ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 5, pp. 765-783 © Pleiades Publishing, Ltd., 2024.
765
REVIEW
CAR Cells beyond Classical CAR T Cells:
Functional Properties and Prospects of Application
Elizaveta P. Minina
1
, Dmitry V. Dianov
1
, Saveliy A. Sheetikov
1
,
and Apollinariya V. Bogolyubova
1,a
*
1
National Medical Research Centre for Hematology, Ministry of Health of the Russian Federation,
125167 Moscow, Russia
a
e-mail: apollinariya.bogolyubova@gmail.com
Received August 14, 2023
Revised November 23, 2023
Accepted December 2, 2023
Abstract— Chimeric antigen receptors (CARs) are genetically engineered receptors that recognize antigens and
activate signaling cascades in a cell. Signal recognition and transmission are mediated by the CAR domains derived
from different proteins. T cells carrying CARs against tumor-associated antigens have been used in the develop-
ment of the CAR T cell therapy, a new approach to fighting malignant neoplasms. Despite its high efficacy in the
treatment of oncohematological diseases, CAR T cell therapy has a number of disadvantages that could be avoided
by using other types of leukocytes as effector cells. CARs can be expressed in a wide range of cells of adaptive and
innate immunity with the emergence or improvement of cytotoxic properties. This review discusses the features
of CAR function in different types of immune cells, with a particular focus on the results of preclinical and clinical
efficacy studies and the safety of potential CAR cell products.
DOI: 10.1134/S0006297924050018
Keywords: chimeric antigen receptor, immunotherapy, cell therapy, CAR, CAR T cell, CAR NK cell
Abbreviations: CAR,chimeric antigen receptor; CARM,macrophage expressing CAR; CAR T,T cell expressing CAR; CBCR,chi-
meric B cell receptor; CIK,cytokine-induced killers; CRS,cytokine release syndrome; DC,dendritic cell; DN T cell,double-neg-
ative T cell; GD2,disialoganglioside GD2; GVHD,graft-versus-host disease; iNKT cell,invariant NKT cell; MAIT cell,mucosal-
associated invariant T cell; MHC,major histocompatibility complex; NK,natural killer cell; NKT cells,natural killer T cell;
NSCAR,non-signaling CAR; TCR,T cell receptor; Treg,regulatory T cell.
* To whom correspondence should be addressed.
INTRODUCTION
A chimeric receptor consisting of the variable
immunoglobulin domain and constant regions of the
T cell receptor (TCR) was first created in 1987 [1].
It recognized the bacterial antigen phosphorylcholine
and was expressed in EL4 lymphoblastic T cells. The
first T lymphocytes with a chimeric antigen receptor
(CAR) were obtained in 1993 [2]. This receptor recog-
nized 2,4,6-trinitrophenol and was one of the first gen-
eration of CARs. It consisted of the extracellular anti-
gen-recognizing single-chain variable fragment (scFv)
linked by a transmembrane region to the intracellular
CD3ζ signaling domain (a fragment of the endogenous
TCR) [3]. CAR T cells expressing the first-generation re-
ceptors were poorly effective against malignant cells
because, despite of their strong cytotoxic properties,
they were easily exhausted [4]. In 2002, second-gen-
eration CARs were obtained. They contained the CD28
costimulatory domain between the signaling and trans-
membrane domains [5]. Second-generation CAR T cells
targeting the CD19 antigen were effective in recogniz-
ing and eliminating B cell tumors in a mouse model
[6]. Since then, CAR T lymphocytes targeting various tu-
mor-associated antigens have been actively developed.
CAR T therapy has been particularly effective in
the treatment of hematological oncological diseases.
To date, the Food and Drug Administration (FDA) has
approved six CAR-T therapies for the treatment of
B-cell neoplasms [7]. So far, CAR-T products that have
MININA et al.766
BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
successfully passed clinical trials and have been ap-
proved by FDA are based on the second-generation
CARs containing CD28 or 4-1BB signaling domains as
costimulatory domains. Other variants of CAR T cells
containing receptors with different immunomodulato-
ry domains are being researched and developed. For
example, third-generation CARs containing two costim-
ulatory domains have been developed, although their
clinical use has been limited due to strong severe side
effects. CAR T cells expressing fourth-generation CARs
also secrete cytokines that increase the persistence of
these cells in the tumor microenvironment [8].
Despite significant success in the treatment of
oncohematological diseases, CAR T cell therapy has a
number of drawbacks. For example, it becomes inef-
fective when malignant cells lose the tumor antigens
targeted by the CARs [8]. CAR T cells are often unable
to infiltrate solid tumors and recognize the antigen.
In addition, the cytotoxic function of CAR T cells is
significantly influenced by the immunosuppressive
tumor microenvironment. Currently, progress in the
treatment of solid tumors with CAR T cells has reached
its limits [9]. Finally, the secretion of pro-inflammatory
cytokines by activated CAR T cells can lead to severe
side effects, such as cytokine release syndrome (CRS)
and neurotoxicity [10], which are due to the rapid ac-
tivation and proliferation of T cells producing pro-in-
flammatory cytokines and typically develop within
one week of CAR T cell administration. It has been sug-
gested that excessive release of pro-inflammatory cy-
tokines increases capillary permeability in the brain,
leading to severe neurological symptoms and even
death, although the molecular mechanisms of neuro-
toxicity in the case of CAR T cell therapy are not ful-
ly understood [10]. The adverse effects of this therapy
are often due to the poor regulation of the activity of
CAR T cells, which can sometimes be activated even
inthe absence of an antigenic stimulus [11].
Fig. 1. Advantages and disadvantages of different types of immune cells in the development of CAR-bearing cell products.
