CAR T cells redirected against tumor-specific antigen glycoforms: can low-sugar antigens guarantee a sweet success? / 医学前沿

Immune-based therapies have experienced a pronounced breakthrough in the past decades as they acquired multiple US Food and Drug Administration (FDA) approvals for various indications. To date, six chimeric antigen receptor T cell (CAR-T) therapies have been permitted for the treatment of certain patients with relapsed/refractory hematologic malignancies. However, several clinical trials of solid tumor CAR-T therapies were prematurely terminated, or they reported life-threatening treatment-related damages to healthy tissues. The simultaneous expression of target antigens by healthy organs and tumor cells is partly responsible for such toxicities. Alongside targeting tumor-specific antigens, targeting the aberrantly glycosylated glycoforms of tumor-associated antigens can also minimize the off-tumor effects of CAR-T therapies. Tn, T, and sialyl-Tn antigens have been reported to be involved in tumor progression and metastasis, and their expression results from the dysregulation of a series of glycosyltransferases and the endoplasmic reticulum protein chaperone, Cosmc. Moreover, these glycoforms have been associated with various types of cancers, including prostate, breast, colon, gastric, and lung cancers. Here, we discuss how underglycosylated antigens emerge and then detail the latest advances in the development of CAR-T-based immunotherapies that target some of such antigens.

[Evaluation of poly [amidoamine] dendrimer surface modification with poly[ethylene glycol] on cytotoxicity reduction]

Objective: Generation 5 poly [amidoamine] dendrimers are promising multipotent gene delivery vectors that provide favorable DNA condensation properties; however, their high toxicity limits their applications. Toxicity of PAMAM dendrimers depends on their type, generation and applied dosage in a way that lower generations [lower than G5 dendrimers] and anionic dendrimers have lower toxicity than higher generations and cationic dendrimers. The aim of this study is to evaluate the effect of PEGylation on toxicity of G5 PAMAM dendrimers

Methods: In this study, to improve their characteristics as gene delivery carriers, G5 PAMAM dendrimers were conjugated to polyethylene glycol molecules [PEG, molecular weight 3500] at three different molar ratios of 10, 20 and 30. Also the number of conjugated PEG chains was quantified using TNBSA and Ellman assays. The effect of different degrees of PEGylation on cytotoxicity and transfection efficiency of modified PAMAM dendrimers toward BT-474 and MCF-10A cell lines were assessed

Results: Compared to unconjugated, PEG conjugated PAMAM dendrimers had lower in vitro cytotoxicity, particularly at higher PEG to PAMAM molar ratios. Among all prepared PEG-PAMAM dendrimers, G5 PAMAM dendrimers that conjugated to PEG at a molar ratio of 10/1 had the highest in vitro transfection rate in both cell lines

Conclusion: Our results showed that these PEG-conjugated PAMAM dendrimers possess a great potential for in vitro gene delivery

[Identification of novel targeting peptides for PC3 cells by the screening of a phage display peptide library]

Objective: Prostate cancer is the second cause of cancer-associated death in men. In recent years, targeted therapy for cancer has attracted the attention of researchers. Targeted therapy leads to a decrease in drug adverse effects. Studies indicate that targeting peptides for cancer cells represent valuable tools for diagnostics and therapeutics. Recently, phage display peptide libraries have been used to identify target peptides to a variety of cancer cells. In the current study, we aim to isolate peptides that target PC3 cells [human prostate adenocarcinoma cells]

Methods: Four rounds of subtractive panning on control cells that included 5637 [bladder], Huh-7 [liver], SW480 [colon], AGS [stomach] and human fibroblast normal in addition to four rounds of positive panning on PC3 [target cell] were performed. Polyclonal phage ELISA was used to evaluate the process of enrichment during biopanning. Subsequently, phage clones were randomly selected from titer plates, amplified by plaque-PCR, and their genomic DNA was sequenced. We conducted bioinformatic analysis for further characterization of the isolated peptides

Results: Several rounds of panning resulted in the enrichment of a number of peptides. The results of polyclonal phage ELISA indicated that the biopanning process was successful. In silico analysis showed the presence of several consensus amino acid motifs in the peptides

