Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning "Two Keys" simultaneously.

BACKGROUND: Macrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo. METHODS: Macrophages were derived from human peripheral blood monocytes by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors. RESULTS: We found that MAG significantly increased macrophage phagocytosis of ES cells in vitro (p < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors. CONCLUSIONS: By turning "two keys" simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES.

M6A-mediated upregulation of lncRNA TUG1 in liver cancer cells regulates the antitumor response of CD8+ T cells and phagocytosis of macrophages.

Tumor immune evasion relies on the crosstalk between tumor cells and adaptive/innate immune cells. Immune checkpoints play critical roles in the crosstalk, and immune checkpoint inhibitors have achieved promising clinical effects. The long non-coding RNA taurine-upregulated gene 1 (TUG1) is upregulated in hepatocellular carcinoma (HCC). However, how TUG1 is upregulated and the effects on tumor immune evasion are incompletely understood. Here, METTL3-mediated m6A modification led to TUG1 upregulation is demonstrated. Knockdown of TUG1 inhibited tumor growth and metastasis, increased the infiltration of CD8+ T cells and M1-like macrophages in tumors, promoted the activation of CD8+ T cells through PD-L1, and improved the phagocytosis of macrophages through CD47. Mechanistically, TUG1 regulated PD-L1 and CD47 expressions by acting as a sponge of miR-141 and miR-340, respectively. Meanwhile, TUG1 interacted with YBX1 to facilitate the upregulation of PD-L1 and CD47 transcriptionally, which ultimately regulated tumor immune evasion. Clinically, TUG1 positively correlated with PD-L1 and CD47 in HCC tissues. Moreover, the combination of Tug1-siRNA therapy with a Pdl1 antibody effectively suppressed tumor growth. Therefore, the mechanism of TUG1 in regulating tumor immune evasion is revealed and can inform existing strategies targeting TUG1 for enhancing HCC immune therapy and drug development.

Beta cells deficient for Renalase counteract autoimmunity by shaping natural killer cell activity.

Type 1 diabetes (T1D) arises from autoimmune-mediated destruction of insulin-producing pancreatic beta cells. Recent advancements in the technology of generating pancreatic beta cells from human pluripotent stem cells (SC-beta cells) have facilitated the exploration of cell replacement therapies for treating T1D. However, the persistent threat of autoimmunity poses a significant challenge to the survival of transplanted SC-beta cells. Genetic engineering is a promising approach to enhance immune resistance of beta cells as we previously showed by inactivating the Renalase (Rnls) gene. Here, we demonstrate that Rnls loss of function in beta cells shapes autoimmunity by mediating a regulatory natural killer (NK) cell phenotype important for the induction of tolerogenic antigen-presenting cells. Rnls-deficient beta cells mediate cell-cell contact-independent induction of hallmark anti-inflammatory cytokine Tgfß1 in NK cells. In addition, surface expression of regulatory NK immune checkpoints CD47 and Ceacam1 is markedly elevated on beta cells deficient for Rnls. Altered glucose metabolism in Rnls mutant beta cells is involved in the upregulation of CD47 surface expression. These findings are crucial to better understand how genetically engineered beta cells shape autoimmunity, giving valuable insights for future therapeutic advancements to treat and cure T1D.

Joint DNA-RNA-based NGS for diagnosis and treatment of a rare CD47-MET fusion lung adenocarcinoma which was immunoresistant and savoltinib-sensitive: a case report.

Targeted therapy and immunotherapy are both important in the treatment of non-small-cell lung cancer (NSCLC). Accurate diagnose and precise treatment are key in achieving long survival of patients. MET fusion is a rare oncogenic factor, whose optimal detection and treatment are not well established. Here, we report on a 32-year-old female lung adenocarcinoma patient with positive PD-L1 and negative driver gene detected by DNA-based next-generation sequencing (NGS). A radical resection of the primary lesion after chemotherapy combined with PD-1 checkpoint inhibitor administration indicated primary immuno-resistance according to her pathological response and rapid relapse. A rare CD47-MET was detected by RNA-based NGS, which was confirmed by fluorescence in situ hybridization. Multiplex immunofluorescence revealed a PD-L1 related heterogeneous immunosuppressive microenvironment with little distribution of CD4+ T cells and CD8+ T cells. Savolitinib therapy resulted in a progression-free survival (PFS) of >12 months, until a new secondary resistance mutation in MET p.D1228H was detected by re-biopsy and joint DNA-RNA-based NGS after disease progression. In this case, CD47-MET fusion NSCLC was primarily resistant to immunotherapy, sensitive to savolitinib, and developed secondary MET p.D1228H mutation after targeted treatment. DNA-RNA-based NGS is useful in the detection of such molecular events and tracking of secondary mutations in drug resistance. To this end, DNA-RNA-based NGS may be of better value in guiding precise diagnosis and individualized treatment in this patient population.

