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1.
Cancers (Basel) ; 14(15)2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35892876

RESUMO

Chimeric antigen receptor (CAR)-modified T-cells (CAR-T) have demonstrated promising clinical benefits against B-cell malignancies. Yet, its application for solid tumors is still facing challenges. Unlike haematological cancers, solid tumors often lack good targets, which are ideally expressed on the tumor cells, but not by the normal healthy cells. Fortunately, receptor tyrosine kinase-like orphan receptor 1 (ROR1) is among a few good cancer targets that is aberrantly expressed on various tumors but has a low expression on normal tissue, suggesting it as a good candidate for CAR-T therapy. Here, we constructed two ROR1 CARs with the same antigen recognition domain that was derived from Zilovertamab but differing in hinge regions. Both CARs target ROR1+ cancer cells specifically, but CAR with a shorter IgG4 hinge exhibits a higher surface expression and better in vitro functionality. We further tested the ROR1 CAR-T in three human solid tumor xenografted mouse models. Our ROR1 CAR-T cells controlled the solid tumor growth without causing any severe toxicity. Our results demonstrated that ROR1 CAR-T derived from Zilovertamab is efficacious and safe to suppress ROR1+ solid tumors in vitro and in vivo, providing a promising therapeutic option for future clinical application.

2.
Front Oncol ; 10: 1326, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903495

RESUMO

Drug development targeting the most frequently mutation G12D of KRAS has great significance. As an attractive immunotherapy, cancer vaccines can overcome binding difficulties of small molecules; however, the weak immunogenicity and production difficulties of reported KRAS mutation vaccines limit their clinical application. To improve antigen-specific immune responses and Anti-Tumor effects on tumors expressing KRAS G12D mutation, we designed recombinant proteins containing KRAS peptide (amino acids 5-21) with G12D (called SP) in two forms: DTT-SP4 and DTSP. DTT-SP4 was constructed by fusing four copies of SP to the C-terminal of the translocation domain of diphtheria toxin (DTT), and DTSP was constructed by grafting SP onto DTT. The two vaccines in combination with aluminum hydroxide (Alum) and cytosine phosphoguanine (CpG) successfully induced conspicuous SP-specific humoral and cellular immune responses, and displayed prominent protective and therapeutic Anti-Tumor effects in mouse CT26 tumor models. Surprisingly, the DTSP-treated group displayed better Anti-Tumor effects in vivo compared with the DTT-SP4-treated and control groups. Moreover, 87.5 and 50% of DTSP-treated mice in the preventive and therapeutic models were tumor free, respectively. Notably, in the DTSP-treated group, the interferon-γ (IFN-γ) expression of T cells in vitro and the T-helper 1 (Th1)-related cytokine expression in tumor tissues indicated that the activated Th1 immune response may be involved in Anti-Tumor activity. Furthermore, DTSP treatment remarkably altered the subpopulation of T cells in splenocytes and tumor-infiltrating lymphocytes. The percentage of effector CD8+ T cells increased, whereas that of immunosuppressive CD4+Foxp3+ T cells remained reduced in the DTSP group. Dramatic tumor-inhibitory effects of DTSP, which is easily prepared, make it a more attractive strategy against KRAS G12D tumors.

3.
Vaccines (Basel) ; 8(2)2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32423130

RESUMO

Blocking inhibitory signaling and engaging stimulatory signaling have emerged as important therapeutic modalities for cancer immunotherapy. This study aimed to investigate immunomodulatory features of three recombinant costimulatory ligand proteins in a mouse model, which are extracellular domains of OX40-ligand (OX40L), 4-1BB-ligand (4-1BBL), or two domains in tandem, fused with the transmembrane domain of diphtheria toxin (DTT), named DTT-COS1, DTT-COS2, and DTT-COS12, respectively. In vitro study showed that DTT-COS1 and DTT-COS12 had immunological activity increasing the ratio of CD8/CD4 T cells. Treatments with DTT-COS1 and DTT-COS12 dramatically generated immune protection against the B16F10 tumor challenge in both prophylactic and therapeutic efficacy. Furthermore, regarding tumor microenvironment (TME) immunomodulation, DTT-COS1 treatment increased the proportion of CD4+ effector T cells (Teff) and decreased the expression of a suppressive cytokine. Meanwhile, DTT-COS12 reduced regulatory T cells (Treg) and improved the level of stimulatory cytokines. In addition, endogenous antibodies against OX40L/4-1BBL were generated, which may help with antitumor responses. Unexpectedly, DTT-COS2 lacked antitumor effects in vitro and in vivo. Importantly, serum analysis of liver-function associated factors and pro-inflammatory cytokines demonstrated that treatments were safe formulations in mice without signs of systemic toxicity. Remarkably, DTT-COS1 and DTT-COS12 are functional immunomodulators for mouse B16F10 melanoma, creating practical preclinical value in cancer immunotherapy.

