Establishment of a murine model of breast cancer expressing human epidermal growth factor receptor 2 (4T1-HER2).

AIM: Although people with HER2-positive breast cancer benefit from approved HER2-targeted therapy, acquiring resistance to the therapies occurs. Animal models can play a part in gaining a deep understanding of such a process and addressing questions concerning developing and improving immunotherapy approaches. MATERIALS AND METHODS: To develop such a model, we transfected murine 4T1 cells with the pCMV6-Neo-HER2 construct and evaluated HER2 expression and its effects on the established cell line behavior in vitro and in vivo. RESULTS: Data illustrated that human HER2 protein was expressed on isolated 4T1-HER2 clones in vitro and in vivo. Except for proliferation over 48 hours, such expression did not change 4T1-HER2 characteristics compared to 4T1 in vitro. Notwithstanding the reduction in proliferation, the rate of tumorigenicity was 90% in challenged mice and Herceptin therapy significantly decreased tumors' growth and metastasis compared to the control group. CONCLUSION: We describe a murine model for HER2-positive breast cancer not only helping shed light on the mechanisms by which the tumor evades antitumor immunity but also playing a key role in making breast cancer more sensitive to novel immunotherapy modalities.

Clinico-Pathological and Prognostic Significance of a Combination of Tumor Biomarkers in Iranian Patients With Breast Cancer.

PURPOSE: Breast cancer is one of the most common cancers in the world. It is a multifaceted malignancy with different histopathological and biological features. Molecular biomarkers play an essential role in accurate diagnosis, classification, prognosis, prediction of treatment response, and cancer surveillance. This study investigated the clinico-pathological and prognostic significance of HER3 and ROR1 in breast cancer samples. METHODS: Tissue microarrays (TMA) were constructed using tissue blocks of 444 Iranian breast cancer patients diagnosed with breast cancer. Overall survival (OS) and disease-free survival (DFS) were assessed after 5 years follow-up. TMA slides were stained with monoclonal antibodies against ROR1, HER3, ER, PR, Ki67, P53, HER2 and CK5/6 using IHC and correlation between the investigated tumor markers and the clinico-pathological parameters of patients were analyzed. RESULTS: Our results showed a significant correlation between ROR1 and ER, PR, HER3, and CK5/6 expression. Additionally, there was a significant correlation between HER3 and ER, PR, HER2, and Ki67 expression. Ki67 was also correlated with HER2 and P53 expression. HER3 expression was significantly correlated with tumor stage, lymph node metastasis, perineural invasion, and multifocal tumors. Furthermore, ROR1 expression was significantly associated with tumor metastasis, lympho-vascular invasion, and perineural invasion. While HER2-HER3 coexpression was significantly associated with poor OS, HER3-ROR1 coexpression was associated with lymph node invasion, lymph node metastasis, and distant metastasis. CONCLUSION: ROR1 and HER3 displayed significant association with different clinic-pathological features and in addition to the other tumor biomarkers could be considered as diagnostic and prognostic biomarkers in breast cancer patients.

Comparison of different transient gene expression systems for the production of a new humanized anti-HER2 monoclonal antibody (Hersintuzumab).

BACKGROUND: Producing therapeutic proteins can be done quickly and on a large scale through Transient Gene Expression (TGE). Chinese hamster ovary (CHO) cell lines are commonly used to achieve this. Although there are few comparative studies, TGE has been observed in suspension-adapted CHO cells. OBJECTIVES: We tested TGE's effectiveness in DG-44, CHO-S, and ExpiCHO-S cell lines with four transfection reagents. METHODS: A design of experiments (DoE) was followed to optimize transfection using a recombinant monoclonal antibody (mAb) construct. To evaluate the efficacy, flow cytometry and ELISA were used. Feeding strategies and temperature shifts were implemented to enhance transfection effectiveness. The quality of the mAb was assessed through ELISA, SDS-PAGE, and proliferation inhibition assays. RESULTS: We adapted all cell lines to grow in suspension using a serum-free medium. Our findings from flow cytometry and ELISA tests indicate that PEI and Pmax reagents had a higher rate of transfection and mAb production than the ExpiCHO commercial transfection reagent. While DG-44 cells had better transfection efficiency than CHO-S and ExpiCHO-S, there was no significant difference between CHO-S and ExpiCHO-S. Our TGE system was more productive at 32 °C than at 37 °C. In the optimized TGE of Pmax-based transfection in DG-44 at 37 and 32 °C, the production level of mAb was more than half of the amount of the commercial ExpiCHO-S expression system. Still, the number of transfected cells was three times higher, making it more efficient. The purified mAb from all transfected cell lines had similar structural and functional properties under different conditions. CONCLUSION: Our research shows that using Pmax and DG-44 cells in the TGE system is a cost-effective and efficient way to produce humanized monoclonal antibodies. We discovered that this method outperforms the ExpiCHO-S kit.

