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Methods in Molecular Biology (Clifton,... 2015First described by Engvall and Perlmann, the enzyme-linked immunosorbent assay (ELISA) is a rapid and sensitive method for detection and quantitation of an antigen using...
First described by Engvall and Perlmann, the enzyme-linked immunosorbent assay (ELISA) is a rapid and sensitive method for detection and quantitation of an antigen using an enzyme-labeled antibody. Besides routine laboratory usage, ELISA has been utilized in medical field and food industry as diagnostic and quality control tools. Traditionally performed in 96-well or 384-well polystyrene plates, the technology has expanded to other platforms with increase in automation. Depending on the antigen epitope and availability of specific antibody, there are variations in ELISA setup. The four basic formats are direct, indirect, sandwich, and competitive ELISAs. Direct ELISA is the simplest format requiring an antigen and an enzyme-conjugated antibody specific to the antigen. This chapter describes the individual steps for detection of a plate-bound antigen using a horseradish peroxidase (HRP)-conjugated antibody and luminol-based enhanced chemiluminescence (ECL) substrate. The methodological approach to optimize the assay by chessboard titration is also provided.
Topics: Antibodies; Antigens, Surface; Binding, Competitive; Enzyme-Linked Immunosorbent Assay; Horseradish Peroxidase; Humans; Immobilized Proteins; Immunoconjugates; Light; Luminescence; Luminol; Polystyrenes; Protein Binding; Sensitivity and Specificity
PubMed: 26160564
DOI: 10.1007/978-1-4939-2742-5_6 -
Methods in Molecular Biology (Clifton,... 2015The enzyme-linked immunosorbent assay (ELISA) is a simple and rapid technique for detecting and quantitating antibodies or antigens attached to a solid surface. Being...
The enzyme-linked immunosorbent assay (ELISA) is a simple and rapid technique for detecting and quantitating antibodies or antigens attached to a solid surface. Being one of the most sensitive immunoassays, ELISA offers commercial value in laboratory research, diagnostic of disease biomarkers, and quality control in various industries. This technique utilizes an enzyme-linked antibody binding to a surface-attached antigen. Subsequently, a substrate is added to produce either a color change or light signal correlating to the amount of the antigen present in the original sample. This chapter provides the procedures required for carrying out indirect ELISA, one of the many forms of ELISA, to detect polystyrene-immobilized antigen. Methodological approaches to optimize this assay technique are also described, a prerequisite for automation and multiplexing.
Topics: Antibodies; Antigens, Surface; Enzyme Assays; Enzyme-Linked Immunosorbent Assay; Humans; Immobilized Proteins; Light; Luminescence; Polystyrenes; Sensitivity and Specificity
PubMed: 26160563
DOI: 10.1007/978-1-4939-2742-5_5 -
Science Immunology Mar 2023Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface...
Chimeric antigen receptor (CAR) T cell therapy relies on T cells that are guided by synthetic receptors to target and lyse cancer cells. CARs bind to cell surface antigens through an scFv (binder), the affinity of which is central to determining CAR T cell function and therapeutic success. CAR T cells targeting CD19 were the first to achieve marked clinical responses in patients with relapsed/refractory B cell malignancies and to be approved by the U.S. Food and Drug Administration (FDA). We report cryo-EM structures of CD19 antigen with the binder FMC63, which is used in four FDA-approved CAR T cell therapies (Kymriah, Yescarta, Tecartus, and Breyanzi), and the binder SJ25C1, which has also been used extensively in multiple clinical trials. We used these structures for molecular dynamics simulations, which guided creation of lower- or higher-affinity binders, and ultimately produced CAR T cells endowed with distinct tumor recognition sensitivities. The CAR T cells exhibited different antigen density requirements to trigger cytolysis and differed in their propensity to prompt trogocytosis upon contacting tumor cells. Our work shows how structural information can be applied to tune CAR T cell performance to specific target antigen densities.
Topics: United States; Humans; Antigens, CD19; Adaptor Proteins, Signal Transducing; Antigens, Surface; B-Lymphocytes; Cell Death
PubMed: 36867678
DOI: 10.1126/sciimmunol.adf1426 -
Cancer Research Oct 2020Identification of tumor-specific cell surface antigens has proven challenging, as the vast majority of tumor-associated antigens are also expressed in normal tissues. In...
