Comparative Immunogenicity and Neutralization Potency of Four Approved COVID-19 Vaccines in BALB/c Mice.

Background: Since the outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), several vaccine candidates have been developed within a short period of time. Although the potency of these vaccines was evaluated individually, their comparative potency was not comprehensively evaluated. Objective: To compare the immunogenicity and neutralization efficacy of four approved COVID-19 vaccines in Iran, including: PastoCovac Plus, Sinopharm, SpikoGen, and Noora in BALB/c mice. Methods: Different groups of female BALB/c mice were vaccinated with three doses of each vaccine. The serum levels of antibodies against the viral receptor binding domain (anti-RBD) and spike (anti-spike) protein as well as the vaccine formulation (anti-vaccine) were evaluated using enzyme-linked immunosorbent assay (ELISA). The neutralization efficacy of these four vaccines was assessed through four neutralization assays: conventional virus neutralization test (cVNT), pseudotype virus neutralization test (pVNT), surrogate virus neutralization test (sVNT), and inhibition flow cytometry. Results: All four vaccines induced seroconversion in vaccinated animals. All vaccines successfully induced high levels of anti-vaccine antibody; however, PastoCovac Plus and Sinopharm vaccines induced significantly higher levels of anti-RBD antibody titer compared to Noora and SpikoGen. Moreover, the results of the antibody response were corroborated by the virus neutralization tests, which revealed very weak neutralization potency by Noora and SpikoGen in all tests. Conclusion: Our results indicate significant immunogenicity and neutralization efficacy induced by PastoCovac Plus and Sinopharm, but not by Noora and SpikoGen. This suggests the need for additional comparative assessment of the potency and efficacy of these four vaccines in vaccinated subjects.

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.

Investigation of Expression Profile of Placenta-specific 1 (PLAC1) in Acute Myeloid and Lymphoid Leukemias.

Background: Placenta-specific 1 (PLAC1) is one of the cancer-testis-placenta antigens that has no expression in normal tissue except placenta trophoblast and testicular germ cells, but is overexpressed in a variety of solid tumors. There is a lack of studies on the expression of PLAC1 in leukemia. We investigated expression of PLAC1 in Acute Myeloid Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL). Methods: In this study, we investigated expression pattern of PLAC1 gene in peripheral blood and bone marrow mononuclear cells of newly-diagnosed patients with AML (n=31) and ALL (n=31) using quantitative real-time PCR. Normal subjects (n=17) were considered as control. The PLAC1 protein expression in the samples were also detected using western blotting. Results: Our data demonstrated that PLAC1 transcripts had 2.7 and 2.9 fold-change increase in AML and ALL, respectively, compared to normal samples. PLAC1 transcript expression was totally negative in all studied normal subjects. Level of PLAC1 mRNA expression in ALL statistically increased compared to normal samples (p=0.038). However, relative mRNA expression of PLAC1 in AML was not significant in comparison to normal subjects (p=0.848). Furthermore, relative mRNA expression of PLAC1 in AML subtypes was not statistically significant (p=0.756). PLAC1 gene expression showed no difference in demographical clinical and para-clinical parameters. Western blotting confirmed expression of PLAC1 in the ALL and AML samples. Conclusion: Considering PLAC1 expression profile in acute leukemia, PLAC1 could be a potential marker in leukemia which needs complementary studies in the future.

Novel mouse monoclonal antibodies against Bordetella pertussis pertactin antigen with versatile applications.

BACKGROUND: Pertussis, or whooping cough, is a highly contagious respiratory disease caused by Bordetella pertussis (BP). Pertactin (PRN) is one of the main immunogenic components of BP and is employed in many commercialized acellular pertussis vaccines (aPVs). Purification of this protein by conventional chromatography methods is challenging and commonly requires multiple laborious processes with low recovery. Using specific monoclonal antibodies (mAbs) for the purification of PRN antigen is expected to yield high purity and recovery of the target molecule. METHODS: Recombinant PRN antigen was used to produce mouse mAbs using hybridoma technology. Structural and functional characteristics of the mAbs were assessed by ELISA, immunoblotting, and flow cytometry. Selected mAbs were employed to purify PRN by affinity chromatography, and the purity and recovery of the purified protein were analyzed by ELISA, SDS-PAGE, and immunoblotting. Moreover, ELISA and flow cytometry techniques were designed using these mAbs to detect PRN in different strains of BP. RESULTS: Five mAbs were produced and selected based on their reactivity with native PRN. Our results demonstrate that purification of PRN by affinity chromatography resulted in a highly pure antigen with 75-85 percent recovery. In addition, ELISA and flow cytometry results indicated that these mAbs could recognize PRN in the bacterial cell walls of different BP strains. CONCLUSION: We successfully produced PRN-specific mAbs and designed an affinity chromatography method to purify PRN antigen with higher purity and recovery than conventional methods. These mAbs could be employed as valuable tools for the detection and purification of PRN for vaccine manufacturing.

