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1.
Virchows Arch ; 484(4): 597-608, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38570364

ABSTRACT

Assessing programmed death ligand 1 (PD-L1) expression on tumor cells (TCs) using Food and Drug Administration-approved, validated immunoassays can guide the use of immune checkpoint inhibitor (ICI) therapy in cancer treatment. However, substantial interobserver variability has been reported using these immunoassays. Artificial intelligence (AI) has the potential to accurately measure biomarker expression in tissue samples, but its reliability and comparability to standard manual scoring remain to be evaluated. This multinational study sought to compare the %TC scoring of PD-L1 expression in advanced urothelial carcinoma, assessed by either an AI Measurement Model (AIM-PD-L1) or expert pathologists. The concordance among pathologists and between pathologists and AIM-PD-L1 was determined. The positivity rate of ≥ 1%TC PD-L1 was between 20-30% for 8/10 pathologists, and the degree of agreement and scoring distribution for among pathologists and between pathologists and AIM-PD-L1 was similar both scored as a continuous variable or using the pre-defined cutoff. Numerically higher score variation was observed with the 22C3 assay than with the 28-8 assay. A 2-h training module on the 28-8 assay did not significantly impact manual assessment. Cases exhibiting significantly higher variability in the assessment of PD-L1 expression (mean absolute deviation > 10) were found to have patterns of PD-L1 staining that were more challenging to interpret. An improved understanding of sources of manual scoring variability can be applied to PD-L1 expression analysis in the clinical setting. In the future, the application of AI algorithms could serve as a valuable reference guide for pathologists while scoring PD-L1.


Subject(s)
Artificial Intelligence , B7-H1 Antigen , Biomarkers, Tumor , Observer Variation , Humans , B7-H1 Antigen/analysis , B7-H1 Antigen/metabolism , Biomarkers, Tumor/analysis , Biomarkers, Tumor/metabolism , Reproducibility of Results , Carcinoma, Transitional Cell/pathology , Carcinoma, Transitional Cell/metabolism , Carcinoma, Transitional Cell/diagnosis , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/metabolism , Urologic Neoplasms/pathology , Urologic Neoplasms/metabolism , Immunohistochemistry/methods , Pathologists , Urothelium/pathology , Urothelium/metabolism
2.
PLoS One ; 10(6): e0129981, 2015.
Article in English | MEDLINE | ID: mdl-26114641

ABSTRACT

Production of pro-inflammatory cytokines by innate immune cells at the early stages of bacterial infection is important for host protection against the pathogen. Many intracellular bacteria, including Francisella tularensis, the agent of tularemia, utilize the anti-inflammatory cytokine IL-10, to evade the host immune response. It is well established that IL-10 has the ability to inhibit robust antigen presentation by dendritic cells and macrophages, thus suppressing the generation of protective immunity. The pathogenesis of F. tularensis is not fully understood, and research has failed to develop an effective vaccine to this date. In the current study, we hypothesized that F. tularensis polarizes antigen presenting cells during the early stages of infection towards an anti-inflammatory status characterized by increased synthesis of IL-10 and decreased production of IL-12p70 and TNF-α in an IFN-É£-dependent fashion. In addition, F. tularensis drives an alternative activation of alveolar macrophages within the first 48 hours post-infection, thus allowing the bacterium to avoid protective immunity. Furthermore, we demonstrate that targeting inactivated F. tularensis (iFt) to Fcγ receptors (FcÉ£Rs) via intranasal immunization with mAb-iFt complexes, a proven vaccine strategy in our laboratories, reverses the anti-inflammatory effects of the bacterium on macrophages by down-regulating production of IL-10. More specifically, we observed that targeting of iFt to FcγRs enhances the classical activation of macrophages not only within the respiratory mucosa, but also systemically, at the early stages of infection. These results provide important insight for further understanding the protective immune mechanisms generated when targeting immunogens to Fc receptors.


Subject(s)
Antigens, Bacterial/immunology , Francisella tularensis/immunology , Receptors, Fc/metabolism , Tularemia/immunology , Tularemia/metabolism , Animals , Antibodies, Monoclonal/immunology , Antigen-Antibody Complex/immunology , Cytokines/metabolism , Disease Models, Animal , Inflammation Mediators/metabolism , Lung/immunology , Lung/metabolism , Lung/pathology , Macrophage Activation/immunology , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Mice , Mice, Knockout , Receptors, IgG/metabolism , Tularemia/microbiology
3.
Infect Immun ; 83(1): 77-89, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25312957

ABSTRACT

Targeting antigens (Ag) to Fcγ receptors (FcγR) intranasally (i.n.) enhances immunogenicity and protection against intracellular and extracellular pathogens. Specifically, we have demonstrated that targeting fixed (inactivated) Francisella tularensis (iFT) organisms to FcR in mice i.n., with MAb-iFT immune complexes, enhances F. tularensis-specific immune responses and protection against F. tularensis challenge. Furthermore, traditional adjuvant is not required. In addition, we have demonstrated that the increased immunogenicity following the targeting of iFT to FcR is due, in part, to enhanced dendritic cell (DC) maturation, enhanced internalization, and processing and presentation of iFT by DCs, as well as neonatal FcR (FcRn)-enhanced trafficking of iFT from the nasal passage to the nasal mucosa-associated lymphoid tissue (NALT). Using this immunization and challenge model, we expanded on these studies to identify specific in vivo immune responses impacted and enhanced by FcR targeting of iFT i.n. Specifically, the results of this study demonstrate for the first time that targeting iFT to FcR increases the frequency of activated DCs within the lungs of MAb-iFT-immunized mice subsequent to F. tularensis LVS challenge. In addition, the frequency and number of gamma interferon (IFN-γ)-secreting effector memory (EM) CD4(+) T cells elicited by F. tularensis infection (postimmunization) is increased in an interleukin 12 (IL-12)-dependent manner. In summary, these studies build significantly upon previously published work utilizing this vaccine platform. We have identified a number of additional mechanisms by which this novel, adjuvant-independent, FcR-targeted mucosal vaccine approach enhances immunity and protection against infection, while further validating its potential as a universal vaccine platform against mucosal pathogens.


Subject(s)
Antibodies, Bacterial/metabolism , Bacterial Vaccines/immunology , Francisella tularensis/immunology , Receptors, Fc/metabolism , Tularemia/prevention & control , Administration, Intranasal , Animals , Antibodies, Monoclonal/metabolism , Antigen Presentation , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , CD4-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Mice, Inbred C57BL , Tularemia/immunology
4.
MAbs ; 5(5): 787-94, 2013.
Article in English | MEDLINE | ID: mdl-23883920

ABSTRACT

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2-8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Chemistry, Pharmaceutical/methods , Drug Discovery/methods , Technology, Pharmaceutical/methods , Antibodies, Monoclonal/chemistry , Chromatography, High Pressure Liquid , Drug Approval/methods , Drug Stability , Humans , Mass Spectrometry , Solubility , Temperature , United States , United States Food and Drug Administration , Viscosity
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