Abstract
Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.
Highlights
The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al, 2019)
Because retroviruses infect proliferating cells (Kusabuka et al, 2016; Chinnasamy et al, 2010; Hu et al, 2017), we first compared three commonly used methods for inducing T cell activation: (i) magnetic beads coated with anti-(α) CD3 antibody (Ab) and αCD28 Ab plus recombinant human IL-2, (ii) plate-immobilized αCD3 Ab along with soluble αCD28 Ab plus hIL-2, and (iii) Concanavalin A plus hIL-2 and hIL-7
Given the enhanced functional properties of CART cells exposed to hIL-7/IL-15 at 48 h after transduction as opposed to hIL-2 alone, we focused on coengineering T cells to constitutively produce murine interleukin-15 (mIL-15)
Summary
The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al, 2019). Another approach to ACT involves the redirection of peripheral blood T cells to tumor antigens by engineering them to express a chimeric antigen receptor (CAR) that triggers cellular activation upon tumor antigen binding. A range of physical (Lanitis et al, 2015) and immunometabolic barriers that can prevent T cell homing, transendothelial migration across tumor blood vessels, engraftment/persistence, and effector function limit the potency of CAR-T cell therapy against solid tumors (Brown et al, 2016; Louis et al, 2011). CAR-T cells can be armed with secretory molecules or additional receptors to support CAR-T cell activity and/or harness endogenous immunity (Adachi et al, 2018; Pegram et al, 2012)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
