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1.
Talanta ; 280: 126766, 2024 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-39191106

RESUMO

Glypican-1 (GPC-1) protein-positive small extracellular vesicles (GPC-1+-sEV) have been proposed as potential biomarkers for early diagnosis of pancreatic cancer. In this study, we present an integrated real-time isolation and detection platform (IRTIDP) to capture and analyze GPC-1+-sEV directly from sera of pancreatic cancer patients. First, CD63 antibody-modified metal-organic framework (MOF) materials were utilized to enrich sEVs with a capture efficiency of 93.93 %. Second, a SERS probe was constructed by Raman reporter 4-MBA and GPC-1 antibody modified SERS active silver nanoparticles (AgNPs), which formed a sandwich complex structure of "MOFs@GPC-1+-sEV@AgNPs-4-MBA" with MOFs-enriched sEVs. The IRTSDP can complete the capture and detection process within 35 min, with a detection limit for 1 GPC-1+-sEV/µL, and linear range between 105∼109 GPC-1+-sEV/mL. Furthermore, this approach has been applied to quantify serum sEV GPC-1 in clinical pancreatic cancer patients. Based on the SERS intensity analysis, pancreatic cancer patients can be distinguished from pancreatic cystadenoma patients and healthy individuals effectively using this innovative platform that provides highly specific and sensitive means for early diagnosis of pancreatic cancer as well as other tumor types.


Assuntos
Vesículas Extracelulares , Glipicanas , Nanopartículas Metálicas , Neoplasias Pancreáticas , Humanos , Glipicanas/sangue , Glipicanas/imunologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/sangue , Vesículas Extracelulares/química , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Prata/química , Estruturas Metalorgânicas/química , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/análise , Limite de Detecção , Tetraspanina 30
2.
Clin Exp Med ; 24(1): 204, 2024 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-39196390

RESUMO

The application of CAR-T cells in solid tumors poses several challenges, including poor T cell homing ability, limited infiltration of T cells and an immunosuppressive tumor environment. In this study, we developed a novel approach to address these obstacles by designing GPC3-specific CAR-T cell that co-express IL-21 and CXCL9 (21 × 9 GPC3 CAR-T cells) and blocking the PD-1 expression on it. The proliferation, cell phenotype, cytokine secretion and cell migration of indicated CAR-T cells were evaluated in vitro. The cytotoxic activities of genetically engineered CAR-T cells were accessed in vitro and in vivo. Compared to conventional GPC3 CAR-T cells, the 21 × 9 GPC3 CAR-T cells demonstrated superior proliferation, cytokine secretion and chemotaxis capabilities in vitro. Furthermore, when combined with PD-1 blockade, the 21 × 9 GPC3 CAR-T cells exhibited enhanced proliferation, cytokine secretion and enrichment of effector T cells such as CTL, NKT and TEM cells. In xenograft tumor models, the PD-1 blocked 21 × 9 GPC3 CAR-T cells effectively suppressed HCC xenograft growth and increased T cell infiltration. Overall, our study successfully generated GPC3 CAR-T cells expressing both IL-21 and CXCL9, demonstrated that combining PD-1 blockade can further enhance CAR-T cell function by promoting proliferation, cytokine secretion, chemotaxis and antitumor activity. These findings present a hopeful and potentially effective strategy for GPC3-positive HCC patients.


Assuntos
Carcinoma Hepatocelular , Quimiocina CXCL9 , Glipicanas , Imunoterapia Adotiva , Interleucinas , Neoplasias Hepáticas , Receptor de Morte Celular Programada 1 , Receptores de Antígenos Quiméricos , Glipicanas/imunologia , Glipicanas/metabolismo , Glipicanas/antagonistas & inibidores , Glipicanas/genética , Interleucinas/metabolismo , Interleucinas/genética , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Animais , Humanos , Imunoterapia Adotiva/métodos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/tratamento farmacológico , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Camundongos , Quimiocina CXCL9/metabolismo , Quimiocina CXCL9/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células , Linhagem Celular Tumoral
3.
Nat Commun ; 15(1): 7141, 2024 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-39164224

