Optimization and Validation of a Harmonized Protocol for Generating Therapeutic-Grade Dendritic Cells in a Randomized Phase II Clinical Trial, Using Two Varied Antigenic Sources.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

Mitochondrial ribosomal small subunit (MRPS) MRPS23 protein-protein interaction reveals phosphorylation by CDK11-p58 affecting cell proliferation and knockdown of MRPS23 sensitizes breast cancer cells to CDK1 inhibitors.

BACKGROUND: Post-translational modification of some mitoribosomal proteins has been found to regulate their functions. MRPS23 has been reported to be overexpressed in various cancers and has been predicted to be involved in increased cell proliferation. Furthermore, MRPS23 is a driver of luminal subtype breast cancer. However, its exact role and function in cancer remains unknown. METHODS AND RESULTS: Our previous study identified protein-protein interactions involving MRPS23 and CDK11A. In this study, we confirmed the interaction of MRPS23 with the p110 and p58 isoforms of CDK11A. Phosphoprotein enrichment studies and in vitro kinase assay using CDK11A/cyclin D3 followed by MALDI-ToF/ToF analysis confirmed the phosphorylation of MRPS23 at N-terminal serine 11 residue. Breast cancer cells expressing the MRPS23 (S11G) mutant showed increased cell proliferation, increased expression of PI3-AKT pathway proteins [p-AKT (Ser47), p-AKT (Thr308), p-PDK (Ser241) and p-GSK-3ß (Ser9)] and increased antiapoptotic pathway protein expression [Bcl-2, Bcl-xL, p-Bcl2 (Ser70) and MCL-1] when compared with the MRPS23 (S11A) mutant-overexpressing cells. This finding indicated the role of MRPS23 phosphorylation in the proliferation and survival of breast cancer cells. The correlation of inconsistent MRPS23 phosphoserine 11 protein expression with CDK11A in the breast cancer cells suggested phosphorylation by other kinases. In vitro kinase assay showed that CDK1 kinase also phosphorylated MRPS23 and that inhibition using CDK1 inhibitors lowered phospho-MRPS23 (Ser11) levels. Additionally, modulating the expression of MRPS23 altered the sensitivity of the cells to CDK1 inhibitors. CONCLUSION: In conclusion, phosphorylation of MRPS23 by mitotic kinases might potentially be involved in the proliferation of breast cancer cells. Furthermore, MRPS23 can be targeted for sensitizing the breast cancer cells to CDK1 inhibitors.

Combination of IDO1high and CCL19low expression in the tumor tissue reduces survival in HPV positive cervical cancer.

The over expression of Indoleamine 2, 3-Dioxygenase (IDO1), an immune checkpoint inhibitor, is well known in cervical cancer. However, its association with chemokine signals promoting cellular infiltration in the cervical tumor microenvironment, is unknown. In the current study, we evaluated the expression and enzymatic activity of IDO1. We also profiled the expression of chemokine ligand-receptors- CCR4-CCL22, CXCR3-CXCL10, CXCR4-CXCL12, and CCR7-CCL19 using immunohistochemistry (IHC), and studied their association with IDO1, statistically. After getting an informed consent, punch biopsy samples were obtained from 105 patients diagnosed with cervical cancer. HPV typing by Sanger sequencing, realtime PCR for quantifying IDO1 mRNA expression, HPLC for determining the K/T ratio and IHC for all the above chemokine receptor-ligand pairs along with IDO1 were performed. We found a significant increase in the expression of IDO1 and K/T levels in early and locally advanced stages when compared to Stage IV disease. Among the chemokine ligand -receptor pairs profiled, we found that high CCL19 marker expression was a good prognostic indicator of patients' disease-free (p = 0.013) and overall survival (p = 0.043). Although we could not identify IDO1 as an independent prognostic factor, we found that high levels of IDO1 expression may further reduce survival outcomes in patients with low CCL19 expression. This could be vital for designing immuno therapeutic interventions targeting IDO1.

Dendritic cells matured with recombinant human sperm associated antigen 9 (rhSPAG9) induce CD4+, CD8+ T cells and activate NK cells: a potential candidate molecule for immunotherapy in cervical cancer.

