Towards canine immunotherapy models: Monoclonal antibodies with redox regulated epitopes targeting TIM3 attenuate Galectin-9 binding

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61389030%3A_____%2F26%3A00646729" target="_blank" >RIV/61389030:_____/26:00646729 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1016/j.ab.2025.116033" target="_blank" >https://doi.org/10.1016/j.ab.2025.116033</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ab.2025.116033" target="_blank" >10.1016/j.ab.2025.116033</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Towards canine immunotherapy models: Monoclonal antibodies with redox regulated epitopes targeting TIM3 attenuate Galectin-9 binding

Popis výsledku v původním jazyce

The TIM3 receptor acts as an immune checkpoint protein. Canine cancers exhibit higher pan-cancer penetrance of TIM3 compared to PD1, highlighting the potential of TIM3 as a compelling target in comparative immuno-oncology. We have used a highly diverse naïve canine scFv phage library to isolate antibodies to canine TIM3. Alternating rounds of biopanning were performed using either Fc or GST tagged canine TIM3 synthesized in mammalian cells. scFv sequences were identified using colony screening and next generation deep sequencing (NGS). The NGS protocol identified lower abundant frequency clones, demonstrating the enhanced depth of repertoire discovery possible using sequence-tag based tracing. Three representative scFv were expressed as mouse-canine chimeric scFv-Fc fusions or as full-length chimeric IgG. These antibodies all bound to the TIM3 receptor using either ELISA or immunoblotting. All the antibodies displayed sensitivity to reducing agents, which indicates the existence of disulfide-stabilized conformational epitopes. Epitope mapping using pepscan libraries suggested that the antibodies recognize a shared structural motif within the IgV domain of TIM3, within β-sheets fixed by disulfide bonds which would form the conformational epitope. Such conformational epitopes might be functional because they overlap with ligand-binding interfaces. Consistent with this, the antibodies attenuated TIM3 binding to Galectin-9. These data affirm that naïve canine scFv antibody libraries can yield self-antigen reactive antibodies to immune blockade receptor antigens. The data also emphasize the value in using native, folded, mammalian expressed receptor antigens to increase the probability of acquiring conformationally sensitive antibodies with potential therapeutic applications in veterinary and human medicine.

Název v anglickém jazyce

Towards canine immunotherapy models: Monoclonal antibodies with redox regulated epitopes targeting TIM3 attenuate Galectin-9 binding

Popis výsledku anglicky

The TIM3 receptor acts as an immune checkpoint protein. Canine cancers exhibit higher pan-cancer penetrance of TIM3 compared to PD1, highlighting the potential of TIM3 as a compelling target in comparative immuno-oncology. We have used a highly diverse naïve canine scFv phage library to isolate antibodies to canine TIM3. Alternating rounds of biopanning were performed using either Fc or GST tagged canine TIM3 synthesized in mammalian cells. scFv sequences were identified using colony screening and next generation deep sequencing (NGS). The NGS protocol identified lower abundant frequency clones, demonstrating the enhanced depth of repertoire discovery possible using sequence-tag based tracing. Three representative scFv were expressed as mouse-canine chimeric scFv-Fc fusions or as full-length chimeric IgG. These antibodies all bound to the TIM3 receptor using either ELISA or immunoblotting. All the antibodies displayed sensitivity to reducing agents, which indicates the existence of disulfide-stabilized conformational epitopes. Epitope mapping using pepscan libraries suggested that the antibodies recognize a shared structural motif within the IgV domain of TIM3, within β-sheets fixed by disulfide bonds which would form the conformational epitope. Such conformational epitopes might be functional because they overlap with ligand-binding interfaces. Consistent with this, the antibodies attenuated TIM3 binding to Galectin-9. These data affirm that naïve canine scFv antibody libraries can yield self-antigen reactive antibodies to immune blockade receptor antigens. The data also emphasize the value in using native, folded, mammalian expressed receptor antigens to increase the probability of acquiring conformationally sensitive antibodies with potential therapeutic applications in veterinary and human medicine.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10609 - Biochemical research methods

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2026

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Analytical Biochemistry

ISSN

0003-2697

e-ISSN

1096-0309

Svazek periodika

711

Číslo periodika v rámci svazku

APR

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

21

Strana od-do

116033

Kód UT WoS článku

001665100000001

EID výsledku v databázi Scopus

2-s2.0-105026986566