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【Objective】 To perform pre-transfusion examination and major crossmatch test using CD47 anti-idiotypic antibody (CD47 AID) (method 1) and reagent lack of anti-IgG4 anti-human globulin(method 2) in patients treated with CD47 monoclonal antibodies, and evaluate the feasibility of method 1 by comparing the transfusion efficacy of patients after cross matching with two methods. 【Methods】 Post-drug samples were collected from 18 clinical subjects treated with CD47 monoclonal antibody in our hospital. Antibody screening and major crossmatch test were performed using method 1 and method 2, and the difference of ΔHb (post-transfusion Hb minus pre-transfusion Hb) was compared after transfusion. The differences in ΔHb after transfusion were analyzed between the test group using method 1 and the control group without CD47 monoclonal antibody using ordinary microcolumn gel method. 【Results】 There was no significant difference in ΔHb between the test group using method 1 and test group using method 2 (8.40±0.71 vs 7.36±0.94, P>0.05). No significant difference was noticed in ΔHb between the test group using method 1 and the control group without CD47 monoclonal antibody (8.40±0.71 vs 6.59±0.77, P>0.05). 【Conclusion】 In the test group, major crossmatch test with method 1 has the same transfusion efficacy as the test with method 2. Method 1 is simple and easy to operate, and the results are objective and accurate. It is recommended to use method 1 for pre-transfusion antibody screening and major crossmatch tests for patients using CD47 monoclonal antibody.
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CD47 is a transmembrane protein widely expressed on cell surface, which is considered as a key molecule for immune escape. With an increasing number of related studies, the role of CD47 and its ligands in immunomodulatory effects has been gradually understood. Recent studies have investigated the role of CD47 in ischemia-reperfusion injury of allogenetic kidney transplantation, rejection and xenotransplantation. Nevertheless, the specific role and the key mechanism remain elusive. In this article, the structure and function of CD47, common CD47 ligands, the relationship between CD47 and kidney transplantation, and the application of CD47 in kidney transplantation were reviewed, the latest research progress of CD47 in kidney transplantation was summarized, and the limitations of current research and subsequent research direction were analyzed, aiming to provide reference for subsequent application of CD47 in allogeneic and kidney xenotransplantation.
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Context: Despite the follow-up protocols developed in non–muscle-invasive bladder cancer patients, progression and recurrence could not be prevented. Aims: We aimed to investigate whether proteins such as OCT-4, CD47, p53, Ki-67, and Survivin, which increase in bladder cancer cells, can be used as prognostic markers for patients with non–muscle-invasive bladder cancer. Settings and Design: The study included a total of 89 patients with newly diagnosed non–muscle-invasive bladder cancer between January 2015 and December 2020. Materials and Methods: Levels of OCT-4, CD47, p53, K?-67, and Survivin proteins in cancer cells were determined with a semi-quantitative immunohistochemical experiment. Pathological data and survival rates were compared according to the staining rates. Statistical Analysis Used: Data obtained in the study were analyzed statistically with SPSS 22.0 (SPSS, Chicago, IL, USA). Results: The mean age of the patients was 64.25 ± 9.91 years, and the median follow-up period was 55 months. Recurrence rate was determined to be 36% (n = 32), and the rate of progression at 40.4% (n = 36). The staining rates were stronger for each marker in the progression group and advanced-stage tumors (p < 0.001). The findings of the multivariate analysis carried out as part of the study showed that older age and higher tumor stage were independent risk factors for recurrence-free survival (HR = 1.048 and 7.074, respectively; P = 0.02). Also, higher tumor stages, diameters, and grades were associated with reduced progression-free survival (HR = 0.105, 0.395, 0.225, respectively; P < 0.05). Conclusions: Although immunohistochemical staining rates are promising, it is more appropriate to use tumor characteristics when assessing survival rate in patients with non–muscle-invasive bladder cancer.
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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.
