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1.
Cell J ; 26(8): 487-495, 2024 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-39380479

RESUMEN

OBJECTIVE: CD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cell malignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developing a bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTagged anti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells. MATERIALS AND METHODS: In this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vector and expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the eluted protein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed in E. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher and SpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nb was tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometry. RESULTS: SpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio of SpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flow cytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is no binding to the CD22- K562 control cells. CONCLUSION: The novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficial expression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22 in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs).

2.
Cancer Cell Int ; 24(1): 304, 2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-39227937

RESUMEN

CAR-T cell therapy is known as an effective therapy in patients with hematological malignancies. Since 2017, several autologous CAR-T cell (auto-CAR-T) drugs have been approved by the US Food and Drug Administration (FDA) for the treatment of some kinds of relapsed/refractory hematological malignancies. However, some patients fail to respond to these drugs due to high manufacturing time, batch-to-batch variation, poor quality and insufficient quantity of primary T cells, and their insufficient expansion and function. CAR-T cells prepared from allogeneic sources (allo-CAR-Ts) can be an alternative option to overcome these obstacles. Recently, several allo-CAR-Ts have entered into the early clinical trials. Despite their promising preclinical and clinical results, there are two main barriers, including graft-versus-host disease (GvHD) and allo-rejection that may decline the safety and efficacy of allo-CAR-Ts in the clinic. The successful development of these products depends on the starter cell source, the gene editing method, and the ability to escape immune rejection and prevent GvHD. Here, we summarize the gene editing technologies and the potential of various cell sources for developing allo-CAR-Ts and highlight their advantages for the treatment of hematological malignancies. We also describe preclinical and clinical data focusing on allo-CAR-T therapy in blood malignancies and discuss challenges and future perspectives of allo-CAR-Ts for therapeutic applications.

3.
Front Immunol ; 14: 1012841, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36761751

RESUMEN

The immune system is essential in recognizing and eliminating tumor cells. The unique characteristics of the tumor microenvironment (TME), such as heterogeneity, reduced blood flow, hypoxia, and acidity, can reduce the efficacy of cell-mediated immunity. The primary goal of cancer immunotherapy is to modify the immune cells or the TME to enable the immune system to eliminate malignancies successfully. Nanobodies, known as single-domain antibodies, are light chain-free antibody fragments produced from Camelidae antibodies. The unique properties of nanobodies, including high stability, reduced immunogenicity, enhanced infiltration into the TME of solid tumors and facile genetic engineering have led to their promising application in cell-mediated immunotherapy. They can promote the cancer therapy either directly by bridging between tumor cells and immune cells and by targeting cancer cells using immune cell-bound nanobodies or indirectly by blocking the inhibitory ligands/receptors. The T-cell activation can be engaged through anti-CD3 and anti-4-1BB nanobodies in the bispecific (bispecific T-cell engagers (BiTEs)) and trispecific (trispecific T-cell engager (TriTEs)) manners. Also, nanobodies can be used as natural killer (NK) cell engagers (BiKEs, TriKEs, and TetraKEs) to create an immune synapse between the tumor and NK cells. Nanobodies can redirect immune cells to attack tumor cells through a chimeric antigen receptor (CAR) incorporating a nanobody against the target antigen. Various cancer antigens have been targeted by nanobody-based CAR-T and CAR-NK cells for treating both hematological and solid malignancies. They can also cause the continuation of immune surveillance against tumor cells by stopping inappropriate inhibition of immune checkpoints. Other roles of nanobodies in cell-mediated cancer immunotherapy include reprogramming macrophages to reduce metastasis and angiogenesis, as well as preventing the severe side effects occurring in cell-mediated immunotherapy. Here, we highlight the critical functions of various immune cells, including T cells, NK cells, and macrophages in the TME, and discuss newly developed immunotherapy methods based on the targeted manipulation of immune cells and TME with nanobodies.


Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/uso terapéutico , Inmunoterapia , Células Asesinas Naturales , Linfocitos T , Receptores Quiméricos de Antígenos/genética , Neoplasias/terapia
4.
Drug Res (Stuttg) ; 69(3): 144-150, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30060264

RESUMEN

BACKGROUNDS: Peptide-based drugs have shown promising results in overcoming the limitations of chemotherapeutic drugs by providing a targeted therapy approach to cancer. However, the response rate of targeted therapies is limited, in large part due to the intra- and inter-heterogeneity of tumors. METHODS: In this study, we engineered a novel chimeric protein composed of the p28 peptide as a tumor-homing killer peptide and apoptin as a killer peptide. We evaluated its cytotoxicity against MCF7 and MDA-MB-231 breast cancer cells and HEK-293 normal cells by the MTT assay. Different linkers were evaluated when designing the chimeric protein. Three-dimensional structure predictions of chimeric proteins with different linkers were carried out by Modeller 9.19, and their validation and analysis were performed by RAMPAGE. RESULTS: Results showed that a cleavable linker, including furin cleavage sites, is preferred over other linkers. The chimeric protein was then successfully expressed in E. coli and purified by affinity chromatography under native conditions, then confirmed by SDS-PAGE and Western blot analysis. Compared with apoptin alone, the chimeric protein showed significantly higher toxicity against breast cancer cell lines in a dose-dependent manner. The IC50 values of the chimeric protein for MCF7 and MDA-MB-231 cells were 38.55 µg/mL and 43.11 µg/mL, respectively. There was no significant cytotoxic effect on the normal HEK293 cell line. CONCLUSION: This study demonstrates that fusion of p28 peptide to a potent protein could provide an effective method for tumor targeting. Further, in vitro and in vivo studies of this novel chimeric protein are underway.


Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Células MCF-7
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