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1. WO2020109355 - METHODS AND KIT FOR ASSAYING LYTIC POTENTIAL OF IMMUNE EFFECTOR CELLS

Publication Number WO/2020/109355
Publication Date 04.06.2020
International Application No. PCT/EP2019/082672
International Filing Date 27.11.2019
IPC
G01N 33/50 2006.01
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
CPC
G01N 33/5011
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
48Biological material, e.g. blood, urine
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
5005involving human or animal cells
5008for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
5011for testing antineoplastic activity
G01N 33/5047
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
48Biological material, e.g. blood, urine
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
5005involving human or animal cells
5008for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
5044involving specific cell types
5047Cells of the immune system
G01N 33/505
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
48Biological material, e.g. blood, urine
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
5005involving human or animal cells
5008for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
5044involving specific cell types
5047Cells of the immune system
505involving T-cells
Applicants
  • INSERM (INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECHERCHE MÉDICALE) [FR]/[FR]
  • UNIVERSITÉ PAUL SABATIER TOULOUSE III [FR]/[FR]
Inventors
  • VALITUTTI, Salvatore
  • FILALI, Liza
  • MUELLER, Sabina
  • PUISSEGUR, Marie-Pierre
Agents
  • INSERM TRANSFERT
Priority Data
18306576.228.11.2018EP
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) METHODS AND KIT FOR ASSAYING LYTIC POTENTIAL OF IMMUNE EFFECTOR CELLS
(FR) MÉTHODES ET KIT DE DOSAGE DU POTENTIEL LYTIQUE DE CELLULES EFFECTRICES IMMUNITAIRES
Abstract
(EN)
To overcome the difficulty in achieving a quantitative assessment of CTL function in clinical settings, the inventors aimed at developing a new method inspired by their knowledge of the CTL/tumor target biology and based on flow cytometry. In particular, to directly detect the earliest steps of tumor cell resistance to CTL attack at the lytic synapse the inventors developed a method to monitor CTL/tumor cells interaction in the presence of FM1-43, a fluorescent lipophilic dye that rapidly intercalates into lipid bilayer during the membrane turnover that accompanies plasma membrane reparation. This assay allows the inventors to measure reparative membrane turnover of tumor cells under CTL attack by FACS analysis at the whole tumor cell population level. They initially applied this approach to compare the response of melanoma cell (D10 cells) to CTL attack as compared to conventional target cells that are sensitive to CTL mediated cytotoxicity (EB V -transformed B cells, JY cells). The methodology allows to rapidly assessing the synaptic resistance of tumor target cells to CTL attack and the intrinsic capacity of CD8+CTL to efficiently kill their target cells. Thus, the present invention relates to methods and kit for assaying lytic potential of immune effector cells and uses thereof in diagnostic assays.
(FR)
Afin de surmonter la difficulté de la réalisation d'une évaluation quantitative de la fonction CTL dans des réglages cliniques, les inventeurs ont pour objectif de développer une nouvelle méthode inspirée de leur connaissance de la biologie de cible de CTL/tumeur et fondée sur la cytométrie en flux. Plus précisément, afin de détecter directement les étapes les plus précoces d'une résistance de cellule tumorale à une attaque CTL au niveau de la synapse lytique, les inventeurs ont mis au point une méthode de surveillance d'une interaction CTL/cellules tumorales en présence de FM1-43, un colorant lipophile fluorescent qui s'intercale rapidement dans la bicouche lipidique pendant le renouvellement de la membrane qui accompagne la réparation de la membrane plasmatique. Ledit dosage permet aux inventeurs de mesurer le renouvellement de membrane réparatrice de cellules tumorales sous une attaque CTL par analyse FACS au niveau de la population de cellules tumorales entière. Ils ont initialement appliqué cette approche pour comparer la réponse d'une cellule de mélanome (cellules D10) à une attaque CTL par comparaison avec des cellules cibles classiques sensibles à la cytotoxicité médiée par CTL (cellules B transformées par EB V, cellules JY). La méthodologie permet d'évaluer rapidement la résistance synaptique de cellules cibles tumorales à une attaque CTL et la capacité intrinsèque de CTL+CD8 à tuer efficacement leurs cellules cibles. Ainsi, la présente invention concerne des méthodes et un kit de dosage du potentiel lytique de cellules effectrices immunitaires et des utilisations correspondantes dans des dosages diagnostiques.
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