Identification of tumor-specific cell surface antigens has proven challenging, as the vast majority of tumor-associated antigens are also expressed in normal tissues. In mesothelioma, we identified a highly specific tumor cell surface antigen that can be targeted for therapy development. Mesothelioma is caused by malignant transformation of the mesothelium, is incurable, and can be categorized into three histologic subtypes: epithelioid, biphasic, and sarcomatoid. To identity novel mesothelioma cell surface antigens with broad subtype coverage and high tissue specificity, we have previously selected phage antibody display libraries on live mesothelioma cells and tissues following counterselection on normal cells and identified a panel of human antibodies that bind all subtypes of mesothelioma, but not normal mesothelium. One of the antibodies, M25, showed high specificity against an antigen we identify here as ALPPL2. IHC on normal human tissues found that ALPPL2 is expressed only on placental trophoblasts, but not on any other normal tissues. This significant tissue specificity and broad tumor type coverage suggest that ALPPL2 could be an excellent cell surface target for therapeutic development against mesothelioma. To evaluate therapeutic potential of ALPPL2 targeting, an ALPPL2-targeted antibody-drug conjugate was developed and demonstrated potent and specific tumor killing and against both epithelioid and sarcomatoid mesothelioma. Thus, ALPPL2 belongs to a rare class of cell surface antigens classified as truly tumor specific and is well suited for therapy development against ALPPL2-expressing tumors. SIGNIFICANCE: These findings identify ALPP2 as a true tumor-specific cell surface antigen whose tissue specificity enables the development of novel therapies.
Topics: Alkaline Phosphatase; Animals; Antigens, Surface; Antineoplastic Agents, Immunological; CHO Cells; Cell Line, Tumor; Cricetulus; Epitopes; Female; GPI-Linked Proteins; Humans; Immunoconjugates; Immunoglobulin G; Male; Mesothelioma, Malignant; Mice, Inbred NOD; Molecular Targeted Therapy; Xenograft Model Antitumor Assays
PubMed: 32868383
DOI: 10.1158/0008-5472.CAN-20-1418 -
Journal of Nanobiotechnology Nov 2023Pancreatic cancer is a highly aggressive malignancy with limited treatment options and a poor prognosis. Trophoblast cell surface antigen 2 (TROP2), a cell surface...
BACKGROUND
Pancreatic cancer is a highly aggressive malignancy with limited treatment options and a poor prognosis. Trophoblast cell surface antigen 2 (TROP2), a cell surface antigen overexpressed in the tumors of more than half of pancreatic cancer patients, has been identified as a potential target for antibody-drug conjugates (ADCs). Almost all reported TROP2-targeted ADCs are of the IgG type and have been poorly studied in pancreatic cancer. Here, we aimed to develop a novel nanobody-drug conjugate (NDC) targeting TROP2 for the treatment of pancreatic cancer.
RESULTS
In this study, we developed a novel TROP2-targeted NDC, HuNb-MMAE, for the treatment of TROP2-positive pancreatic cancer. HuNb-MMAE is characterized by the use of nanobodies against TROP2 and human serum albumin (HSA) and has a drug-antibody ratio of 1. HuNb-MMAE exhibited specific binding to TROP2 and was internalized into tumor cells with high endocytosis efficiency within 5 h, followed by intracellular translocation to lysosomes and release of MMAE to induce cell apoptosis in TROP2-positive pancreatic cancer cells through the caspase-3/9 pathway. In a xenograft model of pancreatic cancer, doses of 0.2 mg/kg and 1 mg/kg HuNb-MMAE demonstrated significant antitumor effects, and a dose of 5 mg/kg even eradicated the tumor.
CONCLUSION
HuNb-MMAE has desirable affinity, internalization efficiency and antitumor activity. It holds significant promise as a potential therapeutic option for the treatment of TROP2-positive pancreatic cancer.
Topics: Humans; Antigens, Surface; Cell Line, Tumor; Immunoconjugates; Pancreatic Neoplasms; Xenograft Model Antitumor Assays; Animals
PubMed: 37932752
DOI: 10.1186/s12951-023-02183-9 -
Clinical Microbiology and Infection :... Jun 2018The present review is part of the ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies. (Review)
Review
ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Agents targeting lymphoid cells surface antigens [I]: CD19, CD20 and CD52).
BACKGROUND
The present review is part of the ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies.
AIMS
To review, from an Infectious Diseases perspective, the safety profile of agents targeting CD19, CD20 and CD52 and to suggest preventive recommendations.
SOURCES
Computer-based MEDLINE searches with MeSH terms pertaining to each agent or therapeutic family.
CONTENT
Although CD19-targeted agents (blinatumomab or inebilizumab) are not associated with an increased risk of infection, they may cause IgG hypogammaglobulinaemia and neutropenia. The requirement for prolonged intravenous infusion of blinatumomab may increase the risk of catheter-associated bloodstream infections. Infection remains the most common non-haematological adverse effect of anti-CD20 monoclonal antibodies, including severe respiratory tract infection, hepatitis B virus (HBV) reactivation and varicella-zoster virus infection. Screening for chronic or resolved HBV infection is recommended for patients receiving anti-CD20 monoclonal antibodies. Antiviral prophylaxis should be offered for 12-18 months to hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative/anti-hepatitis B core antibody (HBc)-positive patients. Anti-Pneumocystis prophylaxis should be considered in patients receiving concomitant chemotherapy, particularly steroids. Alemtuzumab (anti-CD52) increases the risk of infections, in particular among leukaemia and solid organ transplant patients. These populations benefit from anti-Pneumocystis prophylaxis, prevention strategies for cytomegalovirus infection, and screening for HBV, hepatitis C virus and tuberculosis. Antiviral prophylaxis for at least 6-12 months should be provided for HBsAg-positive patients.