RESUMO

Novel chimeric antigen receptor (CAR) T-cell approaches are needed to improve therapeutic efficacy in solid tumors. High-risk neuroblastoma is an aggressive pediatric solid tumor that expresses cell-surface GPC2 and GD2 with a tumor microenvironment infiltrated by CD16a-expressing innate immune cells. Here we engineer T-cells to express a GPC2-directed CAR and simultaneously secrete a bispecific innate immune cell engager (BiCE) targeting both GD2 and CD16a. In vitro, GPC2.CAR-GD2.BiCE T-cells induce GPC2-dependent cytotoxicity and secrete GD2.BiCE that promotes GD2-dependent activation of antitumor innate immunity. In vivo, GPC2.CAR-GD2.BiCE T-cells locally deliver GD2.BiCE and increase intratumor retention of NK-cells. In mice bearing neuroblastoma patient-derived xenografts and reconstituted with human CD16a-expressing immune cells, GD2.BiCEs enhance GPC2.CAR antitumor efficacy. A CAR.BiCE strategy should be considered for tumor histologies where antigen escape limits CAR efficacy, especially for solid tumors like neuroblastoma that are infiltrated by innate immune cells.


Assuntos
Gangliosídeos , Imunidade Inata , Imunoterapia Adotiva , Células Matadoras Naturais , Neuroblastoma , Receptores de Antígenos Quiméricos , Linfócitos T , Neuroblastoma/imunologia , Neuroblastoma/terapia , Neuroblastoma/patologia , Animais , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Camundongos , Gangliosídeos/imunologia , Linhagem Celular Tumoral , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Glipicanas/imunologia , Glipicanas/metabolismo , Microambiente Tumoral/imunologia , Feminino
4.
Front Biosci (Landmark Ed) ; 29(7): 268, 2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-39082348

RESUMO

Liver cancer, primarily hepatocellular carcinoma (HCC), is the second leading cause of cancer-related deaths globally. It is typically characterized by rapid progression, poor prognosis, and high mortality rates. Given these challenges, the search for molecular targets aiding early diagnosis and targeted therapy remains imperative. Glypican 3 (GPC3), a cell-surface glycoprotein, emerges as a promising candidate for addressing HCC Overexpressed in HCC tissues; GPC3 is a credible immunohistochemical marker for liver cancer diagnosis and a potential marker for liquid biopsy through soluble GPC3 in serum. Various immunotherapies targeting GPC3 have been developed, including vaccines, anti-GPC3 immunotoxins, and chimeric antigen receptor-modified cells. This review comprehensively covers the structure, physicochemical properties, biological functions, and clinical applications of GPC3. It explores diagnostic and treatment strategies centered around GPC3, offering hope for improved early detection and targeted therapies in the challenging landscape of HCC.


Assuntos
Biomarcadores Tumorais , Carcinoma Hepatocelular , Glipicanas , Imunoterapia , Neoplasias Hepáticas , Glipicanas/imunologia , Glipicanas/metabolismo , Humanos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Imunoterapia/métodos , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/sangue , Terapia de Alvo Molecular/métodos , Medicina de Precisão/métodos , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico
5.
Sheng Wu Gong Cheng Xue Bao ; 40(7): 2258-2269, 2024 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-39044589

RESUMO

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.


Assuntos
Membrana Externa Bacteriana , Carcinoma Hepatocelular , Glipicanas , Neoplasias Hepáticas , Anticorpos de Cadeia Única , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/química , Animais , Camundongos , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/imunologia , Células Hep G2 , Glipicanas/imunologia , Glipicanas/metabolismo , Glipicanas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Sistemas de Liberação de Medicamentos , Camundongos Nus
6.
Front Med ; 18(4): 708-720, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38833102

RESUMO

CD39 serves as a crucial biomarker for neoantigen-specific CD8+ T cells and is associated with antitumor activity and exhaustion. However, the relationship between CD39 expression levels and the function of chimeric antigen receptor T (CAR-T) cells remains controversial. This study aimed to investigate the role of CD39 in the functional performance of CAR-T cells against hepatocellular carcinoma (HCC) and explore the therapeutic potential of CD39 modulators, such as mitochondrial division inhibitor-1 (mdivi-1), or knockdown CD39 through short hairpin RNA. Our findings demonstrated that glypican-3-CAR-T cells with moderate CD39 expression exhibited a strong antitumor activity, while high and low levels of CD39 led to an impaired cellular function. Methods modulating the proportion of CD39 intermediate (CD39int)-phenotype CAR-T cells such as mdivi-1 and CD39 knockdown enhanced and impaired T cell function, respectively. The combination of mdivi-1 and CD39 knockdown in CAR-T cells yielded the highest proportion of infiltrated CD39int CAR-T cells and demonstrated a robust antitumor activity in vivo. In conclusion, this study revealed the crucial role of CD39 in CAR-T cell function, demonstrated the potential therapeutic efficacy of combining mdivi-1 with CD39 knockdown in HCC, and provided a novel treatment strategy for HCC patients in the field of cellular immunotherapy.