BACKGROUND: Dendritic cell (DC)-based immunotherapy is capable of activating the immune system and in particular tumor-specific cytotoxic T lymphocytes (CTLs) to eradicate the tumor. However, major limitations are the availability of autologous tumor cells as antigenic source and the selection of antigen that may have potential to activate both CD4+ and CD8+ T cells in immune-specific manner. Recently, we reported the expression of sperm associated antigen 9 (SPAG9) that is associated with various types of malignancies including cervical cancer. We examined the recombinant human SPAG9 (rhSPAG9) as an antigenic source for generating efficient DCs to stimulate CD4+ and CD8+ T cell responses for future DCs-based vaccine trials in cervical cancer patients. METHODS: Human monocytes derived DCs were pulsed with different concentrations (250 ng/ml to 1000 ng/ml) of recombinant human SPAG9 (rhSPAG9) and evaluated for their phenotypic and functional ability. The efficacy of DCs primed with 750 ng/ml of rhSPAG9 (SPDCs) was compared with DCs primed with autologous tumor lysates (TLDCs), to induce CD4+, CD8+ T cells and activating NK cells. In addition, we investigated the effect of the chemotherapeutic drug cisplatin on phenotypic and functional potential of SPDCs. RESULTS: Phenotypic and functional characterization of DCs pulsed with 750 ng/ml rhSPAG9 was found to be optimal and effective for priming DCs. SPDCs were also capable of stimulating allogeneic T cells similar to TLDCs. SPDCs showed a statistically insignificant increase in the expression of maturation marker CD83 and migration towards CCL19 and CCL21 compared with TLDCs (CD83; P = 0.4; migration; P = 0.2). In contrast, although TLDCs showed better proliferation and secretion of Th1 cytokines (IL12p40, IL12p70 and IFNγ) compared to SPDCs, this difference was not statistically significant (IL12p40, P = 0.06). Further we also observed that clinical dose of cisplatin (200 µM) treated SPDCs were able to stimulate the proliferation of cytotoxic T lymphocytes without increasing the FOXP3+ Tregs in autologous co-cultures. CONCLUSIONS: In summary, in order to overcome the limitation of the availability of autologous tumor cells as antigenic sources, our present strategy provides an insight to consider rhSPAG9 as a strong immunogen for DC-based immunotherapy for cervical cancer trials and warrants further studies. This is the first report to suggest that rhSPAG9 is an effective antigen for pulsing DCs that are capable of eliciting a potent Th1 response which, in turn, may help in decreasing the tumor burden when used along with a cisplatin based combinatorial regimen for therapeutic intervention.

Autologous cervical tumor lysate pulsed dendritic cell stimulation followed by cisplatin treatment abrogates FOXP3+ cells in vitro.

OBJECTIVE: Dendritic cells (DCs) are administered as immunotherapeutic adjuvants after the completion of standard treatment in most settings. However, our Phase I trial indicated that one patient out of four, who received autologous tumor lysate-pulsed dendritic cell (TLDC) also received cisplatin chemotherapy and experienced complete regression of her lung lesion, continuing to be disease free till date. Hence, the objective of our current study is to evaluate the sustenance or augmentation of immune responses when autologous human papillomavirus positive cervical tumor lysate pulsed DC- are combined with cisplatin, using co-culture assays in vitro. METHODS: Before treatment, peripheral blood and punch biopsy samples were collected from 23 cervical cancer patients after obtaining an informed consent. DC functionality was confirmed through phenotypic and functional assays using autologous peripheral blood mononuclear cells as responders. For cisplatin experiments, the drug was added at 150, 200 (clinical dose equivalent), and 400 µM concentrations to DCs alone or DC-T cell co-cultures. Phenotypic assessment and functional characterization of DCs was done using flow cytometry. Cytokine enzyme-linked immunosorbent assay and interferon (IFN)-γ enzyme-linked immune absorbent spot assays were also performed. RESULTS: The functionality of TLDCs was not compromised upon cisplatin treatment in vitro even at the highest (400 µM) concentration. Even though cisplatin treatment reduced the secretion of IFN-γ and interleukin (IL)-12p40 in co-cultures stimulated with TLDCs, this effect was not significant (p>0.05). A doubling of IFN-γ secretion following cisplatin treatment was observed in at least one of three independent experiments. Additional experiments showed a reduction in both FOXP3+ regulatory T cells and IL-10 levels. CONCLUSION: Our results provide evidence that cisplatin treatment may be given after autologous TLDC administration to maintain or improve a productive anti-tumor response in vaccinated patients.

Identification of novel dysregulated circular RNAs in early-stage breast cancer.