Sujet(s)
Humains , Souris , Animaux , Carcinome pulmonaire non à petites cellules/métabolisme , Tumeurs du poumon/métabolisme , Acrylamides/pharmacologie , Récepteurs ErbB/métabolisme , Lignée cellulaire tumorale , Antigènes CD47/usage thérapeutiqueRÉSUMÉ
【Objective】 To evaluate the interference of anti-CD47 monoclonal antibody on transfusion compatibility detection, in order to establish methods for removing interference and evaluate its efficacy. 【Methods】 Blood samples from 8 patients in our clinical trial who were treated with anti-CD47 monoclonal antibody from Tianjing and Xinda were collected. ABO and Rh blood group antigen identification, direct anti-human globulin test, unexpected antibody screening test and cross-matching test were performed by ZZAP, Gamma-clone(an anti-globulin reagent lacking IgG4) and Immucor Capture-R solid phase agglutination kit. 【Results】 ABO blood group identification of 5 subjects were interfered after treatment with anti-CD47 monoclonal antibody. All 8 subjects showed 2+ to 4+ agglutination intensity on direct anti-human globulin test and 3+ to 4+ on unexpected antibody screening. The results of unexpected antibody screening by Gamma-clone and Immucor Capture-R solid phase agglutination kit were all negative, while the cross-matching test were compatible. Patients with anemia caused by CD47 monoclonal antibody treatment were transfused with 2 U suspension red blood cells, and the evaluation showed that the transfusion was effective. 【Conclusion】 The CD47 monoclonal antibody can interfere with transfusion compatibility detection, and the use of antiglobulin reagents lacking IgG4 and Immucor Capture-R solid phase agglutination kit can remove the interference, with good transfusion efficacy in patients.
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Diffuse large B-cell lymphoma (DLBCL) is a common, highly aggressive and heterogeneous hematologic malignancy in adults. Patients with DLBCL have substantially differences in molecular biological characteristics, clinical manifestations, and prognosis. Increasing evidence shows that the tumor microenvironment plays an important role in the occurrence and development of DLBCL. CD47, an integrin related protein, is overexpressed in DLBCL cells and plays a key role in immune escape of lymphoma. This work reviews the research progress of CD47 in DLBCL TME in terms of CD47-related signal pathway, CD47 role in DLBCL TME, and therapeutic strategies targeting CD47 in DLBCL TME.
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Described as a "don't eat me" signal, CD47 becomes a vital immune checkpoint in cancer. Its interaction with signal regulatory protein alpha (SIRPα) prevents macrophage phagocytosis. In recent years, a growing body of evidences have unveiled that CD47-based combination therapy exhibits a superior anti-cancer effect. Latest clinical trials about CD47 have adopted the regimen of collaborating with other therapies or developing CD47-directed bispecific antibodies, indicating the combination strategy as a general trend of the future. In this review, clinical and preclinical cases about the current combination strategies targeting CD47 are collected, their underlying mechanisms of action are discussed, and ideas from future perspectives are shared.
