Development and Characterization of A Novel SpyTagged Modular Nanobody as A Detection Platform for CD22-Positive Cells.

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).

Recent updates on allogeneic CAR-T cells in hematological malignancies.

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.

Functionalization of Defective Zr Metal-Organic Frameworks for Water Decontamination: Mechanistic Insight into the Competitive Roles of -NH2 and -SH Sites in Removal of As(III) Species.

Direct removal of trivalent arsenic, As(III), arsenite, or H3AsO3, is a great challenge in accessing clean sources of water. Different methodologies and materials were applied in this regard, but among them, direct removal of As(III) species using a metal-organic framework (MOF)-based adsorbent shows a great deal of potential. Although some studies were conducted on As(III) removal using MOFs, studies of functional groups are still quite rare. For this purpose, three novel functionalized defective Zr-MOFs, using UiO-66 [Zr6(OH)4O4(BDC)6, where BDC2- = benzene-1,4-dicarboxylate], were fabricated to investigate the competitive or cooperative roles of the free -NH2 and/or -SH site in the removal of As(III). UiO-66 was functionalized with monocarboxylate linkers, including glycine (Gly, NH2-CH2-COOH), cysteine [Cys, SH(CH2)-NH2(CH)-COOH], and mercaptopropionic acid [Mer, SH-(CH2)2-COOH]. Gly@UiO-66, Cys@UiO-66, and Mer@UiO-66 were applied for the direct removal of As(III) species. Although Cys@UiO-66 is functionalized with both amine and thiol functional groups, Gly@UiO-66 has a higher adsorption capacity (301.4 mg g-1) with respect to As(III), which is among the best reported values. This is due to the fact that (1) the affinity of amine sites in Gly@UiO-66 for As(III) is higher than that of thiol sites in Mer@UiO-66 and (2) Cys@UiO-66 has a very small surface area compared to that of Gly@UiO-66. Mechanistic studies using X-ray photoelectron spectroscopy and vibrational spectroscopy reveal that not only the functionalization and chemical nature of the function but also other parameters such as the protonation-deprotonation mechanisms and chemical state of the function are other critical factors for designing a functional MOF-based adsorbent with high affinity for and maximum capacity with respect to the target analyte.

Functionalization of Defective Zr-MOFs for Water Decontamination: Mechanistic Insight into the Competitive Roles of -NH2 and -SH Sites in the Removal of Hg(II) Ions.

Functional metal-organic frameworks (MOFs), especially those based on sulfur and nitrogen atoms, were frequently applied for the removal of Hg(II) ions. However, a systematic study on the cooperative or competitive roles of -SH and -NH2 functions in the presence of secondary mechanisms (proton transfer and redox) is still rare. In this work, the UiO-66 framework (Zr6(OH)4O4(BDC)6, BDC2- = benzene-1,4-dicarboxylate) was decorated with functional monocarboxylate linkers including glycine (Gly), mercaptopropionic acid (Mer), and cysteine (Cys). Due to the molecular similarity of these functional linkers, the coordination affinity between the amine and thiol sites with Hg(II) ions can be compared, and the effect of proton transfer and redox mechanisms on the possible thiol···Hg(II) and amine···Hg(II) interactions can be investigated. The results show that the Cys@UiO-66 framework can adsorb 1288 mg g-1 of Hg(II), while Mer@UiO-66 and Gly@UiO-66 can adsorb 593 and 313 mg g-1 at pH = 7 and 500 ppm, respectively. This is due to the facts that both the amine and the thiol functions of the Cys@UiO-66 framework show synergism in Hg(II) removal, and the secondary mechanisms reduce the affinity of thiol in Mer@UiO-66 and amine in Gly@UiO-66 frameworks in the removal process of Hg(II) ions. Free -SH sites in Mer@UiO-66 undergo a redox convert to -SO3H groups, and free protonated -NH2 sites in Gly@UiO-66 do not fully deprotonate during Hg(II) removal. Yet, in the case of Cys@UiO-66, free protonated -NH2 sites are fully deprotonated, and free SH sites did not convert to -SO3H groups during Hg(II) removal. These observations show that the redox and proton transfer mechanisms can negatively affect the adsorption capacity of functional MOFs containing free -SH and -NH2 groups. So, not only the functionalization but also control over secondary mechanisms in the removal process are necessary parameters to improve the affinity between functional MOFs and Hg(II) ions.

Tuning spacer length improves the functionality of the nanobody-based VEGFR2 CAR T cell.