Nomenclature: TCR, T cell receptor; CAR, chimeric antigen receptor; NKG2D, activating receptor; MR1, non-canonical major
histocompatibility complex close to classI; CD1,non-canonical major histocompatibility complex close to classI; CRS,cytokine
release syndrome; MHC,major histocompatibility complex; GVHD,graft-versus-host disease.
CAR CELLS BEYOND CLASSICAL CAR T CELLS 767
BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024
To overcome these drawbacks, new strategies for
the CAR T cell generation are being actively being de-
veloped [12, 13], including the use of different (non-
T cells) leukocytes as CAR-bearing cells (Fig. 1). CAR-
expressing cells have been derived from γδ T cells,
regulatory T cells (Tregs), mucosal-associated invariant
T cells (MAIT cells), double-negative T cells (DNTcells),
natural killer (NK) cells, natural killer T cells (NKT
cells), cytokine-induced killers (CIKs), macrophages,
dendritic cells (DCs), and even B lymphocytes.
Preclinical studies have shown that many alterna-
tive immune CAR cells with a high antitumor activity
have fewer or none of the typical drawbacks of CAR
T cells. However, such cells are poorly studied. Only a
few cell products have entered phase I/II clinical tri-
als; some have preclinical data, while others are still
under development. This review discusses the features
of CAR function in different types of immune cells and
the results of available preclinical and clinical studies
of CAR-bearing immune cells.
CARs IN DIFFERENT TYPES
OF IMMUNE CELLS
Below we discuss the properties of different im-
mune cells that could be used in the development of
novel CAR-mediated immunotherapies, focusing on
the results of preclinical and, if available, clinical stud-
ies of CAR expression and efficacy in these cells.
Natural killer (NK) cells are innate immune cells
of the lymphoid lineage. NK cells account approx-
imately 10% of peripheral blood lymphocytes [14].
Unlike T lymphocytes, they recognize malignant and
infected cells with a variety of non-polymorphic acti-
vating and inhibitory receptors. The directionality of
the NK cell response in each case is the result of a bal-
ance of various signals. When the signals from the ac-
tivating receptors outweigh those from the inhibitory
receptors, an NK cell causes a lysis of the target cell.
NK cells express a variety of activating and inhibiting
receptors on their surface, the most important among
them are receptors that recognize the major histocom-
patibility complex MHC I and MHC I-like molecules.
Cells that express these molecules are recognized as
healthy by NK cells [15]. It is important to note that red
blood cells do not carry the ligands (either activating
or inhibiting) for the NK cell receptors on their surface
and therefore are not recognized as targets by these
cells. In addition to MHC, NK cells can recognize other
cell surface ligands, in particular, the stress markers
MICA, MICB, and UL16BP1, due to the presence of spe-
cific receptors, including the NKG2D receptor, which is
also present on the surface of γδ T cells [16]. In addi-
tion, the expression of FcγRIIIa allows NK cells to rec-
ognize and kill cells opsonized by antibodies [17].
The cytotoxic function of NK cells is manifested by
the formation of an immunological synapse between
the NK cell and the target cell and the subsequent se-
cretion of granzyme-containing lytic granules. NK cells
can also kill target cells by inducing programmed cell
death through FasL and TRAIL. Finally, activated NK
cells release a number of pro-inflammatory cytokines,
in particular, interferon γ (IFNγ) and tumor necrosis
factor (TNF) [14].
The cytotoxic properties of NK cells and the lack
of need for the antigen presentation in the MHC con-
text required for the recognition of target cells (unlike
in T cells), have significantly contributed to the devel-
opment of anti-cancer therapies based on genetically
modified NK cells, including those expressing CARs.
In most studies, NK cells have been transduced with
the CAR constructs originally developed for the CAR
T cells therapy. In addition to the CD28 and 4-1BB co-
stimulatory domains, some of the CARs expressed
in NK cells contained the 2B4 costimulatory domain
(Fig. 2). The 2B4 receptor is one of the activating re-
ceptors of the SLAM (signaling lymphocytic activation
molecule) family on NK cells. It is important to note
that CAR constructs originally designed for expression
in T cells can also function in NK cells due to the pres-
ence of common signaling pathways that control cell
activation in both cell types. In particular, the signal-
ing from some activating receptors in NK cells involves
the CD3ζ signaling domain which is intended for use
in T cells [18]. NK cells transduced with the second-
generation 2B4-containing CAR targeting CD5 had a
higher cytotoxic activity against malignant cells and
showed more rapid proliferation and enhanced cyto-
kine production compared to NK cells expressing CARs
with the CD28 domain [19]. In addition to the CD3ζ do-
main, CARs developed for the expression in NK cells
can contain the DAP10 and DAP12 domains, which are
involved in the signaling from a number of activating
NK cell receptors. It has been shown that the antitu-
mor activity observed for the constructs with the CD3ζ
domain was higher than that of those with the DAP10
domain, but lower than the activity of CARs with the
DAP12 domain [20,21]. Cifaldi etal. [22] proposed the
use of DNAM-1 as a part of a CAR adapted to NK cells.
DNAM-1 recognizes the poliovirus receptor (PVR) and
nectin-2, which are expressed on the virus-infected
cells and many malignant cells. Receptors contain-
ing the 2B4 and CD3ζ domains in addition to DNAM-1
cause further activation of NK cells.
It should be emphasized that CAR-expressing NK
cells can potentially exhibit cytotoxic activity against
malignant cells in a CAR-independent manner due to
their own activating receptors, as well as their abili-
ty to recognize cells opsonized by antibodies [20,23].
The presence of a CAR-independent antitumor activ-
ity enhances the efficacy of the CAR NK cell therapy.