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Objective@#To investigate the correlation between CD47 and cervical lesions.@*Methods@#The expression and overall survival of CD47 in cervical cancer was analyzed using TCGA and TCGA Target GTEx databases. Based on GEO databases , the expression of CD47 was analyzed between cervical sample infected by HPV 16/18 and nonHPV. The differences of CD47 expression in the degrees of cervical lesions infected by HPV 16/18 were compared.The patholgical sections of colposcopy biopsy came from 320 patients who were admitted cervical cancer screening ,including HPV typing and E6/E7 mRNA detection. Immunohistochemistry were used to determine CD47 expression , and histochemistry score(H⁃score) was used to quantify the levels of CD47. The correlation among HPV 16/18 , pathological grades and CD47 was analyzed.@*Results@#Bioinformatics tool analysis and immunohistochemistry showed that CD47 was up⁃regulated in HSIL and CC , associated with the prognosis of CC. The expression levels of CD47 in cervical tissues increased significantly after HPV 16/18 infection ( t = 2. 494 , P < 0. 05 ) , and in HPV 16/18 infected tissues , with the increase of pathological grades. The expression levels of CD47 significantly increased(F = 4. 351 , P < 0. 05 ; rs = 0. 278 , P < 0. 001) . The expression of CD47 in the E6/E7 mRNA( + ) was higher than that in the E6/E7 mRNA( - ) ( t = 5. 710 , P < 0. 000 1) , and the copies of E6/E7 were related to the degree of cervical lesions , and were positively correlated with the expression level of CD47 ( F = 15. 557 , P <0. 000 1 ; rs = 0. 649 , P < 0. 000 1) ( rs = 0. 73 , P < 0. 000 1) .@*Conclusion@#CD47 is overexpressed in HSIL and CC after HPV 16/18 infection. The expression levels of CD47 increases with the degree of cervical lesions , and is correlated with E6/E7 mRNA. CD47 may be a potential target for cervical disease progression and therapy.
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Objectives: CD47 is a membrane protein that belongs to the immunoglobulin superfamily and regulates macrophage phagocytosis negatively. As CD47 expression at the cancer cell membrane would inhibit the phagocytic activity of immune cells, it is connected to an unfavorable prognosis in leukemia and malignancies of various solid organs. Materials and Methods: In this study, retrospectively evaluated 72 patients who had been diagnosed with endometrial carcinoma at Pathology Department and had undergone total abdominal hysterectomy and bilateral salpingo-oophorectomy (TAH + BSO) and/or lymphadenectomy. CD47 expression was evaluated in tumorous and nontumor areas in all patients considering cytoplasmic and membranous brown staining in cells. The proportion of expression was evaluated as well as the intensity and an “h score” was obtained. This score was compared with known prognostic parameters. Results: CD47 expressions showed a statistically significant correlation with tumor grade (P < 0.05); however, no significant relationship was observed with myometrial invasion depth and lymph vascular invasion status (P = 0.923 and P = 0.754, respectively). Conclusions: As with other tumors, anti-CD47 antibody may be an alternative treatment option in patients with high-grade endometrial carcinoma.
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CD47 is widely expressed on the cell surface, and combines with signal regulatory protein α (SIRPα) to transmit the "don't eat me" signal, which plays a key role in self-recognition and tumor immune escape. Studies have shown that the high expression of CD47 in different hematologic neoplasms is associated with the occurrence, progression and poor prognosis of tumors. As a new immune checkpoint, CD47 is gradually becoming an effective target for tumor immunotherapy. and various related preclinical and clinical studies for hematologic neoplasms are underway. This article summarizes the application of CD47 in hematologic neoplasms, in order to provide references for the treatment.
