CD47 Blockade Research Articles

PARP inhibition synergizes with CD47 blockade to promote phagocytosis by tumor-associated macrophages in homologous recombination-proficient tumors

AimsPARP inhibitors (PARPi) are known to exert anti-tumor effects in patients with BRCA-mutated (BRCAmut) or homologous recombination (HR)-deficient cancer, but recent clinical investigations have suggested that this treatment may also be beneficial in patients with HR-proficient tumors. In this study, we aimed to investigate how PARPi exerts anti-tumor effects in non-BRCAmut tumors. Main methodsBRCA wild-type, HR-deficient-negative ID8 and E0771 murine tumor cells were treated in vitro and in vivo with olaparib, a clinically approved PARPi. The effects on tumor growth in vivo were determined in immune-proficient and -deficient mice and alterations of immune cell infiltrations were analyzed with flow cytometry. Tumor-associated macrophages (TAMs) were further investigated with RNA-seq and flow cytometry. In addition, we confirmed olaparib's effect on human TAMs. Key findingsOlaparib did not affect HR-proficient tumor cell proliferation and survival in vitro. However, olaparib significantly decreased tumor growth in C57BL/6 and SCID-beige mice (defective in lymphoid development and NK cell activity). Olaparib increased macrophage numbers in the tumor microenvironment, and their depletion diminished the anti-tumor effects of olaparib in vivo. Further analysis revealed that olaparib improved TAM-associated phagocytosis of cancer cells. Notably, this enhancement was not solely reliant on the “Don't Eat Me” CD47/SIRPα signal. In addition, compared to monotherapy, the concomitant administration of αCD47 antibodies with olaparib improved tumor control. SignificanceOur work provides evidence for broadening the application of PARPi in HR-proficient cancer patients and paves the way for developing novel combined immunotherapy to upgrade the anti-tumor effects of macrophages.

Nanoparticle-Mediated CD47-SIRPα Blockade and Calreticulin Exposure for Improved Cancer Chemo-Immunotherapy.

Enabling macrophages to phagocytose tumor cells holds great potential for cancer therapy but suffers from tremendous challenges because the tumor cells upregulate antiphagocytosis molecules (such as CD47) on their surface. The blockade of CD47 alone is insufficient to stimulate tumor cell phagocytosis in solid tumors due to the lack of "eat me" signals. Herein, a degradable mesoporous silica nanoparticle (MSN) is reported to simultaneously deliver anti-CD47 antibodies (aCD47) and doxorubicin (DOX) for cancer chemo-immunotherapy. The codelivery nanocarrier aCD47-DMSN was constructed by accommodating DOX within the mesoporous cavity, while adsorbing aCD47 on the surface of MSN. aCD47 blocks the CD47-SIRPα axis to disable the "don't eat me" signal, while DOX induces immunogenic tumor cell death (ICD) for calreticulin exposure as an "eat me" signal. This design facilitated the phagocytosis of tumor cells by macrophages, which enhanced antigen cross-presentation and elicited efficient T cell-mediated immune response. In 4T1 and B16F10 murine tumor models, aCD47-DMSN generated a strong antitumor effect after intravenous injection by increasing tumor-infiltration of CD8+ T cells. Taken together, this study offers a nanoplatform to modulate the phagocytosis of macrophages for efficacious cancer chemo-immunotherapy.

CD73 and PD-L1 dual blockade amplifies antitumor efficacy of SBRT in murine PDAC models

BackgroundStereotactic body radiotherapy (SBRT) induces immunogenic cell death, leading to subsequent antitumor immune response that is in part counterbalanced by activation of immune evasive processes, for example, upregulation of programmed...

CD73 blockade alleviates intestinal inflammatory responses by regulating macrophage differentiation in ulcerative colitis.

