Article

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

, Dmitry V. Dianov

, Saveliy A. Sheetikov

and Apollinariya V. Bogolyubova

1,a

National Medical Research Centre for Hematology, Ministry of Health of the Russian Federation,

125167 Moscow, Russia

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; CARM,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

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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

inthe 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 classI; CD1,non-canonical major histocompatibility complex close to classI; CRS,cytokine

release syndrome; MHC,major histocompatibility complex; GVHD,graft-versus-host disease.

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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 (DNTcells),

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 etal. [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.

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Fig. 2. Structure of CARs used in the development of products based on different cell types. Nomenclature: NK cells,natural killer

cells; NKT cells,natural killer T cells.

Inaddition, the diversity of activating receptors allows

CAR NK cells to recognize and destroy tumor cells with

an altered phenotype that have survived a long-term

treatment [24]. NK cells treated with exogenous IL-12

and IL-18 acquire a phenotype similar to that of mem-

ory cells. The efficacy of such cells has already been

demonstrated in phase I trials in patients with re-

lapsed or resistant acute myeloid leukemia. Additional

expression of a CAR directed against nucleophosmin1

(NPM1) in these cells increased the efficacy of the

therapy and reduced the incidence of side effects [25].

Anincreased sensitivity of NK cells to reactive oxygen

species (compared to T and B cells) can be reduced by

expression of peroxiredoxin 1, which would promote

the persistence of CAR NK cells in the acidified envi-

ronment of solid tumors [26].

Preclinical and clinical studies have shown that

CAR NK cells lack many of the disadvantages of CAR T

cells. Since NK cells do not carry variable TCRs, their

adoptive transfer does not lead to the development

of the graft-versus-host disease (GVHD) [27, 28]. This

opens up the possibility for the producing off-the-shelf

allogeneic cell preparations suitable for many patients

at the same time. In addition, CAR NK cell transfer

carries almost no risk of CRS or neurotoxicity [29,30].

Finally, CAR NK cells have been shown in preclinical

studies to be effective not only against oncohematolog-

ical diseases, but also in the treatment of solid tumors

[31]. Currently, many developed CAR NK cell prepa-

rations, including those against solid tumors, have

moved from preclinical studies to clinical trials.

Several dozen clinical trials have been registered

to investigate the use of CAR NK cells directed against

various tumor antigens, both as a monotherapy and

in combination with other therapeutic approaches.

For example, in one trial (NCT04847466), the partici-

pants treated with CAR NK cells against PD-L1 also

received the immunostimulant N-803, which induces

proliferation and activation of NK and CD8

T cells, and

pembrolizumab (monoclonal antibody against PD-1).

NK cells sequentially transfected with the chemokine

receptor CXCR4 and a CAR directed against B cell mat-

uration antigen (BCMA) efficiently destroyed multiple

myeloma cells [32]. Currently, all clinical trials of the

CAR NK cell therapy are in phase I/II. Several regis-

tered trials are testing CAR NK cells against oncohema-

tological diseases, such as multiple myeloma (target,

BCMA; NCT05008536), B cell lymphomas (target, CD19;

NCT05379647) and acute lymphoblastic leukemia (tar-

get, CD19; NCT05563545). Clinical trials investigating

the efficacy and safety of CAR NK cells in the treat-

ment of various solid tumors are ongoing: ovarian

cancer (target, claudin 6; NCT05410717); colon cancer

(target, NKGD2L; NCT05213195); pancreatic cancer

(target, ROBO1; NCT03941457); prostate cancer (target,

PSMA; NCT03692663); and others (table). The success-

ful completion of phase I in several clinical trials of

CAR NK cells has demonstrated the high safety of this

type of therapy and the almost complete absence of

side effects [33].

Despite many advantages, CAR NK cell thera-

py has some limitations. CAR NK cells are character-

ized by a low persistence after adoptive transfer: the

lifespan of CAR T cells in a patient’s body can be up

to 10 years [34], while the lifespan of CAR NK cells

does not exceed several weeks [35]. CAR NK cells are

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Some registered CAR NK cell clinical trials

Clinical trial Started in Phase Disease Target

NCT02892695 2016 I/II lymphomas and leukemias CD19

NCT03940833 2019 I/II multiple myeloma BCMA

NCT02742727 2016 I/II lymphomas and leukemias CD7

NCT02944162 2016 I/II acute myeloid leukemia CD33

NCT02839954 2016 I/II solid tumors MUCL

NCT03383978 2017 I glioblastoma HER2

NCT03415100 2018 I metastatic solid tumors NKG2DL

NCT03941457 2019 I/II pancreatic cancer ROBO1

NCT03692663 2018 I prostate cancer PSMA

NCT05410717 2022 I/II ovarian cancer claudin6 (CLDN6)

NCT05194709 2021 I developed solid tumors 5T4

NCT05507593 2022 I non-small cell lung cancer DLL3

difficult to expand ex vivo and do not tolerate freez-

ing and storage well [36]. However, there is hope that

these technical difficulties will be soon be overcome.

In particular, it has been shown that the persistence

of CAR NK cells can be doubled by knocking out the

CISH gene, which encodes the CIS protein, a nega-

tive regulator of the IL-15 signaling pathway. The ab-

sence of CIS in CAR NK cells activates IL-15 secretion

and, since CAR NK cells carry IL-15 receptors on their

surface, causes autocrine activation of the IL-15 sig-

naling pathway. Activation of IL-15-mediated signal-

ing, in turn, promotes the expansion of CAR NK cells

ex vivo, increases their persistence and enhances the

antitumor properties of these cells [37]. Incorporation

of IL-15 into a CAR construct can also improve the cell

metabolic status of the cells and activate the effector

functions of CAR NK cells that are attenuated by the

interaction with metabolically active tumors [38].

Another factor that can reduce the efficacy of CAR

NK therapy is the capture of tumor antigens by the

therapeutic CAR NK cells via the trogocytosis mecha-

nism. After capturing the tumor antigen, CAR NK cells

become the target of the therapy and are destroyed

by other NK cells via the fratricide pathway, while

the amount of tumor antigen on the targeted malig-

nant cells decreases. The risk of fratricide among CAR

NK cells can be reduced by co-expression of activating

CARs directed against the tumor antigen and inhibitory

CARs that recognize antigens specific for NK cells [39].

γδ T cells. Approximately 3.7% of T cells circulat-

ing in the bloodstream have TCRs formed by the γ and

δ chains on their surface [40,41]. Such “non-classical”

(as opposed to “classical” αβ T cells) γδ T cells differ

significantly from αβ T cells, which carry TCRs formed

by the α and β chains and constitute a predominant

population of circulating T cells. Recognition of anti-

gens by αβ T cells is only possible when the antigen

is presented in the context of the MHC, whereas γδ T

cells do not require the involvement of classical MHC

molecules in the antigen recognition, greatly expand-

ing the possibilities for their transfer between differ-

ent organisms and significantly reducing the likelihood

of complications such as GVHD. Relatively safe adop-

tive transfer of γδ T cells is also possible because the

repertoire of γ and δ chains in a population is much

less diverse than the repertoire of α and β chains, so

that γδ T cells tend to recognize molecular signatures

common to different individuals, indicating the devel-

opment of an infectious process or the emergence of

malignant cells. For this reason, they are often thought

to be similar to the cells of the innate immunity. γδ T

cells also resemble innate immune cells in the expres-

sion of Toll-like receptors and receptors similar to the

activating receptors of NK cells, in particular, NKG2D

[42]. However, γδ T cells are also capable of forming

immunological memory and are therefore classified as

components of the adaptive immunity [43]. γδ T cells

can differentiate into Th-like cells and produce a wide

range of cytokines [44].

Most circulating γδ T cells are Vγ9Vδ2 T cells. Their

TCRs consist of the Vγ9 and Vδ2 segments of the γ and

δ chains, respectively. Since Vγ9Vδ2 T cells recognize

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phosphoantigens, synthetic analogs such as zoledron-

ic acid are used to expand Vγ9Vδ2 T cells exvivo [45].

γδ T cells, which have the Vδ1 segment in the TCRs,

localize mainly to the mucosa [23].

The role of γδ T cells in providing antitumor pro-

tection was first demonstrated in mice lacking these

cells. In these mice, chemical mutagens caused early

development of squamous cell carcinoma of the skin

[46]. Later, an important role of γδ T cells in antitumor

immunity has been demonstrated in various tumor

models. In particular, the extent of malignant tumor

infiltration by γδ T cells has been shown to correlate

with a favorable prognosis in many types of cancer,

such as melanoma [47] and gastric cancer [48]. It is be-

lieved that the activity of γδ T cells against malignant

cells is ensured by their cytotoxic properties, as well as

the production of IFNγ and TNF [33]. The cytotoxicity

of γδ T cells is provided by the action of perforin and

granzymes; the cells are also capable of antibody-de-

pendent cellular cytotoxicity. Antitumor activity is in-

herent to both Vγ9Vδ2 and Vδ1 T cells [45, 48, 49].

γδ T cells have both the antitumor and pro-tumor

properties. The pro-tumor effect of γδ T cells is usual-

ly due to the production of interleukin-17 (IL-17) and

some other cytokines. For example, in pancreatic can-

cer, γδ T cells suppress the activity of αβ T cells by se-

creting IL-10 and IL-17 and promoting PD-L1 expres-

sion in tumor cells [50].

The strong intrinsic antitumor activity of γδ T cells,

together with a highly safe adoptive transfer, makes

these cells a promising tool in the immunotherapy of

oncological diseases. Since the transfer of unmodified

γδ T cells to a patient after their exvivo expansion ap-

peared to be safe but ineffective [45], the enhancement

of the antitumor activity of γδ T cells via CAR expres-

sion has been actively investigated. It was shown that

CD19-directed CAR γδ T cells effectively recognized and

destroyed malignant cells, including those that had

lost CD19. The cytotoxic properties of CAR γδ T cells

were enhanced by their treatment with zoledronic

acid [51, 52]. The ability of CAR γδ T lymphocytes to

kill malignant blood cells that have lost antigen is due

to the high non-specific cytotoxicity of γδ T cells to-

wards leukemia cells, which is further enhanced by

zoledronic acid during ex vivo expansion of CAR γδ

T cells. Such retention of cytotoxic properties against

malignant blood cells that have lost the antigen is an

important advantage of CAR γδ T cells [52].

CAR γδ T cells can also be effective against solid

tumors. It has been shown that CAR γδ T cells direct-

ed against the neuroblastoma antigen (GD2) not only

destroy malignant cells, but also present antigens to

and activate αβ T cells in vitro. It is possible that CAR

γδ T cells also present antigens and activate αβ T cells

invivo, but further experiments are needed to clarify

this issue [23].

Most CARs that have been successfully expressed

in γδ T cells are the second-generation receptors and

contain CD28 and CD3ζ as costimulatory and signaling

domains, respectively [23] (Fig. 2). In [53], the first-

generation GD-recognizing CAR with a single intracel-

lular domain DAP10 was introduced into γδ T cells.

Itshould be noted that γδ T cells express of costimula-

tory molecules, such as CD28, CD27, and 4-1BB, which

contribute to the activation of these cells during sig-

naling by CARs [54].

Some studies have used γδ T cells to create a com-

binatorial antigen recognition system acting as logical

operators. For example, Fisher et al. [53] obtained γδ

CAR T cells expressing GD2-recognizing DAP10-CAR and

γδ TCR that bound to phosphoantigens expressed by

malignant (but not normal) cells. These γδ CAR Tcells

were activated only by interaction with the target cells

exposing both antigens recognized by the CAR and

TCR, thus significantly reducing the likelihood of kill-

ing off-target cells.

Fleischer et al. [55] obtained γδ T cells expressing

non- signaling CAR (NSCAR), which lack intracellular ac-

tivation domains and are therefore unable to trigger the

cytotoxic response upon antigen recognition. NSCAR-

expressing γδ T cells can recognize the tumor antigens

CD19 and CD5 and, through this interaction, approach

malignant cells. However, direct antitumor activity re-

quires endogenous MHC-independent cytotoxicity of

γδ T cells. In acute lymphoblastic leukemia, γδ T cells

expressing NSCARs were found to be more cytotoxic

against B and T cells than γδ T cells expressing CARs

against the same antigens (CD19 and CD5). Expression of

NSCARs instead of CARs in αβ T cells did not significant-

ly increase their cytotoxic properties, suggesting that

the molecular mechanisms underlying the enhanced

antitumor activity of γδ T cells expressing NSCARs are

fundamentally different from those of αβ T cells [55].