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Objective:To study the protective anti-radiation effect of inhibiting CD47 expression in the lung tissues of mice and to explore the associated mechanism.Methods:Female C57BL/6 mice ( n = 60) were randomly divided into four groups: normal, blank, negative and positive. The blank group received only whole-lung irradiation; The negative group received whole-lung irradiation and tracheal infusion of adeno-associated virus containing nonsense sequence shRNA; The positive group received whole lung irradiation and tracheal drip containing adeno-associated virus containing shRNA expression of CD47. Fresh blood samples were collected at 24 h, 4 weeks, and 12 weeks post-irradiation, respectively. The expression level of CD47 mRNA was determined by RT-PCR. Determination of hydroxyproline content by alkaline hydrolysis. The LC3 expression level was measured by immunohistochemical staining. Serum transforming growth factor-β 1 (TGF-β 1) and tumor necrosis factor-α (TNF-α) levels were assessed by ELISA. Results:RT-PCR showed that the relative expression level of CD47 mRNA in the lung tissues in the positive group was significantly lower compared to those in the negative group, the normal group, the blank group (24-hour, P were <0.001,<0.001,<0.001, respectively. 4-weeks, P were <0.001,0.003,0.001, respectively. 12-weeks, P were 0.009, 0.002, 0.005, respectively). There were no significant differences in CD47 mRNA expression in the three groups except the positive group (all P>0.05), and there was no significant difference in CD47 mRNA expression with time in each group (all P>0.05).The serum TGF-β1 content was higher in the 24 h, 4-week, and 12-week blank groups ( P were <0.001, 0.003 and 0.003, respectively) and negative groups( P were 0.001, 0.021 and 0.034, respectively) after irradiation than that in the mice in the normal group. At the same time, the serum TNF-α of positive group after irradiation (24 hours, 4 weeks, 12 weeks, P were 0.022, <0.001, <0.001, respectively) were significantly higher than those of the normal group. The content of hydroxyproline in the blank group was significantly higher than that in the normal group (4 weeks, 12 weeks, P were 0.002, <0.001, respectively). Immunohistochemical indications: 24 h after irradiation was higher than the expression of LC3 in mouse lung tissue at 4 weeks and 12 weeks (all P < 0.001). The difference between the negative group and the blank group was not obvious ( P>0.05). Conclusions:Inhibition of CD47 expression can reduce the degree of radiation-induced pneumonia and pulmonary fibrosis probably via enhanced autophagy. CD47 may represent a novel target for the protection of radiation-induced lung injury.
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IMM0306 is a recombinant human signal regulatory protein α-anti-CD20 mouse chimeric antibody fusion protein, intended to treat refractory or recurrent CD20 positive B-cell non-Hodgkin lymphoma (B-NHL) in clinical. In this study, an ELISA method was established to evaluate the pharmacokinetics of IMM0306 in human serum. The experiment was approved by the Ethics Committee of the Cancer Hospital of the Chinese Academy of Medical Sciences (No. CTR20192612). Recombinant human CD47 protein was coated with the plate overnight, blocking the plate with 5% skin milk for 2 h. After washing, 100 μL per well standard and unknown samples were added, and incubated for 1.5 h. After washing, the detection antibody Anti-IMM0306-Biotin was added and incubated for 1 h, and then HRP-labeled streptavidin was added for 1 h, and the color was detected. The optimal concentration of coating reagent was 2 μg·mL-1 by ELISA method, and the optimal dilution of anti-IMM0306-biotin and SA-HRP were 1∶500 and 1∶5 000, respectively. The lower limit of quantitation was 4 ng·mL-1, and the standard curve range was 4-100 ng·mL-1. The verification results of the method meets the corresponding acceptance criteria, and can be used in IMM0306 clinical pharmacokinetic studies.
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【Objective】 To explore the impact of monoclonal anti-CD47(IBI188) on clinical pre-transfusion testing and its solutions, then compare it with monoclonal anti-CD38, so as to develop safe and rational transfusion strategies. 【Methods】 The blood typing, direct antiglobulin testing(DAT) and antibody screening were conducted by standard methods. Red blood cells(RBCs) were treated with fig protease, papain, trypsin and dithiothreitol(DTT) to observe whether the effect of monoclonal anti-CD47 could be eliminated. Cord RBCs and RBCs with different Rh phenotypes were cross-matched; Plasma samples were adsorbed with papain-treated O allogeneic RBCs. 【Results】 ABO reverse typing were affected by monoclonal anti-CD47 treatment, and all serum antibody screening were positive, and their DAT were negative or weakly positive. Neither enzyme nor DTT could weaken the effect of monoclonal anti-CD47 on antibody screening. In saline cross-matching, differences in agglutination intensity were corresponded to differences in CD47 expression on RBCs, but all RBCs agglutinated 2+ to 4+ by polybrene method and anti-human globulin method. Papain treated allogeneic RBCs can remove the monoclonal anti-CD47 in the serum through 3 to 4 rounds of absorption. 【Conclusion】 Monoclonal anti-CD47 interferes with pre-transfusion testing, which can be removed by allogeneic RBCs absorption(not suitable for antibody screening or cross-matching), but not by enzyme or DTT. Blood typing and antibody screening should be conducted before monoclonal anti-CD47 treatment and patients should be transfused with homozygous matched RBCs.