Ulcerative colitis (UC) is a type of inflammatory bowel disease characterized by excessive and persistent inflammation. Intestinal macrophages play a considerable role in regulating inflammatory immune reactions in the gut mucosa. It has previously been reported that CD73 is related to the pathogenesis of inflammatory or immune-related diseases; however, the roles of CD73 in UC remain unclear. In this study, CD73 expression in the inflamed mucosa of patients with UC was examined using reverse transcription-quantitative PCR (RT-qPCR), western blotting, and immunohistochemistry. Adenosine 5'-(α, β-methylene) diphosphate (APCP) was used to block the expression of CD73. Furthermore, the mRNA levels of proinflammatory mediators associated with macrophages following the blocking of CD73 were examined using RT-qPCR. Finally, the regulatory function of CD73 in intestinal inflammation was assessed by administering APCP in a mouse model of dextran sulfate sodium salt (DSS)-induced colitis. Notably, it was found that CD73 expression was significantly increased in the colonic mucosal tissues of patients with UC. Blockade of CD73 inhibited the expression of pro-inflammatory cytokines but promoted the production of anti-inflammatory cytokines in macrophages, while its promotion of M2 macrophage polarization was also verified. In vivo, CD73 blockade markedly alleviated DSS-induced colitis in mice, as characterized by reduced weight loss, reduction in the incidence of diarrhea, and reduced amount of bloody stool. Mechanistically, it was shown that CD73 regulated macrophage differentiation via the NF-κB and ERK signaling pathways. In conclusion, the findings of the present study indicate that CD73 may have a potential impact on the pathogenesis of UC by modulating the immune response of macrophage differentiation; thus, providing a novel pathway for modulating mucosal inflammation in UC.

B7-H3 drives immunosuppression and Co-targeting with CD47 is a new therapeutic strategy in β-catenin activated melanomas.

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/β-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the 'cold tumor' phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by β-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).

Synergistic effect of CD47 blockade in combination with cordycepin treatment against cancer.

Cordycepin is widely considered a direct tumor-suppressive agent. However, few studies have investigated as the effect of cordycepin therapy on the tumor microenvironment (TME). In our present study, we demonstrated that cordycepin could weaken the function of M1-like macrophages in the TME and also contribute to macrophage polarization toward the M2 phenotype. Herein, we established a combined therapeutic strategy combining cordycepin and an anti-CD47 antibody. By using single-cell RNA sequencing (scRNA-seq), we showed that the combination treatment could significantly enhance the effect of cordycepin, which would reactivate macrophages and reverse macrophage polarization. In addition, the combination treatment could regulate the proportion of CD8+ T cells to prolong the progression-free survival (PFS) of patients with digestive tract malignancies. Finally, flow cytometry validated the changes in the proportions of tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs). Collectively, our findings suggested that the combination treatment of cordycepin and the anti-CD47 antibody could significantly enhance tumor suppression, increase the proportion of M1 macrophages, and decrease the proportion of M2 macrophages. In addition, the PFS in patients with digestive tract malignancies would be prolonged by regulating CD8 + T cells.

Cellular Membrane Localization of Innate Immune Checkpoint Molecule CD47 Is Regulated by Radixin in Human Pancreatic Ductal Adenocarcinoma Cells.

In the past decade, immune checkpoint inhibitors have exhibited potent antitumor efficacy against multiple solid malignancies but limited efficacy against pancreatic ductal adenocarcinoma (PDAC). Cluster of differentiation (CD) 47, a member of the immunoglobulin G superfamily, is overexpressed in the surface membrane of PDAC and independently correlates with a worse clinical prognosis. Furthermore, CD47 functions as a dominant macrophage checkpoint, providing a potent "do not eat me" signal to enable cancer cells to evade the innate immune system. Thus, the blockade of CD47 is a promising immunotherapeutic strategy for PDAC. In this study, we determined whether ezrin/radixin/moesin (ERM) family members, which post-translationally modulate the cellular membrane localization of numerous transmembrane proteins by crosslinking with the actin cytoskeleton, contribute to the cellular membrane localization of CD47 in KP-2 cells derived from human PDAC. Immunofluorescence analysis showed that CD47 and ezrin/radixin were highly co-localized in the plasma membrane. Interestingly, gene silencing of radixin but not ezrin dramatically decreased the cell surface expression of CD47 but had little effects on its mRNA level. Furthermore, CD47 and radixin interacted with each other, as determined by a co-immunoprecipitation assay. In conclusion, radixin regulates the cellular membrane localization of CD47 as a scaffold protein in KP-2 cells.