BACKGROUND: The chimeric antigen receptor-expressing T (CAR-T) cells for cancer immunotherapy have obtained considerable clinical importance. CAR T cells need an optimized intracellular signaling domain to get appropriately activated and also for the proper antigen recognition, the length and composition of the extracellular spacer are critical factors. RESULTS: We constructed two third-generation nanobody-based VEGFR2-CARs containing either IgG1 hinge-CH2-CH3 region or hinge-only as long or short extracellular spacers, respectively. Both CARs also contained intracellular activating domains of CD28, OX40, and CD3ζ. The T cells from healthy individuals were transduced efficiently with the two CARs, and showed increased secretion of IL-2 and IFN-γ cytokines, and also CD69 and CD25 activation markers along with cytolytic activity after encountering VEGFR2+ cells. The VEGFR2-CAR T cells harboring the long spacer showed higher cytokine release and CD69 and CD25 expression in addition to a more efficient cytolytic effect on VEGFR2+ target cells. CONCLUSIONS: The results demonstrated that the third-generation anti-VEGFR2 nanobody-based CAR T cell with a long spacer had a superior function and potentially could be a better candidate for solid tumor treatment.

Chimeric antigen receptor-natural killer cells: a promising sword against insidious tumor cells.

Natural killer (NK) cells are a critical component of innate immunity, particularly in initial cancer recognition and inhibition of additional tumor growth or metastasis propagation. NK cells recognize transformed cells without prior sensitization via stimulatory receptors and rapidly eradicate them. However, the protective tumor microenvironment facilitates tumor escaping via induction of an exhaustion state in immune cells, including NK cells. Hence, genetic manipulation of NK cells for specific identification of tumor-associated antigens or a more robust response against tumor cells is a promising strategy for NK cells' tumoricidal augmentation. Regarding the remarkable achievement of engineered CAR-T cells in treating hematologic malignancies, there is evolving interest in CAR-NK cell recruitment in cancer immunotherapy. Innate functionality of NK cells, higher safety, superior in vivo maintenance, and the off-the-shelf potential move CAR-NK-based therapy superior to CAR-T cells treatment. In this review, we have comprehensively discussed the recent genetic manipulations of CAR-NK cell manufacturing regarding different domains of CAR constructs and their following delivery systems into diverse sources of NK cells. Then highlight the preclinical and clinical investigations of CAR-NK cells and examine the current challenges and prospects as an optimistic remedy in cancer immunotherapy.

Nanobodies in cell-mediated immunotherapy: On the road to fight cancer.

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.

Anti-Acinetobacter baumannii single-chain variable fragments show direct bactericidal activity.

Objectives: The high resistance rate of Acinetobacter baumannii and the limited number of available antibiotics have prompted a worldwide effort to develop effective antimicrobial agents. Accordingly, identifying single-chain variable fragment antibodies (scFvs), capable of exerting direct antibacterial activity in an immune system-independent manner, may be making immunocompromised patients more susceptible to A. baumannii infections. Materials and Methods: To isolate bactericidal scFvs targeting A. baumannii, we panned a large human scFv phage display library against whole-cell extensively drug-resistant (XDR) A. baumannii strains grown as biofilm or cultured with human blood or human peripheral blood mononuclear cells plus plasma. The binding of scFv-phages to A. baumannii was assessed by the dot-blot assay. Soluble scFvs, derived from the selected phages, were assessed based on their ability to bind and inhibit the growth of A. baumannii. Results: Five phage clones showed the highest reactivity toward A. baumannii. Among five soluble scFvs, derived from positive phage clones, two scFvs, EB211 and EB279, had high expression yields and displayed strong binding to A. baumannii compared with the controls. Moreover, XDR A. baumannii strains treated with positively-charged scFvs, including EB211, EB279, or a cocktail of EB211 and EB279 (200 µg/ml), displayed lower viability (approximately 50%, 78%, and 40% viability, respectively) compared with PBS-treated bacteria. Conclusion: These results suggest that combining last-resort antibiotics with bactericidal scFvs could provide promising outcomes in immunocompromised individuals with A. baumannii infections.

Immunovirotherapy: The role of antibody based therapeutics combination with oncolytic viruses.