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【Objective】 To discuss the case reports concerning anti-CD47 monoclonal antibody interfere in pre-transfusion testing, so as to find mitigation strategies for this drug interference. 【Methods】 Blood transfusion cases in clinical trials concerning CD47 mAb drugs at home and abroad were retrieved from PubMed, Medline, Web of Science, Wanfang data knowledge service platform and CNKI database. The characteristics and solutions of this drug interfering with pre-transfusion testing were analyzed. 【Results】 A total of 26 cases concerning anti-CD47 mAb interference in pretransfusion testing were retrieved, and 16 valid cases were included in this study (All received HU5F9-G4 as anti-CD47 mAb). After treatment with Hu5F9-G4, the discrepancy between forward and reverse blood typing reached 77% in pre-transfusion testing. Panagglutination was presented in antibody screenings, and all(100%) platelet antibody screenings was interfered. These results indicated that Hu5F9-G4 seriously affected the compatibility test of blood transfusion. Methods of eliminating anti-CD47 interference, as well as their advantages and disadvantages were further analyzed. 【Conclusion】 The advantages and disadvantages of eliminating anti-CD47 interference with pre-transfusion testing was analyzed according to its characteristics, which could provide reference for the laboratory testing.
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【Objective】 To discuss the interference of anti-CD47 in pre-transfusion test and the mitigation measures. 【Methods】 Blood sample of one patient received anti-CD47 treatment was collected to conduct routine serological tests including ABO/Rh phenotype, direct anti-human globulin test, irregular antibody screening, antibody identification and cross-match. Packed platelet from multiple type O blood donors was used to absorb with patient′s plasma. The patient′s plasma was absorbed with CCDee, ccDEE and ccdee red cells, respectively. Anti-IgG monoclonal Gamma-clone which lacks reactivity with human subclass IgG4 was used to perform antibody screening and cross-match. Capture-R was used to perform antibody screening. 【Results】 The direct anti-human globulin test was positive(1+ ), the reactivity in all phases was strong positive(3+ -4+ ). The anti-CD47 was eliminated after platelet and red cells absorption. Antibody screening became negative using Gamma-clone and Capture-R, and cross-match successfully using Gamma-clone. 【Conclusion】 Anti-CD47 monoclonal antibody can interfere with pre-transfusion test and cross matching. To remove the interference of anti-CD47 requires the use of Gamma-clone anti-IgG in the indirect antiglobulin testing or Capture-R.
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CD47 is a member of the immunoglobulin superfamily. It is expressed on various cells and tissues of human, but it is more expressed on tumor cells, especially in various hematopoietic tumors. The combination of CD47 expressed on tumor cells with signal regulator protein α (SIRPα) on macrophages inhibits the phagocytosis of tumors by macrophages. The reaction can lead to tumor immune escape. CD47 has become a new hot spot in tumor research. This article reviews the correlation between the structure and expression of CD47, CD47-SIRPα, CD47-targeting antibody drugs and lymphoma immunotherapy.