Discovery of a novel marker for human granulocytes and tissue macrophages: RTL1 revisited.

Here, retrotransposon-like 1 (RTL1) is introduced as a marker for circulating and tissue neutrophils, tissue macrophages, and tumor-associated macrophages (TAM) and neutrophils (TAN). Anti-RTL1 polyclonal and monoclonal antibodies were produced, and their reactivity was examined by Western blotting (WB), ELISA, and immunostaining of human normal and cancer tissues. The reactivity of the anti-RTL1 antibodies with peripheral blood leukocytes and a panel of hematopoietic cell lines was examined. The generated antibodies specifically detected RTL1 in the WB of the placenta and U937 cells. The polyclonal antibody showed excellent reactivity with tissue-resident macrophages, Hofbauer cells, alveolar and splenic macrophages, Kupffer cells, and inflammatory cells in the tonsil, appendix, and gallbladder. In vitro GM-CSF-differentiated macrophages also showed a high level of intracellular RTL1 expression. TAM and TAN also showed excellent reactivity with this antibody. Almost all circulating granulocytes but not lymphocytes or monocytes expressed RTL1 at their surface. Serial sections of the appendix stained with CD15 and RTL1 and placenta stained with CD68 and RTL1 showed a considerable overlap in RTL1 expression in CD15+ granulocytes and CD68+ macrophages. A small percentage of myelomonocytic cell lines was positive for surface RTL1, while promyelocytic, monocytic, megaloblastic, and lymphoblastic cell lines were negative. Endothelial cells of normal and cancer tissues highly expressed RTL1. RTL1 could be considered a new marker for different normal tissue macrophages, TAM, circulating and tissue neutrophils, and TAN.

Evaluation of the anti-tumor effects of an anti-Human Epidermal growth factor receptor 2 (HER2) monoclonal antibody in combination with CD11b+/Gr-1+ myeloid cells depletion using a recombinant peptibody in 4 T1-HER2 tumor model.

INTRODUCTION: Clinical efficacy of Human Epidermal growth factor Receptor 2 (HER2) targeted strategies is limited due to impaired anti-tumor responses negatively regulated by immunosuppressive cells. We thus, investigated the inhibitory effects of an anti-HER2 monoclonal antibody (1 T0 mAb) in combination with CD11b+/Gr-1+ myeloid cells depletion in 4 T1-HER2 tumor model. METHODS: BALB/c mice were challenged with human HER2-expressing 4 T1 murine breast cancer cell line. A week post tumor challenge, each mouse received 50 µg of a myeloid cells specific peptibody every other day, or 10 mg/kg of 1 T0 mAb two times a week, and their combination for two weeks. The treatments effect on tumor growth was measured by calculating tumor size. Also, the frequencies of CD11b+/Gr-1+ cells and T lymphocytes were measured by flow cytometry. RESULTS: Peptibody treated mice indicated tumor regression and 40 % of the mice eradicated their primary tumors. The peptibody was capable to deplete notably splenic CD11b+/Gr-1+ cells as well as intratumoral CD11b+/Gr-1+ cells (P < 0.0001) and led to an increased number of tumor infiltrating CD8+ T cells (3.3 folds) and also that of resident tumor draining lymph nodes (TDLNs) (3 folds). Combination of peptibody and 1 T0 mAb resulted in enhanced expansion of tumor infiltrating CD4 + and CD8+ T cells which was associated with tumor eradication in 60 % of the mice. CONCLUSIONS: Peptibody is able to deplete CD11b+/Gr-1+ cells and increase anti-tumoral effects of the 1 T0 mAb in tumor eradication. Thus, this myeloid population have critical roles in development of tumors and their depletion is associated with induction of anti-tumoral responses.

Novel Monoclonal Antibodies Specific for Human Ki67 and P53 Tumor Markers in Breast Cancer Tissue Samples.

Background: Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test. Objective: To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes. Methods: Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs. Results: Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer. Conclusion: The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.

