Monoclonal antibodies to human small-cell lung cancer]

Murine monoclonal antibodies to human small cell lung cancer (SCLC) have been developed and partially characterized. Primary hybridoma clones were screened in the indirect immunofluorescence assay (IFA) on alive H417 cells. Then five clones (IgG1, IgG2a, IgG3 and IgM) non-reactive with normal human bone marrow cells and positively reactive with SCLC tumors were selected. The H417.3 antibody is directed against 47-50kD surface antigens of H417 cells. The antibodies are supposed to be applied for the immunodetection of SCLC metastases to bone marrow and immunotoxin preparations.     

The Therapeutic Potential of PI3K/Akt/mTOR Inhibitors in Breast Cancer: Rational and Progress

Breast cancer (BC) is the most commonly diagnosed cancer in women. The PI3K/AKT/mTOR pathway is among the most frequently dysregulated pathways in patients with BC. The activation of this pathway is associated with increased cell growth and clinical outcome, and its overexpression is associated with a poor prognosis. It has been proposed that it may be of importance as a potential therapeutic target in the treatment of BC. The aim of current review is to provide an overview of the potential utility of PI3K/Akt/mTOR inhibitors in patients with BC, with particular emphasis on recent preclinical and clinical studies. J. Cell. Biochem. 119: 213–222, 2018. © 2017 Wiley Periodicals, Inc.     

Monoclonal antibodies to Mycoplasma pneumoniae antigens]

Approaches to obtaining stable mouse hybridomas, capable of producing monoclonal antibodies (McAb) to M. pneumoniae key antigens, were developed. As the result of hybridization experiments, 7 clones were obtained; of these, 4 clones stably synthesized IgG McAb. Clones H1/H9 and H9/B2 synthesized antibodies to thermolabile, proteinase-sensitive K protein, produced by cytoplasmic membranes of M. pneumoniae cells. The molecular weight of this protein was found to be 90 kD. McAb of clone H1/H9, labeled with horse-radish peroxidase and fluorescein isothiocyanate, specifically reacted with M. pneumoniae antigens in the immunofluorescence test and the enzyme immunoassay (EIA). The sensitivity of EIA was 0.25 ng/ml of antigen protein. These data are prerequisites for the development of diagnostic test systems for the detection of M. pneumoniae antigens in different biological substances obtained from patients with respiratory pathology.     

A coordinated cytotoxic effect of IFN-gamma and cross-reactive antibodies in the pathogenesis of Helicobacter pylori gastritis

Background. Helicobacter pylori (H. pylori) infection is associated with chronic infiltration into the stomach by T cells and plasma cells producing IFN‐γ and antibodies of various specificities, respectively. It is unknown whether these lymphocyte‐products may play coordinated roles in the gastric pathology of this infection. Aims. To know how IFN‐γ may relate to anti‐H. pylori antibodies in their roles in pathogenesis, we determined the isotype subclass of those antibodies as well as their cross‐reactivity and cytotoxicity to gastric epithelium. Methods and Results. We infected BALB/c mice with H. pylori (SS1, Sydney Strain 1) and generated monoclonal antibodies, which were comprised of 240 independent clones secreting immunoglobulin and included 80 clones reactive to SS1. Ninety percent of the SS1‐reactive clones had IgG2a isotype. Two clones, 2B10 and 1A9, were cross reactive to cell surface antigens in H. pylori and to antigens of 28 KDa and 42 KDa, respectively, which were present on the cell surface of and shared by both mouse and human gastric epithelial cells. The antigens recognized by these monoclonal antibodies localized a distinctive area in the gastric glands. In the presence of complement, 2B10 showed cytotoxicity to gastric epithelial cells. The effect was dose dependant and augmented by IFN‐γ. Finally, administration of 2B10 to mice with SS1 infection aggravated gastritis by increasing cellular infiltration. Conclusion. IFN‐γ by gastric T cells may participate in pathogenesis of the H. pylori infected stomach by directing an isotype‐switch of anti‐H. pylori antibodies to complement‐binding subclass and by augmenting cytotoxic activity of a certain autoantibody. This may explain a host‐dependent diversity in gastric pathology of the patients with H. pylori infection.     

In Silico Designing a Candidate Vaccine Against Breast Cancer

International Journal of Peptide Research and Therapeutics - Breast cancer (BC) is the most common type of women’s cancer with a prevalence of about 25%, although it is rare in men...     