4.
Front Immunol ; 10: 2472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31749795

RESUMO

Tumor neoantigens are ideal targets for cancer immunotherapy as they are recognized by host immune system as foreigners and can elicit tumor-specific immune responses. However, existing strategies utilizing RNA or long peptides for the neoantigen vaccines render limited immune responses since only 20-30% of neoantigens predicted in silico to bind MHC I molecules are capable of eliciting immune responses with the majority of responding T cells are CD4+. Therefore, it warrants further exploration to enhance neoantigen-specific CD8+ T cells responses. Since neoantigens are naturally weak antigens, we asked whether foreign T help epitopes could enhance their immunogenicity. In present study, we chose 4 weak B16F10 neoantigens as vaccine targets, and fused them to the transmembrane domain of diphtheria toxin, namely DTT-neoAg. Strikingly, the vaccine elicited anti-tumor CD8+ T cells responses and enhanced tumor infiltration of both T cells and NK cells. Impressively, DTT-neoAg vaccine significantly deterred tumor growth with the inhibition rate reached 88% in the preventive model and 100% in the therapeutic model at low dose of tumor challenge. Furthermore, after second challenge with higher dose of tumor cells, 33.3% of the immunized mice remained tumor-free for 6 months in the therapeutic model. Because DTT is a non-toxic domain of diphtheria toxin, it may be not of great concern in terms of safety as a Th epitope provider. Thus, the fusion strategy employed by this study may become a feasible and powerful approach for development of personalized cancer vaccines.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Toxina Diftérica/imunologia , Melanoma Experimental/terapia , Animais , Anticorpos Antineoplásicos/imunologia , Linhagem Celular Tumoral , Feminino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Carga Tumoral
5.
J Ethnopharmacol ; 177: 1-9, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26571086

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Many traditional Chinese medicines (TCM) have been used for hundreds of years for hair blackening and hair nourishing, and now many of them are commonly used in Chinese herbal shampoo to nourish the hair and promote hair growth. AIMS OF THE STUDY: The present study was performed to screen 5α-reductase (5αR) inhibitors from traditional Chinese medicines, evaluate its hair growth promoting activity in vivo, and further investigate its effects on androgen metabolism and the expression of 5αR II in hair follicles. MATERIALS AND METHODS: Nine TCM which were dried, ground and extracted by maceration with 75% ethanol or distilled water were used for screening 5αR inhibitors, and enzymes were extracted from the rat epididymis. The leaves of Platycladus orientalis (L.) Franco was used to evaluate the in vivo anti-androgenic activity. Skin color was observed daily and the hair re-growth was assessed by assigning the hair growth score. The longitudinal sections of hair follicles were used for observing follicle morphology, classifying of distinct stages of hair follicle morphogenesis and calculate the average score. The transverse sections were used for determination of hair follicle counts. Testosterone (T), Dihydrotestosterone (DHT) and Estradiol (E2) levels in serum and skin tissue were detected by ELISA kits. The immunofluorescence assay was used to detect the influence of CP-ext on 5αR expression in dorsal skin. RESULTS: We found the extract of Ganoderma lucidum (GL-ext), Polygonum multiflori (PM-ext), Cacumen platycladi (CP-ext) and Cynomorium songaricum (CS-ext) showed stronger 5αR inhibitory activity. CP-ext (5mg and 2mg/mouse/day) could significantly shorten the time of the dorsal skin darkening and got longhaired (P<0.01), and showed high hair re-growth promoting activity. Furthermore the histological data of hair follicles in each group showed that CP-ext could promote the growth of hair follicle and slowed down hair follicles enter the telogen. What's more CP-ext significantly reduced DHT levels and down-regulated the expression of 5αRⅡin skin (P<0.01). CONCLUSIONS: GL-ext, PM-ext, CP-ext and CS-ext showed strong 5αR inhibitory activity. CP-ext possesses high hair growth promoting activity in the in vivo androgen-sensitive mouse model via inhibiting the 5αR activity, decreasing the DHT levels and in turn suppressing the expression of 5αR. Our study may contribute to the development of a new generation of herbal supplements with clearer material basis of pharmacodynamic for treating androgenic alopecia (AGA).


