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Background Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain–containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. Methods Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. Results Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8 + T cells, CD4 + T cells, and CD56 + natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. Conclusions Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called ‘don’t eat me’ signals—including CD47 1 , programmed cell death ligand 1 (PD-L1) 2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M) 3 . Monoclonal antibodies that antagonize the interaction of ‘don’t eat me’ signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers 4 , 5 . However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown ‘don’t eat me’ signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24–Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy. CD24 interacts with the tumour-associated-macrophage receptor Siglec-10 to inhibit the macrophage-mediated clearance of cancer cells, revealing a new ‘don’t eat me’ signal as a potential target for cancer immunotherapy.

Background Third‐generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) show initial efficacy in EGFR‐mutated lung cancer, but residual disease persists. This study aimed to investigate cluster of differentiation 24 (CD24) as a translational immunotherapeutic target for enhancing third‐generation EGFR‐TKI efficacy. Methods We conducted RNA‐sequencing (RNA‐seq) on drug‐responsive, drug‐tolerant persister, and drug‐resistant cells to identify therapeutic targets to pair with EGFR‐TKIs. For validation, we integrated single‐cell RNA‐seq data from 29 lung cancer specimens and used single‐nucleus RNA‐seq and immunohistochemistry on clinical residual tumor samples following TKI therapy (TKI‐residual). With CRISPR/Cas9, we studied the effect of CD24 on proliferation and phagocytic clearance during EGFR‐TKI treatment. We tested CD24 knockout or ATG‐031 (a first‐in‐class CD24 antibody) with EGFR‐TKIs in vitro, xenografts, and spontaneous lung cancer models. To explore mechanisms, we used DNA affinity precipitation, chromatin immunoprecipitation sequencing, and luciferase assays to identify transcription factors regulating CD24. Co‐immunoprecipitation combined with mass spectrometry and phosphoproteomics were used to study YIN‐YANG‐1 (YY1) S247 phosphorylation's expression and function, while kinase inhibitors assessed upstream phosphorylation of YY1 S247 and its regulation of CD24. Results CD24 expression rose in drug‐responsive, ‐resistant, and ‐tolerant lung cancer cells and post‐EGFR‐TKI treatment clinical specimens. This elevation promoted cell proliferation and shielded tumor cells from macrophage‐mediated phagocytosis. Genetic depletion of CD24 or treatment with ATG‐031 significantly enhanced phagocytosis and tumor eradication in vitro, in xenografts, and in mice harboring EGFRL858R·T790M‐driven spontaneous lung tumors. Furthermore, we revealed that YY1 S247 phosphorylation was responsible for the upregulation of CD24 upon EGFR‐TKI treatment, facilitating YY1 dimerization and the formation of promoter‐enhancer loops that regulate CD24 expression. Conclusions CD24 is a promising target in EGFR‐mutated lung cancers, potentially enhancing efficacy of third‐generation EGFR‐TKIs.

Background In cardiac arrest (CA) patients undergoing cardiopulmonary resuscitation (CPR), neuroinflammation following return of spontaneous circulation (ROSC) contributes to brain ischemia/reperfusion injury and neurological dysfunction. Recent evidence suggested that neuraminidase could exacerbate inflammatory responses by disrupting CD24‐Siglec‐G/10 immune checkpoint axis. As a neuraminidase inhibitor, oseltamivir phosphate (OP) holds potential for immunomodulation beyond its antiviral use. We aimed to investigate the impact and mechanism of OP on neuroinflammation regulation after ROSC. Methods Male pigs were randomized into the sham control group, CPR, and CPR + OP group. CA was induced in pigs through 8 min of untreated ventricular fibrillation. Brains were harvested for assessing serum inflammatory markers and neuronal damage at 24 h after ROSC. BV2 microglial underwent oxygen–glucose deprivation/reperfusion (OGD/R). Effects of OP on inflammatory responses, NF‐κB activation, cell viability, and the CD24‐Siglec‐G/10 interaction were evaluated using immunofluorescence, immunoprecipitation, molecular, and biochemical assays. Results In vivo, OP attenuated pig cerebral microglial activation and neuronal integrity with attenuated neuroinflammation, alongside time‐dependent neuraminidase activity increases. In vitro, OP suppressed OGD/R‐induced microglial NF‐κB activation, reduced pro‐inflammatory cytokine levels, and preserved CD24‐Siglec‐G interaction, correlating with diminished neuraminidase release. Conclusions OP as a repurposed immunomodulator that suppresses microglial‐driven neuroinflammation after CA by preserving sialylation‐dependent CD24‐Siglec‐G/10 interaction. The schematic diagram demonstrates how oseltamivir phosphate (OP, Tamiflu) alleviates cerebral injury and neuroinflammation after cardiac arrest/cardiopulmonary resuscitation (CA/CPR) through the CD24‐Siglec‐G/10 axis. The interaction between CD24 and Siglec‐G/10 via sialic acid restrains NF‐κB p65 nuclear translocation. This molecular inhibitory mechanism is compromised by neuraminidase during ischemia‐reperfusion injury, while OP exerts neuroprotection through targeted inhibition of desialylation, offering therapeutic opportunities.