Assuntos
Apirase , Carcinoma Hepatocelular , Glipicanas , Imunoterapia Adotiva , Neoplasias Hepáticas , Receptores de Antígenos Quiméricos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/genética , Glipicanas/imunologia , Glipicanas/genética , Glipicanas/metabolismo , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/genética , Apirase/metabolismo , Apirase/genética , Humanos , Animais , Camundongos , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Linhagem Celular Tumoral , Antígenos CD/genética , Antígenos CD/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Clin Cancer Res ; 30(16): 3578-3591, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38864848

RESUMO

PURPOSE: Retinoblastoma is the most common intraocular malignancy in children. Although new chemotherapeutic approaches have improved ocular salvage rates, novel therapies are required for patients with refractory intraocular and metastatic disease. Chimeric antigen receptor (CAR) T cells targeting glypican-2 (GPC2) are a potential new therapeutic strategy. EXPERIMENTAL DESIGN: GPC2 expression and its regulation by the E2F1 transcription factor were studied in retinoblastoma patient samples and cellular models. In vitro, we performed functional studies comparing GPC2 CAR T cells with different costimulatory domains (4-1BB and CD28). In vivo, the efficacy of local and systemic administration of GPC2 CAR T cells was evaluated in intraocular and leptomeningeal human retinoblastoma xenograft models. RESULTS: Retinoblastoma tumors, but not healthy retinal tissues, expressed cell surface GPC2, and this tumor-specific expression was driven by E2F1. GPC2-directed CARs with 4-1BB costimulation (GPC2.BBz) were superior to CARs with CD28 stimulatory domains (GPC2.28z), efficiently inducing retinoblastoma cell cytotoxicity and enhancing T-cell proliferation and polyfunctionality. In vivo, GPC2.BBz CARs had enhanced persistence, which led to significant tumor regression compared with either control CD19 or GPC2.28z CARs. In intraocular models, GPC2.BBz CAR T cells efficiently trafficked to tumor-bearing eyes after intravitreal or systemic infusions, significantly prolonging ocular survival. In central nervous system (CNS) retinoblastoma models, intraventricular or systemically administered GPC2.BBz CAR T cells were activated in retinoblastoma-involved CNS tissues, resulting in robust tumor regression with substantially extended overall mouse survival. CONCLUSIONS: GPC2-directed CAR T cells are effective against intraocular and CNS metastatic retinoblastomas.


Assuntos
Glipicanas , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Retinoblastoma , Linfócitos T , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Retinoblastoma/imunologia , Retinoblastoma/patologia , Retinoblastoma/terapia , Camundongos , Receptores de Antígenos Quiméricos/imunologia , Glipicanas/imunologia , Glipicanas/antagonistas & inibidores , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/terapia , Neoplasias do Sistema Nervoso Central/imunologia , Neoplasias do Sistema Nervoso Central/secundário , Neoplasias do Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Feminino
8.
Cytotherapy ; 26(11): 1308-1319, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38904586

RESUMO

BACKGROUND AIMS: Chimeric antigen receptor T (CAR-T) cells targeting single antigens show limited activity against solid tumors due to poor T cell persistence, low efficiency infiltration, and exhaustion together with heterogeneous tumor-associated antigen (TAA) expression. This is also true in high-risk neuroblastoma (HRNB), a lethal pediatric extracranial malignancy. To overcome these obstacles, a combinational strategy using GD2-specific and GPC2-specific CAR-T cells was developed to improve immunotherapeutic efficacy. METHODS: We individually developed GD2-specific and GPC2-specific CARs containing a selective domain (sCAR) which was a peptide of 10 amino acids derived from human nuclear autoantigen La/SS-B. These constructs allowed us to generate two different HRNB antigen-specific CAR-T cells with enhanced biological activity through stimulating sCAR-engrafted T cells via a selective domain-specific monoclonal antibody (SmAb). Binding affinity and stimulation of GD2- and GPC2-specific sCARs by SmAb were measured, and transient and persistent anti-tumor cytotoxicity of GD2sCAR-T and GPC2sCAR-T cells were quantified in neuroblastoma cell lines expressing different TAA levels. The anti-tumor pharmaceutical effects and cellular mechanisms mediated by single or combinational sCAR-T cells were evaluated in vitro and in vivo. RESULTS: GD2- and GPC2-specific sCARs had antigen-specific binding affinity similar to their parental counterparts and were recognized by SmAb. SmAb-mediated stimulation selectively activated sCAR-T proliferation and increased central memory T cells in the final products. SmAb-stimulated sCAR-T cells had enhanced transient cytolytic activity, and combination therapy extended long-term anti-tumor activity in vitro through TNF-α and IL-15 release. Stimulated sCAR-T cells overcame heterogeneous antigen expression in HRNB, and the multi-TAA-targeting strategy was especially efficacious in vivo, inducing apoptosis through the caspase-3/PARP pathway and inhibiting the release of several tumor-promoting cytokines. CONCLUSIONS: These data suggest that combined targeting of multiple TAAs is a promising strategy to overcome heterogenous antigen expression in solid tumors and extend CAR-T cell persistence for HRNB immunotherapy.