Combining CD38 antibody with CD47 blockade is a promising strategy for treating hematologic malignancies expressing CD38.

Background: CD38 and CD47 are expressed in many hematologic malignancies, including multiple myeloma (MM), B-cell non-Hodgkin lymphoma (NHL), B-cell acute lymphoblastic leukemia (ALL), and B-cell chronic lymphocytic leukemia (CLL). Here, we evaluated the antitumor activities of CD38/CD47 bispecific antibodies (BsAbs). Methods: Five suitable anti-CD38 antibodies for co-targeting CD47 and CD38 BsAb were developed using a 2 + 2 "mAb-trap" platform. The activity characteristics of the CD38/CD47 BsAbs were evaluated using in vitro and in vivo systems. Results: Using hybridoma screening technology, we obtained nine suitable anti-CD38 antibodies. All anti-CD38 antibodies bind to CD38+ tumor cells and kill tumor cells via antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Five anti-CD38 antibodies (4A8, 12C10, 26B4, 35G5, and 65A7) were selected for designing CD38/CD47 BsAbs (IMM5605) using a "mAb-trap" platform. BsAbs had higher affinity and binding activity to the CD38 target than those to the CD47 target, decreasing the potential on-target potential and off-tumor effects. The CD38/CD47 BsAbs did not bind to RBCs and did not induce RBC agglutination; thus, BsAbs had much lower blood toxicity. The CD38/CD47 BsAbs had a greater ability to block the CD47/SIRPα signal in CD38+/CD47+ tumor cells than IMM01 (SIRPα Fc fusion protein). Through Fc domain engineering, CD38/CD47 BsAbs were shown to kill tumors more effectively by inducing ADCC and ADCP. IMM5605-26B4 had the strongest inhibitory effect on cellular CD38 enzymatic activity. IMM5605-12C10 had the strongest ability to directly induce the apoptosis of tumor cells. The anti-CD38 antibody 26B4 combined with the SIRPα-Fc fusion proteins showed strong antitumor effects, which were better than any of the mono-therapeutic agents used alone in the NCI-H929 cell xenograft model. The CD38/CD47 BsAbs exhibited strong antitumor effects; specifically, IMM5605-12C10 efficiently eradicated all established tumors in all mice. Conclusion: A panel of BsAbs targeting CD38 and CD47 developed based on the "mAb-tarp" platform showed potent tumor-killing ability in vitro and in vivo. As BsAbs had lower affinity for binding to CD47, higher affinity for binding to CD38, no affinity for binding to RBCs, and did not induce RBC agglutination, we concluded that CD38/CD47 BsAbs are safe and have a satisfactory tolerability profile.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen.

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Photodynamic Therapy Synergizes CD47 Blockade Strategy for Enhanced Antitumor Therapy.

The antitumor strategies based on innate immunity activation have become favored by researchers in recent years. In particular, strategies targeting antiphagocytic signaling blockade to enhance phagocytosis have been widely reported. For example, the addition of prophagocytic signals such as calreticulin could make the strategy significantly more effective. In this study, an antitumor strategy that combines photodynamic therapy (PDT) with CD47 blockade has been reported. This approach promotes the maturation of dendritic cells and the presentation of tumor antigens by PDT-mediated tumor immunogenic cell death, as well as the enhancement of cytotoxic T lymphocyte infiltration in tumor areas and the phagocytic activity of phagocytes. Furthermore, the downregulation and blockage of CD47 protein could further promote phagocytic activity, strengthen the innate immune system, and ultimately elevate the antitumor efficacy and inhibit tumor metastasis.

Evorpacept-Induced Interference and Application of a Novel Mitigation Agent, Evo-NR, in Pretransfusion Testing.

