TYPE Review

PUBLISHED 07 December 2023

DOI 10.3389/fimmu.2023.1302307

CAR-T cell immunotherapy

OPEN ACCESS for ovarian cancer:
EDITED BY
Cristina Maccalli,
Sidra Medicine, Qatar
hushing the silent killer
REVIEWED BY
Alessandro Poggi, Fatemeh Nasiri 1,2†, Khadijeh Farrokhi 3†,
San Martino Hospital (IRCCS), Italy
Degang Song, Pouya Safarzadeh Kozani 1*, Maral Mahboubi Kancha 4,
Janssen Pharmaceuticals, Inc.,
United States
Setareh Dashti Shokoohi 1 and Pooria Safarzadeh Kozani 1*
*CORRESPONDENCE
1
Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University,
Pouya Safarzadeh Kozani Tehran, Iran, 2 Department of Production Platforms & Analytics, Human Health Therapeutics Research
puyasafarzadeh@[Link] Centre, National Research Council Canada, Montreal, QC, Canada, 3 Department of Microbial
Biotechnology, Faculty of Biotechnology, Amol University of Special Modern Technologies,
Pooria Safarzadeh Kozani
Amol, Iran, 4 Department of Medical Nanotechnology, School of Medicine, Shahroud University of
pooriasafarzadeh@[Link]
Medical Sciences, Shahroud, Iran
†
These authors share first authorship

RECEIVED 26 September 2023

ACCEPTED 13 November 2023
PUBLISHED 07 December 2023 As the most lethal gynecologic oncological indication, carcinoma of the ovary
CITATION has been ranked as the 5th cause of cancer-related mortality in women, with a
Nasiri F, Farrokhi K, Safarzadeh Kozani P, high percentage of the patients being diagnosed at late stages of the disease and
Mahboubi Kancha M, Dashti Shokoohi S
a five-year survival of ~ 30%. Ovarian cancer patients conventionally undergo
and Safarzadeh Kozani P (2023)
CAR-T cell immunotherapy for ovarian surgery for tumor removal followed by platinum- or taxane-based
cancer: hushing the silent killer. chemotherapy; however, a high percentage of patients experience tumor
Front. Immunol. 14:1302307.
doi: 10.3389/fimmu.2023.1302307 relapse. Cancer immunotherapy has been regarded as a silver lining in the
treatment of patients with various immunological or oncological indications;
COPYRIGHT
© 2023 Nasiri, Farrokhi, Safarzadeh Kozani, however, mirvetuximab soravtansine (a folate receptor a-specific mAb) and
Mahboubi Kancha, Dashti Shokoohi and bevacizumab (a VEGF-A-specific mAb) are the only immunotherapeutics
Safarzadeh Kozani. This is an open-access
article distributed under the terms of the approved for the treatment of ovarian cancer patients. Chimeric antigen
Creative Commons Attribution License receptor T-cell (CAR-T) therapy has achieved tremendous clinical success in
(CC BY). The use, distribution or
the treatment of patients with certain B-cell lymphomas and leukemias, as well
reproduction in other forums is permitted,
provided the original author(s) and the as multiple myeloma. In the context of solid tumors, CAR-T therapies face
copyright owner(s) are credited and that serious obstacles that limit their therapeutic benefit. Such hindrances include
the original publication in this journal is
cited, in accordance with accepted
the immunosuppressive nature of solid tumors, impaired tumor infiltration, lack
academic practice. No use, distribution or of qualified tumor-associated antigens, and compromised stimulation and
reproduction is permitted which does not persistence of CAR-Ts following administration. Over the past years,
comply with these terms.
researchers have made arduous attempts to apply CAR-T therapy to ovarian
cancer. In this review, we outline the principles of CAR-T therapy and then
highlight its limitations in the context of solid tumors. Ultimately, we focus on
preclinical and clinical findings achieved in CAR-T-mediated targeting of
different ovarian cancer-associated target antigens.