IMPLICATIONS
As there are limited clinical data for many of the reviewed agents, special attention must be given to promptly detect and report emerging infectious complications.
Topics: Antibodies, Monoclonal, Murine-Derived; Antigens, CD19; Antigens, CD20; Antigens, Surface; Biological Therapy; CD52 Antigen; Clinical Trials as Topic; Consensus; Immunocompromised Host; Immunologic Factors; Lymphocytes; Molecular Targeted Therapy; Rituximab; Virus Activation; Virus Diseases
PubMed: 29447988
DOI: 10.1016/j.cmi.2018.02.003 -
Cell Feb 2016T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity...
T cells can be re-directed to kill cancer cells using chimeric antigen receptors (CARs) or T cell receptors (TCRs). This approach, however, is constrained by the rarity of tumor-specific single antigens. Targeting antigens also found on bystander tissues can cause life-threatening adverse effects. A powerful way to enhance ON-target activity of therapeutic T cells is to engineer them to require combinatorial antigens. Here, we engineer a combinatorially activated T cell circuit in which a synthetic Notch receptor for one antigen induces the expression of a CAR for a second antigen. These dual-receptor AND-gate T cells are only armed and activated in the presence of dual antigen tumor cells. These T cells show precise therapeutic discrimination in vivo-sparing single antigen "bystander" tumors while efficiently clearing combinatorial antigen "disease" tumors. This type of precision dual-receptor circuit opens the door to immune recognition of a wider range of tumors. VIDEO ABSTRACT.
Topics: Animals; Antigens, CD19; Antigens, Surface; Bystander Effect; Cell Communication; Cell Line, Tumor; Disease Models, Animal; GPI-Linked Proteins; Humans; Immunotherapy; Jurkat Cells; Lymphocyte Activation; Mesothelin; Mice; Neoplasms; Receptors, Notch; T-Lymphocytes
PubMed: 26830879
DOI: 10.1016/j.cell.2016.01.011 -
Cancer Treatment Reviews Feb 2024Antibody-drug conjugates (ADCs) represent a novel class of molecules composed of a recombinant monoclonal antibody targeted to a specific cell surface antigen,... (Review)
Review
Antibody-drug conjugates (ADCs) represent a novel class of molecules composed of a recombinant monoclonal antibody targeted to a specific cell surface antigen, conjugated to a cytotoxic agent through a cleavable or non-cleavable synthetic linker. The rationale behind the development of ADCs is to overcome the limitations of conventional chemotherapy, such as the narrow therapeutic window and the emergence of resistance mechanisms. ADCs had already revolutionized the treatment algorithm of HER2-positive breast cancer. Currently, emergent non-HER2 targeted ADCs are gaining momentum, with special focus on triple-negative disease therapeutic landscape. Sacituzumab govitecan (SG) is an ADC consisting of a humanized monoclonal antibody hRS7 targeting trophoblast cell surface antigen 2 (Trop2), linked to the topoisomerase I inhibitor SN-38 by a hydrolysable linker. It currently stands as the only non-HER2 targeted ADC that already received approval for the treatment of unresectable locally advanced or metastatic triple negative breast cancer (TNBC) in patients who had received two or more prior systemic therapies, with at least one for advanced disease. The purpose of these review is to analyze the available evidence regarding ADCs in TNBC, alongside with providing an overview on the ongoing and future research horizons in this field.
Topics: Humans; Female; Triple Negative Breast Neoplasms; Breast Neoplasms; Irinotecan; Camptothecin; Immunoconjugates; Antibodies, Monoclonal; Antigens, Surface
PubMed: 38118302
DOI: 10.1016/j.ctrv.2023.102672 -
Journal of Hepatology Nov 2022With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates...
BACKGROUND & AIMS
With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control.
METHODS
We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining, and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly ex vivo.
RESULTS
ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection.
CONCLUSIONS
Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses.
LAY SUMMARY
Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell).
Topics: Antigens, Surface; Antiviral Agents; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Hepatitis B; Hepatitis B virus; Hepatitis B, Chronic; Humans
PubMed: 35716846
DOI: 10.1016/j.jhep.2022.05.041 -
Nature Medicine Aug 2015Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of...
Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 10(9) engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1-LAGE-1 TCR-engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.
Topics: Aged; Antigens, Neoplasm; Antigens, Surface; Female; Genetic Engineering; Humans; Male; Membrane Proteins; Middle Aged; Multiple Myeloma; Receptors, Antigen, T-Cell; Syndecan-1; T-Lymphocytes
PubMed: 26193344
DOI: 10.1038/nm.3910