Heterodimerizacijske domene človeškega izvora za uravnavanje celičnega signaliziranja CAR-T

Jelenčič, Amadej (author), Jerala, Roman (mentor), Golob Urbanc, Anja (comentor)

Terapije s celicami T, ki izražajo himerne antigenske receptorje (ang. chimeric antigen receptor T cells; CAR-T), predstavljajo učinkovit pristop za zdravljenje določenih malignih obolenj. Zaradi resnih stranskih učinkov, ki se pri teh terapijah pojavljajo, je pomemben razvoj strategij za uravnavanje aktivnosti celic CAR-T. Načrtovali in testirali smo pristop inducibilne inhibicije signaliziranja receptorjev CAR na osnovi kemijsko inducirane dimerizacije (ang. chemically induced dimerization; CID). Kot heterodimerizacijski domeni smo uporabili človeški retinol vezavni protein 4 (ang. human retinol binding protein; hRBP4) in načrtovan hRBP4 vezavni protein RS3, dimerizacijo katerih sproži mala molekula A1120. Ugotovili smo, da slednji ligand do koncentracije 10 µM ni citotoksicen za testirane sesalske celice. S testom z razcepljeno luciferazo nam ni uspelo dokazati delovanja sistema CID. Nato smo načrtovali receptorje CAR z znotrajceličnima ali zunajceličnima domenama hRBP4 in RS3, ki naj bi ob dodatku A1120 dimerizirali in k znotrajcelični signalizacijski domeni receptorja CAR pripeljali inhibitorno domeno fosfataze CD45, kar bi inhibiralo nadaljnje signaliziranje. Za preverjanje inducibilne inhibicije smo celice Jurkat, ki so izražale naše konstrukte, gojili s tumorskimi celicami Raji z izraženim tarčnim antigenom CD19, ob odsotnosti in prisotnosti induktorja ter izmerili koncentracijo človeškega interlevkina-2 v supernatantu. Pri nobenem izmed načrtovanih receptorjev CAR s sistemom CID, ob dodatku A1120, niti pri najvišji uporabljenih koncentracijah, nismo opazili inhibicije signaliziranja preko CAR. Ugotavljamo, da sistem CID na osnovi hRBP4 in RS3 ni primeren za namen inhibicije signaliziranja preko receptorjev CAR z inhibitornimi domenami CD45.

• Language: Slovenian
• Keywords: CAR-T celice, CAR, CRS, CID, uravnavanje signaliziranja
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2023
• PID: 20.500.12556/RUL-148534
• COBISS.SI-ID: 162424067
• Publication date in RUL: 26.08.2023

Secondary language

• Language: English
• Title: Heterodimerization domains of human origin for regulation of CAR-T cell signaling

Abstract

Chimeric antigen receptor T-cell (CAR-T) therapies are an effective treatment for certain malignancies. Due to the occurrence of serious side effects, the development of strategies to regulate CAR-T cell activation is crucial. We developed and tested an approach of inducible inhibition of CAR signaling based on the chemically induced dimerization (CID) system consisting of the heterodimerization domains human retinol-binding protein 4 (hRBP4) and modified archaeal protein RS3, which dimerize upon addition of the inducer A1120. We found the latter not to be excessively cytotoxic up to the concentration of 10 µM. Split luciferase assay failed to show the functioning of the CID system. We generated fusion proteins based on intra- and extracellular hRBP4 and RS3 domains that were supposed to dimerize after the addition of A1220, bringing the inhibitory domains of the phosphatase CD45 to the intracellular signaling region of CAR and inhibiting further signaling. To test inducible inhibition, we cocultivated Jurkat cells expressing our fusion proteins with Raji cells expressing the target antigen CD19 in the absence or presence of A1120 and measured the concentration of human interleukin-2 (hIL-2) in the supernatant. We did not observe inhibition upon the addition of 5 or 10 µM A1120 with the constructs containing intra- or extracellular heterodimerization domains. We conclude that the CID system based on hRBP3 and RS3 is not suitable for use in CAR signaling inhibition with CD45 inhibitory domains.

• Keywords: CAR-T cells, CAR, CRS, CID, regulation of signaling