Conclusion: The peptides identified through biopanning can be considered as potential specific binders to PC3 cells. Peptides with specificity binding to target cells can be used for targeted gene and drug delivery to malignant tumor cells. Further analyses of these peptides are required to show their capacity for targeted delivery of various genes and drugs into prostate cancer cells

Production of cyclin D1 specific siRNAs by double strand processing for gene therapy of of esophageal squamous cell carcinoma

[RNA interference] is a new strategy in gene therapy and biotechnology which provides new viewpoints in treatment of different diseases such as cancer and viral diseases. CCND1 which is a key gene in cell cycle is amplified and over expressed in esophageal cancer. The aim of this study was to produce siRNAs for CCND1, the key gene in cell cycle. dsRNA digestion method was applied by using recombinant human dicer enzyme to cleave in vitro transcribed dsRNA into a pool of 22bp siRNA. Total RNA was extracted and cDNA was produced using RT-PCR. T7 promoter was added to both ends of the DNA template by PCR. RNA was produced from both strands of the DNA using T7 RNA polymerase. After annealing both strands, dsRNA was prepared. Finally siRNA pool was produced by dicer treatment. RNA extraction yield from HN5 cell line was 14.69 micro g/106 cell. The results from beta actin control gene confirmed the cDNA integrity. After optimization, T7 promoter adding was confirmed using gel electrophoresis and DNA sequencing. After optimization dsRNA yield was improved. The best incubation condition was 18h. Each microgram of dsRNA yielded 0.5 micro g siRNA. dsRNA digestion method includes several steps in which the product of each step is used as the precursor for the next step. So optimization and increasing the specificity and product yield should be the most important goals of the study, because the yield of each step has a direct relationship with the final product yield namely; siRNA. Optimizing and increasing the yield, dsRNA digestion method could be a rapid, available and profitable method for siRNA generation, and providing large amounts of siRNA

Generation of in-vitro spermatogonial stem cells following genetic manipulation of primordial germ-like cells

Research about potential use of stem cells for the development of germ line cells in vitro had been challenged. In the present study, we reported a novel protocol consisting of cocktail growth factor addition for germ cell differentiation followed by transfection. The cells were purificated based on the expression on the cell surface of a protein. This protein is not present in normal cells of mice and does not interfere with cellular function. This cell surface marker is efficiently recognized by monoclonal antibodies. Bone marrow mesenchymal stem cells derived primordial germ like cells were differentiated to spermatogo-nial stem like cells by inducer cocktail including Retinoic acid [RA]+Leukemia inhibitory factor [LIF]+Basic fibroblast growth factor [bFgF]. Co-culture system was used as a feeder under differentiated cells. A 400 bp fragment of sperm-atogoniaspecific Stra-8 locus was enough to direct gene expression to the germ line stem cells. Stra8-CD4HAglo construct was used for purification of pre-meiotic differentiated cells. Expression of pluripotency [Pou5F1, Nanog, c-Myc] and specific germ cell [Mvh, Piwil2, Stra-8] genes in each stage were analyzed. The purified cells expressed the known molecular markers of PGC-like cells such as Mvh, Piwil2 and Stra-8. The outcomes of qPCR showed that ratio pluripotency of genes expression in selective group significantly decreased [p?0.05] in the initial differentiation process. This results showed that ratio of Pou5F1, Nanog, c-Myc, Mvh, Piwil2 and Stra-8 expression to purified PGC-like cells were 0.41, 0.204, 1.1, 0.003, 0.184 and 2.276, respectively. Treatment of cells with RA affected up regulation of Stra-8. Although, c-Myc gene as an oncogenic gene had significantly increased [p

association between selected RANKL gene polymorphisms and chronic/aggressive periodontitis in Iranian subjects

The primary aim of this study was to investigate the status of RANKL6-7 gene polymorphism in patients with chronic [mild, moderate, severe] and aggressive periodontitis as well as healthy controls. We examined 80 patients for the RANKL6-7 polymorphisms [rs1054016 and rs9567000]. Polymorphism was determined by polymerase chain reaction [PCR] followed by direct sequencing. No statistically significant association was found between the polymorphism in the RANKL 7 gene and periodontal disease [P<0.55]. There was also no polymorphic allele observed in RANKL 6 gene of the study population. We found no association between the studied RANKL polymorphisms and chronic/aggressive periodontitis

