Advanced search
Start date
Betweenand


Functional characterization of anti-CD19 CAR-T lymphocytes polarized for the Th17 phenotype through RORγt overexpression

Full text
Author(s):
Heloisa Brand
Total Authors: 1
Document type: Master's Dissertation
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Lucas Eduardo Botelho de Souza; Niels Olsen Saraiva Câmara; Vanderson Geraldo Rocha
Advisor: Lucas Eduardo Botelho de Souza
Abstract

Adoptive transfer of T cells engineered to express anti-CD19 chimeric antigen receptors (CAR) has resulted in complete remission rates above 80% in patients with B-cell acute lymphoblastic leukemia. However, the clinical response is poor (~30 %) even in other B-cell neoplasms, such as chronic lymphocytic leukemia. Solid tumors have been even more challenging, as CAR-T cells face obstacles such as low persistence, low infiltration and functional exhaustion. As an alternative to increase the effectiveness of CAR-T cells, preclinical evidence indicates that lymphocytes polarized to the T helper 17 phenotype (Th17) are more efficient in eradicating tumors than Th1 cells. Polarization to Th17 in vitro involves culturing the cells with a cocktail of cytokines and neutralizing antibodies, which increases the complexity and the cost of manufacturing. In response to cytokines, the transcription factor retinoic acid gamma 2-related orphan receptor (RORγt) orchestrates the polarization to Th17. Thus, we hypothesized that the co-expression of a CAR and RORγt in human T cells would generate CAR-T cells with a Th17 phenotype. To test our hypothesis, we produced lentiviral vectors containing the genes encoding the anti-CD19 CAR and/or RORγt (for the production of rTh17-CAR). For comparison purposes, we also established a Th17 polarization method through culture with cytokines and antibodies (cTh17). After transduction, we evaluated the cytotoxicity of CAR-T cells in vitro and in vivo against Raji cells modified to express luciferase. In vitro cytotoxicity was monitored during co-culture with Raji cells by bioluminescence quantification, and IL-17 and IFN-γ release was measured by ELISA. During the establishment of Th17 polarization with cytokines, we observed that the use of the RORγt agonist increased the transcription of IL-17A by ~45%, without changing the transcription of RORC2. Therefore, we included the RORγt agonist to the cTh17 production protocol. After transduction to express CAR and/or RORγt, cTh17-CAR, rTh17-CAR and CD4-CAR cells mixed with CD8-CAR cells in a 1:1 ratio showed similar in vitro cytotoxicity against CD19+ lymphoma cells. We also observed that CD4-CAR and cTh17-CAR cells exhibited cytotoxicity in the absence of CD8+ cells. Furthermore, the co-expression of CAR and RORγt generated CAR-T cells with a Th17 profile, defined by IL-17 secretion (~1000 pg/mL/24h). IL-17 secretion by cTh17 cells was ~2000 pg/mL/24h and non-polarized cells showed negligible secretion. In an in vivo model of disseminated lymphoma, rTh17-CAR/CD8-CAR and cTh17-CAR/CD8-CAR populations had a lower antitumor capacity than conventional CD4-CAR/CD8-CAR, indicating that the superior antitumor Th17 activity reported by previous studies may depend on the tumor type. Understanding these particularities is essential to benefit from the therapeutic potential of Th17 cells. We conclude that RORγt overexpression generates cells with a Th17 profile, whose efficacy has been demonstrated in preclinical models of solid neoplasms. Our data establish a simplified strategy for the manufacture of CAR-T cells with Th17 phenotype that can be used to evaluate the therapeutic efficacy of this population in other neoplasms. (AU)

FAPESP's process: 19/18702-8 - Generation and evaluation of antitumor efficiency of CAR-T cells harboring a T helper 17 phenotype
Grantee:Heloisa Brand
Support Opportunities: Scholarships in Brazil - Master