Several ongoing clinical trials are focusing on the

use of γδ CAR T cells in the immunotherapy of different

types of cancer, such as acute myeloid leukemia (tar-

gets, CD33 and CD123; NCT03885076, NCT04796441),

relapsed or resistant CD7

T cell leukemia (target, CD7;

NCT04702841), various B cell oncohematological

diseases (targets, CD20 and CD19; NCT04735471,

NCT02656147, NCT04911478) and relapsed or resistant

solid tumors of various origins (target, NKG2DL,

NCT04107142, NCT05302037). To date, all these clinical

trials are in phaseI, so it is too early to assess the clin-

ical efficacy of γδ CAR T cells. It is believed that the

γδ CAR T cell-based therapy will have an advantage

over conventional CAR T cell therapy by reducing the

risk of developing side effects, such as CRS and neuro-

toxicity [49]. Studies on the function of γδ CAR T cells

invivo have shown that these cells only persist in the

body for a short time [52], which may be a significant

limitation for their clinical application.

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NKT cells are similar to γδ T cells and combine

the properties of adaptive and innate immune cells.

NKT cells represent less than 1% of T cells in the blood

[56] and express αβ TCRs, as well as molecules charac-

teristic of NK cells, such as CD16 (FcγR) and CD56 [57].

Unlike T lymphocytes, NKT cells recognize antigens

only in the context of CD1d, a non-classical MHC mol-

ecule close to MHC classI. NKT cells are usually clas-

sified into invariant NKT (iNKT) cells, or type I NKT

cells, and type II NKT cells. iNKT cells are character-

ized by a restricted TCR repertoire and recognize α-ga-

lactosylceramide (α-GalCer), whereas typeII NKT cells

are characterized by a broader TCR repertoire and do

not recognize α-GalCer [58].

The number of NKT cells in the blood is often re-

duced in patients with various malignancies, partic-

ularly oncohematological ones, and the functions of

these cells (e.g., IFNγ production) are also impaired

[60]. An increased number of NKT cells in the periph-

eral blood, as well as NKT cell infiltration into the tu-

mor tend to correlate with a favorable prognosis for

patients [61, 62]. Only iNKT cells have a pronounced

antitumor effect, whereas type II NKT cells can even

suppress the immune response against malignant

cells. The cytotoxicity of iNKT cells, which is mediat-

ed by granzymes or FasL, is promoted by the recog-

nition of the lipid antigen associated with CD1d [63].

Inaddition to direct lysis of tumor cells, iNKT cells can

modulate the activity of other immune cells (in par-

ticular, DCs, NK cells, B and T lymphocytes) through

the secretion of pro- and anti-inflammatory cytokines,

such as IFNγ, TNF, IL-4, IL-10, and IL-13 [64,  65]. Cyto-

kines released by iNKT cells can also affect the tumor

microenvironment and thus indirectly influence the

immune response against tumors [64]. NKT cells with

a transient CAR expression produced less IL-6 than

transduced CD8

CAR T lymphocytes, while maintain-

ing their cytotoxic activity. For this reason, it has been

suggested that the risk of CRS is lower with CAR NKT

therapy than with CAR-T therapy, but the issue needs

further clarification [66].

Some properties of NKT cells make them partic-

ularly attractive as effectors for CAR therapy. Their

intrinsic antitumor activity (not only the cytotoxic

properties, but also the ability to rearrange the tumor

environment) can complement the CAR-mediated ef-

fect against malignant cells. Since NKT cells recognize

antigens in the context of non-polymorphic CD1d, their

allogeneic adoptive transfer does not cause GVHD, al-

lowing the generation of allogeneic CAR iNKT cells

from the cells of healthy donors [23]. In addition, NKT

cells are able to proliferate rapidly exvivo and are rel-

atively easy to obtain in the quantities required to pro-

duce a clinical product [56].

In the first work on the generation of CAR iNKT

cells, the expressed CAR was directed against the neu-

roblastoma antigen GD2 and contained the 4-1BB and

CD3ζ signaling domains (Fig. 2). 4-1BB was shown to

polarize CAR iNKT cells towards the Th1 phenotype,

enabling these cells to efficiently destroy neuroblasto-

ma cells in vivo and persist in the body for a long time

[67]. CD62L expression has been shown to be a mark-

er of increased persistence and anti-tumor activity of

NKT cells, including those expressing CARs. Adminis-

tration of anti-CD19 CAR NKT cells expressing CD62L

to mice with the B cell lymphoma resulted in a signif-

icant disease regression [68]. The study of the efficacy

of CAR NKT cells recognizing chondroitin sulfate pro-

teoglycan 4 (CSPG4; melanoma cell antigen) showed

that CAR NKT cells destroyed melanoma cells in vitro

no less efficiently than CAR T cells [69]. The generation

of NKT cells expressing the third-generation CARs di-

rected against glypican-3 (hepatocellular carcinoma

cell antigen) were obtained and containing CD28 and

4-1BB costimulatory domains has recently been an-

nounced [70].

Advances in the preclinical application of CAR

NKT cells have led to the initiation of clinical tri-

als for the treatment of neuroblastoma (target, GD2;

NCT03294954, NCT02439788), relapsed or resistant

B cell lymphoma (target, CD19; NCT03774654) and

other B cell neoplasms (target, CD19; NCT05487651).

The small number of CAR NKT clinical trials is proba-

bly due to the insufficient preclinical research. Anoth-

er major limitation for the production of clinical prod-

ucts from CAR NKT cells is the low number of NKT

cells in the peripheral blood.

Regulatory T cells (Tregs) are a specialized subset

of CD4

T lymphocytes with the immunosuppressive

function. Tregs account for 5 to 10% of CD4

Tcells in

the circulation [71]. The inhibitory influence of Tregs

on the effector T cells or antigen-presenting cells can

be direct or indirect. In the first case, Tregs act on

target cells by secreting anti-inflammatory cytokines

(IL-35, IL-10, or TGF-β) or by releasing granzymes from

the lytic granules, resulting in the target cell death.

Indirect mechanisms include targeting of other cells

exposed to Tregs [72].

The ability of Tregs to inhibit effector immune

cells makes them potentially useful for therapeutic

applications and in various conditions associated with

an exaggerated immune response, such as transplanta-

tion. However, the first studies of the therapeutic prop-

erties of polyclonal Tregs showed that their adoptive

transfer induces non-specific tolerance, reduces the

body’s resistance to infection and increases the risk of

developing malignant neoplasms [73]. CAR expression

allows Tregs to selectively attack target tissues while

maintaining the original inhibitory activity of these

cells and avoiding induction of unwanted immunolog-

ical tolerance. The first-generated CAR Tregs were in-

tended for the therapy of colitis in a mouse model [74];

MININA et al.772

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

soon after, the development of the first human CAR

Tregs was reported [75].

In most studies of CAR Tregs, these cells expressed

second-generation CARs with the CD28 or 4-1BB as

costimulatory domains. Systematic analysis of the ac-

tivity of second-generation CARs directed against the

human leukocyte antigen HLA-A2 with different co-

stimulatory domains in Tregs showed that CARs with

CD28 had the most pronounced activity. The suppres-

sive properties of these cells were further increased by

the expression of IL-10 [76]. CAR Tregs with the CD28

domain showed better persistence than CAR Tregs

with the 4-1BB domain (unlike classic CAR T) [77,78].

Tregs expressing third-generation CARs with both CD28

and 4-1BB costimulatory domains are currently being

studied [79-81].

In the decade since the first CAR Treg cells were

generated, several CAR Tregs with different specifici-

ties have been obtained. These cells are characterized

by a high efficacy, stability, and increased persistence

in mouse models [82]. CAR Tregs are also character-

ized by a reduced requirement for IL-2 compared to

CAR T [83]. CAR Treg prevent or reduce the symptoms

of GVHD, disorders associated with the excessive activ-

ity of the immune system, hemophilia, and other dis-

eases in mouse models [73]. Currently, only one CAR

Treg study has entered clinical trial (NCT04817774),

which aims to assess the efficacy and safety of CAR

Treg in kidney transplantation. In this research, CARs

recognize HLA-A2 in the recipient organ, so that CAR

Tregs are attracted to the antigen and suppress the

immune response.

Mucosal-associated T cells. Mucosal-associated in-

variant T cells (MAIT) recognize metabolites of vita-

mins B2 and B9 of bacterial origin in a complex with

the non-classical MHC molecule MR1 [84]. MAIT cells

account for 1 to 8% of peripheral blood T cells [85] and

are localized to mucous membranes and lymphoid tis-

sues. MAIT cell TCRs consist of non-polymorphic α and

β chains; most MAIT cells also express CD8. They also

carry granzyme-containing lytic granules and have

cytotoxic properties [86]. When activated, MAIT cells

secrete the pro-inflammatory cytokines IFNγ, TNF,

and IL-17 [87].

MAIT cells are involved in protection against most-

ly bacterial infections and in the pathogenesis of many

non-communicable diseases, such as autoimmune dis-

orders, inflammatory bowel disease, celiac disease, and

cancer [84]. The level of MAIT cells in the blood decreas-

es in colorectal cancer. MAIT cells migrate into the tu-

mors, where they exert their antitumor cytotoxic effect

by releasing granzymes [88]. Areduction in the number

of MAIT cells is also observed in patients with multiple

myeloma [89]. In some cases, actively migrating MAIT

cells become immunosuppressed by the tumor micro-

environment and lose the ability to secrete IFNγ [90].

The fact that MAIT cells actively infiltrate tumor

microenvironment and have the cytotoxic properties

suggests that they can be used in the development

of novel anti-tumor immunotherapeutic approaches.

Since MAIT cells only recognize antigens associat-

ed with the non-classical MHC class I molecule MR1,

the likelihood of GVHD development when using CAR

MAIT cells would be lower than in the case of cytotox-

ic CAR T cells [23]. Dogan et al. [86] expressed CARs

against CD19 or HER2 antigens in primary human

MAIT cells and showed that the resulting cells were

cytotoxic effects against B cell lymphoma and breast

cancer cells, respectively. The activated CAR MAIT cells

were not inferior to CD8

CAR T lymphocytes in their

cytotoxicity against target cells, but produced signifi-

cantly lower levels of IFNγ and other pro-inflammato-

ry cytokines. This latter observation suggests that the

likelihood of CRS and neurotoxicity following injection

of CAR MAIT cells into the patient’s body may be low-

er than with CAR T cells [87]. Further evaluation of

the safety and antitumor properties of CAR MAIT cells

should be performed in in vivo in laboratory animals

and in clinical trials.

Double negative (DN) T cells. DN T cells are char-

acterized by CD3 expression in the absence of CD4 and

CD8 expression. DN T cells also express CD25 [91], a

molecule that is highly expressed by Tregs. TCRs of DN

T cells can contain both αβ and γδ chains. DN Tcells

make up 3 to 5% of T cells in the blood [92]. They ex-

ert a strong suppressive effect on several groups of

immune cells (CD4

and CD8

T cells, B cells, and NK

cells) both in vitro and in vivo. Thus, DN T cells play

an important role in the prevention of GVHD and the

maintenance of tolerance to allografts and xenografts

[93-95]. Thus, DN T cells can be considered as a non-

canonical subset of Tregs.

Despite their immunosuppressive effect on many

immune cells, DN T cells possess their own MHC-in-

dependent cytotoxic properties. DN T cells can induce

malignant cell death via FasL [96], TRAIL, and other

cytotoxicity-associated surface proteins [97]. They also

express perforin and granzymes and secrete TNF and

IFNγ [97]. In addition to MHC-independent cytotoxici-

ty, DN T cells have other properties, such as the possi-

bility of easy expansion ex vivo [98], lack of extra-tu-

mor cytotoxic activity, and reduced risk of rejection

after allogeneic transfer [99], which allow their use in

immunotherapy of malignant diseases.

