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Cancer Genomics & Proteomics 2024Breast cancer (BC) is the most common malignant disease worldwide. Localized stages of BC can be successfully treated by surgery. However, local recurrence occurs in...
BACKGROUND/AIM
Breast cancer (BC) is the most common malignant disease worldwide. Localized stages of BC can be successfully treated by surgery. However, local recurrence occurs in about 4-10% of patients, requiring systemic treatments that impair the patients' quality of life and shortens life expectancy. Therefore, new therapeutic options are needed, which can be used intraoperatively and contribute to the complete removal of residual tumor cells in the surgical area. In the present study, we describe a cysteine-modified variant of the anti-HER2 antibody trastuzumab, that was coupled to the silicon phthalocyanine photosensitizer dye WB692-CB1 for the photoimmunotherapy (PIT) of BC.
MATERIALS AND METHODS
The cysteine modified trastuzumab variant was cloned and expressed in Expi293F cells. After purification via immobilized affinity chromatography, the antibody was coupled to the dye. Cell binding of the antibody and the antibody dye conjugate was measured by flow cytometry. After incubation of BC cells with the conjugate and activation of the dye by irradiation with red light, cell viability was determined.
RESULTS
The antibody and the conjugate showed specific binding to HER2-expressing BC cells. Treatment of the HER2 BC cell line SK-BR-3 with the conjugate followed by irradiation with a red light dose of 32 J/cm led to complete cell killing within 24 h.
CONCLUSION
Our novel antibody dye conjugate represents a promising candidate for intraoperative treatment of localized BC, aiming to eliminate residual tumor cells in the surgical area and potentially reduce local recurrence, thereby improving recovery prospects for BC patients.
Topics: Humans; Breast Neoplasms; Female; Receptor, ErbB-2; Immunotherapy; Trastuzumab; Phototherapy; Photosensitizing Agents; Cell Line, Tumor
PubMed: 38944426
DOI: 10.21873/cgp.20453 -
Cancer Genomics & Proteomics 2024Clear cell carcinoma is a prevalent histological type of ovarian cancer in East Asia, particularly in Japan, known for its resistance to chemotherapeutic agents and poor...
BACKGROUND/AIM
Clear cell carcinoma is a prevalent histological type of ovarian cancer in East Asia, particularly in Japan, known for its resistance to chemotherapeutic agents and poor prognosis. ARID1A gene mutations, commonly found in ovarian clear cell carcinoma (OCCC), contribute to its pathogenesis. Recent data revealed that the ARID1A mutation is related to better outcomes of cancer immunotherapy. Thus, this study aimed to investigate the immunotherapy treatment susceptibility of OCCC bearing ARID1A mutations.
MATERIALS AND METHODS
Expression of ARID1A was analyzed using western blotting in ovarian cancer cell lines. OCCC cell lines JHOC-9 and RMG-V were engineered to overexpress NY-ESO-1, HLA-A*02:01, and ARID1A. Sensitivity to chemotherapy and T cell receptor-transduced T (TCR-T) cells specific for NY-ESO-1 was assessed in ARID1A-restored cells compared to ARID1A-deficient wild-type cells.
RESULTS
JHOC-9 cells and RMG-V cells showed no expression of ARID1A protein. Overexpression of ARID1A in JHOC-9 and RMG-V cells did not impact sensitivity to gemcitabine. While ARID1A overexpression decreased sensitivity to cisplatin in RMG-V cells, it had no such effect in JHOC-9 cells. ARID1A overexpression reduced the reactivity of NY-ESO-1-specific TCR-T cells, as observed by the IFNγ ESLIPOT assay.
CONCLUSION
Cancer immunotherapy is an effective approach to target ARID1A-deficient clear cell carcinoma of the ovary.
Topics: Humans; Female; Ovarian Neoplasms; Transcription Factors; DNA-Binding Proteins; Adenocarcinoma, Clear Cell; T-Lymphocytes, Cytotoxic; Cell Line, Tumor; Antigens, Neoplasm; HLA-A2 Antigen; Membrane Proteins
PubMed: 38944423
DOI: 10.21873/cgp.20460 -
The Journal of Biological Chemistry Jun 2024Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of...
Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We employed phage display technology to identify novel single-chain variable fragments (scFvs) that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size exclusion chromatography combined with multi-angle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.
PubMed: 38944121
DOI: 10.1016/j.jbc.2024.107507 -
ACS Infectious Diseases Jun 2024The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the...
The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.
PubMed: 38943594
DOI: 10.1021/acsinfecdis.4c00058 -
Communications Biology Jun 2024The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate 'virulence factor present in multiple pathogenic species of the Mollicutes, including the...
The Mycoplasma Immunoglobulin Binding/Protease (MIB-MIP) system is a candidate 'virulence factor present in multiple pathogenic species of the Mollicutes, including the fast-growing species Mycoplasma feriruminatoris. The MIB-MIP system cleaves the heavy chain of host immunoglobulins, hence affecting antigen-antibody interactions and potentially facilitating immune evasion. In this work, using -omics technologies and 5'RACE, we show that the four copies of the M. feriruminatoris MIB-MIP system have different expression levels and are transcribed as operons controlled by four different promoters. Individual MIB-MIP gene pairs of M. feriruminatoris and other Mollicutes were introduced in an engineered M. feriruminatoris strain devoid of MIB-MIP genes and were tested for their functionality using newly developed oriC-based plasmids. The two proteins are functionally expressed at the surface of M. feriruminatoris, which confirms the possibility to display large membrane-associated proteins in this bacterium. However, functional expression of heterologous MIB-MIP systems introduced in this engineered strain from phylogenetically distant porcine Mollicutes like Mesomycoplasma hyorhinis or Mesomycoplasma hyopneumoniae could not be achieved. Finally, since M. feriruminatoris is a candidate for biomedical applications such as drug delivery, we confirmed its safety in vivo in domestic goats, which are the closest livestock relatives to its native host the Alpine ibex.
Topics: Bacterial Vaccines; Mycoplasma; Animals; Bacterial Proteins; Immunoglobulins; Gene Expression Regulation, Bacterial; Mycoplasma Infections; Goats
PubMed: 38942984
DOI: 10.1038/s42003-024-06497-8 -
Nature Chemical Biology Jul 2024Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while...
Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.
Topics: Clostridioides difficile; Anti-Bacterial Agents; Microbial Sensitivity Tests; Vancomycin; Cell Wall; Teichoic Acids; Peptidoglycan; Drug Therapy, Combination; Peptides, Cyclic; Lipopeptides
PubMed: 38942968
DOI: 10.1038/s41589-024-01651-z -
Analytical and Bioanalytical Chemistry Jun 2024Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns...
Accurate diagnostic and serology assays are required for the continued management of the COVID-19 pandemic yet spike protein mutations and intellectual property concerns with antigens and antibodies used in various test kits render comparability assessments difficult. As the use of common, well-characterized reagents can help address this lack of standardization, the National Research Council Canada has produced two protein reference materials (RMs) for use in SARS-CoV-2 serology assays: biotinylated human angiotensin-converting enzyme 2 RM, ACE2-1, and SARS-CoV-2 Omicron BA.4/5 spike protein RM, OMIC-1. Reference values were assigned through a combination of amino acid analysis via isotope dilution liquid chromatography tandem mass spectrometry following acid hydrolysis, and ultraviolet-visible (UV-Vis) spectrophotometry at 280 nm. Vial-to-vial homogeneity was established using UV-Vis measurements, and protein oligomeric status, monitored by size exclusion liquid chromatography (LC-SEC), was used to evaluate transportation, storage, and freeze-thaw stabilities. The molar protein concentration in ACE2-1 was 25.3 ± 1.7 µmol L (k = 2, 95% CI) and consisted almost exclusively (98%) of monomeric ACE2, while OMIC-1 contained 5.4 ± 0.5 µmol L (k = 2) spike protein in a mostly (82%) trimeric form. Glycoprotein molar mass determination by LC-SEC with multi-angle light scattering detection facilitated calculation of corresponding mass concentrations. To confirm protein functionality, the binding of OMIC-1 to immobilized ACE2-1 was investigated with surface plasmon resonance and the resulting dissociation constant, K ~ 4.4 nM, was consistent with literature values.