[Construction of PEG-PAMAM dendrimer-based TAG72-targeting nanocarrier for t-Bid gene delivery to colorectal tumor cells]

The objective of this study is to develop and assess targeted PAMAM-PEG nanocarrier with anti-TAG72 nanobody for t-Bid gene coding construct delivery into the human colonic adenocarcinoma cells. Nanobody [Nb] coding sequence was subcloned into pSJ expression vector for large-scale production and then Nb was purified by Ni++ affinity chromatography. SDS-PAGE and western blot analysis were performed to verify purifiction. PAMAM was reacted with PEG at the ratio 1:2 [mol/mol] and anti-TAG72 Nb at the ratio 1:1 [mol/mol]. Surface charge and size of resulting nanoparticles were evaluated by Malvern zeta sizer and Nanosight. Efficiency of constructed gene carrier for t-Bid, a killer gene, delivery into colonic adenocarcinoma cells in in vitro was assessed using real time PCR and cell counting assays. Production of nanoparticles with the average size of 162 +/- 92 nm and+4.57 +/- 0.52 zeta potential was confirmed by nanosight and Malvern zeta sizer in order. Gel retardation assay result verified efficiency of carrier for pDNA comlexation. Real time PCR results confirmed the target gene overexpression in the cancerous cell lines. The results of this research confirms the efficiency of PAMAM dendrimers for gene transferring, positive effect of PEGylation and targeting of nanoparticles by anti-TAG72 nanobody

[Study on co-administration of GM-CSF, IL-15 on the enhancement of the immunogenicity of HIV-1 P24-Nef expression vector in BALB/c mouse model]

Today, AIDS is considered as a global problem and many efforts to generate an effective vaccine against this disease have been made, but remain inconclusive. DNA vaccines are a member of the new generation of vaccines that can efficiently stimulate the immune system. However, recent findings indicate low immunogenicity for these vaccines and it is believed that these types of vaccines require strategies that could infer more immunogenicity. The employment of adjuvants could be considered as one of the most important methods involved. In this study, a DNA vaccine candidate for HIV P24-Nef is constructed and then using genetic adjuvants IL-15 and GM-CSF, cellular immune responses have been studied. In this study the gene structure of HIV P24-Nef in eukaryotic expression vector was constructed and expression vectors of IL-15 and GM-CSF were used as adjuvants. After inoculation of the candidate vaccine to BALB/c mice, cytokine patterns, lymphocytes proliferation and cytotoxicity were analyzed. Our findings indicate that candidate vaccine significantly stimulated cellular immune responses. The usage of IL-15 and GM-CSF as DNA adjuvants together and separately with candidate vaccine has strengthened cellular immune responses significantly. Co-administration of DNA adjuvants significantly increased cellular immune responses when the ratio of the vaccine dose was more than the adjuvants. The sequences that we selected as candidate vaccine demonstrated good immunogenicity in mouse model and co-administration of IL-15 and GM-CSF DNA adjuvants increased cellular immune response to DNA vaccine construct

[Evaluation of immune responses from immunization of HIV-1 P24-Nef fusion peptide vaccine with monophosphoryl lipid A [MPL] in BALB/c mice]

Several vaccines against HIV have been investigated but none has been approved as an effective HIV vaccine. An approach that could induce stronger immune response against the pathogen is utilizing a multi-epitopic vaccine. This strategy was used in the design of several vaccines and resulted in improved immune responses. In this study a multi-epitopic fusion peptide including parts of HIV-1 Nef and P24 as a vaccine candidate was injected into mice and immune humoral responses measured with total antibody and IgG sub-classes using ELISA. Also measurement of cellular immune responses through evaluation of spleen cells proliferation response using MTT and cytotoxicity by LDH were performed. Finally, the cytokine pattern of IFN-gamma and IL-4 were also determined with ELISA. The results indicate that candidate vaccine stimulated mouse splenic lymphocyte proliferation response and also induced strong cytotoxicity responses. Analysis of humoral immune response has shown that the candidate vaccine has induced specific antibody production mainly of the IgG2a sub-class. Also cytokine pattern evaluation has shown that IFN-gamma secretion was dominant. The use of immunogen and conserved epitopes from P24 and Nef induced strong humoral and cellular immune responses and this construct could be candidate for further studies in animal models