Gene-Gene Interaction Study Between Genetic Polymorphisms of Folate Metabolism and MTR SNPs on Prognostic Features Impact for Breast Cancer

Background:Breast Cancer (BC), the second leading cause of cancer mortality after lung cancer and varied across the world due to genetic and environmental factors. In this study, we evaluated the interaction between the polymorphisms in genes encoding enzymes of folate metabolism: methylenetetrahydrofolate reductase (MTHFR), methionine synthesis reductase (MTR) with the BC prognostic factors.Methods:This study was conducted on 160 Egyptian subjects, 60 controls and 100 cases. Sequencing, RFLP analysis in addition to statistical analysis including Chi‐squared test, haplotype analysis was used to evaluate associations with BC risk and its clinicopathological parameters. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression.Results:Strong significant association with breast cancer risk was observed for the haplotype (T-C-G) of MTHFR C677T/ MTHFR A1289C and MTRA2576G and hormonal receptor expression (ER-/PR-/HER2+), bigger and advanced tumor and metastatic lymph nodes. However, no significant difference was observed for age.Conclusion:The combination of SNPs from MTHFR and MTR genes has a more synergistically genetic effect on BC disease progression. These SNPs could be used as tumor aggressiveness markers among Egyptian females with BC and could help in saving money and time.Key Words: Breast cancer, Methionine synthesis reductase, MTHFR, PCR-RFLP, SNPs     

Increased Expression Levels of WAVE3 Are Associated with the Progression and Metastasis of Triple Negative Breast Cancer

Background

Breast Cancer (BC) is a heterogeneous disease comprised of at least five genetically distinct subtypes, which together form the second leading cause of cancer death in women in the United States. Within BC subtypes, those classified as Triple Negative BCs (TNBCs) exhibit dismal survival rates due to their propensity to develop distant metastases. We have identified the WAVE3 protein, which is a critical regulator of actin cytoskeleton dynamics that are required for the motility and invasion of cancer cells through its activation of the Arp2/3 complex, as a key regulator of the different steps of the invasion-metastasis cascade in BC, especially in the more aggressive TNBCs. Our published studies have also shown that elevated expression levels of WAVE3 in the TNBC cell lines directly contribute to their increased invasion and metastasis potentials both in vitro and in vivo in murine models of BC metastasis.

Methodology/Principal Findings

Herein, we utilized both immunohistochemistry (IHC) of primary human BC tumors as well as quantitative real-time RT-PCR of WAVE3 in the peripheral blood of BC patients to clearly establish that WAVE3 is a predictive marker of overall BC patients’ survival. High levels of WAVE3 were predictive for reduced distant recurrence-free survival as well as for decreased disease-specific mortality. Our analysis of WAVE3 expression levels in the peripheral blood of BC patients showed that WAVE3 is highly expressed in the blood of patients who developed metastatic breast cancer compared to those who did not. WAVE3 expression was also highly upregulated in the blood of BC patients with the more aggressive TNBC subtype.

Conclusions

Together, these findings establish WAVE3 as a novel marker for increased risk of breast-cancer-specific mortality and for the metastatic potential of the TNBCs, and also identify WAVE3 as an attractive therapeutic target for the treatment of metastatic BC.     

Breast cancer stem cells: The role of sex steroid receptors

Breast cancer(BC) is the most common cancer among women, and current available therapies often have high success rates. Nevertheless, BC might acquire drug resistance and sometimes relapse. Current knowledge about the most aggressive forms of BC points to the role of specific cells with stem properties located within BC, the so-called "BC stem cells"(BCSCs). The role of BCSCs in cancer formation, growth, invasiveness, therapy resistance and tumor recurrence is becoming increasingly clear. The growth and metastatic properties of BCSCs are regulated by different pathways, which are only partially known. Sex steroid receptors(SSRs), which are involved in BC etiology and progression, promote BCSC proliferation, dedifferentiation and migration. However, in the literature,there is incomplete information about their roles. Particularly, there are contrasting conclusions about the expression and role of the classical BC hormonal biomarkers, such as estrogen receptor alpha(ERα), together with scant,albeit promising information concerning ER beta(ERβ) and androgen receptor(AR) properties that control different transduction pathways in BCSCs. In this review, we will discuss the role that SRs expressed in BCSCs play to BC progression and recurrence and how these findings have opened new therapeutic possibilities.     