The possibility of CAR expression in DN T cells

has been poorly investigated. In 2022, Vasic etal. [99]

reported the generation of DN T cells with the CD19-

directed CAR and compared their antitumor proper-

ties with traditional CAR T cells directed against the

same antigen. The authors found that the CAR DN

Tcells were not inferior to conventional CAR T cells in

their in vitro and in vivo anti-tumor properties, but did

CAR CELLS BEYOND CLASSICAL CAR T CELLS 773

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

not cause GVHD [99]. However, these results require

independent confirmation by other investigators.

Cytokine-induced killer (CIK) cells are a hetero-

geneous group of CD8

T cells that are phenotypically

and functionally close to NK cells [100, 101]. CIKs are

derived from CD3

T lymphocytes that begin to express

CD56 during expansion. Initially, CIKs were derived

from the lymphokine-activated killer cells (LAKs, lym-

phocytes with the ability to lyse tumor cells after in-

cubation with IL-2 [102]) during the optimization of

the expansion protocol. LAKs had already been ob-

tained in the 1980s [103], but despite their strong cy-

totoxic properties against tumor cells, the difficulties

in expanding these cells exvivo have limited their use.

The timed addition of IFNγ, IL-2, and anti-CD3 mono-

clonal antibody (which had the mitogenic effect) to

LAKs led to the development of CIKs [104]. Currently,

these cells can be produced relatively cheaply from

peripheral or umbilical cord blood T cells.

The cytotoxic effect of CIKs requires the involve-

ment of the NKG2D receptor and is manifested by the

release of the contents of granzyme-containing lytic

granules. NKG2D recognizes stress-induced molecules,

such as UL16BP and MICA/MICB, on the surface of tar-

get cells [105]. As TCR signaling is also possible, so the

use of CIKs has for a long time been limited to autol-

ogous therapy due to the fear of acute GVHD. How-

ever, it has been recently been shown that the use of

donor-based CIKs after hematopoietic stem cells al-

lotransplantation is relatively safe, which may be due

to the short persistence of terminally differentiated

CD3

CD56

cells and the reduced expression of some

homing receptors [106].

Due to their strong cytotoxic properties, rapid

ex vivo expansion, tumor cell recognition via NKG2D,

and safety of allogeneic adoptive transfer, CIKs are

considered as promising effectors for CAR cell thera-

py [107]. Several preclinical studies have demonstrat-

ed the efficacy of CAR CIKs against hematological and

solid tumors. For example, CIKs expressing a CAR di-

rected against CD19 and containing CD28/4-1BB and

CD3ζ domains efficiently destroyed B cell acute lym-

phoblastic leukemia cells [108,109]. CIKs bearing CARs

to relevant tumor antigens effectively destroyed colon

cancer cells, acute myeloid leukemia cells, soft tissue

sarcoma cells, and cells of other tumor [107]. Expres-

sion of CARs in CIKs leads to increased production of

IFNγ and TNF compared to CIKs that do not express

CARs [110,111].

As of 2022, there is only one clinical trial inves-

tigating the efficacy and safety of CD19-directed CAR

CIKs in acute lymphoblastic leukemia (NCT03389035).

Further studies are therefore needed to provide infor-

mation on the therapeutic efficacy of CAR CIKs.

Macrophages as specialized phagocytic cells that

heavily infiltrate the stroma of solid tumors and are

an important component of the tumor microenviron-

ment. Following recognition of foreign agents by in-

nate immune receptors (mainly Toll-like and NOD-like

receptors), macrophages become activated and start to

produce pro-inflammatory cytokines (TNF, IL-1β, IL-6,

IL-12, and IL-23) that affect many cells in the tumor mi-

croenvironment and in particular promote the antitu-

mor activity of T cells and NK cells. Macrophages with

the pro-inflammatory properties have the so-called

M1 phenotype, whereas macrophages with M2 phe-

notype inhibit inflammation and the development of

antitumor T cell immunity and promote tumor growth

by producing IL-4, IL-5, and IL-13 cytokines. The ma-

jor physiological role of M2 macrophages is wound

healing [112]. Macrophages in the tumor microenvi-

ronment are known as tumor-associated macrophages

(TAMs). The TAMs with M2 phenotype promote tumor

growth and metastasis and has the immunosuppres-

sive properties [66]. Therefore, polarization of TAMs

towards the M1 phenotype, which contributes to the

destruction of neoplasia, is an important component

of the anti-tumor immunotherapy using these cells.

The phagocytic capacity of macrophages can be

enhanced by the expression of CARs bearing intracel-

lular domains that trigger phagocytosis-activating sig-

naling. In the one of the first studies focusing on CAR

macrophages (CAR Ms), primary human monocytes

were transduced with a CAR directed against carci-

noembryonic antigen (CEA) and bearing CD64 (FcγRI)

as a signaling domain. The resulting CAR Ms showed

antitumor activity in vitro and in vivo, although the

molecular mechanism of this activity remained un-

clear [113]. Further studies showed that expression

of CARs containing the phagocytic receptor Megf10 or

FcγR as signaling domains promoted the phagocytic

activity of macrophages [114, 115]. Phagocytosis was

also activated by the expression of CARs with the CD3ζ

signaling domain, which is homologous to FcγRI.

Macrophages expressing CARs directed against

CD19 with Megf10 or FcγR domains efficiently de-

stroyed CD19-positive tumor cells. Phagocytosis was

only observed for a small fraction of malignant cells,

while the majority were destroyed by trogocytosis

[116]. Zhang etal. [116] derived macrophages express-

ing second-generation anti-CD19 CAR and possessing

CD3ζ signaling and 4-1BB costimulatory domains from

the induced pluripotent stem cells (iPSCs). These mac-

rophages phagocytized tumor cells in culture and pro-

duced pro-inflammatory cytokines, but had little effect

on the tumor growth invivo [117].

It has been shown that the process of CAR M gen-

eration itself induces their polarization into pro-in-

flammatory cells. In particular, it was shown that trans-

duction with a CAR-encoding adenoviral vector and

subsequent CAR expression polarized macrophages

towards the M1 phenotype, which was maintained

MININA et al.774

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

in the tumor microenvironment [118]. In addition,

CAR Ms are capable of cross-presenting tumor anti-

gens after phagocytosis. Macrophages expressing CARs

directed against HER2 (breast cancer antigen) phago-

cytized tumor cells and presented the processed an-

tigens. CAR Ms have been shown to activate systemic

antitumor immunity [119]. A single administration of

CAR Ms in a mouse model of ovarian cancer signifi-

cantly prolonged animal survival and slowed (but did

not completely suppress) tumor development [115].

Nevertheless, the ability of CAR Ms to transform the

tumor microenvironment into a pro-inflammatory one

makes them an additional tool in the CAR T cell thera-

py or other immunotherapies.

Macrophages can produce matrix metallopro-

teinases that cause significant changes in the tumor

extracellular matrix and affect the architecture of the

neoplasm, which can be exploited in the CAR therapy.

Expression of CARs directed against HER2 and contain-

ing CD147 as a transmembrane/intracellular domain in

macrophages stimulated the secretion of matrix metal-

loproteinases by these cells. Using a mouse model of

breast cancer, it was shown that administration of these

CAR Ms led to the tumor matrix rearrangement, which

promoted T cell infiltration into the tumor. Levels of

CRS-associated pro-inflammatory cytokines (IFNγ, TNF,

and IL-6) in the blood of mice with breast cancer were

reduced, while the levels of IL-12 and IFNγ in the tumor

were increased [120]. It is reasonable to assume that

CAR M therapy is associated with a reduced risk of CRS.

At present, there is not enough information to

draw conclusions about the efficacy and safety of CAR

M therapy in humans. The fact that macrophages are

one of the main cell types of that trigger the CRS re-

quires a detailed review of the safety of CAR M thera-

py. In addition, peripheral blood monocytes are hetero-

geneous, and there is a possibility that CAR Ms derived

from these cells may migrate to healthy tissues more

often than to the tumor when used systemically [23],

which may lead to significant side effects in different

organ systems. To date, only one HER2-overexpressing

CAR M against solid tumors (NCT04660929) has en-

tered the clinical trial.

Dendritic cells. As “professional” antigen-present-

ing cells, DCs are involved in the maturation of naive

T cells and the reactivation of memory T cells. During

antigen presentation to T cells, DCs produce cytokines

that modulate T cell activity. When present in the tu-

mor microenvironment, DCs can both induce immune

tolerance and contribute to the development of the an-

titumor immune response [121,122]. In addition, DCs

are able to cross-present tumor antigens to the cyto-

toxic CD8

 T cells, thereby promoting the antitumor

response [123].

The potential of CAR-expressing DCs in controlling

of the antitumor activity of CAR T cells has been poor-

ly investigated. Suh et al. [123] investigated the pos-

sibility of using CAR-expressing DCs (CAR DCs) to at-

tract to the tumor microenvironment and to activate

CAR T cells through the cytokine secretion. Expression

of CARs against CD33 on DCs promoted the homing

of these cells to the bone marrow in mice with acute

myeloid leukemia. Mice receiving CAR DCs and CAR

T cells targeting CD33 had significantly elevated lev-

els of IL-12, IFNγ, and TNF, and the overall survival of

these animals was longer than that of mice receiving

CAR T cells alone. Thus, co-administration of CAR DCs

and CAR T cells significantly increased the efficiency of

malignant cell destruction [124]. The safety of clinical

application of CAR DCs should be investigated, as IL-12

secreted in high concentrations may have a systemic

toxic effect [125]. To date, there are no registered clini-

cal trials focusing on CAR DCs.

B cells. In addition to T cells and innate immune

cells, B cells are also of interest for CAR technology.

Because of their ability to differentiate into long-lived

antibody-secreting plasma cells, B lymphocytes could

become a safe and controlled source of therapeutic

monoclonal antibodies. By expressing CARs against tu-

mor antigens in these cells, targeted antibody delivery

can avoid the side effects of systemic antibody admin-

istration.

Despite these prospects, the studies on the CAR-ex-

pressing B cells and their therapeutic use are current-

ly limited. The possibility of lentiviral transduction of

B cells and subsequent expression of CARs in them

was described in 2018, when a patient experienced a

relapse of B cell lymphoma caused by a single B cell

clone accidentally transduced with a CAR [126]. Soon

after, Pesch etal. [126] demonstrated that genes encod-

ing chimeric B cell receptors (CBCRs) could be insert-

ed into B cells using the CRISPR/Cas9 genome editing

technology. The CBCR used in this study contained the

transmembrane region CD28 and the BCR signaling do-

main CD79β instead of the classical CD3ζ. Introducing

of the gene encoding this CBCR into primary mouse

B cells resulted in the abundant presence of CBCR on

their surface, allowing the cells to recognize antigens

without the involvement of their own B cell receptors

[127]. The antitumor activity and the therapeutic effi-

cacy of CAR-expressing B cells, as well as their safety

in humans, are currently unknown.

ONE TARGET – MANY TYPES OF CAR CELLS

Early studies focused on the antitumor effects of

CAR-expressing leukocytes of different types have used

a small set of tumor-associated antigens, including

CD19, GD2 and others as CAR targets. At present, CAR

cells that effectively recognize tumor antigens have

been derived from different types of white blood cells.

CAR CELLS BEYOND CLASSICAL CAR T CELLS 775

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

The generation of CAR cells that recognize the same

tumor antigen from different cell sources could pro-

vide scope for selecting the optimal therapy for each

patient, as the effector properties of different groups

of white blood cells are different.

Four out of the six FDA-approved CAR T cell ther-

apies target the CD19 antigen and are designed to treat

B cell neoplasias. Despite proven high efficacy, the

anti- CD19 CAR T cell therapy is associated with serious

side effects, primarily CRS and neurotoxicity, in a sig-

nificant proportion of patients. For example, the use

of axicabtagene ciloleucel (CAR T cells with the CD28

costimulatory domain and CD3ζ signaling domain)

causes CRS in 93% of patients and neurotoxicity in

67% [128,129]. To date, CAR cells that efficiently rec-

ognize and destroy CD19-positive malignant cells have

been derived from γδ T cells [52], MAIT cells [87], NKT

cells [68], NK cells [29], DN T cells [100], CIKs [108]

and macrophages [114, 115]. Tregs expressing CD19

have been engineered to suppress the antibody pro-

duction and prevent GVHD [130].