Ischemic stroke is well-known for its high mortality and morbidity, but its treatment remains to be explored due to the current limitations. For example, severe neuroinflammation occurs after ischemic stroke; however, effective neuroinflammatory inhibitors are still lacking. Thus, the development of new therapeutic targets of inhibiting neuroinflammation is urgent. CD24 is a small heavy glycosylated protein, which plays a critical role in neural development and acts as an inflammatory suppressor in tumors and autoimmune diseases. But the role of CD24 in ischemic stroke remains unknown. The role of CD24 in ischemic stroke should be explored. Additionally, the potential relationship between the H S donor, S-propargyl-cysteine (SPRC) and CD24 in ischemic stroke should be revealed. Mechanism studies have been performed both in vitro and in vivo to verify the CD24-mediated inflammation and migration. SPRC has been applied to treat ischemic stroke, and its potential association with CD24 has been studied. The overexpression of CD24 can inhibit the nuclear factor kappa B (NF-κB) inflammatory signaling pathway and promote the migration ability of M2 microglia cells via Src/Fak/Pyk2 signaling pathway in an inflammatory model of BV2 cells. SPRC can upregulate the level of endogenous H S via cystathionase-β-synthase (CBS) and it indirectly plays a role in upregulating CD24. CD24 could be a potential target of inhibiting neuroinflammation. SPRC reduces inflammation in ischemic stroke by regulating the CD24/Iκ-Bα/NF-κB inflammatory signaling pathway and improves the migration ability of M2 microglia via CD24/Src/Fak/Pyk2 signaling pathway, which further alleviates the inflammatory response at the lesion.

Increased expression of CD24 and MET, markers for cancer stem‐like cells (CSCs), are each associated with ovarian cancer severity. However, whether CD24 and MET are co‐expressed in ovarian CSCs and, if so, how they are related to CSC phenotype manifestation remains unknown. Our immunohistochemistry analysis showed that the co‐expression of CD24 and MET was associated with poorer patient survival in ovarian cancer than those without. In addition, analyses using KM plotter and ROC plotter presented that the overexpression of CD24 or MET in ovarian cancer patients was associated with resistance to platinum‐based chemotherapy. In our miRNA transcriptome and putative target genes analyses, miR‐181a was downregulated in CD24‐high ovarian cancer cells compared to CD24‐low and predicted to bind to CD24 and MET 3'UTRs. In OV90 and SK‐OV‐3 cells, CD24 downregulated miR‐181a expression by Src‐mediated YY1 activation, leading to increased expression of MET. And, CD24 or MET knockdown or miR‐181a overexpression inhibited the manifestation of CSC phenotypes, cellular quiescence‐like state and chemoresistance, in OV90 and SK‐OV‐3 cells: increased colony formation, decreased G0/G1 phase cell population and increased sensitivity to Cisplatin and Carboplatin. Our findings suggest that CD24‐miR‐181a‐MET may consist of a signalling route for ovarian CSCs, therefore being a combinatory set of markers and therapeutic targets for ovarian CSCs. CD24‐mediated signalling route for ovarian cancer stem‐like phenotype manifestation. In the absence of CD24‐mediate signalling, MET mRNA is degraded by miR‐181a. On the other hand, in the presence of CD24‐mediate signalling, the YY1 activated by Src suppresses the transcription of miR‐181a, which leads to MET expression. MET may induce the cellular quiescence‐like state and its following chemoresistance in ovarian cancer cells.

Overexpression of CD24 is an independent prognostic factor for breast cancer. Recently, two polymorphisms in the CD24 gene were linked to disease risk and progression in autoimmune diseases. Here, we evaluated the clinical relevance of these polymorphisms with respect to their potential to predict a pathologic complete response (pCR) to neoadjuvant chemotherapy (NCT) for primary breast cancer (PBC), one of the strongest prognostic factors in this setting. A total of 257 patients were randomized to either doxorubicin/cyclophosphamide (AC) or doxorubicin/pemetrexed (AP), both followed by docetaxel (Doc) as NCT for T2-4 N0-2 M0 PBC as part of an international, multicenter, randomized phase II trial. CD24 polymorphisms were analyzed on germ line DNA and correlated with clinicopathologic variables and pCR. No significant associations were found between either of the polymorphisms and any of the clinicopathologic variables. In a multivariate analysis, CD24 Val/Val genotype was the only significant predictor of pCR (OR: 4.97; P  = 0.003). The predictive potential was significant in both treatment arms and in the hormone receptor–positive subgroup. There was no correlation between CD24 3′UTR (TG/Del) genotype and pCR. We did not observe any association between CD24 genotype and CD24 protein expression or in vitro chemosensitivity, but there was a significant correlation between CD24 Val/Val and intratumoral lymphocyte aggregates. In conclusion, CD24 Ala/Val SNP is a strong and independent predictor of pCR after NCT for PBC and may affect immune functions rather than tumor characteristics. Further evaluation of the CD24 function and validation of its predictive potential are clearly warranted.