Introduction: Evorpacept is a CD47-blocking agent currently being developed for the treatment of various cancers. Interference by evorpacept in pretransfusion compatibility testing has been reported at limited plasma concentrations. Although various mitigation strategies have been proposed, none are practical. This in vitro study assessed evorpacept-induced interference at extended concentrations and investigated the capability of a novel mitigation agent, Evo-NR. Methods: Antibody screening tests were performed on evorpacept-spiked plasma with (anti-E and anti-Jka) or without alloantibodies at evorpacept concentrations up to 2,000 µg/mL using manual gel cards and automated analyzers. Evorpacept-coated red blood cells (RBCs) (rr [ce/ce], Fy[a+b-], S-s+) were tested by direct antiglobulin testing (DAT) and antigen typing using anti-Fyb and anti-S reagents at indirect antiglobulin testing (IAT) phase. Evo-NR was used to resolve the interference in plasma and RBC samples. Flow cytometry was used to assess the mitigation effects. Results: Evorpacept-spiked plasma showed panreactive interference in antibody screening tests using manual gel cards (2+ to 3+) and automated analyzers (4+). A carryover effect was also observed in the automated analyzers. The use of a 3- to 6-fold molar excess of Evo-NR effectively resolved the interference in the plasma and enabled accurate alloantibody identification. Although the reduction in evorpacept binding to RBCs was identified via flow cytometry, Evo-NR was incapable of resolving the serologic interference observed in DAT and antigen typing at IAT phase. Discussion: Evorpacept showed constant panreactivity and a carryover effect at high concentrations. Evo-NR successfully resolved the interference in the plasma samples and could be considered a practical and efficient mitigation solution. Implementation of Evo-NR has the potential to support RBC transfusion for patients undergoing evorpacept treatment.

A Perspective on the CD47-SIRPA Axis in High-Risk Neuroblastoma.

Neuroblastoma is a pediatric cancer with significant clinical heterogeneity. Despite extensive efforts, it is still difficult to cure children with high-risk neuroblastoma. Immunotherapy is a promising approach to treat children with this devastating disease. We have previously reported that macrophages are important effector cells in high-risk neuroblastoma. In this perspective article, we discuss the potential function of the macrophage inhibitory receptor SIRPA in the homeostasis of tumor-associated macrophages in high-risk neuroblastoma. The ligand of SIRPA is CD47, known as a "don't eat me" signal, which is highly expressed on cancer cells compared to normal cells. CD47 is expressed on both tumor and stroma cells, whereas SIRPA expression is restricted to macrophages in high-risk neuroblastoma tissues. Notably, high SIRPA expression is associated with better disease outcome. According to the current paradigm, the interaction between CD47 on tumor cells and SIRPA on macrophages leads to the inhibition of tumor phagocytosis. However, data from recent clinical trials have called into question the use of anti-CD47 antibodies for the treatment of adult and pediatric cancers. The restricted expression of SIRPA on macrophages in many tissues argues for targeting SIRPA on macrophages rather than CD47 in CD47/SIRPA blockade therapy. Based on the data available to date, we propose that disruption of the CD47-SIRPA interaction by anti-CD47 antibody would shift the macrophage polarization status from M1 to M2, which is inferred from the 1998 study by Timms et al. In contrast, the anti-SIRPA F(ab')2 lacking Fc binds to SIRPA on the macrophage, mimics the CD47-SIRPA interaction, and thus maintains M1 polarization. Anti-SIRPA F(ab')2 also prevents the binding of CD47 to SIRPA, thereby blocking the "don't eat me" signal. The addition of tumor-opsonizing and macrophage-activating antibodies is expected to enhance active tumor phagocytosis.

The biological roles of CD47 in ovarian cancer progression.

Ovarian cancer is one of the most lethal malignant tumors, characterized by high incidence and poor prognosis. Patients relapse occurred in 65-80% after initial treatment. To date, no effective treatment has been established for these patients. Recently, CD47 has been considered as a promising immunotherapy target. In this paper, we reviewed the biological roles of CD47 in ovarian cancer and summarized the related mechanisms. For most types of cancers, the CD47/Sirpα immune checkpoint has attracted the most attention in immunotherapy. Notably, CD47 monoclonal antibodies and related molecules are promising in the immunotherapy of ovarian cancer, and further research is needed. In the future, new immunotherapy regimens targeting CD47 can be applied to the clinical treatment of ovarian cancer patients.

Evaluation of stored red blood cell quality after washing using immune indices.