Preclinical studies and studies in small groups of

patients, have shown that anti-CD19 CAR cells, which

are different from the cytotoxic T cells, have a strong

antitumor effect with fewer side effects than the CAR

T cell therapy. However, many of the cell products

mentioned above have not yet reached full-scale clin-

ical trials, while those that have are still in the early

stages of clinical testing, so it is too early to draw con-

clusions about their safety and efficacy.

Two other FDA-approved CAR T cell therapies,

idecabtagene vicleucel and ciltacabtagene autoleucel,

which target the BCMA antigen, have been developed

for the treatment of multiple myeloma. As with the an-

ti-CD19 CAR T therapy, the high efficacy of these drugs

is associated with a significant risk of CRS or neuro-

toxicity: 76 and 42% for idecabtagene vicleucel and

92 and 20% for ciltacabtagene autoleucel, respectively

[131,132]. It has been reported that CAR NK cells tar-

geting BCMA have been obtained [133], but have not

yet entered clinical trials.

There are also CAR cells targeting the same tu-

mor-associated antigen, derived from different types

of white blood cells, intended for the therapy of solid

tumors. For example, GD2-recognizing CAR cells have

been derived from γδ T cells and NKT cells. Currently,

GD2-recognizing CAR NKT cells are in phaseI clinical

trials, so the safety and efficacy of both cell products

remain to be investigated.

CONCLUSION

Over the past 20 years, the CAR T cell approach

has been marked by a recognized success in the treat-

ment of hematologic cancers, and six CAR T cell ther-

apies have been approved for clinical use by the FDA.

Despite the drawbacks of “traditional” CAR T cell ther-

apy, the possibility of generating CAR cells from other

types of white blood cells has long been overlooked.

The results of preclinical studies of the efficacy and

safety of CAR cells in cell culture and mouse models

suggest a wide range of applications for CAR cells in

the treatment of human cancer, including solid tu-

mors. Data on the expression of CARs against different

targets and in different leukocyte subsets are summa-

rized in Table 1 in the article by Qin et al. [33]. Cur-

rently, existing cellular products are only in phaseI/II

of clinical trials, while the vast majority of therapeutic

drugs based on CAR-expressing immune cells have not

yet progressed beyond the preclinical studies.

Although alternative CAR cell products have sig-

nificant advantages over traditional CAR T cells (e.g.,

reduced risk of CRS and neurotoxicity), the latter are

still superior in a number of parameters. Since the sig-

naling pathways underlying the activity of CARs are

different in different groups of adaptive and innate

immune cells, the choice of costimulatory and signal-

ing domains is crucial for the development of CARs in-

tended for the expression in different types of immune

cells. Inclusion of a domain that is not sufficiently func-

tional in a particular cell type can lead to a reduction

in the CAR activity and therapeutic efficacy. For ex-

ample, in macrophages, the Megf10 signaling domain

in the CAR construct is more efficient than the CD3ζ

domain, which has shown optimal activity in T cells.

Therefore, each new CAR-expressing cell may require

“customization” of the receptors and identification of

domains that provide the required level of signaling

from the activated receptor. In the case of traditional

CAR T cells, the choice of domains for CARs has been

much better studied. In addition, some immune cells

are difficult to expand ex vivo, and obtaining sufficient

numbers of these cells can be expensive and time-con-

suming, significantly limiting the ability to produce

clinically relevant products. However, in some cases, it

is possible to find alternative sources of white blood

cells that can at least partially overcome the problem

of cell expansion. For example, CAR NK cells may be

derived from cell lines and iPSCs [20]; CAR Ms may

be derived from monocytes and iPSCs [116]. However,

this method of generating CAR cells may be associat-

ed with a potential risk of tumor formation due to the

high oncogenic potential of iPSCs, and therefore its

safety needs to be further investigated.

Despite significant limitations associated with the

development of CAR-mediated therapies based on dif-

ferent types of white blood cells, these cells have good

prospects in the immunotherapy of solid tumors. The

use of CAR T cells in the treatment of solid tumors has

been largely ineffective due to the insufficient infiltra-

tion of CAR T cells into the tumor, suppressive effects

MININA et al.776

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

of the tumor microenvironment, and heterogeneity

and frequent loss of tumor antigens. Preclinical stud-

ies have shown that many alternative CAR-expressing

immune cells successfully destroy solid tumors. Some

cells, in particular CAR-Ms, can transform tumor mi-

croenvironment into an inflammatory environment

that promotes tumor elimination. The issue of infil-

tration of CAR-expressing immune cells into solid tu-

mors has also not been resolved. Efficient tumor in-

filtration of CAR cells has only been demonstrated for

NK cells and macrophages [117]. Nevertheless, it can

be assumed that CAR leukocytes will become effective

agents for the treatment of solid tumors when used in

combination with other therapeutic approaches.

In conclusion, CAR cell therapy based on different

types of immune cells is moving towards its clinical

application. However, it may be years before alterna-

tive CAR cells become effective, safe, and routinely used

tools in the treatment of cancer and other diseases.

Contributions. E.P.M. and A.V.B. developed the

concept of the review; E.P.M. prepared the manuscript;

S.A.Sh. created illustrations; D.V.D. and A.V.B. edited the

manuscript.

Ethics declarations. This work does not contain

any studies involving human and animal subjects.

Theauthors of this work declare that they have nocon-

flicts of interest.

REFERENCES

1. Kuwana,Y., Asakura,Y., Utsunomiya,N., Nakanishi,M.,

Arata,Y., Itoh, S., Nagase,F., and Kurosawa,Y. (1987)

Expression of chimeric receptor composed of immuno-

globulin-derived Vregions and T-cell receptor- derived

C regions, Biochem. Biophys. Res. Commun., 149, 960-

968, doi:10.1016/0006-291X(87)90502-X.

2. Eshhar, Z., Waks, T., Gross, G., and Schindler, D. G.

(1993) Specific activation and targeting of cytotoxic

lymphocytes through chimeric single chains consist-

ing of antibody-binding domains and the γ or ζ sub-

units of the immunoglobulin and T-cell receptors,

Proc. Natl. Acad. Sci. USA, 90, 720-724, doi: 10.1073/

pnas.90.2.720.

3. Sadelain, M., Brentjens, R., and Riviere, I. (2013)

The basic principles of chimeric antigen recep-

tor design, Cancer Discov., 3, 388-398, doi: 10.1158/

2159-8290.CD-12-0548.

4. Gomes-Silva, D., Mukherjee, M., Srinivasan, M.,

Krenciute,G., Dakhova,O., Zheng, Y., Cabral, J.M.S.,

Rooney, C. M., Orange, J. S., Brenner, M. K., and Ma-

monkin, M. (2017) Tonic 4-1BB costimulation in chi-

meric antigen receptors impedes T cell survival and

is vector-dependent, Cell Rep., 21, 17-26, doi:10.1016/

j.celrep.2017.09.015.

5. Maher, J., Brentjens, R. J., Gunset, G., Rivière, I., and

Sadelain,M. (2002) Human T-lymphocyte cytotoxicity

and proliferation directed by a single chimeric TCRζ/

CD28 receptor, Nat. Biotechnol., 20, 70-75, doi:10.1038/

nbt0102-70.

6. Brentjens, R. J., Latouche, J. B., Santos, E., Marti, F.,

Gong, M. C., Lyddane, C., King, P. D., Larson, S.,

Weiss,M., Riviere,I., and Sadelain,M. (2003) Eradica-

tion of systemic B-cell tumors by genetically targeted

human T lymphocytes co-stimulated by CD80 and in-

terleukin-15, Nat. Med., 9, 279-286, doi:10.1038/nm827.

7. List of FDA approved cell and gene therapies, URL:

https://www.fda.gov/vaccines-blood-biologics/cellular-

gene-therapy-products/approved-cellular-and-gene-

therapy-products.

8. Morgan, R. A., Yang, J. C., Kitano, M., Dudley, M. E.,

Laurencot, C.M., and Rosenberg, S.A. (2010) Case re-

port of a serious adverse event following the adminis-

tration of Tcells transduced with a chimeric antigen

receptor recognizing ERBB2, Mol. Ther., 18, 843-851,

doi:10.1038/mt.2010.24.

9. D’Aloia, M. M., Zizzari, I. G., Sacchetti, B., Pierelli, L.,

and Alimandi, M. (2018) CAR-T cells: the long and

winding road to solid tumors, Cell Death Dis., 9, 282,

doi:10.1038/s41419-018-0278-6.

10. Larson, R.C., and Maus, M.V. (2021) Recent advances

and discoveries in the mechanisms and functions of

CAR Tcells, Nat. Rev. Cancer, 21, 145-161, doi:10.1038/

s41568-020-00323-z.

11. Long, A.H., Haso, W.M., Shern, J.F., Wanhainen, K.M.,

Murgai, M., Ingaramo, M., Smith, J. P., Walker, A. J.,

Kohler, M.E., Venkateshwara, V.R., Kaplan, R.N., Pat-

terson, G.H., Fry, T.J., Orentas, R.J., and Mackall, C.L.

(2015) 4-1BB costimulation ameliorates Tcell exhaus-

tion induced by tonic signaling of chimeric antigen re-

ceptors, Nat. Med., 21, 581-590, doi:10.1038/nm.3838.

12. Hong, M., Clubb, J. D., and Chen, Y. Y. (2020) Engi-

neering CAR-T cells for next-generation cancer ther-

apy, Cancer Cell, 38, 473-488, doi: 10.1016/j.ccell.

2020.07.005.

13. Rafiq,S., Hackett, C.S., and Brentjens, R.J. (2020) En-

gineering strategies to overcome the current road-

blocks in CAR Tcell therapy, Nat. Rev. Clin. Oncol., 17,

147-167, doi:10.1038/s41571-019-0297-y.

14. Meza Guzman, L. G., Keating, N., and Nicholson,

S. E. (2020) Natural killer cells: tumor surveillance

and signaling, Cancers (Basel), 12, 952, doi: 10.3390/

cancers12040952.

15. Kumar,S. (2018) Natural killer cell cytotoxicity and its

regulation by inhibitory receptors, Immunology, 154,

383-393, doi:10.1111/imm.12921.

16. Fauriat, C., Long, E. O., Ljunggren, H. G., and Bry-

ceson, Y. T. (2010) Regulation of human NK-cell cy-

tokine and chemokine production by target cell

recognition, Blood, 115, 2167-2176, doi: 10.1182/

blood-2009-08-238469.

CAR CELLS BEYOND CLASSICAL CAR T CELLS 777

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

17. Lo Nigro, C., Macagno, M., Sangiolo, D., Bertolacci-

ni,L., Aglietta, M., and Merlano, M. C. (2019) NK-me-

diated antibody-dependent cell-mediated cytotoxicity

in solid tumors: biological evidence and clinical per-

spectives, Ann. Transl. Med., 7, 105, doi:10.21037/atm.

2019.01.42.

18. Tyshchuk, E. V., Mikhailova, V. A., Selkov, S. A., and

Sokolov, D. I. (2021) Natural killer cells: origin, phe-

notype, function, Medical Immunology (Russia), 23,

1207-1228, doi:10.15789/1563-0625-NKC-2330.

19. Xu,Y., Liu,Q., Zhong,M., Wang,Z., Chen,Z., Zhang,Y.,

Xing,H., Tian,Z., Tang,K., Liao,X., Rao,Q., Wang,M.,

and Wang, J. (2019) 2B4 costimulatory domain en-

hancing cytotoxic ability of anti-CD5 chimeric antigen

receptor engineered natural killer cells against Tcell

malignancies, J.Hematol. Oncol., 12, 49, doi: 10.1186/

s13045-019-0732-7.

20. Xie, G., Dong, H., Liang, Y., Ham, J. D., Rizwan, R.,

and Chen, J. (2020) CAR-NK cells: a promising cellu-

lar immunotherapy for cancer, EBioMed., 59, 102975,

doi:10.1016/j.ebiom.2020.102975.