Despite the fact that the new drugs and targeted therapies have been approved for cancer therapy during the past 30 years, the majority of cancer types are still remain challenging to be treated. Due to the tumor heterogeneity, immune system evasion and the complex interaction between the tumor microenvironment and immune cells, the great majority of malignancies need multimodal therapy. Unfortunately, tumors frequently develop treatment resistance, so it is important to have a variety of therapeutic choices available for the treatment of neoplastic diseases. Immunotherapy has lately shown clinical responses in malignancies with unfavorable outcomes. Oncolytic virus (OV) immunotherapy is a cancer treatment strategy that employs naturally occurring or genetically-modified viruses that multiply preferentially within cancer cells. OVs have the ability to not only induce oncolysis but also activate cells of the immune system, which in turn activates innate and adaptive anticancer responses. Despite the fact that OVs were translated into clinical trials, with T-VECs receiving FDA approval for melanoma, their use in fighting cancer faced some challenges, including off-target side effects, immune system clearance, non-specific uptake, and intratumoral spread of OVs in solid tumors. Although various strategies have been used to overcome the challenges, these strategies have not provided promising outcomes in monotherapy with OVs. In this situation, it is increasingly common to use rational combinations of immunotherapies to improve patient benefit. With the development of other aspects of cancer immunotherapy strategies, combinational therapy has been proposed to improve the anti-tumor activities of OVs. In this regard, OVs were combined with other biotherapeutic platforms, including various forms of antibodies, nanobodies, chimeric antigen receptor (CAR) T cells, and dendritic cells, to reduce the side effects of OVs and enhance their efficacy. This article reviews the promising outcomes of OVs in cancer therapy, the challenges OVs face and solutions, and their combination with other biotherapeutic agents.

Development of a human phage display-derived anti-PD-1 scFv antibody: an attractive tool for immune checkpoint therapy.

BACKGROUND: The PD-1 checkpoint pathway plays a major role in tumor immune evasion and the development of the tumor microenvironment. Clinical studies show that therapeutic antibodies blocking the PD-1 pathway can restore anti-tumor or anti-virus immune responses by the reinvigoration of exhausted T cells. Because of the promising results of anti-PD-1 monoclonal antibodies in cancer treatment, autoimmune disorders, and infectious diseases, the PD-1 has emerged as an encouraging target for different diseases. RESULTS: In the present study, we employed a human semi-synthetic phage library for isolation of some scFvs against the extracellular domain of PD-1 protein by panning process. After the panning, a novel anti-PD-1 scFv (SS107) was found that exhibited specific binding to PD-1 antigen and stimulated Jurkat T cells. The selected anti-PD-1 scFv could restore the production of IL-2 and IFN-γ by Jurkat T cells that were co-cultured with PD-L1 positive tumor cells. CONCLUSION: This anti-PD-1 scFv with high specificity and the ability to reactivate exhausted T cells has the potential to be developed as an anti-cancer agent or to be used in combination with other therapeutic approaches.

Bactericidal fully human single-chain fragment variable antibodies protect mice against methicillin-resistant Staphylococcus aureus bacteraemia.

OBJECTIVES: The increasing prevalence of antibiotic-resistant Staphylococcus aureus, besides the inadequate numbers of effective antibiotics, emphasises the need to find new therapeutic agents against this lethal pathogen. METHODS: In this study, to obtain antibody fragments against S. aureus, a human single-chain fragment variable (scFv) library was enriched against living methicillin-resistant S. aureus (MRSA) cells, grown in three different conditions, that is human peripheral blood mononuclear cells with plasma, whole blood and biofilm. The antibacterial activity of scFvs was evaluated by the growth inhibition assay in vitro. Furthermore, the therapeutic efficacy of anti-S. aureus scFvs was appraised in a mouse model of bacteraemia. RESULTS: Three scFv antibodies, that is MEH63, MEH158 and MEH183, with unique sequences, were found, which exhibited significant binding to S. aureus and reduced the viability of S. aureus in in vitro inhibition assays. Based on the results, MEH63, MEH158 and MEH183, in addition to their combination, could prolong the survival rate, reduce the bacterial burden in the blood and prevent inflammation and tissue destruction in the kidneys and spleen of mice with MRSA bacteraemia compared with the vehicle group (treated with normal saline). CONCLUSION: The combination therapy with anti-S. aureus scFvs and conventional antibiotics might shed light on the treatment of patients with S. aureus infections.

High performance of ultrasonic-assisted synthesis of two spherical polymers for enantioselective catalysis.