Abstract 4433: Galectin-3 synergizes with CD47 to suppress phagocytosis and T cell immunity in peritoneal metastases of gastric adenocarcinoma

Abstract Peritoneal metastasis (PC), the common sites of gastric adenocarcinoma (GAC) metastasis, remains resistant to therapies available in the clinics and new target is strived to be found. CD47 is one of the major players in conveying the “don’t eat me” signal upon binding its receptor SIRPα on myeloid cells which evade innate immune responses. Previously clinical studies on the blockade of CD47 alone resulted in limited clinical benefits suggesting that other target(s) must be inhibited simultaneously with CD47 for gaining advantage. Here, we found that CD47 was highly expressed on peritoneal malignant cells and its overexpression was a poor prognosticator. Additionally, we observed that Galectin-3 (Gal-3) was highly enriched in tumor cluster and was positively correlated with CD47 expression in short survival of patients revealed by scRNA-seq and metastatic GAC samples. Moreover, we found double positive of Gal-3 and CD47 significantly associated with diffuse type, poor differentiation, and tumor relapse. Mechanistically, Depletion of Gal-3 in mouse tumor and human tumor cells reduced the levels of CD47 through inhibition c-Myc expression and its binding to the promoter of CD47. Additionally, tumor-derived Gal-3 were found critical for tumor progression accompanied with protected GAC cells from phagocytosis by macrophages. Gal-3 deficiency resulted in a marked reduction in M2 macrophages and elevated T cell effector activity. Dual blockade of Gal-3 and CD47 significantly decreased tumor growth by promoted phagocytosis, repolarization of macrophage and boosted T cell immune responses in the tumor microenvironment. Our data uncover that Gal-3 synergizes with CD47 to suppress phagocytosis and orchestrates immunosuppressed milieu in the tumor microenvironment warrants combination therapeutic strategy in GAC patients with peritoneal metastases. Citation Format: Yibo Fan, Shumei Song, Yuan Li, Shilpa Dhar, Jiankang Jin, Xiaodao Yao, Ruiping Wang, Ailing Wang, Melissa Pool Pizzi, Jingjing Wu, Katsuhiro Yoshimura, Lang Ma, Samir Hanash, George Calin, Linghua Wang, Michael Curran, Jaffer Ajani. Galectin-3 synergizes with CD47 to suppress phagocytosis and T cell immunity in peritoneal metastases of gastric adenocarcinoma. [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 4433.

Abstract 1255: Glucose dependent CD73+ CAFs enforce a tumor metabolic barrier that promotes T cell exclusion