Assuntos
Gangliosídeos , Glipicanas , Imunoterapia Adotiva , Neuroblastoma , Receptores de Antígenos Quiméricos , Neuroblastoma/terapia , Neuroblastoma/imunologia , Humanos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/genética , Animais , Gangliosídeos/imunologia , Gangliosídeos/metabolismo , Imunoterapia Adotiva/métodos , Camundongos , Linhagem Celular Tumoral , Glipicanas/imunologia , Glipicanas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos de Neoplasias/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Acta Pharmacol Sin ; 45(9): 1937-1950, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38750075

RESUMO

Chimeric antigen receptor-expressing T (CAR-T) cells induce robust antitumor responses in patients with hematologic malignancies. However, CAR-T cells exhibit only limited efficacy against solid tumors such as hepatocellular carcinoma (HCC), partially due to their limited expansion and persistence. CD8+ T cells, as key components of the adaptive immune response, play a central role in antitumor immunity. Aerobic glycolysis is the main metabolic feature of activated CD8+ T cells. In the tumor microenvironment, however, the uptake of large amounts of glucose by tumor cells and other immunosuppressive cells can impair the activation of T cells. Only when tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment have a glycolytic advantage might the effector function of T cells be activated. Glucose transporter type 1 (GLUT1) and acylglycerol kinase (AGK) can boost glycolytic metabolism and activate the effector function of CD8+ T cells, respectively. In this study, we generated GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK for the treatment of HCC. GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK specifically and effectively lysed GPC3-positive tumor cells in vitro in an antigen-dependent manner. Furthermore, GLUT1 or AGK overexpression protected CAR-T cells from apoptosis during repeated exposures to tumor cells. Compared with second-generation CAR-T cells, GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK exhibited greater CD8+ T-cell persistence in vivo and better antitumor effects in HCC allograft mouse models. Finally, we revealed that GLUT1 or AGK maintained anti-apoptosis ability in CD8+ T cells via activation of the PI3K/Akt pathway. This finding might identify a therapeutic strategy for advanced HCC.


Assuntos
Carcinoma Hepatocelular , Transportador de Glucose Tipo 1 , Glipicanas , Neoplasias Hepáticas , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Animais , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Transportador de Glucose Tipo 1/metabolismo , Humanos , Camundongos , Glipicanas/metabolismo , Glipicanas/imunologia , Imunoterapia Adotiva/métodos , Linhagem Celular Tumoral , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Microambiente Tumoral , Apoptose
10.
J Immunother Cancer ; 11(1)2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36631162

RESUMO

BACKGROUND: Although most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their cancer. Therapeutic strategies to target MRD may benefit these children. We developed a new chimeric antigen receptor (CAR) targeting glypican-2 (GPC2) and conducted iterative preclinical engineering of the CAR structure to maximize its anti-tumor efficacy before clinical translation. METHODS: We evaluated different GPC2-CAR constructs by measuring the CAR activity in vitro. NOD-SCID mice engrafted orthotopically with human NB cell lines or patient-derived xenografts and treated with human CAR T cells served as in vivo models. Mechanistic studies were performed using single-cell RNA-sequencing. RESULTS: Applying stringent in vitro assays and orthotopic in vivo NB models, we demonstrated that our single-chain variable fragment, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H.BBζ) elicits the best preclinical anti-NB activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. Finally, we also showed that the CT3.28H.BBζ CAR we developed was more potent than a recently clinically tested GD2-targeted CAR to control NB growth in vivo. CONCLUSION: Given the robust preclinical activity of CT3.28H.BBζ, these results form a promising basis for further clinical testing in children with NB.