Chiral polymers have aroused great attention in among chiral supramolecular materials based on their features. Herein, for the first time, the synthesis of chiral polymeric composites (CMNPs/1,4-Zbtb & 1,3-Zbtb) have been reported with entrapment through three strategies: ultrasonic irradiation, solvothermal, and mechanical stirring. According to the obtained results, it is found that ultrasound-assisted synthesis can be considered as an inexpensive and efficient method than the others, from the point ofviewof energy and time consuming. In this strategy, encapsulation of chiral magnetic nanoparticles (CMNPs) by using tetrazole-based polymers (Zbtbs) happens, in-situly. These chiral sphere-like inorganic-organic polymers can be considered as core and shell composites with catalytic activity due to their acidic (semi unsaturated Zn: open metal sites) and basic (abundant basic nitrogens) centers. In these structures, the unprecedented chirality induction can happen from the core to shell by non-covalent interaction, easily. They could catalyze symmetric oxidation and asymmetric henry condensation to give chiral ß-nitroalkanol. Circular dichroism and chiral gas chromatography were used to characterize the produced enantiomers. These chiral polymeric materials can be considered as unique acid-base bifunctional catalysts with efficient properties such as high stability, enantiomeric excess, enantioselectivity to the main product, and protecting from CMNPs leaching.

Kinetic and thermodynamic study of c-Met interaction with single chain fragment variable (scFv) antibodies using phage based surface plasmon resonance.

Mesenchymal epithelial transition factor (c-Met) has been recently regarded as an attractive target for the treatment of cancer. Our previous study showed that c-Met-specific single chain fragment variables (scFvs) can be considered as a promising therapy for cancer, however, their molecular interaction with c-Met protein have not been assessed. Accordingly, in the current study we aim to evaluate the kinetic and thermodynamic properties of c-Met interaction with these scFvs as anticancer agents by means of surface plasmon resonance (SPR) technique. Phage-scFvs were immobilized on the 11-mercaptoundecanoic acid gold chips after carboxylic groups activation by N-ethyl-N-(3-diethylaminopropyl) carbodiimide/N-hydroxysuccinimide and, then the c-Met binding to each scFvs (ES1, ES2, and ES3) at different concentrations (ranging from 20 to 665 µM) was explored. Kinetic studies revealed that ES1 has the highest affinity (KD = 3.36 × 10-8) toward its target at 25°C. Calculation of thermodynamic parameters also showed positive values for enthalpy and entropy changes, which was representative of hydrophobic forces between c-Met and ES1. Furthermore, the positive value of Gibbs free energy indicated that c-Met binding to ES1 was enthalpy-driven. Taken together, we concluded that produced ES1 can be applied as promising scFv-based therapy for diagnosis or targeting of c-Met in various cancers.

Targeting the 4-1BB costimulatory molecule through single chain antibodies promotes the human T-cell response.

BACKGROUND: Adoptive T-cell therapy (ACT) using autologous tumor-reactive T lymphocytes has considerable potential for cancer immunotherapy. In ACT, T cells are isolated from cancer patients and then stimulated and expanded in vitro by cytokines and costimulatory molecules. 4-1BB is an important costimulatory protein belonging to the TNF receptor superfamily. It is involved in T-cell survival, proliferation and activation. Agonistic anti-4-1BB monoclonal antibodies have been introduced as appropriate tools for ACT. METHODS: Here, various single-chain fragment variable (scFv) antibodies were used to activate T cells isolated from peripheral blood via immune magnetic isolation. The T cells were stimulated with IL-2 and anti-CD-3 mAb and then treated with agonistic anti-4-1BB scFvs. The results showed the remarkable effects of anti-41BB scFvs on the functional properties of T cells, including their activation, proliferation and cytokine production. The flow cytometry analysis revealed a considerable increase in the expression of the T-cell activation marker CD69. Moreover, T-cell proliferation was evidenced in treated cells by CFSE labeling compared to the control groups. RESULT: Anti-4-1BB scFvs significantly increased IFN-γ and IL-2 mRNA and protein expression in T cells, but exhibited no stimulatory effect on IL-4 expression. These findings show that anti-4-1BB scFvs could evoke a Type I immune response. CONCLUSIONS: Our results demonstrate that targeting the 4-1BB molecule using agonistic scFvs could be an effective strategy for T-cell stimulation as part of an ACT approach to cancer treatment.