Abstract Trafficking of T lymphocytes from the lymph nodes to the tumor microenvironment is a critical process of the tumor immunity cycle to elicit cytotoxic anti-tumor responses driven by CD8+ T cells. However, some tumors termed “immune excluded” recruit lymphocytes to the tumor site, but the lymphocytes are unable to penetrate the tumor parenchyma and localize primarily in the peritumoral region. In soft tissue sarcoma patients, most tumors are poorly infiltrated by T cells, which is associated with a poor response to immunotherapies. It has been described that cancer associated fibroblasts (CAFs) are enriched in immune excluded tumors and may directly block the migration of T cells via the production of dense extracellular matrix or by forging an immunosuppressive niche. We generated two models of undifferentiated pleomorphic sarcoma (UPS) that recapitulate the “immune excluded” and “inflamed” microenvironments observed in sarcoma patients. These syngeneic models rely on p53KO mesenchymal stem cells overexpressing either Ccne1 or Vgll3, which are frequently amplified in UPS patients. These models differ in their overall proportion of infiltrating TILs, and specifically T cells, making them ideal for comparative studies to investigate the mechanisms driving T cell exclusion in the TME. Using single-cell RNA-sequencing, we identified a population of CAFs expressing Nt5e, encoding CD73, which are spatially enriched in the peritumoral region of immune excluded Ccne1 tumors and closely associate with CD8+ T cells located at the tumor margin. Using transwell invasion assays, we show that CD73+ CAFs but not CD73- CAFs are able to block the migration of activated T cells towards tumor cells, even in the presence of CXCL10. Further, we show that Nt5e CAFs are enriched for signatures of glucose metabolism, and hypoxia, thus we hypothesized that CD73+ CAFs may block the migration of T cells into tumors by forging a nutrient poor metabolic barrier around the tumor. To test this, we treated Ccne1 tumors with BAY-876, a GLUT1 inhibitor and observed a significant accumulation of infiltrating CD8+ T cells compared to controls. GLUT1 treated CAFs expressed significantly less Nt5e, indicating that CD73 may play a role in the maintenance of glucose metabolism in CAFs. Furthermore, blockade of CD73 in CD73+ CAFs decreases the expression of the glucose transporter, Glut1. All together, these data suggest that CD73 may serve as a marker of glucose dependent CAFs that alter the metabolic niche to block T cell infiltration into tumors. Citation Format: Marina Broz, Emily Ko, Jinfen Xiao, Marco DeSimone, Roberta Piras, Kristin Ishaya, Xen Ping Hoi, Jlenia Guarnerio. Glucose dependent CD73+ CAFs enforce a tumor metabolic barrier that promotes T cell exclusion [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 1255.

Abstract 2938: Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy

Abstract The overexpression of human epidermal growth factor receptor 2 (HER2) has been identified in a variety of solid tumors including breast cancer (BC), gastric cancer and is associated with tumor recurrence, brain metastasis, and an overall poor prognosis. As such, HER2 has become a major target in the research and development of anticancer drugs. CD47, an integrin-associated protein, is expressed in many human cancer cells, especially upregulated preferentially in HER2-expressing cells. Co-expression of HER2 and CD47 is more frequently detected in recurrent BC patients with poor prognosis when comparing with the counterpart primary tumors. IMM2902 is a first-in-class, clinical-stage, novel recombinant bispecific mAb-Trap fusion protein targeting both CD47 and HER2 simultaneously. The binding activities of IMM2902, measured as EC50, to total CD47+ and HER2+ in BT474, NCI-N87 and SKOV-3 cancer cell lines are 16, 11 and 5 nM respectively. The affinity of IMM2902 to HER2 is about 15 times higher than that of CD47. IMM2902 not only prevents the engagement of CD47 with SIRPα and hence blocks “don’t eat me” signal of CD47/SIRPα, but also induces strong antibody-dependent cell-mediated cytotoxicity (ADCC, EC50 = 0.0045 - 0.0544 nM) and antibody-dependent cellular phagocytosis (ADCP, EC50 = 0.05 - 0.13 nM) activity with specifically engineered IgG1 Fc fragment. Most importantly, IMM2902 induces accelerated HER2 degradation in tumor tissues. Despite its high affinity to CD47, IMM2902 does not binds to CD47 on erythrocyte membrane, therefore, unlike conventional CD47 mAb, IMM2902 demonstrated a minimal impact on red blood cells (RBC). In preclinical research, IMM2902 has demonstrated a much stronger antitumor activity with excellent tolerability in a mouse model of human breast cancer (dose range 3.5 - 10 mg/kg) and a mouse model of human gastric cancer (dose range 1 -18 mg/kg) than that of trastuzumab (targeting HER2 only) alone, IMM01 (a SIRPα-Fc fusion protein) alone, and the combination of trastuzumab and IMM01. In HER2-low SUN-1 xenograft models, IMM2902 has showed a strong tumor growth inhibition at doses between 2 to 18 mg/kg. Intriguingly, IMM2902 also has exhibited potent tumor killing activity against breast cancer resistant to trastuzumab (HCC-1954 and patient-derived xenografts herceptin-resistant model) at doses between 3.5 to 10 mg/kg. Given the strong preclinical antitumor activity as well as the favorable safety profile, IMM2902 may serve as a potent immunotherapy for HER2-expressing cancers via dual blockade of CD47 and HER2. A Phase 1 clinical trial exploring safety, tolerability, and preliminary efficacy of IMM2902 in patients with HER2-expressing advanced solid tumors is currently ongoing in both China (CXSL2101035) and USA (NCT05076591). Citation Format: Binglei Zhang, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Frank X. Gan, Wenzhi Tian, Fan Zhang, Yongping Song. Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy [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 2938.