Washed red blood cells (RBCs) can be used to treat immune-related diseases. However, whether the washing process changes the quality of RBCs and affects the curative effect of transfusion therapy remains unclear. We retrospectively analysed the clinical data of patients who received blood transfusion. The physiological and biochemical parameters of RBCs were tested on an automated haematology-biochemical analyser. CD47 and phosphatidylserine (PS) plasma membrane expression were analysed using flow cytometry. Morphological changes in RBCs were observed using scanning electron microscopy. The results showed that the curative effect on patients who received washed RBCs was weaker than that on those who received non-washed RBCs. Physiological and biochemical parameters of RBCs were not significantly different. RBC immune indices changed significantly after washing. The expression of "don't eat me" signals was weakened, whereas the intensity of "eat me" signals was enhanced. This study suggests that the current use of physiological and biochemical parameters as indicators to evaluate the quality of RBCs may not be comprehensive and that evaluation of the real status of RBCs requires other effective parameters. Immune molecules in RBCs are expected to become supplementary markers for evaluating RBC quality.

CD47, a novel YAP target gene, contributes to hepatic stellate cell activation and liver fibrosis induced by high-fat diet.

Activated hepatic stellate cells (HSCs) have been widely recognized as a primary source of pathological myofibroblasts, leading to the accumulation of extracellular matrix and liver fibrosis. CD47, a transmembrane glycoprotein expressed on the surface of various cell types, has been implicated in non-alcoholic fatty liver disease. However, the precise role of CD47 in HSC activation and the underlying regulatory mechanisms governing CD47 expression remain poorly understood. In this study, we employed single-cell RNA sequencing analysis to investigate CD47 expression in HSCs from mice subjected to a high-fat diet. CD47 silencing in HSCs markedly inhibited the expression of fibrotic genes and promoted apoptosis. Mechanistically, we found that Yes-associated protein (YAP) collaborates with TEAD4 to augment the transcriptional activation of CD47 by binding to its promoter region. Notably, disruption of the interaction between YAP and TEAD4 caused a substantial decrease in CD47 expression in HSCs and reduced the development of high-fat diet-induced liver fibrosis. Our findings highlight CD47 as a critical transcriptional target of YAP in promoting HSC activation in response to a high-fat diet. Targeting the YAP/TEAD4/CD47 signaling axis may hold promise as a therapeutic strategy for liver fibrosis.

A novel anti-CD47 protein antibody and toll-like receptor agonist complex detects tumor surface CD-47 changes in early stage lung cancer by in vivo imaging.

CD47, a cell surface protein known for inhibiting phagocytosis, plays a critical role in the tumor microenvironment (TME) and is a potential biomarker for cancer. However, directly applying αCD47, a hydrophilic macromolecular antibody that targets CD47, in vivo for cancer detection can have adverse effects on normal cells, cause systemic toxicities, and lead to resistance against anti-cancer therapies. In this study, we developed a novel complex incorporating aluminum-based metal-organic frameworks (Al-MOF) loaded with indocyanine green (ICG), αCD47, and resiquimod (R848), a hydrophobic small molecule Toll-like receptor 7/8 (TLR7/8) agonist. Upon activation with an infrared 808 nm laser, the nanocomposites exhibited photothermal effects that triggered the release of the loaded reagents, induced ROS production, and induced changes in the TME. This led to the polarization of immune-suppressive M2 macrophages towards an immune-stimulatory M1 phenotype, promoted dendritic cell (DC) maturation, and enabled mature DCs to facilitate antigen presentation, T-cell activation, and critical roles in tumor immunity. Furthermore, in vivo imaging successfully detected the specific binding of αCD47 with CD47 on tumor cells. Overall, the complex composed of αCD47 antibody and toll-like receptor agonist showed promising efficacy in both tumor diagnosis and therapy, providing a potential strategy for detecting early lung cancer and modulating the tumor microenvironment for improved treatment outcomes.

The cGAS-STING pathway drives inflammation in Usual Interstitial Pneumonia, phagocytosis could prevent inflammation but is inhibited by the don't eat me signal CD47.