Inhibitory effects of Lactobacillus salivarius and Lactobacillus crispatus isolated from chicken gastrointestinal tract on Salmonella enteritidis and Escherichia coli growth

Probiotics are live cultures of microbes; often lactic acid bacteria, but also some other species, which when fed to animals, improve their health and growth through altering the intestinal microbial balance. In the present research, healthy chickens' gastrointestinal [GI] tracts were screened for the presence of lactic acid bacteria with probiotic properties. The probiotic properties of the isolates taken from different parts of the GI tract were evaluated. They were examined for resistance to 2% [w/v] bile salts and acidic pH, capability to adhere to the intestinal epithelium and inhibitory effects on the growth of Salmonella enteritidis and Escherichia coli. Fermentation profile analyses and sequencing data of the conserved 16S rRNA genes showed that from a total of five selected clones, four clones isolated from the duodenum and caeca were Lactobacillus salivarius and the fifth clone, isolated from the duodenum, was Lactobacillus crispatus. All the selected clones were able to adhere to the chicken's epithelial cells. The lactobacilli isolated from different parts of the GI tract had probiotic properties suitable for use in animal feed. Due to the inhibitory effects of the isolated lactic acid bacteria on the growth of pathogenic microbiota, it can be concluded that these bacteria are good candidates for treatment of chicken GI infectious diseases

Design a chimeric promoter containing Survivin-like core promoter in order to transcriptional targeting of breast cancer

The greatest challenge in cancer gene therapy is to achieve the high specificity and efficiency in targeting of cancer cells. Because the goal of cancer gene therapy is to eradicate cancer cells, many therapeutic genes could be detrimental if unintentionally expressed in normal cells. Using promoter of the genes which are expressed specifically in cancer cells or have much more expression in cancer cells than normal cells, is very noticeable tool in cancer gene therapy [CGT]. In this study we were searching for cancer specific promoter which could highly express therapeutic gene. In order to apply a cancer specific promoter for creating a CGT construct, a promoter which have 34% similarity to Survivin core promoter was amplified from human genome by using Nested-PCR. Survivin is a member of anti-apoptotic gene and its over-expression was observed in up to 70% of breast cancers. This gene fragment contains two transcriptional binding sites which were similar to Survivin promoter according to the evaluation of Promoter Scan, EPD, Transfac, Compel and TRRD program. These binding sites were recognized by STAT1 and E2F transcription factors. This promoter was cloned into pCDNA3.1/Hygro plasmid in along with hypoxia and estrogen modules and pro-apoptotic gene tBid. Semi-quantitative RT-PCR results of transfected cancer cells showed that this gene fragment [Survivin like promoter] have relatively same potential as CMV promoter to direct tBid gene expression. Utilization of chimeric promoter containing Survivin like promoter could be a promising tool in killing cancer cells naturally by inducing apoptosis. This construct is highly effective in transcriptional targeting of tBid in comparison to control construct

Designing and development of a silencer vector based on second generation of RNA interference [RNAi] technique to study downregulation of steroid receptor RNA activator [SRA]

RNA interference [RNAi] is the most potent technique for gene silencing in eukaryotic cellular system at transcriptomic level. Genetic disorders and cancers are important targets for therapeutic development of this technique. In order to bypass the temporary dpwnregulation by siRNA, a new generation of shRNA named shRNAmir has developed. Silencing construct with structure similar to microRNA [shRNAmir], mimics a natural microRNA pathway inside the cell. Steroid receptor RNA activator [SRA] is one of the regulators of steroid receptor like ER. Prostate, uterus and breast tissue express a low level of SRA, there is an increase of expression during their tumorgenesis. So SRA may participate in tumorgenesis or proliferation of tumors. We used RNAi technique to silence expression of SRA. The SRA silencer was designed and constructed by Soe-PCR, then cloned into an expression vector pEGFPCl. Human breast cancer [MCF7] cells were transfected with silencer plasmid then the changes in the SRA expression estimated by Real-Time PCR at 24, 72 hours and after l0days. The results showed about 60% decrease in relative expression of SRA gene, after 72 hours and 10 days, which shows that shRNAmir-SRA could successfully knockdown the expression of target gene. It seems that the designed shRNAmir may be a suitable tool for a variety of applications because it could stably knockdown the expression of target gene