Sulfotransferase 2B1b in human breast: differences in subcellular localization in African American and Caucasian women

Breast cancer (BC) is the most commonly diagnosed cancer among American women; however, the development of post-menopausal BC is significantly lower in African Americans as compared to Caucasians. Hormonal stimulation is important in BC development and differences in the conversion of dehydroepiandrosterone (DHEA) into estrogens may be involved in the lower incidence of post-menopausal BC in African American women. DHEA sulfation by sulfotransferase 2B1b (SULT2B1b) is important in regulating the conversion of DHEA into estrogens in tissues. SULT2B1b is localized in both cytosol and nuclei of some tissues including cancerous and associated-normal breast tissue. Immunohistochemical staining was used to evaluate the total expression and subcellular localization of SULT2B1b in African American and Caucasian breast tissues. Cell fractionation, immunoblot analysis and sulfation assays were used to characterize the subcellular expression and activity of SULT2B1b in BC tissues and T-47D breast adenocarcinoma cells. Immunohistochemical analysis of SULT2B1b showed that African Americans had a significantly greater amount of SULT2B1b in epithelial cells of associated-normal breast tissue as compared to Caucasians. Also, more SULT2B1b in African American associated-normal breast epithelial cells was localized in the nuclei than in Caucasians. Equivalent levels of SULT2B1b were detected in breast adenocarcinoma tissues from both African American and Caucasian women. Nuclei isolation and immunoblot analysis of both BC tissue and human T-47D breast adenocarcinoma cells demonstrated that SULT2B1b is present in nuclei and cytoplasm.     

Identification of Tumor-Associated Antigens from Medullary Breast Carcinoma by a Modified SEREX Approach

Medullary breast carcinoma (MBC) is a relatively rare malignancy with heavy lymphocytic infiltration that despite cytologically anaplastic features and high mitotic index has more favorable prognosis than other types of breast cancer. Lymphocytic infiltration of tumors reflects ongoing immune response against tumor antigens which could represent a great interest as potential targets for cancer immunotherapy. The search for MBC antigens by SEREX methodology has not been successful due to a very high titer of false positive clones, representing immunoglobulin genes. Here, we describe a novel approach for generating cDNA expression libraries from MBC tumor samples which are depleted of IgG cDNA clones and, therefore, are suitable for the identification of novel tumor-associated antigens (TAA) by SEREX approach. Modified methodology allowed us to isolate a panel of known and novel TAA which are currently under further investigation.     

CAR-T Cell Therapy for Breast Cancer: From Basic Research to Clinical Application

Breast cancer rises as the most commonly diagnosed cancer in 2020. Among women, breast cancer ranks first in both cancer incidence rate and mortality. Treatment resistance developed from the current clinical therapies limits the efficacy of therapeutic outcomes, thus new treatment approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy developed from adoptive T cell transfer, which typically uses patients'' own immune cells to combat cancer. CAR-T cells are armed with specific antibodies to recognize antigens in self-tumor cells thus eliciting cytotoxic effects. In recent years, CAR-T cell therapy has achieved remarkable successes in treating hematologic malignancies; however, the therapeutic effects in solid tumors are not up to expectations including breast cancer. This review aims to discuss the development of CAR-T cell therapy in breast cancer from preclinical studies to ongoing clinical trials. Specifically, we summarize tumor-associated antigens in breast cancer, ongoing clinical trials, obstacles interfering with the therapeutic effects of CAR-T cell therapy, and discuss potential strategies to improve treatment efficacy. Overall, we hope our review provides a landscape view of recent progress for CAR-T cell therapy in breast cancer and ignites interest for further research directions.     

Glycan-masking hemagglutinin antigens from stable CHO cell clones for H5N1 avian influenza vaccine development

Refocusing of B-cell responses can be achieved by preserving the overall fold of the antigen structure but selectively mutating the undesired antigenic sites with additional N-linked glycosylation motifs for glycan masking the vaccine antigen. We previously reported that glycan-masking recombinant H5 hemagglutinin (rH5HA) antigens on residues 83, 127, and 138 (g127 + g138 or g83 + g127 + 138 rH5HA) elicited broader neutralizing antibodies and protection against heterologous clades/subclades of high pathogenic avian influenza H5N1 viruses. In this study, we engineered the stably expressing Chinese hamster ovary (CHO) cell clones for producing the glycan-masking g127 + g138 and g83 + g127 + g138 rH5HA antigens. All of these glycan-masking rH5HA antigens produced in stable CHO cell clones were found to be mostly oligomeric structures. Only the immunization with the glycan-masking g127 + g138 but not g83 + g127 + g138 rH5HA antigens elicited more potent neutralizing antibody titers against four out of five heterologous clades/subclades of H5N1 viral strains. The increased neutralizing antibody titers against these heterologous viral strains were correlated with the increased amounts of stem-binding antibodies, only the glycan-masking g127 + g138 rH5HA antigens can translate into more protection against live viral challenges. The stable CHO cell line-produced glycan-masking g127 + g138 rH5HA can be used for H5N1 subunit vaccine development.     