Potential role of EBV and Toll-like receptor 9 ligand in patients with systemic lupus erythematosus.

SLE is a multisystem autoimmune disease characterized by multiple immunological abnormalities including production of autoantibodies. While the etiology of SLE is largely unknown, it is generally accepted that both genetic and environmental factors contribute to disease risk and immune dysregulation. Production of IFN-α is important for protecting the host against infections; however, over stimulation of innate immune pathways can induce autoimmune disease. Environmental factors, particularly Epstein-Barr virus (EBV), have been proposed to play an important role in SLE disease. Improper engagement of Toll-like receptor (TLR) pathways by endogenous or exogenous ligands may lead to the initiation of autoimmune responses and tissue injury. EBV is shown to be a potent stimulant of IFN-α by TLR signaling cascades. Given the highlighted role of IFN-α in SLE pathogenesis and potential role of EBV infection in this disease, the present study is aimed at exploring the in vitro effects of EBV infection and CPG (either alone or in combination) on IFN-α. We also examined the expression level of CD20 and BDCA-4 and CD123 in PBMCs in 32 SLE patients and 32 healthy controls. Our results showed PBMCs treated with CPG-induced higher levels of IFN-α and TLR-9 gene expression fold change compared to cells treated with either EBV or EBV-CPG. Moreover, PBMCs treated with CPG produced significantly higher IFN-α concentration in supernatant compared to cells treated with EBV but not EBV-CPG. Our results further highlight the potential role of EBV infection and TLRs in SLE patients although more studies are warranted to ascertain the global imprint that EBV infection can have on immune signature in patients with SLE.

A Novel Fc-Engineered Anti-HER2 Bispecific Antibody With Enhanced Antitumor Activity.

Human epidermal growth factor receptor 2 (HER2) overexpression has been demonstrated in a variety of cancers. Targeted therapy with anti-HER2 monoclonal antibodies (mAbs) has been approved as a therapeutic modality. Despite the efficacy of mAbs in tumor treatment, many patients do not benefit from this therapeutic platform. Fragment crystallizable (Fc) engineering is a common approach to improve the efficacy of therapeutic mAbs. Five Fc-engineered mAbs have so far been approved by FDA. We have recently developed an anti-HER2 bispecific mAb, BiHT, constructed from variable domains of trastuzumab, and our novel humanized anti-HER2 mAb, hersintuzumab. BiHT displayed promising antitumor activity as potently as the combination of the parental mAbs. Here, we aimed to modify the Fc of BiHT to improve its therapeutic efficacy. The Fc-engineered BiHT (MBiHT) bound to recombinant HER2 and its subdomains with an affinity similar to BiHT. It also recognized native HER2 on different cell lines, inhibited their proliferation, downregulated HER2 expression, and suppressed downstream signaling pathways similar to BiHT. Compared with BiHT, MBiHT displayed enhanced antibody-dependent cellular cytotoxicity activity against various tumor cell lines. It also inhibited the growth of ovarian xenograft tumors in nude mice more potently than BiHT. Our findings suggest that MBiHT could be a potent therapeutic candidate for the treatment of HER2-overexpressing cancer types.

Potent anti-tumor immune response and tumor growth inhibition induced by HER2 subdomain fusion protein in a mouse tumor model.

PURPOSE: Several approaches have so far been employed to establish anti-tumor immunity by targeting HER2 protein. Active immunization with recombinant HER2 subdomains has previously been demonstrated to induce potent immune response and tumor growth inhibition. In the present study, we investigated the immunogenicity and tumor inhibitory effect of a fusion protein consisting of human HER2 extracellular subdomain (ECD-DI + II) together with T-helper cell epitopes of Tetanus toxin (p2 and p30). METHODS: BALB/c mice were immunized with two recombinant proteins (DI + II and p2p30-DI + II) emulsified in 4 different adjuvants. Anti-DI + II antibody response, cytokine profile, frequency of splenic CD25+FOXP3+ regulatory T cells (Tregs) and CD8+CD107a+ cytotoxic T lymphocytes (CTLs) were assessed in the immunized mice. To assess the anti-tumor effect, the immunized mice were subcutaneously challenged with HER2-overexpressing tumor cells and the tumor growth was determined. RESULTS: Both recombinant proteins were able to induce comparable levels of ECD-DI + II-specific antibodies. Immunization with p2p30-DI + II resulted in a significant increase in the level of Interferon-gamma (IFN-γ) secretion compared to DI + II protein and significantly higher frequency of CTLs and lower frequency of Tregs. The number of mice that remained tumor-free until day 120 was significantly higher in p2p30-DI + II vaccinated groups. CONCLUSIONS: Our data suggest that the p2p30-DI + II fusion protein together with CpG adjuvant induces more potent anti-tumor immune responses in a mouse tumor model. Accordingly, this formulation might be considered as a potential immunotherapeutic approach in HER2+ cancers.