Assuntos
Glipicanas , Neuroblastoma , Receptores de Antígenos Quiméricos , Animais , Criança , Humanos , Camundongos , Antígenos CD28 , Gangliosídeos , Glipicanas/imunologia , Glipicanas/uso terapêutico , Imunoterapia Adotiva/métodos , Camundongos Endogâmicos NOD , Camundongos SCID , Neuroblastoma/metabolismo , Neuroblastoma/terapia , Receptores de Antígenos Quiméricos/genética
11.
Sci Rep ; 12(1): 12312, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35853994

RESUMO

Currently, ERY974, a humanized IgG4 bispecific T cell-redirecting antibody recognizing glypican-3 and CD3, is in phase I clinical trials. After a first-in-human clinical trial of an anti-CD28 agonist monoclonal antibody resulting in severe life-threatening adverse events, the minimal anticipated biological effect level approach has been considered for determining the first-in-human dose of high-risk drugs. Accordingly, we aimed to determine the first-in-human dose of ERY974 using both the minimal anticipated biological effect level and no observed adverse effect level approaches. In the former, we used the 10% effective concentration value from a cytotoxicity assay using the huH-1 cell line with the highest sensitivity to ERY974 to calculate the first-in-human dose of 4.9 ng/kg, at which maximum drug concentration after 4 h of intravenous ERY974 infusion was equal to the 10% effective concentration value. To determine the no observed adverse effect level, we conducted a single-dose study in cynomolgus monkeys that were intravenously infused with ERY974 (0.1, 1, and 10 µg/kg). The lowest dose of 0.1 µg/kg was determined as the no observed adverse effect level, and the first-in-human dose of 3.2 ng/kg was calculated, considering body surface area and species difference. For the phase I clinical trial, we selected 3.0 ng/kg as a starting dose, which was lower than the first-in-human dose calculated from both the no observed adverse effect level and minimal anticipated biological effect level. Combining these two methods to determine the first-in-human dose of strong immune modulators such as T cell-redirecting antibodies would be a suitable approach from safety and efficacy perspectives.Clinical trial registration: JapicCTI-194805/NCT05022927.


Assuntos
Anticorpos Biespecíficos , Glipicanas , Linfócitos T , Anticorpos Biespecíficos/administração & dosagem , Relação Dose-Resposta Imunológica , Glipicanas/imunologia , Humanos , Linfócitos T/imunologia
12.
Cancer Cell ; 40(1): 53-69.e9, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-34971569

RESUMO

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.


Assuntos
Glipicanas/imunologia , Imunoterapia Adotiva , Neuroblastoma/tratamento farmacológico , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Linhagem Celular Tumoral , Glipicanas/metabolismo , Humanos , Imunoterapia/métodos , Neuroblastoma/patologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
13.
J Immunother Cancer ; 9(12)2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34916256

RESUMO

BACKGROUND: Glypican-3 (GPC-3) is an oncofetal protein that is highly expressed in various solid tumors, but rarely expressed in healthy adult tissues and represents a rational target of particular relevance in hepatocellular carcinoma (HCC). Autologous chimeric antigen receptor (CAR) αß T cell therapies have established significant clinical benefit in hematologic malignancies, although efficacy in solid tumors has been limited due to several challenges including T cell homing, target antigen heterogeneity, and immunosuppressive tumor microenvironments. Gamma delta (γδ) T cells are highly cytolytic effectors that can recognize and kill tumor cells through major histocompatibility complex (MHC)-independent antigens upregulated under stress. The Vδ1 subset is preferentially localized in peripheral tissue and engineering with CARs to further enhance intrinsic antitumor activity represents an attractive approach to overcome challenges for conventional T cell therapies in solid tumors. Allogeneic Vδ1 CAR T cell therapy may also overcome other hurdles faced by allogeneic αß T cell therapy, including graft-versus-host disease (GvHD). METHODS: We developed the first example of allogeneic CAR Vδ1 T cells that have been expanded from peripheral blood mononuclear cells (PBMCs) and genetically modified to express a 4-1BB/CD3z CAR against GPC-3. The CAR construct (GPC-3.CAR/secreted interleukin-15 (sIL)-15) additionally encodes a constitutively-secreted form of IL-15, which we hypothesized could sustain proliferation and antitumor activity of intratumoral Vδ1 T cells expressing GPC-3.CAR. RESULTS: GPC-3.CAR/sIL-15 Vδ1 T cells expanded from PBMCs on average 20,000-fold and routinely reached >80% purity. Expanded Vδ1 T cells showed a primarily naïve-like memory phenotype with limited exhaustion marker expression and displayed robust in vitro proliferation, cytokine production, and cytotoxic activity against HCC cell lines expressing low (PLC/PRF/5) and high (HepG2) GPC-3 levels. In a subcutaneous HepG2 mouse model in immunodeficient NSG mice, GPC-3.CAR/sIL-15 Vδ1 T cells primarily accumulated and proliferated in the tumor, and a single dose efficiently controlled tumor growth without evidence of xenogeneic GvHD. Importantly, compared with GPC-3.CAR Vδ1 T cells lacking sIL-15, GPC-3.CAR/sIL-15 Vδ1 T cells displayed greater proliferation and resulted in enhanced therapeutic activity. CONCLUSIONS: Expanded Vδ1 T cells engineered with a GPC-3 CAR and sIL-15 represent a promising platform warranting further clinical evaluation as an off-the-shelf treatment of HCC and potentially other GPC-3-expressing solid tumors.