BACKGROUND: Usual Interstitial Pneumonia (UIP) a fibrosing pneumonia is associated with idiopathic pulmonary fibrosis, chronic autoimmune disease (AID), or hypersensitivity pneumonia. Oxygen radicals, due to tobacco smoke, can damage DNA and might upregulate PARP1. Cytosolic DNA from dying pneumocytes activate cytosolic GMP-AMP-synthase-stimulator of interferon genes (cGAS-STING) pathway and TREX1. Prolonged inflammation induces senescence, which might be inhibited by phagocytosis, eliminating nuclear debris. We aimed to evaluate activation of cGAS-STING-TREX1 pathway in UIP, and if phagocytosis and anti-phagocytosis might counteract inflammation. METHODS: 44 cases of UIP with IPF or AID were studied for the expression of cGAS, pSTING, TREX1 and PARP1. LAMP1 and Rab7 expression served as phagocytosis markers. CD47 protecting phagocytosis and p16 to identify senescent cells were also studied. RESULTS: Epithelial cells in remodeled areas and macrophages expressed cGAS-pSTING, TREX1; epithelia but not macrophages stained for PARP1. Myofibroblasts, endothelia, and bronchial/bronchiolar epithelial cells were all negative except early myofibroblastic foci expressing cGAS. Type II pneumocytes expressed cGAS and PARP1, but less pSTING. TREX1 although expressed was not activated. Macrophages and many regenerating epithelial cells expressed LAMP1 and Rab7. CD47, the 'don't-eat-me-signal', was expressed by macrophages and epithelial cells including senescence cells within the remodeled areas. CONCLUSIONS: The cGAS-STING pathway is activated in macrophages and epithelial cells within remodeled areas. LikelyTREX1 because not activated cannot sufficiently degrade DNA fragments. PARP1 activation points to smoking-induced oxygen radical release, prolonging inflammation and leading to fibrosis. By expressing CD47 epithelial cells within remodeled areas protect themselves from being eliminated by phagocytosis.

Intravenous Administration of Anti-CD47 Antibody Augments Hematoma Clearance, Mitigates Acute Neuropathology, and Improves Cognitive Function in a Rat Model of Penetrating Traumatic Brain Injury.

Traumatic brain injury (TBI)-induced intracerebral hematoma is a major driver of secondary injury pathology such as neuroinflammation, cerebral edema, neurotoxicity, and blood-brain barrier dysfunction, which contribute to neuronal loss, motor deficits, and cognitive impairment. Cluster of differentiation 47 (CD47) is an antiphagocytic cell surface protein inhibiting hematoma clearance. This study was designed to evaluate the safety and efficacy of blockade of CD47 via intravenous (i.v.) administration of anti-CD47 antibodies following penetrating ballistic-like brain injury (PBBI) with significant traumatic intracerebral hemorrhage (tICH). The pharmacokinetic (PK) profile of the anti-CD47 antibody elicited that antibody concentration decayed over 7 days post-administration. Blood tests and necropsy analysis indicated no severe adverse events following treatment. Cerebral hemoglobin levels were significantly increased after injury, however, anti-CD47 antibody administration at 0.1 mg/kg resulted in a significant reduction in cerebral hemoglobin levels at 72 h post-administration, indicating augmentation of hematoma clearance. Immunohistochemistry assessment of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1) demonstrated a significant reduction of GFAP levels in the lesion core and peri-lesional area. Based on these analyses, the optimal dose was identified as 0.1 mg/kg. Lesion volume showed a reduction following treatment. Rotarod testing revealed significant motor deficits in all injured groups but no significant therapeutic benefits. Spatial learning performance revealed significant deficits in all injured groups, which were significantly improved by the last testing day. Anti-CD47 antibody treated rats showed significantly improved attention deficits, but not retention scores. These results provide preliminary evidence that blockade of CD47 using i.v. administration of anti-CD47 antibodies may serve as a potential therapeutic for TBI with ICH.

Structural analysis of light chain-driven bispecific antibodies targeting CD47 and PD-L1.

In contrast to natural antibodies that rely mainly on the heavy chain to establish contacts with their cognate antigen, we have developed a bispecific antibody format in which the light chain (LC) drives antigen binding and specificity. To better understand epitope-paratope interactions in this context, we determined the X-ray crystallographic structures of an antigen binding fragment (Fab) in complex with human CD47 and another Fab in complex with human PD-L1. These Fabs contain a κ-LC and a λ-LC, respectively, which are paired with an identical heavy chain (HC). The structural analysis of these complexes revealed the dominant contribution of the LCs to antigen binding, but also that the common HC provides some contacts in both CD47 and PD-L1 Fab complexes. The anti-CD47 Fab was affinity optimized by diversifying complementary-determining regions of the LC followed by phage display selections. Using homology modeling, the contributions of the amino acid modification to the affinity increase were analyzed. Our results demonstrate that, despite a less prominent role in natural antibodies, the LC can mediate high affinity binding to different antigens and neutralize their biological function. Importantly, Fabs containing a common variable heavy (VH) domain enable the generation of bispecific antibodies retaining a truly native structure, maximizing their therapeutic potential.

Clinicopathological analysis of immunohistochemical CD47 and signal-regulatory protein-α expression in Extranodal Natural killer/T-cell lymphoma.