21. Töpfer, K., Cartellieri, M., Michen, S., Wiedemuth, R.,

Muller, N., Lindemann, D., Bachmann, M., Fussel,M.,

Schackert, G., and Temme, A. (2015) DAP12-based

activating chimeric antigen receptor for NK cell tu-

mor immunotherapy, J. Immunol., 194, 3201-3212,

doi:10.4049/jimmunol.1400330.

22. Cifaldi, L., Melaiu, O., Giovannoni, R., Benvenu-

to, M., Focaccetti, C., Nardozi, D., Barillari, G., and

Bei, R. (2023) DNAM-1 chimeric receptor-engineered

NKcells: a new frontier for CAR-NK cell-based immu-

notherapy, Front. Immunol., 14, 1197053, doi:10.3389/

fimmu.2023.1197053.

23. Oei, V.Y.S., Siernicka,M., Graczyk-Jarzynka,A., Hoel,

H. J., Yang, W., Palacios, D., Almasbak, H., Bajor, M.,

Clement,D., Brandt,L., Onfelt, B., Goodridge,J., Win-

iarska, M., Zagozdzon, R., Olweus, J., Kyte, J. A., and

Malmberg, K. J. (2018) Intrinsic functional potential

of NK-cell subsets constrains retargeting driven by

chimeric antigen receptors, Cancer Immunol. Res., 6,

467-480, doi:10.1158/2326-6066.CIR-17-0207.

24. Xia,J., Minamino,S., and Kuwabara,K. (2020) CAR-ex-

pressing NKcells for cancer therapy: a new hope, Bios-

ci. Trends, 14, 354-359, doi:10.5582/bst.2020.03308.

25. Dong, H., Ham, J. D., Hu, G., Xie, G., Vergara, J., Li-

ang,Y., Ali,A., Tarannum,M., Donner,H., Baginska,J.,

Abdulhamid,Y., Dinh,K., Soiffer, R.J., Ritz,J., Glimch-

er, L. H., Chen, J., and Romee, R. (2022) Memory-like

NKcells armed with a neoepitope-specific CAR exhibit

potent activity against NPM1 mutated acute myeloid

leukemia, Proc. Natl. Acad. Sci. USA, 119, e2122379119,

doi:10.1073/pnas.2122379119.

26. Klopotowska, M., Bajor, M., Graczyk-Jarzynka, A.,

Kraft,A., Pilch,Z., Zhylko,A., Firczuk,M., Baranows-

ka, I., Lazniewski, M., Plewczynski, D., Goral, A.,

Soroczynska, K., Domagala, J., Marhelava, K., Slu-

sarczyk, A., Retecki, K., Ramji, K., Krawczyk, M.,

Temples, M. N., Sharma, B., Lachota, M., Netskar, H.,

Malmberg, K.-J., Zagozdzon, R., and Winiarska, M.

(2022) PRDX-1 supports the survival and antitumor

activity of primary and CAR-modified NK cells under

oxidative stress, Cancer Immunol. Res., 10, 228-244,

doi:10.1158/2326-6066.CIR-20-1023.

27. Ruggeri, L., Capanni, M., Urbani, E., Perruccio, K.,

Shlomchik, W.D., Tosti,A., Posati,S., Rogaia,D., Fras-

soni,F., Aversa,F., Martelli, M.F., and Velardi,A. (2002)

Effectiveness of donor natural killer cell aloreactivi-

ty in mismatched hematopoietic transplants, Science,

295, 2097-2100, doi:10.1126/science.1068440.

28. Miller, J. S., Soignier, Y., Panoskaltsis-Mortari, A.,

McNearney, S. A., Yun, G. H., Fautsch, S. K., McKen-

na, D., Le, C., Defor, T. E., Burns, L. J., Orchard, P. J.,

Blazar, B. R., Wagner, J. E., Slungaard, A., Weisdorf,

D.J., Okazaki, I.J., and McGlave, P.B. (2005) Success-

ful adoptive transfer and invivo expansion of human

haploidentical NKcells in patients with cancer, Blood,

105, 3051-3057, doi:10.1182/blood-2004-07-2974.

29. Liu, E., Marin, D., Banerjee, P., Macapinlac, H. A.,

Thompson, P., Basar, R., Nassif Kerbauy, L., Over-

man, B., Thall, P., Kaplan, M., Nandivada, V., Kaur, I.,

Nunez Cortes,A., Cao,K., Daher,M., Hosing,C., Cohen,

E. N., Kebriaei, P., Mehta, R., Neelapu, S., Nieto, Y.,

Wang, M., Wierda, W., Keating, M., Champlin, R.,

Shpall, E., and Rezvani, K. (2020) Use of CAR-trans-

duced natural killer cells in CD19-positive lymphoid

tumors, N. Engl. J. Med., 382, 545-553, doi: 10.1056/

NEJMoa1910607.

30. Xiao,L., Cen,D., Gan,H., Sun,Y., Huang,N., Xiong,H.,

Jin, Q., Su, L., Liu, X., Wang, K., Yan, G., Dong, T.,

Wu,S., Zhou,P., Zhang, J., Liang,W., Ren,J., Teng, Y.,

Chen, C., and Xu, X. H. (2019) Adoptive transfer of

NKG2D CAR mRNA-engineered natural killer cells in

colorectal cancer patients, Mol. Ther., 27, 1114-1125,

doi:10.1016/j.ymthe.2019.03.011.

31. Wrona,E., Borowiec,M., and Potemski,P. (2021) CAR-

NK cells in the treatment of solid tumors, Int.J. Mol.

Sci., 22, 5899, doi:10.3390/ijms22115899.

32. Ng, Y. Y., Du, Z., Zhang, X., Chng, W. J., and Wang, S.

(2022) CXCR4 and anti-BCMA CAR co-modified natural

killer cells suppress multiple myeloma progression in

a xenograft mouse model, Cancer Gene Ther., 29, 475-

483, doi:10.1038/s41417-021-00365-x.

33. Qin, V.M., D’Souza,C., Neeson, P.J., and Zhu, J.J. (2021)

Chimeric antigen receptor beyond CAR-T cells, Can-

cers (Basel), 13, 404, doi:10.3390/cancers13030404.

34. Melenhorst, J. J., Chen, G. M., Wang, M., Porter, D.L.,

Chen, C., Collins, M. A., Gao, P., Bandyopadhyay, S.,

Sun,H., Zhao,Z., Lundh,S., Pruteanu-Malinici,I., No-

bles, C.L., Maji,S., Frey, N.V., Gill, S.I., Loren, A.W.,

Tian, L., Kulikovskaya, I., Gupta, M., Ambrose, D. E.,

Davis, M. M., Fraietta, J. A., Brogdon, J. L., Young,

R. M., Chew, A., Levine, B. L., Siegel, D.L., Alanio,C.,

MININA et al.778

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

Wherry,E.J., Bushman, F.D., Lacey, S.F., Tan,K., and

June, C. H. (2022) Decade-long leukaemia remissions

with persistence of CD4

CAR T cells, Nature, 602,

503-509, doi:10.1038/s41586-021-04390-6.

35. Sabbah, M., Jondreville, L., Lacan, C., Norol, F., Vieil-

lard, V., Roos-Weil, D., and Nguyen, S. (2022) CAR-NK

cells: a chimeric hope or a promising therapy? Cancers

(Basel), 14, 3839, doi:10.3390/cancers14153839.

36. Ebrahimiyan, H., Tamimi, A., Shokoohian, B., Mi-

naei, N., Memarnejadian, A., Hossein-Khannazer, N.,

Hassan,M., and Vosough,M. (2022) Novel insights in

CAR-NK cells beyond CAR-T cell technology; promis-

ing advantages, Int. Immunopharmacol., 106, 108587,

doi:10.1016/j.intimp.2022.108587.

37. Daher,M., Basar,R., Gokdemir,E., Baran,N., Uprety,N.,

Nunez Cortes, A.K., Mendt,M., Kerbauy, L.N., Baner-

jee, P.P., Shanley,M., Imahashi,N., Li,L., Lim, F.L.W.I.,

Fathi,M., Rezvan,A., Mohanty,V., Shen,Y., Shaim,H.,

Lu,J., Ozcan,G., Ensley,E., Kaplan,M., Nandivada,V.,

Bdiwi,M., Acharya,S., Xi,Y., Wan,X., Mak,D., Liu,E.,

Jiang, X.R., Ang,S., Muniz-Feliciano,L., Li,Y., Wang,J.,

Kordasti, S., Petrov, N., Varadarajan, N., Marin, D.,

Brunetti, L., Skinner, R. J., Lyu, S., Silva, L., Turk, R.,

Schubert, M.S., Rettig, G.R., McNeill, M.S., Kurgan,G.,

Behlke, M. A., Li, H., Fowlkes, N. W., Chen, K., Kono-

pleva,M., Champlin, R.E., Shpall, E.J., and Rezvani,K.

(2021) Targeting a cytokine checkpoint enhances the

fitness of armored cord blood CAR-NK cells, Blood,

137, 624-636, doi:10.1182/blood.2020007748.

38. Pulè, M. A., Straathof, K. C., Dotti, G., Heslop, H. E.,

Rooney, C. M., and Brenner, M. K. (2005) A chime-

ric T cell antigen receptor that augments cytokine

release and supports clonal expansion of primary

human T cells, Mol. Ther., 12, 933-941, doi: 10.1016/

j.ymthe.2005.04.016.

39. Li,Y., Basar,R., Wang,G., Liu,E., Moyes, J.S., Li,L., Ker-

bauy, L.N., Uprety,N., Fathi,M., Rezvan,A., Banerjee,

P. P., Muniz-Feliciano, L., Laskowski, T. J., Ensley, E.,

Daher,M., Shanley,M., Mendt,M., Acharya,S., Liu,B.,

Biederstadt,A., Rafei,H., Guo,X., Garcia, L.M., Lin,P.,

Ang,S., Marin,D., Chen,K., Bover,L., Champlin, R.E.,

Varadarajan, N., Shpall, E. J., and Rezvani, K. (2022)

KIR-based inhibitory CARs overcome CAR-NK cell tro-

gocytosis-mediated fratricide and tumor escape, Nat.

Med., 28, 2133-2144, doi:10.1038/s41591-022-02003-x.

40. Li, L., Mohanty, V., Dou, J., Huang, Y., Banerjee, P. P.,

Miao,Q., Lohr, J.G., Vijaykumar,T., Frede,J., Knoech-

el, B., Muniz-Feliciano, L., Laskowski, T. J., Liang, S.,

Moyes, J.S., Nandivada, V., Basar, R., Kaplan, M., Da-

her,M., Liu,E., Li,Y., Acharya,S., Lin,P., Shanley,M.,

Rafei,H., Marin,D., Mielke,S., Champlin, R.E., Shpall,

E. J., Chen, K., and Rezvani, K. (2023) Loss of meta-

bolic fitness drives tumor resistance after CAR-NK

cell therapy and can be overcome by cytokine engi-

neering, Sci. Adv., 9, eadd6997, doi: 10.1126/sciadv.

add6997.

41. Papadopoulou, M., Sanchez Sanchez, G., and Ver-

mijlen, D. (2020) Innate and adaptive γδ T cells:

how, when, and why, Immunol. Rev., 298, 99-116,

doi:10.1111/imr.12926.

42. Simões, A.E., Di Lorenzo,B., and Silva-Santos,B. (2018)

Molecular determinants of Target cell recognition by

human γδTcells, Front. Immunol., 9, 929, doi:10.3389/

fimmu.2018.00929.

43. Chien, Y. H., Meyer, C., and Bonneville, M. (2014)

γδTcells: first line of defense and beyond, Annu. Rev.

Immunol., 32, 121-155, doi:10.1146/annurev-immunol-

032713-120216.

44. Deng,J., and Yin,H. (2022) Gamma delta (γδ) Tcells in

cancer immunotherapy; where it comes from, where

it will go? Eur.J. Pharmacol., 919, 174803, doi:10.1016/

j.ejphar.2022.174803.