Abstract 2980: HX009, a first-in-class PD1xCD47 BsAb, demonstrated anti-AML activity in PDX models

Abstract Acute myeloid leukemia (AML) is an aggressive leukemia of myeloid lineage with different subtypes (global annual mortality ~150,000). The first-line treatment is usually induction chemotherapies, followed by further chemo-/radiation therapies or stem cell transplant, etc. Immune checkpoint inhibitor (ICIs), e.g. PD1 blocker, although a great success for treating many solid tumors, has yet to show convincing activity in AML. CD47, a “don’t eat me” receptor frequently found on the surface of tumor cells, including hematological malignances such as AML, prevents phagocytosis by interacting to its ligand, SIRPα, on the surface of macrophages. CD47 is also now shown as a key immune checkpoint for both innate and adoptive immunity (Liu et al., 2018). CD47 targeting, actively being tested for MSD and AML in clinics, encounters challenges for the reported toxicities (e.g. anemia/thrombocytopenia) as well as inadequate efficacy (Feng et al., 2019). Majority of investigational CD47 targeting agents in the clinics are combination therapies with other modality in order to increase the likelihood of success. To this end, it is prudent to develop agents with dual targeting agents, particularly for both the innate and adoptive immune checkpoints. We describe a novel specially designed/constructed bi-specific antibody (BsAb), HX009, targeting CD47 and PD1 for the treatment of cancers. We tested HX009 in several preclinical AML-PDXs (patient-derived xenograft models) via systemic engraftment: AM8096 (CD47lo, H-score of 55 by IHC), AM7577 (CD47hi, H-score of 237)(An et al., 2017) and AM5512 (CD47hi, H-score of 198). All three PDXs responded to 10mg/kg twice weekly dose of HX009 (IP) as measured either by leukemic burden in peripheral blood at different timepoints, and in both spleen and bone marrow at the termination, or by survival. Since there is no T-cell function in xenograft models, all anti-leukemic activities observed should be attributed to the CD47 blockade, not PD1 blockade. AM7577 model responded significantly better than AM8096, likely due to the significantly higher expression of CD47 and also better than Ara-C standard of care (SOC) treatment at 3mg/kg. In contrast, AM8096 responded even less to HX009 than Ara-C. In contrast, in the subcutaneous transplanted AML cell line-derived model, MV4-11, there is little anti-tumor activity observed, although there is significant expression of CD47 on the tumor cells. In conclusion, our data seems to suggest that HX009 could be a candidate immunotherapy for CD47hi AML, with CD47 expression being a positive predictive biomarker, warranting further evaluation. Citation Format: Xiaoyu An, Jinping Liu, Hang Ke, Lingxin Xiong, Xiaolong Tu, Lei Zhang, Faming Zhang, Henry Li. HX009, a first-in-class PD1xCD47 BsAb, demonstrated anti-AML activity in PDX models [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 2980.