BackgroundAside from immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1), intervention of CD47/Sirpα mediated ‘don’t eat me’ signal between macrophage and tumor cell is considered as a promising therapeutic approach for cancer immunotherapy. Compared with CD47, the novel immune checkpoint CD24/Siglec-10 can also deliver ‘don’t eat me’ signal and CD24 shows much lower expression level in normal tissue which might avoid unwanted side effects.MethodsCell-based phage display biopanning and D-amino acid modification strategy were used to identify the CD24/Siglec-10 blocking peptide. Cell-based blocking assay and microscale thermophoresis assay were used to validate the blocking and binding activities of the peptide. Phagocytosis and co-culture assays were used to explore the in vitro function of the peptide. Flow cytometry was performed to assess the immune microenvironment after the peptide treatment in vivo.ResultsA CD24/Siglec-10 blocking peptide (CSBP) with hydrolysis-resistant property was identified. Surprisingly, we found that CSBP could not only block the interaction of CD24/Siglec-10 but also PD-1/PD-L1. CSBP could induce the phagocytosis of tumor cell by both the macrophages and monocytic myeloid-derived suppressor cells (M-MDSCs), which can further activate CD8+ T cells. Besides, combination of radiotherapy and CSBP synergistically reduced tumor growth and altered the tumor microenvironment in both anti-PD-1-responsive MC38 and anti-PD-1-resistant 4T1 tumor models.ConclusionsIn summary, this is the first CD24/Siglec-10 blocking peptide which blocked PD-1/PD-L1 interaction as well, functioned via enhancing the phagocytosis of tumor cells by macrophages and M-MDSCs, and elevating the activity of CD8+ T cells for cancer immunotherapy.

Type I interferon is widely used for antiviral therapy, yet has yielded disappointing results toward chronic HBV infection. Here we identify that PEG-IFNα-2b therapy toward persistent infection in humans is a double-edged sword of both immunostimulation and immunomodulation. Our studies of this randomised trial showed persistent PEG-IFNα-2b therapy induced large number of CD24 + CD38 hi B cells and launched a CD24 + CD38 hi B cells centered immunosuppressive response, including downregulating functions of T cells and NK cells. Patients with low induced CD24 + CD38 hi B cells have achieved an improved therapeutic effect. Specifically, using the anti-CD24 antibody to deplete CD24 + CD38 hi B cells without harming other B cell subsets suggest a promising strategy to improve the therapeutic effects. Our findings show that PEG-IFNα-2b therapy toward persistent infection constitutes an immunomodulation effect, and strategies to identifying the molecular basis for the antiviral versus immunomodulatory effects of PEG-IFNα-2b to selectively manipulate these opposing activities provide an opportunity to ameliorate anti-virus immunity and control viral infection.

BackgroundTumor-associated macrophages (TAMs) abundantly infiltrate tumors and possess potent antitumor capabilities. “Don‘t eat me” signals like CD47 allow tumors to evade macrophages and proliferate unchecked. CD47 is upregulated in many tumors and interacts with the SIRPα expressed on macrophages to restrict effector function. Similarly, CD24 interacts with the Siglec-10 on TAMs to inhibit engulfment. Despite their potential, there is still a lack of effective therapeutics targeting macrophages. Recent clinical trials targeting CD47 have demonstrated limited efficacy and significant side effects in solid tumors, primarily due to the expression of CD47 on healthy cells such as red blood cells (RBCs). We therefore developed novel anti-CD47 variable domain of heavy chain of heavy-chain antibodies (vHHs) with strong ligand-blocking activity while demonstrating minimal binding to RBCs and incorporated these vHHs to generate an anti-CD47/CD24 bispecific antibody that preserves Fc-effector function and achieves improved tumor targeting while maintaining the blockade of antiphagocytic signals elicited by both CD47 and CD24.MethodsYeast display was employed to generate vHHs targeting CD47 and fully human monoclonal antibodies against CD24, respectively. The antigen binding epitopes of the vHHs to CD47 were predicted using AlphaFold3. Bispecific antibodies were designed, constructed, and characterized in vitro. Antitumor efficacy was evaluated in a human immune cell reconstitution mouse model, while safety was evaluated using a humanized syngeneic mouse model. Furthermore, the underlying mechanisms and alterations in tumor microenvironment were explored ex vivo.ResultsVHHs targeting CD47 and a fully human antibody against CD24 were identified, all exhibiting potent ligand-blocking activity. The bispecific antibody BiAb-103C, engineered on a human IgG1 scaffold, had strong binding to CD47+CD24+ tumor cells and could effectively inhibit the CD47-SIRPα interaction. Fc-effector activity was observed towards CD24 (but not CD47) single-positive cells to promote phagocytosis and antibody-dependent cellular cytotoxicity of CD47+CD24+ tumor cells. In mice, antibody candidates demonstrated notable antitumor activity alongside favorable safety observations.ConclusionsOur study presents the discovery of an anti-CD47/CD24 bispecific antibody that offers a promising therapeutic strategy to address the challenges associated with both the efficacy and safety of CD47-targeting agents, offers insight into macrophage-driven cancer immunotherapy, and could potentially provide a therapeutic option for patients non-responsive to immunotherapy.