Introduction
Genetic modification of T cells to express a chimeric antigen receptor
(CAR) has emerged as an effective therapeutic treatment for patients
with B cell hematological malignancies over the last years. CAR T cells
are generated from peripheral T cells isolated from blood of patients.
Based on the differential expression of CD62L, CCR7, CD45RA and CD45RO,
these peripheral T cells can be divided into five subsets: naive T
(TN) cells, which are antigen-unexperienced, effector T
(TEFF) cells, which migrate to sites of inflammation and
promote pathogen clearance, and memory T cells, which persist long-term
to allow protection against subsequent infections. Memory T cells
include stem cell memory (TSCM), central memory
(TCM) and effector memory (TEM) cells .
In humans, T cell differentiation follows a linear progression where
less differentiated cells give rise to more differentiated progeny:
TN > TSCM >
TCM > TEM >
TEFF. During differentiation of TNtowards TEFF cells, the proliferative potential and
memory functions are declining, while effector functions increase.
Notably, the two markers, CD62L and CCR7 are only expressed on
TN and early differentiated (TSCM and
TCM) cells. During T cell isolation and subsequent
cultivation, cells are usually activated using cytokines and stimulating
antibodies to induce T cell proliferation and survival. In the past,
IL-2 was most frequently used for cytokine support, thereby driving T
cell cultures towards terminally differentiated T cells. More recently,
IL-7 and IL‑15 are applied for T cell cultures in an effort to maintain
a more naïve- or memory-like T cell phenotype.
Despite its promising results, CAR T cell therapy still needs to
overcome various hurdles to become standard therapy for all patients in
need. Automated processes have been developed to address the complicated
manufacturing process. However, the most suitable T cell phenotype for
CAR-mediated tumor therapy is a matter of debate. In general, naive and
early memory T cells, are favored for cellular immunotherapy products
due to their higher plasticity, longer persistence and greater
capability to proliferate and differentiate into highly cytolytic
effector cells. Along this line, a beneficial antitumoral function and
cell persistence was associated with a high amount of less
differentiated CAR T cells not only in patients with B-cell malignancies
but also in patients with neuroblastoma.
For the generation of CAR T cell products, lentiviral vectors (LVs)
pseudotyped with the glycoprotein of the vesicular stomatitis virus
(VSV-G), harboring a broad tropism, are commonly used. Optimizing gene
delivery through engineering of vector particles offers the potential to
improve and simplify genetic modification of T cells. In this regard,
receptor-targeted LVs (RT-LVs) specifically transducing CD3, CD4 or CD8
T cells have been described. All three vector types were recently shown
to mediate the generation of CAR T cells directly in vivo in
humanized mouse models. RT-LVs use a cell surface protein of choice as
entry receptor, which can be achieved through pseudotyping with
engineered glycoproteins from paramyxoviruses displaying a
receptor-specific targeting domain, such as a single-chain antibody
fragment (scFv) or designed ankyrin repeat molecule (DARPin). However,
the T cell specific LVs available so far do not discriminate between the
differentiation phenotype and exhaustion status of T cells.
Here we describe the generation of a RT-LV that is specific for a T cell
marker expressed on less differentiated T cells: CD62L. The specificity
of this vector was mediated by displaying a CD62L-specific scFv on
measles virus (MV)-based RT-LVs. The resulting CD62L-LV mediated
efficient gene delivery and preserved a higher degree of less
differentiated CAR T cells upon long-term culture. CAR T cells generated
through short-term incubation with CD62L-LV controlled tumor burden in
an in vivo setting.