Immunology on 20 February 2021 by Chen, J., Cao, X., et al.

PubMed

Evasion of immunosurveillance is critical for cancer initiation and development. The expression of "don't eat me" signals protects cancer cells from being phagocytosed by macrophages, and the blockade of such signals demonstrates therapeutic potential by restoring the susceptibility of cancer cells to macrophage-mediated phagocytosis. However, whether additional self-protective mechanisms play a role against macrophage surveillance remains unexplored. Here, we derived a macrophage-resistant cancer model from cells deficient in the expression of CD47, a major "don't eat me" signal, via a macrophage selection assay. Comparative studies performed between the parental and resistant cells identified self-protective traits independent of CD47, which were examined with both pharmacological or genetic approaches in in vitro phagocytosis assays and in vivo tumor models for their roles in protecting against macrophage surveillance. Here we demonstrated that extracellular acidification resulting from glycolysis in cancer cells protected them against macrophage-mediated phagocytosis. The acidic tumor microenvironment resulted in direct inhibition of macrophage phagocytic ability and recruitment of weakly phagocytic macrophages. Targeting V-ATPase which transports excessive protons in cancer cells to acidify extracellular medium elicited a pro-phagocytic microenvironment with an increased ratio of M1-/M2-like macrophage populations, therefore inhibiting tumor development and metastasis. In addition, blockade of extracellular acidification enhanced cell surface exposure of CD71, targeting which by antibodies promoted cancer cell phagocytosis. Our results reveal that extracellular acidification due to the Warburg effect confers immune evasion ability on cancer cells. This previously unrecognized role highlights the components mediating the Warburg effect as potential targets for new immunotherapy harnessing the tumoricidal capabilities of macrophages. Copyright © 2021 Chen, Cao, Li, Zhao, Chen, Lai, Muend, Nossa, Wang, Guo, Ye, Lee and Feng.