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Objective To modify CD47 nanobody with the self-folding peptide human cartilage oligomeric matrix protein (COMP48) so as to enhance its affinity to CD47 antigen. Methods The fusion sequences of COMP48 and CD47 nanobody (VHHB1) were designed and synthesized, and the recombinant plasmid pET22b-VHHB1-COMP48 was constructed and transformed into E. coli BL21 (DE3) to induce expression of the fusion protein. The binding specificity and affinity of the fusion protein and the antigen CD47 were detected by Western Blot, indirect enzyme-linked immunosorbent assay (ELISA) and non-competitive ELISA. Results The recombinant VHHB1-COMP48 was expressed in BL21(DE3) by inducing with 1 mmol/L IPTG and purified at 90%homogenous in IMAC. Western Blot results showed that the recombinant protein VHHB1-COMP48 specifically binds to antigen CD47 but not to unrelated protein. The indirect ELISA and non-competitive ELISA results showed that the affinity of the conjugated recombinant protein VHHB1-COMP48 was enhanced compared to that of the non-conjugated nanobody, and the difference was statistically significant ( P<0 . 01 ) . Through non-competitive ELISA , the constants of affinity and dissociation constants were 6.97 ×107 L/mol and 1.434 ×10-8 mol/L, respectively. Conclusions The affinity of the nanobody for the antigen can be improved by conjugating a human cartilage matrix protein (COMP48) after the nanobody.
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@# CD47是细胞表面高度糖化的穿膜蛋白,是一种“别吃我”信号,可与信号调节蛋白α(SIRPα)等形成CD47-SIRPα抑制 信号复合体,从固有免疫和适应性免疫两方面同时逃避机体的免疫监视。研究发现,CD47在血液肿瘤和多种实体瘤中高表达, 通过与巨噬细胞上的SIRPα配体结合,启动一系列抑制性的信号转导而躲避吞噬,其高水平表达既能促进肿瘤细胞的生长又能 促进肿瘤细胞的转移。通过抗CD47抗体阻断CD47-SIRPα信号通路,达到抑制肿瘤细胞的免疫逃逸,增强巨噬细胞的吞噬作用 和适应性免疫应答,是免疫治疗肿瘤的新途径。目前,国内外开展了越来越多靶向CD47-SIRPα的药物或抗体的基础研究和临床 试验,有望从抗体分子设计和重组蛋白等方面解决靶向CD47抗肿瘤治疗时发生的贫血和输液相关不良反应等问题。本文就 CD47的分子结构与生理功能、CD47-SIRPα表达调控机制、CD47抗肿瘤治疗研究现状以及靶向CD47导致的相关生物安全性问 题和解决方案等方面进行综述, 为CD47新靶点的基础研究和临床应用提供参考。
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With the development and clinical application of immunological checkpoint drugs such as pro- grammed cell death protein-1(PD-1)/programmed cell death-ligand 1(PD-L1)and cytotoxic T lymphocyte-associated an- tigen-4(CTLA-4), immunotherapy has gradually become one of the most effective clinical treatment methods. Cluster of differentiation 47(CD47), as a natural immune checkpoint molecule, is highly expressed in almost all human cancers (solid tumors and hematological tumors), affecting tumor progression and metastasis, and is involved in the apoptosis, proliferation, adhesion, migration and other processes. Therefore, CD47 has become an important target for the study of human tumors. At present, a number of preclinical studies and clinical trials of the drugs targeting CD47 antibodies and Fc fusion protein are being actively carried out in various countries. This review briefly introduces the structure, function and related diseases of CD47 and focuses mainly on the development and clinical application strategy of CD47 therapeutic antibody drugs.
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@#Using the genetic code extension technology, the immunogenic amino acid, p-nitrophenylalanine, was introduced into the universal T cell epitope and then fused with the fragment of the extracellular region of the immune checkpoint molecular CD47(19-140)to construct a vaccine targeting CD47. The CD47-NitraTh vaccine elicited high titer antibody in BALB/c mice, significantly inhibited CT26 colon cancer cells growth, and increased the ratio of spleen CD4+ T cells and CD8+ T cells. Meanwhile, it promoted the polarization of naï ve T cells to Th1 cells. Notably, CD47-NitraTh not only increased the proportion of tumour-infiltrating lymphocytes but also reduced the proportion of Treg cells in tumour tissues, which means that CD47-NitraTh vaccine can remodel the tumour immunosuppressive microenvironment. The results of this study suggested that CD47-NitraTh can be used as an effective tumour vaccine candidate.