Preclinical Assessment of Immunogenicity and Protectivity of Novel ROR1 Fusion Proteins in a Mouse Tumor Model.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a new tumor associated antigen (TAA) which is overexpressed in several hematopoietic and solid malignancies. The present study aimed to produce and evaluate different fusion proteins of mouse ROR1 (mROR1) to enhance immunogenicity and protective efficacy of ROR1. Four ROR1 fusion proteins composed of extracellular region of mROR1, immunogenic fragments of TT as well as Fc region of mouse IgG2a were produced and employed to immunize Balb/C mice. Humoral and cellular immune responses and anti-tumor effects of these fusion proteins were evaluated using two different syngeneic murine ROR1+ tumor models. ROR1-specific antibodies were induced in all groups of mice. The levels of IFN-γ, IL-17 and IL-22 cytokines in culture supernatants of stimulated splenocytes were increased in all groups of immunized mice, particularly mice immunized with TT-mROR1-Fc fusion proteins. The frequency of ROR1-specific CTLs was higher in mice immunized with TT-mROR1-Fc fusion proteins. Finally, results of tumor challenge in immunized mice showed that immunization with TT-mROR1-Fc fusion proteins completely inhibited ROR1+ tumor cells growth in two different syngeneic tumor models until day 120 post tumor challenge. Our preclinical findings, for the first time, showed that our fusion proteins could be considered as a potential candidate vaccine for active immunotherapy of ROR1-expressing malignancies.

Myeloid-derived suppressor cells (MDSCs) depletion by cabozantinib improves the efficacy of anti-HER2 antibody-based immunotherapy in a 4T1-HER2 murine breast cancer model.

BACKGROUND: Clinical trials using Cabozantinib have shown promising results in metastatic breast cancer. This efficacy mainly results from removing and/or polarization of tumor-promoting myeloid cells. Nevertheless, whether such myeloid-derived suppressor cells (MDSCs) depletion can be used to improve the efficacy of anti-HER2 antibodies in early breast cancer has not been defined yet. METHODS: BALB/c mice were inoculated with 4T1 and 4T1-HER2 murine tumor cell lines, and after 7 days, the mice were divided into different groups. Cabozantinib was orally administrated for 15 consecutive days, and anti-HER2 monoclonal antibody (mAb) 1 T0 was intraperitoneally injected twice a week. Tumor size was measured every other day. RESULTS: Our findings indicated that Cabozantinib combined with anti-HER2 mAb dramatically reduced tumor growth and increased tumor rejection (p = 0.0001). Flow cytometry analysis showed MDSC population decreased in TME, lymph nodes, and spleens by roughly 20%, 0.8%, and 35%, respectively. Myeloid suppressive phenotype was altered through inhibition of the expression of immunosuppressive factor Arg-1. Cytokine profiling of different groups indicated that the level of INF-γ was approximately two times higher than that in the control group, and IL-17 increased compared to the control group. However, IL-4 level was significantly reduced in the groups treated with Cabozantinib. These could bring about a 10% increase in CD8+ infiltration into the tumor bed and activation of tumor-draining lymph nodes and splenic T-lymphocytes. CONCLUSION: Collectively, our data provide pre-clinical evidence for using Cabozantinib to reshape the primary TME, which can enhance the effectiveness of anti-HER2 mAb immunotherapy in primary breast cancer.

Immunization with placenta-specific 1 (plac1) induces potent anti-tumor responses and prolongs survival in a mouse model of melanoma.