Immunostimulation with chemotherapy of a ruthenium-arene complex via blockading CD47 signal in chronic myelogenous leukemia cells

Combination of novel immunomodulation and traditional chemotherapy has become a new tendency in cancer treatment. Increasing evidence suggests that blocking the “don't eat me” signal transmitted by the CD47 can promote the phagocytic ability of macrophages to cancer cells, which might be promising for improved cancer chemoimmunotherapy. In this work, we conjugated CPI-alkyne modified by Devimistat (CPI-613) with ruthenium-arene azide precursor Ru-N3 by copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to construct Ru complex CPI-Ru. CPI-Ru exhibited satisfactory cytotoxicity towards the K562 cells while nearly non-toxic towards the normal HLF cells. CPI-Ru has been demonstrated to cause severe damage to mitochondria and DNA, ultimately inducing cancer cell death through the autophagic pathway. Moreover, CPI-Ru could significantly downregulate the expression of CD47 on the surface of K562 accompanied by the enhanced immune response by targeting the blockade of CD47. This work provides a new strategy for utilizing metal-based anticancer agents to block CD47 signal to achieve chemoimmunotherapy in chronic myeloid leukemia treatment.

Abstract P5-08-14: Blockade of CD47/Thrombospondin-1 signaling increases glycolytic metabolism as a protective mechanism against chemotherapy-associated cardiac injury in a model of Triple-Negative Breast Cancer

Abstract Due to advances in diagnosis and treatment, cancer–related mortality has decreased, and by the year 2030, there will be 22 million cancer survivors in the United States. This success comes with an increased incidence of serious adverse effects, mainly in the cardiovascular system. While new treatment modalities are emerging for Triple-negative breast cancer (TNBC), most current strategies include anthracycline-based regimens to manage disease. Therefore, novel strategies are needed to overcome anthracycline-induced cardiotoxicities in this patient population. Activation of the TSP1/CD47 signaling axis is implicated in the progression of heart failure, with reported increases in TSP1 levels following myocardial infarction. Therefore, we examined the potential of CD47 blockade as a strategy to prevent cardiac injury as a consequence of cancer chemotherapy. Our data in a syngeneic orthotopic breast cancer model shows that blockade of CD47 using an in vivo anti-sense phosphodiesterase morpholino (PMO) preserved ejection fraction, fractional shortening, and cardiac output when compared to DOX treatment while preserving oncologic efficacy of chemotherapy. To determine a potential mechanism of cardioprotection, hearts of control and CD47 PMO-treated mice were subjected to RNA sequencing. Gene set enrichment analysis (GSEA) showed significant positive enrichment for metabolic pathways including pyruvate metabolism (NES= 2.3 , p< 0.002), and oxidative phosphorylation (NES=2.0, p< 0.01). During cardiac insult, metabolic flexibility of cardiomyocytes results in metabolic reprogramming from fatty acid oxidation to a glycolytic mechanism to overcome injury. Thus, DOX-associated cardiotoxicity may be mediated by an increase in TSP1 and a decrease in glycolysis, leading to the inability to overcome acute cellular stress. In vitro cellular bioenergetics, analysis revealed that TSP1 caused a dose-dependent reduction in glycolytic flux and glycolytic capacity in cardiac myoblast. This, coupled with preserved cardiac viability of cardiac cells treated with CD47 PMO in the presence of DOX, suggests that TSP1 may act through CD47 to prevent cardiac cell metabolic reprogramming needed to overcome injury. Furthermore, anti-sense experiments with siRNAs to Glut-4 and Hexokinase-II showed that the protection conferred by CD47 is mediated by activating these proteins. Therefore our studies suggest that the TSP1/CD47 axis may be central to the interplay of metabolism to preserve cardiac tissue integrity; thus, targeting this pathway may prevent the onset of chronic cardiac disease due to chemotherapy in cancer patients. Citation Format: Steven M. Bronson, Jessica D. Mackert, Mitra Kooshki, Adam S. Wilson, Nildris Cruz-Diaz, Pierre L Triozzi, Alexandra Thomas, Katherine L. Cook, David R. Soto-Pantoja. Blockade of CD47/Thrombospondin-1 signaling increases glycolytic metabolism as a protective mechanism against chemotherapy-associated cardiac injury in a model of Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-08-14.