Antigens in immune complexes from patients with breast cancer

Summary Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (¹²⁵Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). ¹²⁵Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified.     

HER2-positive circulating tumor cells in breast cancer

Purpose

Circulating Tumor Cells (CTCs) detection and phenotyping are currently evaluated in Breast Cancer (BC). Tumor cell dissemination has been suggested to occur early in BC progression. To interrogate dissemination in BC, we studied CTCs and HER2 expression on CTCs across the spectrum of BC staging.

Methods

Spiking experiments with 6 BC cell lines were performed and blood samples from healthy women and women with BC were analyzed for HER2-positive CTCs using the CellSearch®.

Results

Based on BC cell lines experiments, HER2-positive CTCs were defined as CTCs with HER2 immunofluoresence intensity that was at least 2.5 times higher than the background. No HER2-positive CTC was detected in 42 women without BC (95% confidence interval (CI) 0–8.4%) whereas 4.1% (95%CI 1.4–11.4%) of 73 patients with ductal/lobular carcinoma in situ (DCIS/LCIS) had 1 HER2-positive CTC/22.5 mL, 7.9%, (95%CI 4.1–14.9%) of 101 women with non metastatic (M0) BC had ≥1 HER2-positive CTC/22.5 mL (median 1 cell, range 1–3 cells) and 35.9% (95%CI 22.7–51.9%) of 39 patients with metastatic BC had ≥1 HER2-positive CTC/7.5 mL (median 1.5 cells, range 1–42 cells). In CTC-positive women with DCIS/LCIS or M0 BC, HER2-positive CTCs were more commonly detected in HER2-positive (5 of 5 women) than HER2-negative BC (5 of 12 women) (p = 0.03).

Conclusion

HER2-positive CTCs were detected in DCIS/LCIS or M0 BC irrespective of the primary tumor HER2 status. Nevertheless, their presence was more common in women with HER2-positive disease. Monitoring of HER2 expression on CTCs might be useful in trials with anti-HER2 therapies.     

Specific Schistosoma mansoni rat T cell clones. I. Generation and functional analysis in vitro and in vivo

In an attempt to determine the role of schistosome-specific T cells in the immune mechanisms developed during schistosomiasis, Schistosoma mansoni-specific T cells and clones were generated in vitro and some of their functions analyzed in vitro and in vivo in the fischer rat model. The data presented here can be summarized as follows: a) Lymph node cells (LNC) from rats primed with the excretory/secretory antigens-incubation products (IPSm) of adult worms proliferate in vitro only in response to the homologous schistosome antigens and not to unrelated antigens (Ag) such as ovalbumin (OVA) or Dipetalonema viteae and Fasciola hepatica parasite extracts. b) After in vitro restimulation of the primed LNC population with IPSm in the presence of antigen-presenting cells (APC) and maintenance in IL 2-containing medium, the frequency of IPSm-specific T cells is increased and the T cells can be restimulated only in the presence of APC possessing the same major histocompatibility complex (MHC) antigens. c) Following appropriate limiting dilution assays (LDA) (1 cell/well), 10 IPSm-specific T cell clones were obtained, and two of four maintained in culture were tested for their helper activity because they expressed only the W3/13+ W3/25+ surface phenotypes. d) The two highly proliferating IPSm-specific T cell clones (G5 and E23) exhibit an IPSm-dependent helper activity, as shown by the increase in IgG production by IPSm-primed B cells. e) IPSm-T cell clone (G5) as well as IPSm-T cell lines when injected in S. mansoni-infested rats can exert an in vivo helper activity, which is characterized by an accelerated production of IgG antibodies specific for the previously identified 30 to 40 kilodaltons (kd) schistosomula surface antigens (Ag). As recent studies have demonstrated that rat monoclonal antibodies recognize some incubation products of adult S. mansoni as well as one of the 30 to 40 kd schistosomula surface antigens, and taking into account the fact that the T cell clones here studied were restimulated either with IPSm or with schistosomulum Ag, it appears that such IPSm-specific T cell clones could be involved in the concomitant immunity mechanisms.     