45. Sebestyen,Z., Prinz,I., Dechanet-Merville,J., Silva-San-

tos, B., and Kuball, J. (2020) Translating gammadel-

ta(γδ) Tcells and their receptors into cancer cell ther-

apies, Nat. Rev. Drug Discov., 19, 169-184, doi:10.1038/

s41573-019-0038-z.

46. Girardi,M., Oppenheim, D.E., Steele, C.R., Lewis, J.M.,

Glusac,E., Filler,R., Hobby,P., Sutton,B., Tigelaar, R.E.,

and Hayday, A.C. (2001) Regulation of cutaneous ma-

lignancy by gammadelta Tcells, Science, 294, 605-609,

doi:10.1126/science.1063916.

47. Donia,M., Ellebaek,E., Andersen, M.H., Straten, P.T.,

and Svane, I. M. (2012) Analysis of Vdelta1 T cells

in clinical grade melanoma-infiltrating lympho-

cytes, Oncoimmunology, 1, 1297-1304, doi: 10.4161/

onci.21659.

48. Wang,J., Lin,C., Li,H., Li,R., Wu,Y., Liu,H., Zhang,H.,

He,H., Zhang,W., and Xu,J. (2017) Tumor-infiltrating

γδTcells predict prognosis and adjuvant chemother-

apeutic benefit in patients with gastric cancer, On-

coimmunology, 6, e1353858, doi: 10.1080/2162402X.

2017.1353858.

49. Silva-Santos, B., Mensurado, S., and Coffelt, S. B.

(2019) γδ T cells: pleiotropic immune effectors with

therapeutic potential in cancer, Nat. Rev. Cancer, 19,

392-404, doi:10.1038/s41568-019-0153-5.

50. Gunderson, A.J., Kaneda, M.M., Tsujikawa,T., Nguyen,

A. V., Affara, N. I., Ruffell, B., Gorjestani, S., Liudahl,

S.M., Truitt,M., Olson,P., Kim,G., Hanahan,D., Tem-

pero, M.A., Sheppard,B., Irving,B., Chang, B.Y., Var-

ner, J. A., and Coussens, L. M. (2016) Bruton tyrosine

kinase-dependent immune cell cross-talk drives pan-

creas cancer, Cancer Discov., 6, 270-285, doi: 10.1158/

2159-8290.CD-15-0827.

51. Deniger, D.C., Switzer,K., Mi,T., Maiti,S., Hurton,L.,

Singh, H., Huls, H., Olivares, S., Lee, D.A., Champlin,

R.E., and Cooper, L.J. (2013) Bispecific T-cells express-

ing polyclonal repertoire of endogenous γδT-cell re-

ceptors and introduced CD19-specific chimeric an-

tigen receptor, Mol. Ther., 21, 638-647, doi: 10.1038/

mt.2012.267.

CAR CELLS BEYOND CLASSICAL CAR T CELLS 779

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

52. Rozenbaum, M., Meir, A., Aharony, Y., Itzhaki, O.,

Schachter,J., Bank,I., Jacoby,E., and Besser, M.J. (2020)

Gamma-delta CAR-T cells show CAR-directed and in-

dependent activity against leukemia, Front. Immunol.,

11, 1347, doi:10.3389/fimmu.2020.01347.

53. Fisher, J., Abramowski, P., Wisidagamage Don, N. D.,

Flutter, B., Capsomidis, A., Cheung, G. W., Gustafs-

son, K., and Anderson, J. (2017) Avoidance of on-tar-

get off-tumor activation using a co-stimulation-only

chimeric antigen receptor, Mol. Ther., 25, 1234-1247,

doi:10.1016/j.ymthe.2017.03.002.

54. Morandi, F., Yazdanifar, M., Cocco, C., Bertaina, A.,

and Airoldi,I. (2020) Engineering the bridge between

innate and adaptive immunity for cancer immuno-

therapy: focus on γδ T and NK cells, Cells, 9, 1757,

doi:10.3390/cells9081757.

55. Fleischer, L. C., Becker, S.A., Ryan, R.E., Fedanov,A.,

Doering, C. B., and Spencer, H. T. (2020) Non-signal-

ing chimeric antigen receptors enhance antigen-di-

rected killing by γδ T cells in contrast to αβ T cells,

Mol. Ther. Oncolytics, 18, 149-160, doi: 10.1016/j.omto.

2020.06.003.

56. Ali, A.K., Tarannum,M., and Romee,R. (2021) Is adop-

tive cellular therapy with non-T-cell immune effec-

tors the future? Cancer J., 27, 168-175, doi: 10.1097/

PPO.0000000000000517.

57. Vivier, E., and Anfossi, N. (2004) Inhibitory NK-cell

receptors on Tcells: witness of the past, actors of the

future, Nat. Rev. Immunol., 4, 190-198, doi: 10.1038/

nri1306.

58. Kriegsmann, K., Kriegsmann, M., von Bergwelt-Bail-

don,M., Cremer,M., and Witzens-Harig,M. (2018) NKT

cells– new players in CAR cell immunotherapy? Eur.J.

Haematol., 101, 750-757, doi:10.1111/ejh.13170.

59. Yoneda,K., Morii,T., Nieda,M., Tsukaguchi,N., Ama-

no,I., Tanaka,H., Yagi,H., Narita,N., and Kimura,H.

(2005) The peripheral blood Valpha24

NKT cell

numbers decrease in patients with haematopoiet-

ic malignancy, Leuk. Res., 29, 147-152, doi: 10.1016/

j.leukres.2004.06.005.

60. Tahir, S.M., Cheng,O., Shaulov,A., Koezuka,Y., Bubley,

G.J., Wilson, S.B., Balk, S.P., and Exley, M. A. (2001)

Loss of IFN-gamma production by invariant NKTcells

in advanced cancer, J. Immunol., 167, 4046-4050,

doi:10.4049/jimmunol.167.7.4046.

61. Tachibana, T., Onodera, H., Tsuruyama, T., Mori, A.,

Nagayama, S., Hiai, H., and Imamura, M. (2005) In-

creased intratumor Valpha24-positive natural kill-

er T cells: a prognostic factor for primary colorec-

tal carcinomas, Clin. Cancer Res., 11, 7322-7327,

doi:10.1158/1078-0432.CCR-05-0877.

62. Schneiders, F. L., de Bruin, R. C., van den Eertwegh,

A.J., Scheper, R. J., Leemans, C. R., Brakenhoff, R. H.,

Langendijk, J. A., Verheul, H. M., de Gruijl, T. D.,

Molling, J.W., and van der Vliet, H.J. (2012) Circulat-

ing invariant natural killer T-cell numbers predict

outcome in head and neck squamous cell carcinoma:

updated analysis with 10-year follow-up, J. Clin. On-

col., 30, 565-567, doi:10.1200/jco.2011.39.3975.

63. Bassiri, H., Das, R., Guan, P., Barrett, D.M., Brennan,

P. J., Banerjee, P. P., Wiener, S. J., Orange, J. S., Bren-

ner, M.B., Grupp, S.A., and Nichols, K.E. (2014) iNKT

cell cytotoxic responses control T-lymphoma growth

in vitro and in vivo, Cancer Immunol. Res., 2, 59-69,

doi:10.1158/2326-6066.CIR-13-0104.

64. Nair, S., and Dhodapkar, M. V. (2017) Natural killer

Tcells in cancer immunotherapy, Front. Immunol., 8,

1178, doi:10.3389/fimmu.2017.01178.

65. McEwen-Smith, R. M., Salio, M., and Cerundolo, V.

(2015) The regulatory role of invariant NKT cells in

tumor immunity, Cancer Immunol. Res., 3, 425-435,

doi:10.1158/2326-6066.CIR-15-0062.

66. Cortés-Selva, D., Dasgupta, B., Singh, S., and Grew-

al, I. S. (2021) Innate and innate-like cells: the fu-

ture of chimeric antigen receptor (CAR) cell thera-

py, Trends Pharmacol. Sci., 42, 45-59, doi: 10.1016/j.

tips.2020.11.004.

67. Heczey, A., Liu, D., Tian, G., Courtney, A. N., Wei, J.,

Marinova, E., Gao, X., Guo, L., Yvon, E., Hicks, J.,

Liu,H., Dotti,G., and Metelitsa, L.S. (2014) Invariant

NKT cells with chimeric antigen receptor provide

a novel platform for safe and effective cancer im-

munotherapy, Blood, 124, 2824-2833, doi: 10.1182/

blood-2013-11-541235.

68. Tian, G., Courtney, A. N., Jena, B., Heczey, A., Liu, D.,

Marinova,E., Guo,L., Xu,X., Torikai,H., Mo, Q., Dot-

ti, G., Cooper, L. J., and Metelitsa, L. S. (2016) CD62L

NKT cells have prolonged persistence and antitu-

mor activity in vivo, J. Clin. Invest., 126, 2341-2355,

doi:10.1172/JCI83476.

69. Simon, B., Wiesinger, M., Marz, J., Wistuba-Ham-

precht, K., Weide, B., Schuler-Thurner, B., Schul-

er, G., Dorrie, J., and Uslu, U. (2018) The generation

of CAR-transfected natural killer T cells for the im-

munotherapy of melanoma, Int. J. Mol. Sci., 19, 2365,

doi:10.3390/ijms19082365.

70. Shaik, R. S., Rathi, P., Courtney, A., Schneller, N.,

Guo, L., Barragan, G., Zhang, C., Xu, X., Sumazin, P.,

Metelitsa, L., and Heczey, A. (2022) Glypican-3-spe-

cific CAR-NKT cells overexpressing BATF3 mediate

potent antitumor activity against hepatocellular car-

cinoma, J. Clin. Oncol., 40, e14521, doi: 10.1200/jco.

2022.40.16_suppl.e14521.

71. Itoh,M., Takahashi,T., Sakaguchi,N., Kuniyasu,Y., Shi-

mizu, J., Otsuka, F., and Sakaguchi, S. (1999) Thymus

and autoimmunity: production of CD25

CD4

natural-

ly anergic and suppressive T cells as a key function

of the thymus in maintaining immunologic self-tol-

erance, J. Immunol., 162, 5317-5326, doi: 10.4049/

jimmunol.162.9.5317.

72. Romano, M., Fanelli,G., Albany, C. J., Giganti, G., and

Lombardi, G. (2019) Past, present, and future of reg-

MININA et al.780

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

ulatory T cell therapy in transplantation and au-

toimmunity, Front. Immunol., 10, 43, doi: 10.3389/

fimmu.2019.00043.

73. Arjomandnejad, M., Kopec, A. L., and Keeler, A. M.

(2022) CAR-T regulatory (CAR-Treg) cells: engineering

and applications, Biomedicines, 10, 287, doi: 10.3390/

biomedicines10020287.

74. Elinav, E., Waks, T., and Eshhar, Z. (2008) Redirec-

tion of regulatory T cells with predetermined spec-

ificity for the treatment of experimental colitis in

mice, Gastroenterology, 134, 2014-2024, doi: 10.1053/

j.gastro.2008.02.060.

75. Hombach, A. A., Kofler, D., Rappl, G., and Abken, H.

(2009) Redirecting human CD4

CD25

regulatory

Tcells from the peripheral blood with pre-defined tar-

get specificity, Gene Ther., 16, 1088-1096, doi:10.1038/

gt.2009.75.

76. Dawson, N.A.J., Rosado-Sánchez,I., Novakovsky, G.E.,

Fung, V.C.W., Huang,Q., McIver,E., Sun,G., Gillies,J.,

Speck,M., Orban, P.C., Mojibian,M., and Levings, M.K.

(2020) Functional effects of chimeric antigen receptor

co-receptor signaling domains in human regulatory

T cells, Sci. Transl. Med., 12, eaaz3866, doi: 10.1126/

scitranslmed.aaz3866.

77. Mohseni, Y. R., Saleem, A., Tung, S. L., Dudreuilh, C.,

Lang,C., Peng,Q., Volpe,A., Adigbli,G., Cross,A., Hes-

ter, J., Farzaneh, F., Scotta, C., Lechler, R. I., Issa, F.,

Fruhwirth, G. O., and Lombardi, G. (2021) Chimeric

antigen receptor-modified human regulatory T cells

that constitutively express IL-10 maintain their phe-

notype and are potently suppressive, Eur.J. Immunol.,

51, 2522-2530, doi:10.1002/eji.202048934.