Novel neutralizing mouse-human chimeric monoclonal antibodies against the SARS-CoV-2 receptor binding domain.

Introduction. Neutralizing antibodies have been widely used for the prophylaxis and treatment of COVID-19.Hypothesis. The major target for these neutralizing antibodies is the receptor-binding domain (RBD) of the viral spike protein.Aim. In the present study, we developed and characterized three neutralizing chimeric mouse-human mAbs for potential therapeutic purposes.Methodology. Light and heavy chain variable region genes of three mouse mAbs (m4E8, m3B6, and m1D1) were amplified and ligated to human Cγ1 and Cκ constant region genes by PCR. After cloning into a dual promoter mammalian expression vector, the final constructs were transiently expressed in DG-44 cells and the purified chimeric antibodies were characterized by ELISA and Western blotting. The neutralizing potency of the chimeric mAbs was determined by three different virus neutralization tests including sVNT, pVNT, and cVNT.Results. All three recombinant chimeric mAbs display human constant regions and are able to specifically bind to the RBD of SARS-CoV-2 with affinities comparable to the parental mAbs. Western blot analysis showed similar epitope specificity profiles for both the chimeric and the parental mouse mAbs. The results of virus neutralization tests (sVNT, pVNT, and cVNT) indicate that c4E8 had the most potent neutralizing activity with IC50 values of 1.772, 0.009, and 0.01 µg ml-1, respectively. All chimeric and mouse mAbs displayed a similar pattern of reactivity with the spike protein of the SARS-CoV-2 variants of concern (VOC) tested, including alpha, delta, and wild-type.Conclusion. The chimeric mAbs displayed neutralizing potency similar to the parental mouse mAbs and are potentially valuable tools for disease control.

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.

Cross-Reactivity of HBe Antigen-Specific Polyclonal Antibody with HBc Antigen.

Hepatitis B virus (HBV) infection is a major health problem worldwide and causes almost one million deaths annually. The HBV core gene codes for two related antigens, known as core antigen (HBcAg) and e-antigen (HBeAg), sharing 149 residues but having different amino- and carboxy-terminals. HBeAg is a soluble variant of HBcAg and a clinical marker for determining the disease severity and patients' screening. Currently available HBeAg assays have a shortcoming of showing cross-reactivity with HBcAg. In this study, for the first time, we evaluated whether HBcAg-adsorbed anti-HBe polyclonal antibodies could specifically recognize HBeAg or still show cross-reactivity with HBcAg. Recombinant HBeAg was cloned in pCold1 vector and successfully expressed in Escherichia coli and after purification by Ni-NTA resin was used to generate polyclonal anti-HBe antibodies in rabbit. Purified HBeAg was further characterized by assessing its reactivity with anti-HBe in the sera of chronically infected patients and HBeAg-immunized rabbit. Sera from patients with chronic HBV infection, containing anti-HBe, specifically reacted with recombinant HBeAg, implying antigenic similarity between the prokaryotic and native HBeAg in the serum of HBV-infected patients. In addition, the designed enzyme-linked immunosorbent assay (ELISA) with rabbit anti-HBe polyclonal antibodies could detect recombinant HBeAg with high sensitivity, while high cross-reactivity with HBcAg was observed. It is noteworthy that HBcAg-adsorbed anti-HBe polyclonal antibodies still showed high cross-reactivity with HBcAg, implying that due to the presence of highly similar epitopes in both antigens, HBcAg-adsorbed polyclonal antibodies cannot differentiate between the two antigens.

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.

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.

Diagnostic performance of a novel antigen-capture ELISA for the detection of SARS-CoV-2.

