T lymphocyte engineering for the recognition of acute lymphoid leukemia

Author(s)

Carolina Dias Carlos

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-01-29

Examining board members

Pedro Otavio de Campos Lima; José Andrés Yunes; Sang Won Han; Fábio Cesar Gozzo; Jane Zveiter de Moraes

• Advisor: Pedro Otavio de Campos Lima

Abstract

Introduction: Acute lymphoid leukemia (ALL) is the most commom form of childhood cancer, representing 31% of all tumors. The conventional treatment in children with ALL is based on chemotherapeutic regimens which have been presenting good results with cure rates around 80% to 90%. Despite this, 10-20% of the children present failure of response to the first category therapy, as well as the occurence of desease relapse. The immunotherapy with genetically modified T-cells has been shown promising results to the relapse treatment. However, there are some limiting aspects to incorporate it to the regular therapeutic conduct, such as: the great amount of vector to be produced, the elevated number of cells to be transduced, notwithstanding the in vivo expansion, the elevated cost of the vectors production/purification and lymphocytes transduction and susceptibility to apoptosis of the injected lymphocytes, specially in a tumoral microenvironment. Objective: Before that, we proposed to optimize the use of the glycolytic pathway of these anti-CD19 CAR T-cells, in order to standardize and improve the bioenergetic profile, making these lymphocytes more effective in responding and effector useful life. Methods: In our strategy, T-cells were genetically modified in ex vivo with lentivirals to induce CAR anti- CD19 expression and the glucose transport Glut-1. Those reprogrammed cells were studied in effector function tests in vitro. Results: We succeeded in the CAR anti-CD19 expression in the T- cells surface, with good transduction rates between 40 and 70%. The T-cells population modified with CAR anti-CD19-GLUT1 owns an increased expression of the GLUT1 transgene in the membrane, generating a bigger internalization of the glucose. These lymphocytes also exhibited increased utilization of the glycolytic pathway, increased basal respiration, maximum respiration capacity and respiratory reserve. There was increase in INF-gama production and proved to be with a specific cytotoxic effector activity acknowledging and eliminating the leukemic target CD19+. They also showed a bigger apoptosis resistance in glucose limiting conditions and a bigger proliferative advantage in response to stimulus with the target cell Nalm-6. Conclusions: A global analysis of the results indicates that the coexpression of the glucose conveyor GLUT1 with the chimeric receptor anti-CD19, promotes a bioenergetic advantage, produzing a more efficient effector activity profile and may make this cellular immunotherapy more accessible to leukemia patients. However, this approach should be tested in vivo its clinical relevance. (AU)

• FAPESP's process: 15/03009-4 - Engineering of T lymphocytes for the recognition of Acute Lymphoblastic Leukemia.
• Grantee: Carolina Dias Carlos
• Support Opportunities: Scholarships in Brazil - Doctorate