78. Boroughs, A. C., Larson, R. C., Choi, B. D., Bouffard,

A. A., Riley, L. S., Schiferle, E., Kulkarni, A. S., Cetru-

lo, C.L., Ting,D., Blazar, B.R., Demehri,S., and Maus,

M. V. (2019) Chimeric antigen receptor costimulation

domains modulate human regulatory Tcell function,

JCI Insight, 5, e126194, doi:10.1172/jci.insight.126194.

79. Fu, R.Y., Chen, A.C., Lyle, M.J., Chen, C.Y., Liu, C.L.,

and Miao, C. H. (2020) CD4

T cells engineered with

FVIII-CAR and murine Foxp3 suppress anti-fac-

tor VIII immune responses in hemophilia a mice,

Cell Immunol., 358, 104216, doi: 10.1016/j.cellimm.

2020.104216.

80. Arjomandnejad, M., Sylvia, K., Blackwood, M., Nix-

on,T., Tang,Q., Muhuri,M., Gruntman, A.M., Gao,G.,

Flotte, T. R., and Keeler, A. M. (2021) Modulating im-

mune responses to AAV by expanded polyclonal T-regs

and capsid specific chimeric antigen receptor T-regu-

latory cells, Mol. Ther. Methods Clin. Dev., 23, 490-506,

doi:10.1016/j.omtm.2021.10.010.

81. Rana,J., and Biswas,M. (2020) Regulatory Tcell thera-

py: current and future design perspectives, Cell Immu-

nol., 356, 104193, doi:10.1016/j.cellimm.2020.104193.

82. MacDonald, K.G., Hoeppli, R.E., Huang,Q., Gillies,J.,

Luciani, D. S., Orban, P. C., Broady, R., and Levings,

M. K. (2016) Alloantigen-specific regulatory T cells

generated with a chimeric antigen receptor, J.Clin. In-

vest., 126, 1413-1424, doi:10.1172/JCI82771.

83. D’Souza, C., Chen, Z., and Corbett, A. J. (2018) Re-

vealing the protective and pathogenic potential of

MAIT cells, Mol. Immunol., 103, 46-54, doi: 10.1016/

j.molimm.2018.08.022.

84. Howson, L. J., Salio, M., and Cerundolo, V. (2015)

MR1-restricted mucosal-associated invariant T cells

and their activation during infectious diseases, Front.

Immunol., 6, 303, doi:10.3389/fimmu.2015.00303.

85. Le Bourhis, L., Dusseaux, M., Bohineust, A., Bes-

soles, S., Martin, E., Premel, V., Core, M., Sleurs, D.,

Serriari, N. E., Treiner, E., Hivroz, C., Sansonetti, P.,

Gougeon, M.L., Soudais,C., and Lantz,O. (2013) MAIT

cells detect and efficiently lyse bacterially-infected ep-

ithelial cells, PLoS Pathog., 9, e1003681, doi: 10.1371/

journal.ppat.1003681.

86. Dogan, M., Karhan, E., Kozhaya, L., Placek, L.,

Chen, X., Yigit, M., and Unutmaz, D. (2022) Engi-

neering human mucosal associated invariant T

(MAIT) cells with chimeric antigen receptors for

cancer immunotherapy, J. Immunol., 209, 1523-1531,

doi:10.1101/2022.07.28.501764.

87. Won, E. J., Ju, J. K., Cho, Y. N., Jin, H. M., Park, K. J.,

Kim, T. J., Kwon, Y.-S., Kee, H. J., Kim, J. C., Kee, S.-J.,

and Park, Y.-W. (2016) Clinical relevance of circulating

mucosal-associated invariant T cell levels and their

anti-cancer activity in patients with mucosal-associ-

ated cancer, Oncotarget, 7, 76274-76290, doi:10.18632/

oncotarget.11187.

88. Gherardin, N.A., Loh,L., Admojo,L., Davenport, A.J.,

Richardson,K., Rogers,A., Darcy, P.K., Jenkins, M.R.,

Prince, H. M., Harrison, S.J., Quach,H., Fairlie, D.P.,

Kedzierska, K., McCluskey, J., Uldrich, A. P., Neeson,

P. J., Ritchie, D. S., and Godfrey, D. I. (2018) Enumer-

ation, functional responses and cytotoxic capacity

of MAIT cells in newly diagnosed and relapsed mul-

tiple myeloma, Sci. Rep., 8, 4159, doi: 10.1038/s41598-

018-22130-1.

89. Reantragoon, R., Boonpattanaporn, N., Corbett, A. J.,

and McCluskey,J. (2016) Mucosal-associated invariant

Tcells in clinical diseases, As. Pac.J. Allergy Immunol.,

34, 3-10.

90. Zhang, Z.-X., Yang, L., Young, K. J., Dutemple, B., and

Zhang, L. I. (2000) Identification of a previously un-

known antigen-specific regulatory T cell and its

mechanism of suppression, Nat. Med., 6, 782-789,

doi:10.1038/77513.

91. Wu, Z., Zheng, Y., Sheng, J., Han, Y., Yang, Y., Pan, H.,

and Yao, J. (2022) CD3

CD4

–

CD8

–

(Double-Negative)

T cells in inflammation, immune disorders and can-

cer, Front. Immunol., 13, 816005, doi: 10.3389/fimmu.

2022.816005.

92. Zhang, Z.-X., Yang,L., Young, K.J., and Zhang,L. (2001)

Suppression of alloimmune responses in vitro and

CAR CELLS BEYOND CLASSICAL CAR T CELLS 781

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

invivo by CD3CD8CD4 regulatory Tcells, Transplant.

Proc., 33, 84-85, doi:10.1016/s0041-1345(00)01915-1.

93. Young, K.J., DuTemple,B., Phillips, M.J., and Zhang,L.

(2003) Inhibition of graft-versus-host disease by dou-

ble-negative regulatory T cells, J.Immunol., 171, 134-

141, doi:10.4049/jimmunol.171.1.134.

94. Chen, W., Ford, M. S., Young, K. J., Cybulsky, M. I.,

and Zhang, L. (2003) Role of double-negative reg-

ulatory T cells in long-term cardiac xenograft sur-

vival, J. Immunol., 170, 1846-1853, doi: 10.4049/

jimmunol.170.4.1846.

95. Chen, J., Hu,P., Wu,G., and Zhou,H. (2019) Antipan-

creatic cancer effect of DNT cells and the underlying

mechanism, Pancreatology, 19, 105-113, doi: 10.1016/

j.pan.2018.12.006.

96. Chen, X., Wang, D., and Zhu,X. (2022) Application of

double-negative T cells in haematological malignan-

cies: recent progress and future directions, Biomark.

Res., 10, 11, doi:10.1186/s40364-022-00360-w.

97. Merims, S., Li, X., Joe, B., Dokouhaki, P., Han, M.,

Childs, R. W., Wang, Z. Y., Gupta, V., Minden, M. D.,

and Zhang, L. (2011) Anti-leukemia effect of ex vivo

expanded DNT cells from AML patients: a potential

novel autologous T-cell adoptive immunotherapy,

Leukemia, 25, 1415-1422, doi:10.1038/leu.2011.99.

98. Lee, J.B., Kang,H., Fang,L., D’Souza,C., Adeyi,O., and

Zhang, L. (2019) Developing allogeneic double-neg-

ative Tcells as a novel off-the-shelf adoptive cellular

therapy for cancer, Clin. Cancer Res., 25, 2241-2253,

doi:10.1158/1078-0432.CCR-18-2291.

99. Vasic,D., Lee, J.B., Leung,Y., Khatri,I., Na,Y., Abate-Da-

ga,D., and Zhang,L. (2022) Allogeneic double-negative

CAR-T cells inhibit tumor growth without off-tumor

toxicities, Sci. Immunol., 7, eabl3642, doi: 10.1126/

sciimmunol.abl3642.

100. Gao, X., Mi, Y., Guo, N., Xu, H., Xu, L., Gou, X., and

Jin,W. (2017) Cytokine-induced killer cells as pharma-

cological tools for cancer immunotherapy, Front. Im-

munol., 8, 774, doi:10.3389/fimmu.2017.00774.

101. Grimm, E.A., Mazumder,A., Zhang, H.Z., and Rosen-

berg, S.A. (1982) Lymphokine-activated killer cell phe-

nomenon. Lysis of natural killer-resistant fresh solid

tumor cells by interleukin 2-activated autologous hu-

man peripheral blood lymphocytes, J.Exp. Med., 155,

1823-1841, doi:10.1084/jem.155.6.1823.

102. Rosenberg, S. A., Spiess, P., and Lafreniere, R. (1986)

A new approach to the adoptive immunotherapy of

cancer with tumor-infiltrating lymphocytes, Science,

233, 1318-1321, doi:10.1126/science.3489291.

103. Cappuzzello,E., Sommaggio,R., Zanovello,P., and Ro-

sato, A. (2017) Cytokines for the induction of antitu-

mor effectors: the paradigm of cytokine-induced kill-

er (CIK) cells, Cytokine Growth Factor Rev., 36, 99-105,

doi:10.1016/j.cytogfr.2017.06.003.

104. Diefenbach,A., Jamieson, A.M., Liu, S.D., Shastri,N.,

and Raulet, D.H. (2000) Ligands for the murine NKG2D

receptor: expression by tumor cells and activation of

NKcells and macrophages, Nat. Immunol., 1, 119-126,

doi:10.1038/77793.

105. Merker,M., Wagner,J., Kreyenberg,H., Heim,C., Mos-

er, L. M., Wels, W. S., Bonig, H., Ivics, Z., Ullrich, E.,

Klingebiel, T., Bader, P., and Rettinger, E. (2020)

ERBB2-CAR-engineered cytokine-induced killer cells

exhibit both CAR-mediated and innate immunity

against high-risk rhabdomyosarcoma, Front. Immu-

nol., 11, 581468, doi:10.3389/fimmu.2020.581468.

106. Rotolo, R., Leuci, V., Donini, C., Cykowska, A., Gam-

maitoni, L., Medico, G., Valabrega, G., Aglietta, M.,

and Sangiolo, D. (2019) CAR-based strategies beyond

T lymphocytes: integrative opportunities for cancer

adoptive immunotherapy, Int. J. Mol. Sci., 20, 2839,

doi:10.3390/ijms20112839.

107. Marin,V., Dander, E., Biagi, E., Introna, M., Fazio, G.,

Biondi,A., and D’Amico,G. (2006) Characterization of

in vitro migratory properties of anti-CD19 chimeric

receptor-redirected CIK cells for their potential use in

B-ALL immunotherapy, Exp. Hematol., 34, 1219-1229,

doi:10.1016/j.exphem.2006.05.004.

108. Marin, V., Kakuda, H., Dander, E., Imai, C., Campa-

na,D., Biondi,A., and D’Amico,G. (2007) Enhancement

of the anti-leukemic activity of cytokine induced killer

cells with an anti-CD19 chimeric receptor delivering a

4-1BB-zeta activating signal, Exp. Hematol., 35, 1388-

1397, doi:10.1016/j.exphem.2007.05.018.

109. Marin, V., Pizzitola, I., Agostoni, V., Attianese, G. M.,

Finney, H., Lawson, A., Pule, M., Rousseau, R., Bion-

di, A., and Biagi, E. (2010) Cytokine-induced killer

cells for cell therapy of acute myeloid leukemia: im-

provement of their immune activity by expression of

CD33-specific chimeric receptors, Haematologica, 95,

2144-2152, doi:10.3324/haematol.2010.026310.

110. Magnani, C. F., Mezzanotte, C., Cappuzzello, C., Bar-

dini,M., Tettamanti, S., Fazio,G., Cooper, L. J. N., Da-

stoli,G., Cazzaniga,G., Biondi,A., and Biagi,E. (2018)

Preclinical efficacy and safety of CD19CAR cytokine-

induced killer cells transfected with sleeping beauty

transposon for the treatment of acute lymphoblastic

leukemia, Hum. Gene Ther., 29, 602-613, doi: 10.1089/

hum.2017.207.