PURPOSE: Melanoma is a malignant and metastatic form of skin cancer, which is not diagnosed in early stages of the disease. Nowadays, immunotherapy is changing the treatment landscape for metastatic melanoma. Placenta-specific1 (PLAC1) is a cancer-testis-placenta (CTP) antigen with differential expression in melanoma tissues. Here, we evaluated the potential of plac1 to induce anti-cancer immune responses as well as to prevent cancer development in a mouse model of melanoma. METHODS: Two proteins containing full extracellular domain (ED) of mouse plac1+KDEL3 and full ED of mouse plac1+ tetanus toxin P2 and P30+ pan DR epitope (PADRE) â€‹+ â€‹KDEL3 were produced and injected in mice to evaluate their capacity to induce anti-cancer immune responses as well as their potential to prevent melanoma development. Induction of plac1-specific humoral and cellular responses as well as tumor-associated parameters were tested in a series of 36 mice. RESULTS: Sera of mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 contained antibodies able to react with surface plac1 in B16F10 â€‹cells. Both proteins induced proliferative cellular immune responses against B16F10 â€‹cells and plac1-specific cytotoxic T cells (CTL) and CD107a â€‹+ â€‹CTL responses, which was higher in mice immunized with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3. Splenocytes of mice vaccinated with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 exerted a significant cytotoxicity against B16F10 â€‹cells. Vaccination with ED â€‹+ â€‹P2P30+PADRE â€‹+ â€‹KDEL3 significantly delayed B16F10-induced tumor onset, reduced tumor growth, and increased survival. Tumors induced by B16F10 expressed plac1 in vivo. CONCLUSION: Our results pave the way for development of effective melanoma preventive vaccine in humans, although further studies are needed.

Generation and Characterization of Novel Diagnostic Mouse Monoclonal Antibodies Against Human Estrogen Receptor Alpha and Progesterone Receptor.

BACKGROUND: Estrogen and progesterone regulate the growth and development of several human cells and tissues. Their corresponding receptors (ER and PR) are important diagnostic and prognostic indicators for cancers of the breast and reproductive organs. Immunohistochemical analysis of ER and PR is the current standard method for evaluating the expression of these receptors in different cancers. Nonetheless, there is a significant lack of reproducibility of IHC results in laboratories worldwide, necessitating to develop more sensitive and specific antibodies for ER and PR IHC staining. METHODS: ER and PR-specific monoclonal antibodies (MAbs) were generated by immunizing mice with synthetic peptides from ERα and PR. The isotypes and affinity constants of the selected MAbs were determined, and their specificities were assessed by peptide-specific ELISA, IHC, Western-blot analysis, and flow cytometry. In addition, the reactivity of generated MAbs was compared with that of the commercially-available anti-ER and anti-PR antibodies in IHC using normal and cancerous tissue sections. Moreover, 200 breast cancer tissue samples were stained using the newly generated MAbs along with commercial antibodies by IHC, and the sensitivity, specificity and accuracy of our MAbs were evaluated. RESULTS: Among different MAbs generated in this study, two anti-ER and one anti-PR MAbs specifically detected the target antigens in normal and cancerous tissues in IHC. Further analyses confirmed the specificity of the MAbs in Western blotting and flow cytometry using a panel of ER and PR positive cell lines. The sensitivity, specificity and accuracy calculated for clone 1B9 (anti-ER) were 92.3%, 94.8% and 93%, and for clone 3D6 (anti-PR) were 93.0%, 94.3% and 93.5%, respectively. CONCLUSION: Our novel anti-ER and PR MAbs could be considered as suitable tools for diagnostic and research purposes.

Cloning, expression and characterization of a peptibody to deplete myeloid derived suppressor cells in a murine mammary carcinoma model.

BACKGROUND: Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we expressed and characterized a peptibody which had previously been defined by phage display technique capable of recognizing and depleting murine MDSCs. MATERIALS AND METHODS: Using splicing by overlap extension (SOE) PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. RESULTS: The purified peptibody appeared as a 70 KDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P < 0.05). CONCLUSION: In this study, a peptibody capable of depleting intratumoral MDSCs, was successfully expressed and purified. Our results imply that it could be considered as a potential tool for research on cancer immunotherapy.

Optimization of Expression and Purification of Recombinant Mouse plac1.

