Generation and characterization of antibody fragments against a receptor expressed on regulatory T cells
(2015) KIM820 20142Department 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)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/5426152
- author
- Holgersson, Stina LU
- supervisor
- organization
- course
- KIM820 20142
- year
- 2015
- 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}}, language = {{eng}}, note = {{Student Paper}}, title = {{Generation and characterization of antibody fragments against a receptor expressed on regulatory T cells}}, year = {{2015}}, }