Analysis of Gut Microbiota in Patients with Breast Cancer and Benign Breast Lesions

Breast cancer (BC) and benign breast lesions (BBLs) are common diseases in women worldwide. The gut microbiota plays a vital role in regulating breast diseases’ formation, progression, and therapy response. Hence, we explored the structure and function of gut microflora in patients with BC and BBLs. A cohort of 66 subjects was enrolled in the study. Twenty-six subjects had BC, 20 subjects had BBLs, and 20 matched healthy controls. High throughput 16S ribosomal RNA (16S rRNA) gene sequencing technology was used to determine the microbial community structure. Compared with healthy individuals, BC patients had significantly lower alpha diversity indices (Sobs index, p = 0.019; Chao1 index, p = 0.033). Sobs and Chao1 indices were also lower in patients with BBLs than healthy individuals, without statistical significance (p = 0.279, p = 0.314, respectively). Both unweighted and weighted UniFrac analysis showed that beta diversity differed significantly among the three groups (p = 3.376e–14, p < 0.001, respectively). Compared with healthy individuals, the levels of Porphyromonas and Peptoniphilus were higher in BC patients (p = 0.004, p = 0.007, respectively), whereas Escherichia and Lactobacillus were more enriched in the benign breast lesion group (p < 0.001, p = 0.011, respectively). Our study indicates that patients with BC and BBLs may undergo significant changes in intestinal microbiota. These findings can help elucidate the role of intestinal flora in BC and BBLs patients. Open in a separate window     

Monoclonal antibody-beta-lactamase conjugates for the activation of a cephalosporin mustard prodrug.

Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a site-specific manner by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified beta-lactamases from Bacillus cereus (BC beta L) and Escherichia coli (EC beta L) catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the beta-lactam ring of CM is hydrolyzed. The Km and Vmax values were 5.7 microM and 201 mumol/min per mg for BC beta L and 43 microM and 29 mumol/min per mg for EC beta L, respectively. Conjugates of BC beta L were prepared by combining the F(ab')2 fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BC beta L. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab')2 monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BC beta L followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BC beta L or 1F5-BC beta L enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody-beta-lactamase conjugate.     

Bovine monoclonal alloantibodies to blood group antigens prepared by murine x bovine or (murine x bovine) x bovine interspecies fusions.

In order to obtain monoclonal alloantibodies against bovine blood group antigens, lymph node cells from calves immunized with bovine red blood cells (RBC) were fused with either murine NSO/1 myeloma cells or a HAT sensitive murine x bovine heterohybridoma cell line. Both fusion partners resulted in heterohybridoma cell lines, producing monoclonal alloantibodies against bovine red blood cell antigens. Several clones produced antibodies against identical antigens and some of these clones have been further analysed. The antibodies produced by these selected cell lines have been compared with conventional polyclonal antisera used in bovine blood typing service. Thus extensive tests--including the ISAG Comparison Tests 1989/90 and 1991/92--have proved that monoclonal alloantibodies specific for the internationally recognized bovine red cell antigens A2, I1, O1, Q, A', B', Q', C1, R1, X1, S and Z have been produced. The Q, A', B', and C1 antibodies react weakly with certain phenogroups, whereas the A2, I1, O1, Q', R1, X1, S and Z antibodies have proved to be excellent blood typing reagents and have now substituted the polyclonal antisera in routine bovine blood typing in our laboratory.     

Plasma Proteomic Profiling in Hereditary Breast Cancer Reveals a BRCA1-Specific Signature: Diagnostic and Functional Implications

Background

Breast cancer (BC) is a leading cause of death among women. Among the major risk factors, an important role is played by familial history of BC. Germ-line mutations in BRCA1/2 genes account for most of the hereditary breast and/or ovarian cancers. Gene expression profiling studies have disclosed specific molecular signatures for BRCA1/2-related breast tumors as compared to sporadic cases, which might help diagnosis and clinical follow-up. Even though, a clear hallmark of BRCA1/2-positive BC is still lacking. Many diseases are correlated with quantitative changes of proteins in body fluids. Plasma potentially carries important information whose knowledge could help to improve early disease detection, prognosis, and response to therapeutic treatments. The aim of this study was to develop a comprehensive approach finalized to improve the recovery of specific biomarkers from plasma samples of subjects affected by hereditary BC.

Methods

To perform this analysis, we used samples from patients belonging to highly homogeneous population previously reported. Depletion of high abundant plasma proteins, 2D gel analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis were used into an integrated approach to investigate tumor-specific changes in the plasma proteome of BC patients and healthy family members sharing the same BRCA1 gene founder mutation (5083del19), previously reported by our group, with the aim to identify specific signatures.

Results

The comparative analysis of the experimental results led to the identification of gelsolin as the most promising biomarker.

Conclusions

Further analyses, performed using a panel of breast cancer cell lines, allowed us to further elucidate the signaling network that might modulate the expression of gelsolin in breast cancer.