PubMed: 38942955
DOI: 10.1007/s00216-024-05413-7 -
Cell Death & Disease Jun 2024S100a8/a9, largely released by polymorphonuclear neutrophils (PMNs), belongs to the S100 family of calcium-binding proteins and plays a role in a variety of inflammatory...
S100a8/a9, largely released by polymorphonuclear neutrophils (PMNs), belongs to the S100 family of calcium-binding proteins and plays a role in a variety of inflammatory diseases. Although S100a8/a9 has been reported to trigger endothelial cell apoptosis, the mechanisms of S100a8/a9-induced endothelial dysfunction during sepsis require in-depth research. We demonstrate that high expression levels of S100a8/a9 suppress Ndufa3 expression in mitochondrial complex I via downregulation of Nrf1 expression. Mitochondrial complex I deficiency contributes to NAD-dependent Sirt1 suppression, which induces mitochondrial disorders, including excessive fission and blocked mitophagy, and mtDNA released from damaged mitochondria ultimately activates ZBP1-mediated PANoptosis in endothelial cells. Moreover, based on comprehensive scRNA-seq and bulk RNA-seq analyses, S100A8/A9 neutrophils are closely associated with the circulating endothelial cell count (a useful marker of endothelial damage), and S100A8 is an independent risk factor for poor prognosis in sepsis patients.
Topics: Calgranulin A; Neutrophils; Sepsis; Humans; Calgranulin B; Mitochondria; Electron Transport Complex I; Endothelial Cells; Animals; Mice; Male; Human Umbilical Vein Endothelial Cells; Mitophagy; Mice, Inbred C57BL; Apoptosis
PubMed: 38942784
DOI: 10.1038/s41419-024-06849-6 -
Virology Jun 2024African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly...
African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly expressed during the early phase of virus replication, it can be used as a target protein for early diagnosis. In this study, the I73R protein of ASFV was expressed, and we successfully prepared a novel monoclonal antibody (mAb), 8G11D7, that recognizes this protein. Through both indirect immunofluorescence and Western blotting assays, we demonstrated that 8G11D7 can detect ASFV strains. By evaluating the binding of the antibody to a series of I73R-truncated peptides, the definitive epitope recognized by the monoclonal antibody 8G11D7 was determined to be DKTNTIYPP . Bioinformatic analysis revealed that the antigenic epitope had a high antigenic index and conservatism. This study contributes to a deeper understanding of ASFV protein structure and function, helping establish ASFV-specific detection method.
PubMed: 38941747
DOI: 10.1016/j.virol.2024.110145 -
ACS Chemical Biology Jun 2024Chemokines are an important family of small proteins integral to leukocyte recruitment during inflammation. Dysregulation of the chemokine-chemokine receptor axis is...
Chemokines are an important family of small proteins integral to leukocyte recruitment during inflammation. Dysregulation of the chemokine-chemokine receptor axis is implicated in many diseases, and both chemokines and their cognate receptors have been the targets of therapeutic development. Analysis of the antigen-binding regions of chemokine-binding nanobodies revealed a sequence motif suggestive of tyrosine sulfation. Given the well-established importance of post-translational tyrosine sulfation of receptors for chemokine affinity, it was hypothesized that the sulfation of these nanobodies may contribute to chemokine binding and selectivity. Four nanobodies (16C1, 9F1, 11B1, and 11F2) were expressed using amber codon suppression to incorporate tyrosine sulfation. The sulfated variant of 16C1 demonstrated significantly improved chemokine binding compared to the non-sulfated counterpart, while the other nanobodies displayed equipotent or reduced affinity upon sulfation. The ability of tyrosine sulfation to modulate chemokine binding, both positively and negatively, could be leveraged for chemokine-targeted sulfo-nanobody therapeutics in the future.
PubMed: 38941516
DOI: 10.1021/acschembio.4c00230