BACKGROUND AND AIMS: The coronavirus disease 2019 (COVID-19) pandemic is a serious health problem worldwide. Early virus detection is essential for disease control and management. Viral antigen detection by ELISA is a cost-effective, rapid, and accurate antigen diagnostic assay which could facilitate early viral detection. METHOD: An antigen-capture sandwich ELISA was developed using novel nucleocapsid (NP)-specific mouse monoclonal antibodies (MAbs). The clinical performance of the assay was assessed using 403 positive and 150 negative respiratory samples collected during different SARS-CoV-2 variants outbreaks in Iran. RESULTS: The limit of detection of our ELISA assay was found to be 43.3 pg/ml for recombinant NP. The overall sensitivity and specificity of this assay were 70.72% (95% CI: 66.01-75.12) and 100% (95% CI: 97.57-100), respectively, regardless of Ct values and SARS-CoV-2 variants. There was no significant difference in our assay sensitivity for the detection of Omicron subvariants compared to Delta variant. Assay sensitivity for the BA.5 Omicron subvariant was calculated as 91.89% (95% CI: 85.17-96.23) for samples with Ct values < 25 and 82.70% (95% CI: 75.19-88.71) for samples with Ct values < 30. CONCLUSION: Our newly developed ELISA method is reasonably sensitive and highly specific for detection of SARS-CoV-2 regardless of the variants and subvariants of the virus.

Cancer Is Associated with the Emergence of Placenta-Reactive Autoantibodies.

Placenta-specific antigens are minimally expressed or unexpressed in normal adult tissues, while they are widely expressed in cancer. In the course of carcinogenesis, a vast array of autoantibodies (AAbs) is produced. Here, we used a quantitative approach to determine the reactivity of AAbs in the sera of patients with breast (BrC: N = 100, 100% female, median age: 51 years), gastric (GC: N = 30, 46.6% female, median age: 57 years), bladder (BC: N = 29, 34.4% female, median age: 57 years), and colorectal (CRC: N = 34, 41.1% female, median age: 51 years) cancers against first-trimester (FTP) and full-term placental proteome (TP) in comparison with age- and sex-matched non-cancer individuals. Human-on-human immunohistochemistry was used to determine reactive target cells in FTP. The effect of pregnancy on the emergence of placenta-reactive autoantibodies was tested using sera from pregnant women at different trimesters of pregnancy. Except for BC, patients with BrC (p < 0.0284), GC (p < 0.0002), and CRC (p < 0.0007) had significantly higher levels of placenta-reactive AAbs. BrC (p < 0.0001) and BC (p < 0.0409) in the early stages triggered higher autoantibody reactivity against FTP. The reactivities of BrC sera with FTP did not show an association with ER, PR, or HER2 expression. Pregnancy in the third trimester was associated with the induction of TP- and not FTP-reactive autoantibodies (=0.018). The reactivity of BrC sera with placental proteins was found to be independent of gravidity or abortion. BrC sera showed a very strong and specific pattern of reactivity with scattered cells beneath the syncytiotrophoblast layer. Our results reinforce the concept of the coevolution of placentation and cancer and shed light on the future clinical application of the placental proteome for the non-invasive early detection and treatment of cancer.

Proteome Analysis of Adult Acute Lymphoblastic Leukemia by Two-dimensional Blue Native/Sodium Dodecyl Sulfate Gel Electrophoresis.

Background: Despite the significant progress in the treatment of Acute Lymphoblastic Leukemia (ALL) in children, it still remains as one of the most challenging malignancies in adults. Identification of new biomarkers may improve the management of adult ALL. Proteins expressed on the cell surface can be considered as disease-associated biomarkers with potential for diagnosis and targeted therapies. Thus, membrane proteome studies give essential information about the disease-related biomarkers. Methods: We applied 2-dimensional blue-native SDS-PAGE technique followed by MALDI-TOF/TOF-mass spectrometry to study the cell membrane proteome of peripheral blood mononuclear cells of adult B-ALL patients in comparison to that of the healthy controls. Results: Sixty seven differentially expressed protein spots were detected, among them 52 proteins were found to be up-regulated but the other 15 proteins were down-regulated in B-ALL. Five differentially expressed proteins, involved in energy metabolism pathways, were detected in B-ALL patients compared to the healthy control group. Conclusion: Differentially expressed proteins provide an insight into the molecular biology of B-ALL. Further studies must be done to confirm our data to be considered as potential targets for detection and treatment of B-ALL.

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.

Nucleolin; A tumor associated antigen as a potential lung cancer biomarker.