Immunogenicity of a new HIV-1 DNA Construct in a BALB/c mouse model

Cell mediated immunity, especially cytotoxic T cell responses against HIV-1 infection, plays a critical role in controlling viral replication and disease progression. DNA vaccine is a novel technology which is known to stimulate strong cellular immune responses. Many DNA vaccines have been tested for HIV infection but there is still no effective vaccine against this infection. Construction of a vaccine consisting of multiple conserved and immunogenic epitopes may increase vaccine efficacy. In the present study a DNA vaccine candidate constructed from HIV-1 P24-Nef was evaluated and cellular immune responses were assessed in murine BALB/c model. HIV-1 P24-Nef gene was cloned in PCDNA3.1 expression vector. Mice were immunized with DNA construct and IL-4 and IFN-gamma evaluation was per-formed using ELISPOT. Cytotoxicity response was evaluated with Granzyme B ELIS-POT assay and lymphocyte proliferation was evaluated with LTT assay. Analysis of immune responses showed that, compared to control groups, the candidate vaccine induced production of higher levels of both IL-4 and IFN-gamma [p<0.05]. Cytotoxicity and lymphocyte proliferation responses of mice vaccinated with the candidate vaccine were significantly increased compared to control groups [p<0.05]. HIV-1 P24-Nef DNA construct displayed strong immunogenicity in a murine model

[Cloning and expression of vascular endothelial growth factor receptor-1 in Escherichia coli and analysis of its growth inhibition effect on human umbilical vein endothelial cells]

Angiogenesis a process that results in neo-vascularization is an essential stage in growth of solid tumors and the formation of metastases. Vascular endothelial growth factor [VEGF] and its receptors, VEGFR1 [Flt-1] and VEGFR2 [KDR/Flk-1], are the major regulators for tumor angiogenesis. Recent studies showed that second domain of VEGFR-1is a key factor for VEGF/VEGFR-1 interaction. In this study, after RNA purification and cDNA synthesis, the second domain of VEGFR-1 [VEGFR-1-II] was amplified by PCR and cloned in T/A cloning vector. In order to increase the expression of the protein, we sub-cloned the gene into pET22b[+] and transformed the construct in Rosseta-gami 2, an efficient host for expression. The expression was induced by IPTG and confirmed by SDS-PAGE and Western Blotting. The recombinant protein was purified by IMAC column and the growth inhibition of human umbilical vein endothelial cells [HUVEC] was analyzed by the recombinant protein. The results of SDS-PAGE and Blotting confirmed the protein purification accuracy. The recombinant protein concentration was determined by Bradford protocol. The results showed that nearly 300 micri g/L VEGFR-1-II protein was produced. The function of this protein was confirmed by inhibition of HUVEC cells growth. Since this protein inhibited the angiogenesis in vitro it may be consider as an efficient anti-angiogenesis factor

Cloning and expression of recombinant camelid single-domain antibody in tobacco

Antibodies provide a suitable tool in fundamental research and their high affinity and specificity make them invaluable for diagnostic and therapeutic applications. A promising alternative to conventional antibodies are the heavy chain antibodies [VHH] of Camelidae having short length, high solubility and stability are preferred to other antibody derivatives. In this study, our goal was production of recombinant VHH antibody fragments [against cancer associated mucin, MUC1] in tobacco plants. The VHH gene cDNA was cloned in TA vector and then subcloned into a plant expression binary vector pBI 121. The VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. The presence of VHH gene in transformed plants was confirmed by PCR. Western blot analysis showed that the recombinant VHH protein was expressed in tobacco plant. ELISA results with MUC1 antigen confirmed that the biological activity and antigen-specific responses of the plant derived VHH protein compare favorably with that of the parent recombinant antibodies. This is the first report of production of camelied VHH antibody against tumor specific antigen from two-humped camel [Camelus bactrianus] in plants