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Generation and characterization of antibody fragments against a receptor expressed on regulatory T cells

Holgersson, Stina LU (2015) KIM820 20142
Department of Immunotechnology
Abstract
A receptor upregulated on regulatory T cells, in this work referred to as receptor X, is a candidate target for cancer treatment. An antibody targeting this receptor could potentially suppress and/or deplete regulatory T cells and hereby lead to activation of effector T cells and tumor regression. In this work fully human scFv fragments targeting receptor X were isolated from a phage display library. Antibodies were isolated against both the human and the murine variant of the receptor using three different selection strategies per receptor variant and three consecutive pannings per strategy. Various combinations of soluble proteins and cells expressing X were used as targets in the selections. ELISA and flow cytometry demonstrated strong... (More)
A receptor upregulated on regulatory T cells, in this work referred to as receptor X, is a candidate target for cancer treatment. An antibody targeting this receptor could potentially suppress and/or deplete regulatory T cells and hereby lead to activation of effector T cells and tumor regression. In this work fully human scFv fragments targeting receptor X were isolated from a phage display library. Antibodies were isolated against both the human and the murine variant of the receptor using three different selection strategies per receptor variant and three consecutive pannings per strategy. Various combinations of soluble proteins and cells expressing X were used as targets in the selections. ELISA and flow cytometry demonstrated strong enrichment of target-specific phages after the third panning in all strategies. Binding properties of individual soluble scFv were analyzed in a primary and, for a subset of binders, a secondary screening. In the primary screening cell binding specificity was assessed using Fluorometric Microvolume Assay Technology resulting in an active frequency above 50% for all selection strategies. In the secondary screening both cell binding specificity (retest of primary screening) and protein binding specificity using ELISA were determined. A vast majority of clones were confirmed as cell binders (95%) and most clones were also protein binders (75%). DNA sequencing indicated high diversity among isolated hit clones. Unique hit clones will be converted into IgG format and further analyzed in functional in vitro and in vivo assays. (Less)
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author
Holgersson, Stina LU
supervisor
organization
course
KIM820 20142
year
type
H2 - Master's Degree (Two Years)
subject
keywords
immunotechnology, immunotherapy, cancer, antibodies
language
English
id
5426152
date added to LUP
2015-06-10 16:50:48
date last changed
2015-06-10 16:50:48
@misc{5426152,
  abstract     = {A receptor upregulated on regulatory T cells, in this work referred to as receptor X, is a candidate target for cancer treatment. An antibody targeting this receptor could potentially suppress and/or deplete regulatory T cells and hereby lead to activation of effector T cells and tumor regression. In this work fully human scFv fragments targeting receptor X were isolated from a phage display library. Antibodies were isolated against both the human and the murine variant of the receptor using three different selection strategies per receptor variant and three consecutive pannings per strategy. Various combinations of soluble proteins and cells expressing X were used as targets in the selections. ELISA and flow cytometry demonstrated strong enrichment of target-specific phages after the third panning in all strategies. Binding properties of individual soluble scFv were analyzed in a primary and, for a subset of binders, a secondary screening. In the primary screening cell binding specificity was assessed using Fluorometric Microvolume Assay Technology resulting in an active frequency above 50% for all selection strategies. In the secondary screening both cell binding specificity (retest of primary screening) and protein binding specificity using ELISA were determined. A vast majority of clones were confirmed as cell binders (95%) and most clones were also protein binders (75%). DNA sequencing indicated high diversity among isolated hit clones. Unique hit clones will be converted into IgG format and further analyzed in functional in vitro and in vivo assays.},
  author       = {Holgersson, Stina},
  keyword      = {immunotechnology,immunotherapy,cancer,antibodies},
  language     = {eng},
  note         = {Student Paper},
  title        = {Generation and characterization of antibody fragments against a receptor expressed on regulatory T cells},
  year         = {2015},
}