Background: Placenta-specific 1 (PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression in a wide range of cancer cells from different histological origins. The production of recombinant human PLAC1 has already been optimized; however, no study has been reported so far on the production and purification of mouse plac1. In this study, mouse plac1 expression and purification was optimized in a prokaryotic system and the effects of the generated proteins on inducing humoral responses in mice were investigated. Methods: A fusion protein containing full extracellular domain of mouse plac1, immunostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main plac1), and the same fragment without immunostimulatory peptides (control plac1) was produced. To optimize production and purification steps, different parameters including bacterial strain, cultivation temperature, cultivation time, IPTG concentration, culture medium, and also different buffers for purification of the recombinant proteins were tested. After confirming the identity of recombinant plac1 proteins with Western Blotting (WB) and ELISA assays, these proteins were subcutaneously injected in mice with Freund's adjuvant and the anti-plac1 antibody response was detected by ELISA. Results: The optimal expression level of main and control plac1 was obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 mM IPTG after 24 hr of induction at 15°C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1 antibody titer was significantly higher in sera of mice immunized with main compared to control plac1. Conclusion: In this study, an optimized protocol for production and purification of mouse plac1 was reported and it was shown that insertion of immunostimulatory peptides in gene construct could efficiently enhance humoral immune responses against mouse plac1, which could potentially augment cellular immune responses against plac1 leading to more effective anti-cancer responses.

Identification of immunodominant epitopes on nucleocapsid and spike proteins of the SARS-CoV-2 in Iranian COVID-19 patients.

Given the emergence of SARS-CoV-2 virus as a life-threatening pandemic, identification of immunodominant epitopes of the viral structural proteins, particularly the nucleocapsid (NP) protein and receptor-binding domain (RBD) of spike protein, is important to determine targets for immunotherapy and diagnosis. In this study, epitope screening was performed using a panel of overlapping peptides spanning the entire sequences of the RBD and NP proteins of SARS-CoV-2 in the sera from 66 COVID-19 patients and 23 healthy subjects by enzyme-linked immunosorbent assay (ELISA). Our results showed that while reactivity of patients' sera with reduced recombinant RBD protein was significantly lower than the native form of RBD (P < 0.001), no significant differences were observed for reactivity of patients' sera with reduced and non-reduced NP protein. Pepscan analysis revealed weak to moderate reactivity towards different RBD peptide pools, which was more focused on peptides encompassing amino acids (aa) 181-223 of RBD. NP peptides, however, displayed strong reactivity with a single peptide covering aa 151-170. These findings were confirmed by peptide depletion experiments using both ELISA and western blotting. Altogether, our data suggest involvement of mostly conformational disulfide bond-dependent immunodominant epitopes in RBD-specific antibody response, while the IgG response to NP is dominated by linear epitopes. Identification of dominant immunogenic epitopes in NP and RBD of SARS-CoV-2 could provide important information for the development of passive and active immunotherapy as well as diagnostic tools for the control of COVID-19 infection.

Differential tumor inhibitory effects induced by HER3 extracellular subdomain-specific mouse monoclonal antibodies.

PURPOSE: The therapeutic potential of targeting the human epidermal growth factor receptor-3 (ErbB3/HER3) has long been ignored due to impaired tyrosine kinase function and low expression level in tumor cells compared with EGFR and HER2. Although recent investigations have explored the potential benefit of HER3 targeting and several anti-HER3 agents have been developed, there is still a critical need to design and produce more efficient therapeutics. This study was designed to develop tumor inhibitory monoclonal antibodies (MAbs) against different extracellular subdomains of HER3. METHODS: Distinct extracellular subdomains of HER3 (DI+II and DIII+IV) were utilized to produce MAbs by hybridoma technology. Biochemical and functional characteristics of these MAbs were then investigated by various methodologies, including immunoblotting, flow cytometry, cell proliferation, cell signaling, and enzyme-linked immunosorbent assays. RESULTS: Four anti-DI+II and six anti-DIII+IV MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-DI+II and four anti-DIII+IV MAbs recognized the native form of HER3 by immunoblotting. Four MAbs recognized the membranous HER3 by flow cytometry leading to induction of different levels of receptor internalization and subsequent degradation. Results of cell proliferation assays using these MAbs indicated that they differentially inhibited proliferation of HER3-expressing cancer cells and showed considerable synergistic effects in combination with trastuzumab. Selected MAb with the highest inhibitory effect significantly inhibited the phosphorylation of AKT and ERK1/2 molecules. CONCLUSION: Some of the anti-HER3 MAbs produced in this study displayed tumor inhibitory function and may be considered promising candidates for future HER3-targeted cancer therapy.

Cross talk between hepatitis B virus and innate immunity of hepatocytes.