Assuntos
Carcinoma Hepatocelular/terapia , Glipicanas/imunologia , Imunoterapia Adotiva/métodos , Interleucina-15/imunologia , Neoplasias Hepáticas/terapia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos Quiméricos/imunologia , Animais , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Proliferação de Células , Feminino , Humanos , Leucócitos Mononucleares , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Cell Rep Med ; 2(7): 100344, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34337560

RESUMO

Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2's immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2.


Assuntos
Epitopos/química , Epitopos/metabolismo , Glipicanas/imunologia , Imunoconjugados/farmacologia , Neoplasias Pulmonares/patologia , Neuroblastoma/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Animais , Efeito Espectador/efeitos dos fármacos , Compartimento Celular , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dano ao DNA , Feminino , Humanos , Camundongos Endogâmicos C57BL , Camundongos SCID , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteínas Oncogênicas/metabolismo , Conformação Proteica
15.
Cell Rep Med ; 2(6): 100297, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34195677

RESUMO

Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.


Assuntos
Anticorpos Monoclonais/farmacologia , Glipicanas/genética , Neoplasias do Sistema Nervoso/terapia , Neuroblastoma/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Éxons , Feminino , Expressão Gênica , Glipicanas/antagonistas & inibidores , Glipicanas/química , Glipicanas/imunologia , Humanos , Imunoterapia Adotiva/métodos , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias do Sistema Nervoso/genética , Neoplasias do Sistema Nervoso/mortalidade , Neoplasias do Sistema Nervoso/patologia , Neuroblastoma/genética , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Análise de Sequência de RNA , Análise de Sobrevida , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
16.
J Hematol Oncol ; 14(1): 118, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34325726

RESUMO

Although chimeric antigen receptor (CAR)-engineered T cells have shown great success in the treatment of B cell malignancies, this strategy has limited efficacy in patients with solid tumors. In mouse CAR-T cells, IL-7 and CCL19 expression have been demonstrated to improve T cell infiltration and CAR-T cell survival in mouse tumors. Therefore, in the current study, we engineered human CAR-T cells to secrete human IL-7 and CCL19 (7 × 19) and found that these 7 × 19 CAR-T cells showed enhanced capacities of expansion and migration in vitro. Furthermore, 7 × 19 CAR-T cells showed superior tumor suppression ability compared to conventional CAR-T cells in xenografts of hepatocellular carcinoma (HCC) cell lines, primary HCC tissue samples and pancreatic carcinoma (PC) cell lines. We then initiated a phase 1 clinical trial in advanced HCC/PC/ovarian carcinoma (OC) patients with glypican-3 (GPC3) or mesothelin (MSLN) expression. In a patient with advanced HCC, anti-GPC3-7 × 19 CAR-T treatment resulted in complete tumor disappearance 30 days post intratumor injection. In a patient with advanced PC, anti-MSLN-7 × 19 CAR-T treatment resulted in almost complete tumor disappearance 240 days post-intravenous infusion. Our results demonstrated that the incorporation of 7 × 19 into CAR-T cells significantly enhanced the antitumor activity against human solid tumor. Trial registration: NCT03198546. Registered 26 June 2017, https://clinicaltrials.gov/ct2/show/NCT03198546?term=NCT03198546&draw=2&rank=1.