111. Jarosz-Biej, M., Kaminska, N., Matuszczak, S., Ci-

chon, T., Pamula-Pilat, J., Czapla, J., Smolarczyk, R.,

Skwarzynska,D., Kulik,K., and Szala,S. (2018) M1-like

macrophages change tumor blood vessels and mi-

croenvironment in murine melanoma, PLoS One, 13,

e0191012, doi:10.1371/journal.pone.0191012.

112. Biglari, A., Southgate, T. D., Fairbairn, L. J., and Gil-

ham, D. E. (2006) Human monocytes expressing a

CEA-specific chimeric CD64 receptor specifically tar-

get CEA-expressing tumour cells in vitro and invivo,

Gene Ther., 13, 602-610, doi:10.1038/sj.gt.3302706.

113. Morrissey, M. A., Williamson, A. P., Steinbach, A. M.,

Roberts, E.W., Kern,N., Headley, M.B., and Vale, R.D.

MININA et al.782

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

(2018) Chimeric antigen receptors that trigger phago-

cytosis, Elife, 7, e36688, doi:10.7554/eLife.36688.

114. Klichinsky, M., Ruella, M., Shestova, O., Lu, X. M.,

Best,A., Zeeman,M., Schmierer,M., Gabrusiewicz,K.,

Anderson, N.R., Petty, N.E., Cummins, K.D., Shen,F.,

Shan,X., Veliz,K., Blouch,K., Yashiro-Ohtani,Y., Ken-

derian, S.S., Kim, M.Y., O’Connor, R.S., Wallace, S.R.,

Kozlowski, M.S., Marchione, D.M., Shestov,M., Garcia,

B.A., June, C.H., and Gill,S. (2020) Human chimeric

antigen receptor macrophages for cancer immuno-

therapy, Nat. Biotechnol., 38, 947-953, doi: 10.1038/

s41587-020-0462-y.

115. Cannac,M., Nikolic,J., and Benaroch,P. (2022) Cancer

immunotherapies based on genetically engineered

macrophages, Cancer Immunol. Res., 10, 1156-1166,

doi:10.1158/2326-6066.CIR-22-0030.

116. Zhang, L., Tian, L., Dai, X., Yu, H., Wang, J., Lei, A.,

Zhu, M., Xu, J., Zhao, W., Zhu, Y., Sun, Z., Zhang, H.,

Hu, Y., Wang, Y., Xu, Y., Church, G. M., Huang, H.,

Weng, Q., and Zhang, J. (2020) Pluripotent stem cell-

derived CAR-macrophage cells with antigen-depen-

dent anti-cancer cell functions, J.Hematol. Oncol., 13,

153, doi:10.1186/s13045-020-00983-2.

117. Hagemann,T., Lawrence,T., McNeish,I., Charles, K.A.,

Kulbe,H., Thompson, R.G., Robinson, S.C., and Balk-

will, F.R. (2008) “Re-educating” tumor-associated mac-

rophages by targeting NF-kappaB, J. Exp. Med., 205,

1261-1268, doi:10.1084/jem.20080108.

118. Niu,Z., Chen,G., Chang,W., Sun,P., Luo,Z., Zhang,H.,

Zhi,L., Guo,C., Chen,H., Yin,M., and Zhu, W. (2021)

Chimeric antigen receptor-modified macrophages

trigger systemic anti-tumour immunity, J.Pathol., 253,

247-257, doi:10.1002/path.5585.

119. Zhang, W., Liu, L., Su, H., Liu, Q., Shen, J., Dai, H.,

Zheng, W., Lu, Y., Zhang, W., Bei, Y., and Shen, P.

(2019) Chimeric antigen receptor macrophage ther-

apy for breast tumours mediated by targeting the tu-

mour extracellular matrix, Br.J. Cancer, 121, 837-845,

doi:10.1038/s41416-019-0578-3.

120. Williford, J.M., Ishihara,J., Ishihara,A., Mansurov,A.,

Hosseinchi,P., Marchell, T.M., Potin,L., Swartz, M.A.,

and Hubbell, J.A. (2019) Recruitment of CD103

den-

dritic cells via tumor-targeted chemokine delivery en-

hances efficacy of checkpoint inhibitor immunothera-

py, Sci. Adv., 5, eaay1357, doi:10.1126/sciadv.aay1357.

121. Maier,B., Leader, A.M., Chen, S.T., Tung,N., Chang,C.,

LeBerichel,J., Chudnovskiy,A., Maskey,S., Walker,L.,

Finnigan, J. P., Kirkling, M. E., Reizis, B., Ghosh, S.,

D’Amore, N. R., Bhardwaj, N., Rothlin, C. V., Wolf, A.,

Flores, R., Marron, T., Rahman, A. H., Kenigsberg, E.,

Brown, B.D., and Merad,M. (2020) Aconserved den-

dritic-cell regulatory program limits antitumour

immunity, Nature, 580, 257-262, doi: 10.1038/s41586-

020-2134-y.

122. Hildner,K., Edelson, B.T., Purtha, W.E., Diamond,M.,

Matsushita, H., Kohyama, M., Calderon, B., Schraml,

B. U., Unanue, E.R., Diamond, M.S., Schreiber, R. D.,

Murphy, T. L., and Murphy, K. M. (2008) Batf3 defi-

ciency reveals a critical role for CD8α

dendritic cells

in cytotoxic T cell immunity, Science, 322, 1097-1100,

doi:10.1126/science.1164206.

123. Suh, H. C., Pohl, K. A., Termini, C., Kan, J., Timmer-

man, J. M., Slamon, D. J., and Chute, J. P. (2018) Bio-

engineered autologous dendritic cells enhance car

t cell cytotoxicity by providing cytokine stimulation

and intratumoral dendritic cells, Blood, 132, 3693,

doi:10.1182/blood-2018-99-115296.

124. Zhang,L., Morgan, R.A., Beane, J. D., Zheng,Z., Dud-

ley, M. E., Kassim, S.H., Nahvi, A.V., Ngo, L. T., Sher-

ry, R. M., Phan, G. Q., Hughes, M.S., Kammula, U.S.,

Feldman, S. A., Toomey, M. A., Kerkar, S. P., Restifo,

N.P., Yang, J.C., and Rosenberg, S.A. (2015) Tumor-in-

filtrating lymphocytes genetically engineered with an

inducible gene encoding interleukin-12 for the immu-

notherapy of metastatic melanoma, Clin. Cancer Res.,

21, 2278-2288, doi:10.1158/1078-0432.CCR-14-2085.

125. Ruella,M., Xu,J., Barrett, D.M., Fraietta, J. A., Reich,

T. J., Ambrose, D. E., Klichinsky, M., Shestova, O., Pa-

tel, P.R., Kulikovskaya,I., Nazimuddin,F., Bhoj, V.G.,

Orlando, E.J., Fry, T.J., Bitter,H., Maude, S.L., Levine,

B.L., Nobles, C.L., Bushman, F.D., Young, R.M., Schol-

ler,J., Gill, S.I., June, C.H., Grupp, S.A., Lacey, S.F., and

Melenhorst, J.J. (2018) Induction of resistance to chi-

meric antigen receptor T cell therapy by transduction

of a single leukemic B cell, Nat. Med., 24, 1499-1503,

doi:10.1038/s41591-018-0201-9.

126. Pesch,T., Bonati,L., Kelton,W., Parola,C., Ehling, R.A.,

Csepregi,L., Kitamura,D., and Reddy, S.T. (2019) Mo-

lecular design, optimization, and genomic integration

of chimeric Bcell receptors in murine B cells, Front.

Immunol., 10, 2630, doi:10.3389/fimmu.2019.02630.

127. Neelapu, S.S., Locke, F.L., Bartlett, N.L., Lekakis, L.J.,

Miklos, D. B., Jacobson, C. A., Braunschweig, I., Olu-

wole, O.O., Siddiqi,T., Lin,Y., Timmerman, J.M., Stiff,

P. J., Friedberg, J. W., Flinn, I. W., Goy, A., Hill, B. T.,

Smith, M.R., Deol,A., Farooq,U., McSweeney,P., Mu-

noz,J., Avivi,I., Castro, J.E., Westin, J.R., Chavez, J.C.,

Ghobadi,A., Komanduri, K.V., Levy,R., Jacobsen, E.D.,

Witzig, T. E., Reagan, P., Bot, A., Rossi, J., Navale, L.,

Jiang,Y., Aycock, J., Elias, M., Chang, D., Wiezorek,J.,

and Go, W.Y. (2017) Axicabtagene ciloleucel CAR T-cell

therapy in refractory large B-cell lymphoma, N.Engl. J.

Med., 377, 2531-2544, doi:10.1056/NEJMoa1707447.

128. Schuster, S. J., Svoboda, J., Chong, E. A., Nasta, S. D.,

Mato, A.R., Anak,O., Brogdon, J.L., Pruteanu-Malini-

ci,I., Bhoj, V., Landsburg,D., Wasik,M., Levine, B.L.,

Lacey, S. F., Melenhorst, J. J., Porter, D. L., and June,

C. H. (2017) Chimeric antigen receptor T cells in re-

fractory B-cell lymphomas, N.Engl. J.Med., 377, 2545-

2554, doi:10.1056/NEJMoa1708566.

129. Imura, Y., Ando, M., Kondo, T., Ito, M., and Yoshimu-

ra,A. (2020) CD19-targeted CAR regulatory Tcells sup-

CAR CELLS BEYOND CLASSICAL CAR T CELLS 783

BIOCHEMISTRY (Moscow) Vol. 89 No. 5 2024

press B cell pathology without GvHD, JCI Insight, 5,

e136185, doi:10.1172/jci.insight.136185.

130. Raje, N., Berdeja, J., Lin, Y., Siegel, D., Jagannath, S.,

Madduri, D., Liedtke, M., Rosenblatt, J., Maus, M. V.,

Turka,A., Lam, L.P., Morgan, R.A., Friedman,K., Mas-

saro, M., Wang, J., Russotti, G., Yang, Z., Campbell, T.,

Hege, K., Petrocca, F., Quigley, M. T., Munshi, N., and

Kochenderfer, J.N. (2019) Anti-BCMA CAR T-cell ther-

apy bb2121 in relapsed or refractory multiple my-

eloma, N. Engl. J. Med., 380, 1726-1737, doi: 10.1056/

NEJMoa1817226.

131. Berdeja, J. G., Madduri, D., Usmani, S. Z., Jakubowi-

ak,A., Agha, M., Cohen, A.D., Stewart, A. K., Hari,P.,

Htut,M., Lesokhin, A., Deol,A., Munshi, N.C., O’Don-

nell, E., Avigan, D., Singh, I., Zudaire, E., Yeh, T. M.,

Allred, A. J., Olyslager, Y., Banerjee, A., Jackson, C. C.,

Goldberg, J. D., Schecter, J. M., Deraed, W., Zhuang,

S. H., Infante, J., Geng, D., Wu, X., Carrasco-Alfonso,

M.J., Akram,M., Hossain, F., Rizvi,S., Fan,F., Lin, Y.,

Martin,T., and Jagannath,S. (2021) Ciltacabtagene au-

toleucel, a B-cell maturation antigen-directed chimeric

antigen receptor T-cell therapy in patients with re-

lapsed or refractory multiple myeloma (CARTITUDE-1):

a phase 1b/2 open-label study, Lancet, 398, 314-324,

doi:10.1016/S0140-6736(21)00933-8.

132. Wang, X., Jasinski, D.L., Medina, J.L., Spencer, D.M.,

Foster, A.E., and Bayle, J.H. (2020) Inducible MyD88/

CD40 synergizes with IL-15 to enhance antitumor

efficacy of CAR-NK cells, Blood Adv., 4, 1950-1964,

doi:10.1182/bloodadvances.2020001510.

133. Shin, M.H., Oh,E., Kim,Y., Nam, D.H., Jeon, S.Y., Yu,

J. H., and Minn, D. (2023) Recent advances in CAR-

based solid tumor immunotherapy, Cells, 12, 1606,

doi:10.3390/cells12121606.

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