BACKGROUND: The interaction between CD47 and signal-regulatory protein-alpha (SIRPα) inhibits phagocytosis, and their clinicopathological characteristics have been evaluated in various diseases. However, the significance of CD47 and SIRPα expression, as well as the combined effect, in Extranodal Natural killer/T-cell Lymphoma (ENKTL) remains uncertain. METHODS: In total, 76 newly diagnosed ENKTL patients (mean age 49.9 years, 73.7% male) were included in this study. CD47 and SIRPα expression were examined by immunohistochemistry. Survival analyses were conducted through Kaplan-Meier curves and the Cox regression model. RESULTS: Seventy-one (93.4%) cases were categorized as the CD47 positive group and 59 (77.6%) cases were categorized as the SIRPα positive group. CD47-negative cases had more advanced-stage illness (P = 0.001), while SIRPα-positive cases showed significantly lower levels of high-density lipoprotein (P < 0.001). In univariable analysis, CD47, SIRPα expression, and their combination were significantly associated with prognosis (P < 0.05). In multivariable analysis, only positive SIRPα expression remained significantly associated with superior overall survival (Hazard ratio [HR] 0.446; 95% confidence interval [CI] 0.207-0.963; P = 0.004). Furthermore, SIRPα expression could re-stratify the survival of patients in ECOG (< 2), advanced CA stage, PINK (HR), CD38-positive, PD1-positive, and CD30-positive groups. CONCLUSIONS: SIRPα status was a potential independent prognostic factor for ENKTL. The prognostic significance of CD47 expression and the interaction between CD47 and SIRPα in ENKTL need further investigation.

Mechanism of efferocytosis in atherosclerosis.

Atherosclerosis (AS) is a chronic inflammatory vascular disease that occurs in the intima of large and medium-sized arteries with the immune system's involvement. It is a common pathological basis for high morbidity and mortality of cardiovascular diseases. Abnormal proliferation of apoptotic cells and necrotic cells leads to AS plaque expansion, necrotic core formation, and rupture. In the early stage of AS, macrophages exert an efferocytosis effect to engulf and degrade apoptotic, dead, damaged, or senescent cells by efferocytosis, thus enabling the regulation of the organism. In the early stage of AS, macrophages rely on this effect to slow down the process of AS. However, in the advanced stage of AS, the efferocytosis of macrophages within the plaque is impaired, which leads to the inability of macrophages to promptly remove the apoptotic cells (ACs) from the organism promptly, causing exacerbation of AS. Moreover, upregulation of CD47 expression in AS plaques also protects ACs from phagocytosis by macrophages, resulting in a large amount of residual ACs in the plaque, further expanding the necrotic core. In this review, we discussed the molecular mechanisms involved in the process of efferocytosis and how efferocytosis is impaired and regulated during AS, hoping to provide new insights for treating AS.

Is overexpression of CD163 and CD47 in tumour cells of breast carcinoma implicated in the recruitment of tumour-associated macrophages (TAMs) in tumour microenvironment? immunohistochemical prognostic study.

BACKGROUND: Now, targeted therapy and immunotherapy are promoted. tumour -Associated Macrophages (TAMs) are an essential component of immune-response in breast cancer(BC) with prognostic controversy. Additionally, their recruiting factors are still obscure. Purpose:This study aimed to evaluate the prognostic significance of CD163 and CD47 in BC of No Special Type (BC-NST) and to explore their suggested role in recruiting TAMs. MATERIAL AND METHODS: This immunohistochemical study was conducted on 91 archival specimens of breast cases. Immunoreactivity scores were correlated with TAMs density, clinicopathological data, and survival. RESULTS: Revealed the highest CD163 expression was detected in the pure DCIS group (p = 0.016), while the highest CD47 expression and high TAMs density were reported in the invasive group (p = 0.008, and p = 0.002 respectively) followed by the DCIS group. In IC-NSTs the CD163 and CD47 scores were associated with poor prognostic parameters like(high grade, advanced stage, distant metastasis, ER negativity,Ki67 index, post-surgical chemotherapy, poor NPI group, high mitotic count, dense infiltration of TAMs, shorter OS). Also, CD47 was associated with the dens infiltration of TAMs in DCIS (p = 0.001). There was a significant correlation between tumour cell expression of CD163 and CD47 in IC-NSTs and DCIS (p = 0.002 and p = 0.009 respectively). CONCLUSIONS: High CD163 and CD47 expressions in both DCIS andIBC are intimately associated, significantly associated with poor prognosis and are important provoking factors of TAMs.