Assuntos
Quimiocina CCL19/imunologia , Proteínas Ligadas por GPI/análise , Glipicanas/análise , Imunoterapia Adotiva/métodos , Interleucina-7/imunologia , Neoplasias/terapia , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Feminino , Proteínas Ligadas por GPI/imunologia , Glipicanas/imunologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Mesotelina , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/terapia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Linfócitos T/imunologia , Resultado do Tratamento
17.
Mol Cancer Ther ; 20(9): 1713-1722, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34224365

RESUMO

Cholangiocarcinoma is a highly malignant cancer. Many patients need systemic chemotherapy to prevent tumor development and recurrence; however, their prognosis is poor due to the lack of effective therapy. Therefore, a new treatment option is urgently required. We recently identified glypican-1 (GPC1) as a novel cancer antigen of esophageal squamous cell carcinoma. We also demonstrated the efficacy and safety of GPC1-targeted ADC (GPC1-ADC) conjugating anti-GPC1 mAb possessing high internalization activity with monomethyl auristatin F (MMAF), which is a potent tubulin polymerizing inhibitor. In this study, we confirmed that GPC1 was highly expressed in cholangiocarcinoma cells and tissues. IHC analysis of 49 extrahepatic cholangiocarcinoma patient tumor specimens revealed high expression of GPC1 in 47% of patients. These patients demonstrated significantly poorer prognosis compared with the low-expression group in terms of disease-free survival and overall survival (P < 0.05). GPC1 was also expressed in tumor vessels of cholangiocarcinoma, but not on the vessels of nontumor tissues. MMAF-conjugated GPC1-ADC showed potent tumor growth inhibition against GPC1-positive cholangiocarcinoma cells in vitro and in vivo In a GPC1 knockout xenograft model, GPC1-ADC partially inhibited tumor growth. Vascular endothelial cells in tumor tissues of GPC1-negative xenograft mice expressed GPC1 and were arrested in the G2-M phase of cell cycle by GPC1-ADC. GPC1-ADC exhibits direct as well as indirect antitumor effects via inhibition of tumor angiogenesis. Our preclinical data highlight GPC1-ADC as a promising therapy for GPC1-positive cholangiocarcinoma.


Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Glipicanas/antagonistas & inibidores , Imunoconjugados/farmacologia , Neovascularização Patológica/prevenção & controle , Animais , Apoptose , Neoplasias dos Ductos Biliares/irrigação sanguínea , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Proliferação de Células , Colangiocarcinoma/irrigação sanguínea , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Feminino , Glipicanas/imunologia , Humanos , Camundongos , Camundongos SCID , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Immunother Cancer ; 9(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33833049

RESUMO

BACKGROUND: Glypican-3 (GPC3), a cell surface glycoprotein that is pathologically highly expressed in hepatocellular carcinoma (HCC), is an attractive target for immunotherapies, including chimeric antigen receptor (CAR) T cells. The serum GPC3 is frequently elevated in HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the function of cell-surface GPC3 as a negative regulator. Therefore, it would be worth investigating the potential influence of antigen shedding in anti-GPC3 CAR-T therapy for HCC. METHODS: In this study, we constructed two types of CAR-T cells targeting distinct epitopes of GPC3 to examine how sGPC3 influences the activation and cytotoxicity of CAR-T cells in vitro and in vivo by introducing sGPC3 positive patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines. RESULTS: Both humanized YP7 CAR-T cells and 32A9 CAR-T cells showed GPC3-specific antitumor functions in vitro and in vivo. The existence of sGPC3 significantly inhibited the release of cytokines and the cytotoxicity of anti-GPC3 CAR-T cells in vitro. In animal models, mice carrying Hep3B xenograft tumors expressing sGPC3 exhibited a worse response to the treatment with CAR-T cells under both a low and high tumor burden. sGPC3 bound to CAR-T cells but failed to induce the effective activation of CAR-T cells. Therefore, sGPC3 acted as dominant negative regulators when competed with cell surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an inhibitory effect on CAR-T cells in HCC. CONCLUSIONS: We provide a proof-of-concept study demonstrating that GPC3 shedding might cause worse response to CAR-T cell treatment by competing with cell surface GPC3 for CAR-T cell binding, which revealed a new mechanism of tumor immune escape in HCC, providing a novel biomarker for patient enrolment in future clinical trials and/or treatments with GPC3-targeted CAR-T cells.