Blockade of the Immune Checkpoint CD47 by TTI-621 Potentiates the Response to Anti−PD-L1 in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is an incurable and cosmetically disfiguring disease associated with microenvironmental signals. We investigated the effects of CD47 and PD-L1 immune checkpoint blockades, as a strategy for targeting both innate and adaptive immunity. CIBERSORT analysis identified the immune-cell composition in the CTCL tumor microenvironment and the immune checkpoint expression profile for each immune-cell gene cluster from CTCL lesions. We investigated the relationship between MYC and CD47 and PD-L1 expression and found that MYC short hairpin RNA knockdown and MYC functional suppression by TTI-621 (SIRPαFc) and anti-PD-L1 (durvalumab) in CTCL cell lines reduced the expression of CD47 and PDL1 mRNA and protein as measured by qPCR and flow cytometry, respectively. Invitro, blockade of the CD47-SIRPα interaction with TTI-621 increased the phagocytic activity of macrophages against CTCL cells and enhanced CD8+ T-cell-mediated killing in a mixed leucocyte reaction. Moreover, TTI-621 synergized with anti-PD-L1 in macrophages reprogram to M1-like phenotypes and inhibited CTCL cell growth. These effects were mediated by cell death-related pathways, including apoptosis, autophagy, and necroptosis. Collectively, our findings show that CD47 and PD-L1 are critical regulators of immune surveillance in CTCL and that dual targeting of CD47 and PD-L1 will provide insight into tumor immunotherapy for CTCL.

RAGA prevents tumor immune evasion of LUAD by promoting CD47 lysosome degradation

CD47 is a macrophage-specific immune checkpoint protein acting by inhibiting phagocytosis. However, the underlying mechanism maintaining CD47 protein stability in cancer is not clear. Here we show that CD47 undergoes degradation via endocytosis/lysosome pathway. The lysosome protein RAGA interacts with and promotes CD47 lysosome localization and degradation. Disruption of RAGA blocks CD47 degradation, leading to CD47 accumulation, high plasma membrane/intracellular CD47 expression ratio and reduced phagocytic clearance of cancer cells. RAGA deficiency promotes tumor growth due to the accumulation of CD47, which sensitizes the tumor to CD47 blockade. Clinical analysis shows that RAGA and CD47 proteins are negatively correlated in lung adenocarcinoma patient samples. High RAGA protein level is related to longer patient survival. In addition, RAGAhighCD47low patients show the longest overall survival. Our study thereby not only reveals a mechanism by which RAGA regulates CD47 lysosome degradation, but also suggests RAGA is a potential diagnostic biomarker of lung adenocarcinoma.

Potentiating antibody-dependent killing of cancers with CAR T cells secreting CD47-SIRPα checkpoint blocker

AbstractChimeric antigen receptor (CAR) T-cell therapy has shown success in the treatment of hematopoietic malignancies; however, relapse remains a significant issue. To overcome this, we engineered “Orexi” CAR T cells to locally secrete a high-affinity CD47 blocker, CV1, at the tumor and treated tumors in combination with an orthogonally targeted monoclonal antibody. Traditional CAR T cells plus the antibody had an additive effect in xenograft models, and this effect was potentiated by CAR T-cell local CV1 secretion. Furthermore, OrexiCAR-secreted CV1 reversed the immunosuppression of myelomonocytoid cells both in vitro and within the tumor microenvironment. Local secretion of the CD47 inhibitor bypasses the CD47 sink found on all cells in the body and may prevent systemic toxicities. This combination of CAR T-cell therapy, local CD47 blockade, and orthogonal antibody may be a combinatorial strategy to overcome the limitations of each monotherapy.