Innate immunity plays a major role in controlling viral infections. Recent exploration of sodium taurocholate co-transporting polypeptide receptor as specific hepatitis B virus (HBV) receptor in human hepatocytes has provided appropriate cell culture tools to study the innate immunity of hepatocytes and its cross talk with HBV. In this review, we give a brief update on interaction between HBV and innate immunity using the currently available in vitro cellular models that support the complete life cycle of HBV. We will discuss how HBV can act as a 'stealth' virus to counteract the innate immune responses mediated by the pathogen recognition receptors of hepatocytes and escape the first line of surveillance of the host immune system. We give an overview of the cellular components of innate immunity that present in these in vitro models and discuss how activating these innate immunity components may contribute to the eradication of HBV infection.

Overall Status of Epstein-Barr virus Infection, IFN-a, and TLR-7/9 in Patients with Systemic Lupus Erythematous.

BACKGROUND: Systemic lupus erythematous (SLE) is a multisystem autoimmune disorder. While studying the pathogenesis of SLE is prevalent, both infectious and non-infectious elements are regarded to exert an important impact on the disease's development. OBJECTIVE: To explore the overall status of EBV, TLR7, TLR9, and IFN-α gene expression in 32 patients suffering from SLE and 32 healthy controls. METHODS: Plasma and PBMCs were separated from fresh whole blood. To measure EBV DNA load and mRNA levels of IFN-a, TLR-7 and9 in PBMCs, molecular techniques were employed. The production of IFN-α, ds-DNA IgG antibody, and EBNA-1 IgG levels were also measured in plasma by ELISA. RESULTS: SLE patients showed significantly higher EBV load (p=0.001) and transcriptional levels of TLR7 (p=0.0001), IFN-α (p=0.0001), and TLR9 (p=0.0001) than controls. Moreover, the plasma levels of IFN-α (p=0.0002) and EBNA-1specific IgG antibodies (p=0.01) were significantly higher in SLE patients. CONCLUSION: The results stressed on the potential role of EBV infection and TLRs in SLE patients although more research is needed to determine the global impact that EBV infection can have on immune signature in patients with SLE.

Endometrial mesenchymal stem/stromal cells: The Enigma to code messages for generation of functionally active regulatory T cells.

BACKGROUND: Regulatory T cells (Tregs) play an important role in fine-tuning of immune responses and are pivotal for a successful pregnancy. Recently, the importance of mesenchymal stem cells in regulation of immune responses in general and Tregs in particular has been highlighted. Here, we hypothesized that menstrual stromal/stem cells (MenSCs) contribute to uterine immune system regulation through induction of functionally active Tregs. METHODS: MenSCs were collected from 18 apparently healthy women and characterized. Bone marrow mesenchymal stem cells (BMSCs) served as a control. The effect of MenSCs on proliferation of anti-CD3/CD28-stimulated T CD4 + cells and generation of Tregs with or without pre-treatment with mitomycin C, IFN-γ and IL-1ß was evaluated by flow cytometry. The potential role of IDO, PGE2, IL-6, IL-10, and TGF-ß on proliferation of T CD4 + cells and generation of Tregs was assessed using blocking antibodies or agents. IDO activity was evaluated in MenSCs and BMSCs culture supernatants by a colorimetric assay. IL-10 and IFN-γ production in MenSCs-primed T CD4 + was measured using intracellular staining. To investigate the functional properties of Tregs induced by MenSCs, Treg cells were isolated and their functional property to inhibit proliferation of anti-CD3/CD28-stimulated PBMCs was assessed by flow cytometry. RESULTS: According to the results, proliferation of T CD4 + lymphocytes was enhanced in the presence of MenSCs, while pre-treatment of MenSCs with pro-inflammatory cytokines reversed this effect. PGE2 and IDO were the major players in MenSCs-induced T cell proliferation. Non-treated MenSCs decreased the frequency of Tregs, whereas after pre-treatment with IFN-γ and IL-1ß, they induced functional Tregs with ability to inhibit the proliferation of anti-CD3/CD28-stimulated PBMCs. This effect was mediated through IL-6, IL-10, TGF-ß and IDO. IFN-γ/IL-1ß-treated MenSCs induced IL-10 and IFN-γ production in CD4 + T cells. CONCLUSION: Collectively, these findings indicate that immunomodulatory impact of menstrual blood stem cells (MenSCs) on generation of Tregs and inhibition of T cells proliferation is largely dependent on pre-treatment with IFN-γ and IL-1ß. This is the first report on immunomodulatory impact of MenSCs on Tregs and highlights the pivotal role of endometrial stem cells in regulation of local endometrial immune responses.