Breast cancer is a major cause of cancer-related death in women worldwide. Non-coding RNAs are a potential resource to be used as an early diagnostic biomarker for breast cancer. Circular RNAs are a recently identified group of non-coding RNA with a significant role in disease development with potential utility in diagnosis/prognosis in cancer. In this study, we identified 26 differentially expressed circular RNAs associated with early-stage breast cancer. RNA sequencing and two circRNA detection tools (find_circ and DCC) were used to understand the circRNA expression signature in breast cancer. We identified hsa_circ_0006743 (circJMJD1C) and hsa_circ_0002496 (circAPPBP1) to be significantly up-regulated in early-stage breast cancer tissues. Co-expression analysis identified four pairs of circRNA-miRNA (hsa_circ_0023990 : hsa-miR-548b-3p, hsa_circ_0016601 : hsa_miR-1246, hsa_circ_0001946 : hsa-miR-1299 and hsa_circ_0000117:hsa-miR-502-5p) having potential interaction. The miRNA target prediction and network analysis revealed mRNA possibly regulated by circRNAs. We have thus identified circRNAs of diagnostic implications in breast cancer and also observed circRNA-miRNA interaction which could be involved in breast cancer development.

Production and characterization of monoclonal antibodies against recombinant extracellular domain of CD99.

BACKGROUND AND OBJECTIVE: CD99/MIC2 gene product is a heavily glycosylated transmembrane protein which plays a major role in homotypic cell adhesion, apoptosis of double positive T cells and vesicular protein trafficking. It is over expressed in various cancers and has been considered as an ideal therapeutic target. The present study focused at developing monoclonal antibodies against the extracellular domain (ECD) of CD99 using hybridoma technology. MATERIALS AND METHODS: In order to generate monoclonal antibodies, the recombinant ECD of CD99 was used for immunizing the mice. Resulting hybridomas were screened through indirect ELISA. Clones which gave high absorbance values were sub cloned by limiting dilution followed by isotype determination, IP, WB and FACS. The monoclonal antibody 547F2 4F12 was purified from culture supernatant using FPLC and further screened using IF. Finally, the antibodies were validated for specificity using siRNA knock-down. RESULTS: We were able to establish stable hybridoma clones secreting CD99 antibodies. The antibodies reacted with both the recombinant ECD as well as the wild type CD99 and their isotype's were determined as IgM. CONCLUSION: Based on these results, we propose that the purified monoclonal antibody 547F2 4F12 could be possibly used for targeting tumors which over express CD99.

In silico and in vitro screening of small molecule Inhibitors against SYT-SSX1 fusion protein in synovial sarcoma.

Synovial sarcoma (SS) is characterized by a tumour specific chromosomal translocation t(X;18) (p11;q11) which results in the formation of SYT-SSX1 fusion protein. This fusion protein represents a clear therapeutic target and molecules specifically targeting SYT-SSX1 fusion protein are currently not available. In this study, SYT-SSX1 fusion protein sequence was retrieved from Uniprot and 3D structure was generated using I-TASSER modeling program. A structure based computational screening approach has been employed using Glide docking software to identify potential SYT-SSX1 small molecule inhibitors that bind to the junction region of the fusion protein. The obtained inhibitors were further filtered based on the docking score and ADME/T properties. Ten best fit compounds were chosen for in vitro studies. The anti-proliferative activities of these 10 compounds were screened in Yamato, ASKA (carries SYT-SSX1 fusion protein) and other sarcoma cell lines such as A673, 143B to understand the specificity of inhibition of the chosen compounds. The in vitro activity was compared against HEK293 cell lines. The compound 5-fluoro-3-(1-phenyl-1H-tetraazol-5-yl)-1H-indole (FPTI) was found to be selectively cytotoxic in synovial sarcoma cell lines (Yamato and ASKA) and this compound also showed insignificant anti proliferative activity on other cell lines. Further, target gene expression study confirmed that FPTI treatment down-regulated SYT-SSX1 and modulated its downstream target genes. Cell cycle analysis revealed the involvement of an apoptotic mechanism of cell death. Further experimental validations may elucidate the therapeutic potentials of FPTI against SYT-SSX1 fusion protein.

Immunotherapy for cervical cancer: Can it do another lung cancer?