CD47-targeted immunotherapy unleashes antitumour immunity in Epstein-Barr virus-associated gastric cancer

The aims of this study were to enhance the antitumour immunity in Epstein-Barr virus-associated gastric cancer (EBVaGC). We performed RNA-seq analysis to compare the differential expression genes between EBVaGC and EBV-negative gastric cancer (EBVnGC) patients. The expression levels of CD68, CD163 and CD47 were analyzed by immunohistochemistry. Different subsets of macrophages were investigated by a coincubation model. The effects of CD47 blockade were also detected. The expression levels of CD68, CD163 and CD47 were significantly higher in EBVaGC, and were associated with poor prognoses. Macrophages coincubated with EBV+ AGS cells tended to be immunosuppressed, which could be reversed by CD47 deficiency or blocking CD47. EBV resulted in cGAS-STING pathway activation, which stimulated CD47 expression and inhibited macrophage phagocytosis. Anti-CD47 therapy activated cGAS-STING signaling, which was responsible for production of IFN-β, resulting in activation of antitumour immunity. Our results provide a promising new strategy for CD47-targeted immunotherapy in EBVaGC.

A phase II multi-arm study of magrolimab combinations in patients with relapsed/refractory multiple myeloma.

Magrolimab is a monoclonal antibody that blocks CD47, a 'do not eat me' signal overexpressed on tumor cells. CD47 is overexpressed in multiple myeloma (MM), which contributes to its pathogenesis. Preclinical studies have shown that CD47 blockade induces macrophage activation, resulting in elimination of myeloma cells, and that there is synergy between magrolimab and certain anticancer therapies. These findings suggest that magrolimab-based combinations may have a therapeutic benefit in MM. This phase II study investigates magrolimab in combination with commonly used myeloma therapies in patients with relapsed/refractory MM and includes a safety run-in phase followed by a dose-expansion phase. Primary end points include the incidence of dose-limiting toxicities and adverse events (safety run-in) and the objective response rate (dose expansion).

189P IL-10 enhances macrophage phagocytosis of cancer cells in response to CD47 blockade and opsonizing antibodies

There is an urgent need to improve the safety and efficacy of immunotherapy, which has focused primarily on the adaptive immune system. Macrophages are the most common infiltrating immune cells in tumors and they can be directed to kill cancer cells by targeting macrophage-specific immune checkpoints, such as the CD47/SIRPa axis. CD47 serves as a “don’t eat me” signal on cancer cells and binds to SIRPa on macrophages to inhibit phagocytosis. Cytokines can influence macrophage polarization states, but how cytokines influence macrophage activation, particularly in the setting of macrophage checkpoint blockade, remains unknown.

CD47 blockade improves the therapeutic effect of osimertinib in non-small cell lung cancer.

The third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib (OSI) has been approved as the first-line treatment for EGFR-mutant non-small cell lung cancer (NSCLC). This study aims to explore a rational combination strategy for enhancing the OSI efficacy. In this study, OSI induced higher CD47 expression, an important anti-phagocytic immune checkpoint, via the NF-κB pathway in EGFR-mutant NSCLC HCC827 and NCI-H1975 cells. The combination treatment of OSI and the anti-CD47 antibody exhibited dramatically increasing phagocytosis in HCC827 and NCI-H1975 cells, which highly relied on the antibody-dependent cellular phagocytosis effect. Consistently, the enhanced phagocytosis index from combination treatment was reversed in CD47 knockout HCC827 cells. Meanwhile, combining the anti-CD47 antibody significantly augmented the anticancer effect of OSI in HCC827 xenograft mice model. Notably, OSI induced the surface exposure of "eat me" signal calreticulin and reduced the expression of immune-inhibitory receptor PD-L1 in cancer cells, which might contribute to the increased phagocytosis on cancer cells pretreated with OSI. In summary, these findings suggest the multidimensional regulation by OSI and encourage the further exploration of combining anti-CD47 antibody with OSI as a new strategy to enhance the anticancer efficacy in EGFR-mutant NSCLC with CD47 activation induced by OSI.