Assuntos
3-Oxo-5-alfa-Esteroide 4-Desidrogenase/metabolismo , Alopecia/induzido quimicamente , Cupressaceae/química , Medicamentos de Ervas Chinesas/farmacologia , Cabelo/efeitos dos fármacos , Testosterona/toxicidade , 3-Oxo-5-alfa-Esteroide 4-Desidrogenase/genética , Animais , Medicamentos de Ervas Chinesas/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cabelo/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley
6.
Cell Res ; 25(11): 1237-49, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26391698

RESUMO

The newly-emerging Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans. Despite global efforts, the potential for an associated pandemic in the future cannot be excluded. The development of effective counter-measures is urgent. MERS-CoV-specific anti-viral drugs or vaccines are not yet available. Using the spike receptor-binding domain of MERS-CoV (MERS-RBD) to immunize mice, we identified two neutralizing monoclonal antibodies (mAbs) 4C2 and 2E6. Both mAbs potently bind to MERS-RBD and block virus entry in vitro with high efficacy. We further investigated their mechanisms of neutralization by crystallizing the complex between the Fab fragments and the RBD, and solved the structure of the 4C2 Fab/MERS-RBD complex. The structure showed that 4C2 recognizes an epitope that partially overlaps the receptor-binding footprint in MERS-RBD, thereby interfering with the virus/receptor interactions by both steric hindrance and interface-residue competition. 2E6 also blocks receptor binding, and competes with 4C2 for binding to MERS-RBD. Based on the structure, we further humanized 4C2 by preserving only the paratope residues and substituting the remaining amino acids with the counterparts from human immunoglobulins. The humanized 4C2 (4C2h) antibody sustained similar neutralizing activity and biochemical characteristics to the parental mouse antibody. Finally, we showed that 4C2h can significantly abate the virus titers in lungs of Ad5-hCD26-transduced mice infected with MERS-CoV, therefore representing a promising agent for prophylaxis and therapy in clinical settings.


Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/tratamento farmacológico , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/uso terapêutico , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/química , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/química , Camundongos , Camundongos Endogâmicos BALB C
7.
Cell Host Microbe ; 16(3): 328-37, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-25211075

RESUMO

The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) is phylogenetically closely related to the bat coronaviruses (BatCoVs) HKU4 and HKU5. However, the evolutionary pathway of MERS-CoV is still unclear. A receptor binding domain (RBD) in the MERS-CoV envelope-embedded spike protein specifically engages human CD26 (hCD26) to initiate viral entry. The high sequence identity in the viral spike protein prompted us to investigate if HKU4 and HKU5 can recognize hCD26 for cell entry. We found that HKU4-RBD, but not HKU5-RBD, binds to hCD26, and pseudotyped viruses embedding HKU4 spike can infect cells via hCD26 recognition. The structure of the HKU4-RBD/hCD26 complex revealed a hCD26-binding mode similar overall to that observed for MERS-RBD. HKU4-RBD, however, is less adapted to hCD26 than MERS-RBD, explaining its lower affinity for receptor binding. Our findings support a bat origin for MERS-CoV and indicate the need for surveillance of HKU4-related viruses in bats.


Assuntos
Quirópteros/virologia , Infecções por Coronavirus/metabolismo , Coronavirus/metabolismo , Dipeptidil Peptidase 4/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Animais , Coronavirus/classificação , Coronavirus/genética , Coronavirus/isolamento & purificação , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/genética , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Receptores Virais/química , Receptores Virais/genética , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
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