Engineered Jurkat Cells for Targeting Prostate-Specific Membrane Antigen on Prostate Cancer Cells by Nanobody-Based Chimeric Antigen Receptor

Background: Recently, modification of T cells with chimeric antigen receptor (CAR) has been an attractive approach for adoptive immunotherapy of cancers. Typically, CARs contain a single-chain variable domain fragment (scFv). Most often, scfvs are derived from a monoclonal antibody of murine origin and may be a trigger for host immune system that leads to the T-cell clearance. Nanobody is a specific antigen-binding fragment derived from camelid that has great homology to human VH and low immunogenic potential. Therefore, in this study, nanobody was employed instead of scFv in CAR construct. Methods: In this study, a CAR was constructed based on a nanobody against PSMA (NBPII-CAR). At first, Jurkat cells were electroporated with NBPII-CAR, and then flow cytometry was performed for NBPII-CAR expression. For functional analysis, CAR T cells were co-cultured with prostate cancer cells and analyzed for IL-2 secretion, CD25 expression, and cell proliferation. Results: Flow cytometry results confirmed the expression of NBPII-CAR on the transfected Jurkat cells. Our data showed the specificity of engineered Jurkat cells against prostate cancer cells by not only increasing the IL-2 cytokine (about 370 pg/ml) but also expressing the T-cell activation marker CD25 (about 30%). In addition, proliferation of engineered Jurkat cells increased nearly 60% when co-cultured with LNCaP (PSMA+), as compared with DU145 (PSMA-). Conclusion: Here, we describe the ability of nanobody-based CAR to recognize PSMA that leads to the activation of Jurkat cells. This construct might be used as a promising candidate for clinical applications in prostate cancer therapy.

T cell engineered with a novel nanobody-based chimeric antigen receptor against VEGFR2 as a candidate for tumor immunotherapy.

Solid tumors that are responsible for more than 85% of cancer death cases need angiogenesis for their growth and metastasis. Among antiangiogenic therapies, targeting the vascular endothelial growth factor receptor 2 (VEGFR2) that is over-expressed on tumor vasculatures has been a promising strategy. In this study, we developed a second generation nanobody (VHH)-based CAR T cell targeting VEGFR2-expressing tumor cells. The CAR T cell was developed by linking the anti-VEGFR2 VHH to a spacer, and signaling domains of CD28 and CD3 ζ. The T cells were activated with anti-CD3 plus rIL-2 and electroporated with a plasmid encoding the CAR construct. The expression of activation markers, CD69 and CD25, on CAR T cells upon coculturing with VEGFR2-expressing cells were 41% and 48%, and the IL-2 and IFN-γ production were 470 pg/mL and 360 pg/mL, respectively. The expression of degranulation marker, CD107a, was 30% and the cytotoxic activity of the CAR T cells reached to more than 30% with E:T ratio of 9:1. The anti-VEGFR2 CAR but not mock T cells mediated specific lysis of 293-KDR cells expressing human VEGFR2 and might be considered as a candidate for adoptive T-cell immunotherapy of solid tumors. © 2019 IUBMB Life, 71(9):1259-1267, 2019.

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA+ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.

Myc Decoy Oligodeoxynucleotide Inhibits Growth and Modulates Differentiation of Mouse Embryonic Stem Cells as a Model of Cancer Stem Cells.

BACKGROUND: Myc (c-Myc) alone activates the embryonic stem cell-like transcriptional module in both normal and transformed cells. Its dysregulation might lead to increased cancer stem cells (CSCs) population in some tumor cells. OBJECTIVE: In order to investigate the potential of Myc decoy oligodeoxynucleotides for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of CSCs. To our best of knowledge this is the first report outlining the application of Myc decoy in transcription factor decoy "TFD" strategy for inducing differentiation in mESCs. METHODS: A 20-mer double-stranded Myc transcription factor decoy and scrambled oligodeoxynucleotides (ODNs) were designed, analyzed by electrophoretic mobility shift (EMSA) assay and transfected into the mESCs under 2 inhibitors (2i) condition. Further investigations were carried out using fluorescence and confocal microscopy, cell proliferation and apoptosis analysis, alkaline phosphatase and embryoid body formation assay, real-time PCR and western blotting. RESULTS: EMSA data showed that Myc decoy ODNs bound specifically to c-Myc protein. They were found to be localized in both cytoplasm and nucleus of mESCs. Our results revealed the potential capability of Myc decoy ODNs to decrease cell viability by (16.1±2%), to increase the number of cells arrested in G0/G1 phases and apoptosis by (14.2±3.1%) and (12.1±3.2%), respectively regarding the controls. Myc decoy could also modulate differentiation in mESCs despite the presence of 2i/LIF in our medium the presence of 2i/LIF in our medium. CONCLUSION: The optimized Myc decoy ODNs approach might be considered as a promising alternative strategy for differentiation therapy investigations.

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.