Cervical cancer, although preventable, is still the second most common cancer among women worldwide. In developing countries like India, where screening for cervical cancer is virtually absent, most women seek treatment only at advanced stages of the disease. Although standard treatment is curative in more than 90% of women during the early stages, for stage IIIb and above this rate drops to 50% or less. Hence, novel therapeutic adjuvants are required to improve survival at advanced stages. Lung cancer has shown the way forward with the use of Immunotherapeutic interventions as standard line of treatment in advanced stages. In this review, we provide an overview of mechanisms of immune evasion, strategies that can be employed to boost the immune system in order to improve the overall survival of the patients and summarize briefly the clinical trials that have been completed or that are underway to bring therapeutic vaccines for cervical cancer to the clinics.

A polypeptide from the junction region sequence of EWS-FLI1 inhibits Ewing's sarcoma cells, interacts with the EWS-FLI1 and partner proteins.

The EWS-FLI1 chimeric protein uniquely expressed in Ewing's sarcoma has an obligate role in its aetiology. In our previous report we showed that ectopic expression of the DNA sequences form the junction region (a.a 251-280) can inhibit Ewing's sarcoma cell growth. In the present report, we introduced a peptide (TAT/NLS/EWS-PEP) comprising of thirty amino acids spanning the junction in conjunction with HIV-1-trans-activating (TAT) and nuclear localization signal sequence (NLS). Peptide uptake and localization studies revealed presence of peptide in ~99% of transduced cells and in the nucleus. Peptide transfection induced cytotoxicity relative to untreated and TAT-NLS peptide treated Ewing's sarcoma cells. The peptide inhibited clonogenicity, cell cycle, bromo-deoxy uridine (BrdU) uptake and invasion capacity of treated cells. The treatment also affected epithelial to mesenchymal transition (EMT) markers and EWS-FLI1 target gene expression levels. Co-immunoprecipitation experiments involving ectopically expressed full-length EWS-FLI1 protein and the peptide revealed an interaction. Additionally, we found that peptide interaction also occurs with the protein-GGAA microsatellite sequences complex known to contain EWS-FLI1. Further, in the pull-down assay, the peptide was found to interact with proteins known to potentially interact with EWS-FLI1. Based on these results we conclude that peptide could be applied in targeting EWS-FLI1 protein.

Dendritic cells primed with HPV positive cervical tumor lysate are superior to unprimed DCs in migratory capacity and induce a potent Th1 response.

In this study, we assessed the efficacy of tumor lysate primed and unprimed monocyte derived mature dendritic cells (DCs) to trigger an effective anti-tumor immune response in cervical cancer patients who tested positive for human papilloma virus (HPV) DNA. Lysate primed and unprimed DCs were assessed for the expression of CD80, CD86, CD40, HLADR and CD83. The ability of DCs to migrate in response to the chemokines CCL19 and 21 as well as their ability to secrete IL12p40 was investigated. Mixed lymphocyte proliferation assays were used to assess DC stimulatory capacity and their ability to generate a Th1 response. Our results showed no difference in phenotypic expression between primed and unprimed DCs but both had significantly increased expression of the activation marker CD83 when compared to immature DCs. Importantly, the primed DCs showed significant (P value=0.03) IL-12p40 secretion and a superior migratory capacity towards CC19 and CCL21 (P value=0.04) compared to unprimed DCs even after cytokine withdrawal. Primed DCs showed superior stimulation of T cell proliferation (allogeneic and autologous) and secretion of IFN gamma (IFN-γ) than the unprimed DCs. Hence whole tumor lysate primed mature DCs could be potent immunotherapeutic adjuvants to standard treatment for cervical cancer.

Development and clinical evaluation of dendritic cell vaccines for HPV related cervical cancer--a feasibility study.

Human papillomavirus infection (HPV) and HPV related immune perturbation play important roles in the development of cervical cancer. Since mature dendritic cells (DCs) are potent antigen-presenting cells (APC), they could be primed by HPV antigens against cervical cancers. In this study we were able to generate, maintain and characterize, both phenotypically and functionally, patient specific dendritic cells in vitro. A randomized Phase I trial with three arms--saline control (arm I), unprimed mature DC (arm II) and autologous tumor lysate primed mature DC (arm III) and fourteen patients was conducted. According to WHO criteria, grade 0 or grade one toxicity was observed in three patients. One patient who received tumor lysate primed dendritic cells and later cis-platin chemotherapy showed a complete clinical response of her large metastatic disease and remained disease free for more than 72 months. Our findings indicate that DC vaccines hold promise as adjuvants for cervical cancer treatment and further studies to improve their efficacy need to be conducted.