Assuntos
Biomarcadores Tumorais/antagonistas & inibidores , Carcinoma Hepatocelular/terapia , Glipicanas/antagonistas & inibidores , Imunoterapia Adotiva , Neoplasias Hepáticas/terapia , Receptores de Antígenos Quiméricos/genética , Linfócitos T/transplante , Animais , Ligação Competitiva , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/imunologia , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Feminino , Glipicanas/sangue , Glipicanas/imunologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Ativação Linfocitária , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Estudo de Prova de Conceito , Ligação Proteica , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Sci Rep ; 11(1): 3731, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33580090

RESUMO

Glypican-3 (GPC3) is a tumor associated antigen expressed by hepatocellular carcinoma (HCC) cells. This preclinical study evaluated the efficacy of a theranostic platform using a GPC3-targeting antibody αGPC3 conjugated to zirconium-89 (89Zr) and yttrium-90 (90Y) to identify, treat, and assess treatment response in a murine model of HCC. A murine orthotopic xenograft model of HCC was generated. Animals were injected with 89Zr-labeled αGPC3 and imaged with a small-animal positron emission/computerized tomography (PET/CT) imaging system (immuno-PET) before and 30 days after radioimmunotherapy (RIT) with 90Y-labeled αGPC3. Serum alpha fetoprotein (AFP), a marker of tumor burden, was measured. Gross tumor volume (GTV) and SUVmax by immuno-PET was measured using fixed intensity threshold and manual segmentation methods. Immuno-PET GTV measurements reliably quantified tumor burden prior to RIT, strongly correlating with serum AFP (R2 = 0.90). Serum AFP was significantly lower 30 days after RIT in 90Y-αGPC3 treated animals compared to those untreated (p = 0.01) or treated with non-radiolabeled αGPC3 (p = 0.02). Immuno-PET GTV measurements strongly correlated with tumor burden after RIT (R2 = 0.87), and GTV of animals treated with 90Y-αGPC3 was lower than in animals who did not receive treatment or were treated with non-radiolabeled αGPC3, although this only trended toward statistical significance. A theranostic platform utilizing GPC3 targeted 89Zr and 90Y effectively imaged, treated, and assessed response after radioimmunotherapy in a GPC3-expressing HCC xenograft model.


Assuntos
Carcinoma Hepatocelular/terapia , Sistemas de Liberação de Medicamentos/métodos , Glipicanas/imunologia , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Glipicanas/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Camundongos , Camundongos Nus , Tomografia por Emissão de Pósitrons/métodos , Medicina de Precisão/métodos , Radioimunoterapia , Radioisótopos/farmacologia , Compostos Radiofarmacêuticos , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto , Radioisótopos de Ítrio/farmacologia , Zircônio/farmacologia
20.
Immunotherapy ; 13(5): 371-385, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33525928

RESUMO

Aim: Two peptide cocktail vaccines using glypican-3, WD-repeat-containing protein up-regulated in hepatocellular carcinoma (HCC) and nei endonuclease VIII-like three epitopes were evaluated in advanced HCC in two Phase I studies. Patients & methods: Study 1 evaluated dose-limiting toxicities (DLTs) of peptides 1-3 (HLA-A24-restricted) and study 2 evaluated DLTs of peptides 1-6 (HLA-A24 or A02-restricted). Results: Overall, 18 and 14 patients were enrolled in studies 1 and 2, respectively. No DLTs were observed up to 7.1 mg of the vaccine cocktail. No complete response/partial response was observed. Stable disease was reported in nine and five patients with a disease control rate of 52.9% and 35.7% in studies 1 and 2, respectively. Conclusion: Both vaccines showed good tolerability and potential usefulness against HCC. Clinical trial registration: JapicCTI-121933; JapicCTI-142477.


Assuntos
Vacinas Anticâncer/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Proteínas de Transporte/imunologia , Cílios/imunologia , Glipicanas/imunologia , Neoplasias Hepáticas/tratamento farmacológico , N-Glicosil Hidrolases/imunologia , Adulto , Idoso , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Determinação de Ponto Final , Epitopos/administração & dosagem , Epitopos/efeitos adversos , Epitopos/imunologia , Feminino , Antígenos HLA-A/imunologia , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Citotóxicos/imunologia , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologia
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