Lung cancer is a primary cause of mortality in many communities. The poor prognosis and clinical outcome of this cancer are mostly attributable to its advanced stage upon diagnosis, and as a result, it places a significant cost on public health across the globe. The majority of patients experience severe adverse effects from conventional therapies that involve nonspecific invasion of both healthy and malignant cells. Furthermore, no particular tumor marker has been developed to evaluate the patients' status and prognosis. NCL as one of the vital nuclear proteins is involved in various cellular activities, including ribosome assembly and rRNA processing. Research have shown that following malignant transformation in lung cancer cells, both the cytosolic and plasma membrane levels of this protein rise dramatically. Furthermore, signaling generated by the surface nucleolin significantly enhances tumor proliferation, differentiation, and angiogenesis. On the other hand, findings showed that altering the size and other properties of tumor cells may influence the expression pattern of nucleolin. Therefore, in the current study, we intend to review the role of nucleolin in the development and progression of lung cancer cells and also evaluate its potential as a prognostic, therapeutic as well as diagnostic marker in lung cancer patients.

Optimization of an Efficient Cell Culture Hepatitis B Infection System for Assessment of Hepatitis B Virus Neutralizing Monoclonal Antibodies.

BACKGROUND: Human polyclonal plasma-derived hepatitis B immunoglobulin (HBIG) is currently used for immunoprophylaxis of HBV infection. The development of virus-neutralizing monoclonal antibodies (MAbs) requires the use of optimized cell culture systems supporting HBV infection. OBJECTIVE: This study aims to optimize the hepatitis B virus infectivity of NTCP-reconstituted HepG2 (HepG2-NTCP) cells to establish an efficient system to evaluate the HBV-neutralizing effect of anti-HBs MAbs. METHODS: Serum-derived HBV (sHBV) and cell culture-derived HBV (ccHBV) were simultaneously used for the optimization of HBV infection in HepG2-NTCP cells by applying different modifications. RESULTS: Our results for the first time showed that in addition to human serum, monkey serum could significantly improve ccHBV infection, while fetal and adult bovine serum as well as duck and sheep serum did not have a promotive effect. In addition, sHBV and ccHBV infectivity are largely similar except that adding 5% of PEG, which is commonly used to improve in vitro infection of ccHBV, significantly reduced sHBV infection. We showed that a combination of spinoculation, trypsinization, and also adding human or monkey serum to HBV inoculum could significantly improve the permissivity of HepG2-NTCP cells to HBV infection compared with individual strategies. All anti-HBs MAbs were able to successfully neutralize both ccHBV and sHBV infection in our optimized in vitro system. CONCLUSION: Our study suggests different strategies for improving ccHBV and sHBV infection in HepG2-NTCP cells. This cell culture-based system allows assessment of HBV neutralizing MAbs and may also prove to be valuable for the analysis of other HBV neutralizing therapeutics.

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.

Epitope mapping of severe acute respiratory syndrome coronavirus 2 neutralizing receptor binding domain-specific monoclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the outbreak led to the coronavirus disease 2019 (COVID-19) pandemic. Receptor binding domain (RBD) of spike (S) protein of SARS-CoV-2 is considered as a major target for immunotherapy and vaccine design. Here, we generated and characterized a panel of anti-RBD monoclonal antibodies (MAbs) isolated from eukaryotic recombinant RBD-immunized mice by hybridoma technology. Epitope mapping was performed using a panel of 20-mer overlapping peptides spanning the entire sequence of the RBD protein from wild-type (WT) Wuhan strain by enzyme-linked immunosorbent assay (ELISA). Several hybridomas showed reactivity toward restricted RBD peptide pools by Pepscan analysis, with more focus on peptides encompassing aa 76-110 and 136-155. However, our MAbs with potent neutralizing activity which block SARS-CoV-2 spike pseudovirus as well as the WT virus entry into angiotensin-converting enzyme-2 (ACE2) expressing HEK293T cells showed no reactivity against these peptides. These findings, largely supported by the Western blotting results suggest that the neutralizing MAbs recognize mainly conformational epitopes. Moreover, our neutralizing MAbs recognized the variants of concern (VOC) currently in circulation, including alpha, beta, gamma, and delta by ELISA, and neutralized alpha and omicron variants at different levels by conventional virus neutralization test (CVNT). While the neutralization of MAbs to the alpha variant showed no substantial difference as compared with the WT virus, their neutralizing activity was lower on omicron variant, suggesting the refractory effect of mutations in emerging variants against this group of neutralizing MAbs. Also, the binding reactivity of our MAbs to delta variant showed a modest decline by ELISA, implying that our MAbs are insensitive to the substitutions in the RBD of delta variant. Our data provide important information for understanding the immunogenicity of RBD, and the potential application of the novel neutralizing MAbs for passive immunotherapy of SARS-CoV-2 infection.