KEYWORDS

cancer immunotherapy, chimeric antigen receptor, ovarian cancer, solid tumors,

adoptive cell therapy

Frontiers in Immunology 01 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

1 Introduction respectively (7). The standard of care for the treatment of ovarian
cancer is surgery, radiation therapy, and chemotherapy using
Ovarian cancer is a type of malignant tumor involving the ovary platinum-based chemotherapeutic agents (8–10). Such treatment
tissue. It is originally derived from the ovary itself but it can also modalities can only be clinically beneficial in patients diagnosed
originate from other structures in the vicinity of an ovary including with early-stage diseases. In the case of late-stage diagnosis, patients
the fallopian tubes (1, 2). Ovarian cancer is the most fatal will experience refectory or recurrent disease, and disease-free
gynecologic neoplasm (3). This often called “silent killer” type of intervals will be shorter in such cases.
cancer is among cancers with relatively poor prognosis mainly Accumulating evidence indicates that tumor-infiltrating
because it is not accurately diagnosed until it reaches its late and lymphocytes (TILs) have remarkable clinical significance and
advanced stages generally due to its vague and/or common clinical prognostic value in various types of solid tumors, revealing their
symptoms (3, 4). Statistics indicate that more than 70% of ovarian immunogenic nature (11–14). Ovarian cancer is also among
cancer cases are not diagnosed before stage III or IV, and the five- malignancies in which TILs play an important role in the clinical
year survival rate for patients is reported to be around 47% (5). response and clinical outcomes of the patients. In detail, there are
According to estimations, around 19,000 new cases of ovarian statistically significant differences in the distributions of overall
cancer are diagnosed annually and around 12,000 ovarian cancer- survival (OS) and progression-free survival (PFS) in ovarian cancer
related mortality occur each year (6). In terms of classification, patients based on the presence or absence of TILs (15). According to
ovarian cancer is categorized into three main types, epithelial, germ a study, the five-year OS rate for ovarian cancer patients with and
cell, and sex-cord-stromal (Figure 1) (7). Epithelial ovarian cancer is without TILs was 38 and 4.5%, respectively (15). Moreover, it has
the most common type among the diagnosed cases accounting for been reported that the five-year OS rate for ovarian cancer patients
around 95% of all the cases. This type has four subtypes including with a complete response after chemotherapy using platinum-based
serous, endometrioid, mucinous, and clear cell. Serous epithelial agents or debulking was 73.9 and 11.9% in patients with and
ovarian cancer is also categorized into high-grade serous without TILs, respectively (15). Research findings also indicate
carcinomas (HGSC) or low-grade serous carcinomas (LGSC). that ovarian cancer patients with TILs exhibit increased
HGSC is the most common subtype of epithelial ovarian cancer intratumoral expression of INF-g, IL-2, and T-cell-associated
accounting for around 70%. This is while LGSC, endometrioid, chemokines alongside having postponed disease recurrence and
mucinous, and clear cell account for around 5, 10, 3, and 10%, death while patients with no TIL have profiles of elevated levels of

FIGURE 1
A simplified schematic of the ovarian cancer types and subtypes.

Frontiers in Immunology 02 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

vascular endothelial growth factor (VEGF) expression (15). Such EpCAM, etc., are the most investigated ones. Recently, researchers
findings point out the fact that ovarian cancer has an immunogenic have developed CAR-T products and applied them to ovarian
nature; therefore, immune-based therapies could be leveraged as c an c e r f o r t h e r ap e u t i c p u r po s e s . I n t h i s ar t i c l e , w e
potent treatment modalities. comprehensively and in a detailed manner review CAR-Ts
Cancer immunotherapy has given hope to patients with developed and investigated for the treatment of ovarian cancer in
advanced oncological and immunological indications over the different experimental stages (focusing on different ovarian cancer
past 40 years. Since then, multiple platforms of this highly cell lines, various cell line- or patient-derived xenograft-based
effective treatment strategy have been established, and the United preclinical animal models, or individuals with different stages of
States Food and Drug Administration (US FDA) has granted ovarian cancer) (Figure 3). We will also detail how researchers
permission to a large number of immunotherapeutics for medical develop counterstrategies to overcome various limitations of CAR-
use. Monoclonal antibodies (mAbs; with the first approval in 1986), T therapy in ovarian cancer, including poor CAR-T trafficking and
T-cell-redirecting bispecific antibodies (TRBAs; with the first persistence, as well as low antigen density of tumor cells.
approval in 2014), antibody-drug conjugates (ADCs; with the first
approval in 2000), and chimeric antigen receptor T lymphocytes
(CAR-Ts; with the first approval in 2017) are examples of how 2 Immunotherapy in ovarian cancer
cancer immunotherapy has changed the landscape of cancer
treatment in an effective and targeted fashion (16–19). To date, The advent of targeted immunotherapy has been considered a
six CAR-T products (Figure 2) and more than a hundred mAbs silver lining in the treatment of cancer patients. With fewer adverse
have been given the green light for clinical use by the US FDA. events and higher remission rates, this treatment modality is slowly
CAR-Ts are T lymphocytes modified to surface-display CARs moving to the front line of treatments for patients with cancer.
by the means of which their cytotoxicity is redirected against cells Among different types of immunotherapies, mAb-based and T-cell-
proficient in the expression of tumor-associated antigens (TAAs) or based therapies are considered the most clinically efficient ones. In
tumor-specific antigens (TSAs) (20). To date, numerous research the past decade, mAb-based cancer treatment modalities have been
teams around the world have put an enormous amount of effort into a hot topic for the treatment of both hematologic malignancies and
the assessment of CAR-T for the treatment of a wide range of solid tumors. Since the FDA approval of rituximab for the treatment
hematologic cancers (including B-cell leukemias and lymphomas, of patients with chemotherapy regimen-resistant B-cell non-
MM, as well as T-cell neoplasms) and solid tumors (including, Hodgkin lymphomas in 1997, many other mAbs have also been
gliomas, gastrointestinal cancers, thyroid cancer, prostate cancer, granted permission for medical use in the field of cancer treatment
breast cancer, lung cancer, cervical cancer, head and neck cancer, (21). mAb-based therapies have proven efficient for the treatment of
and ovarian cancer). Among the targeted antigens, CD19, BCMA, a wide range of solid tumors in which other types of immune-based
CD22, CD20, CD30, CD123, HER2, EGFR, VEGF-R, Claudin, PD- therapies may provide minimal clinical benefit. This is why mAbs
L1, B7-H3, GD2 TROP-2, MUC1, MUC16, CGRP, CD38, SLAMF7, are considered a cornerstone in the treatment of many types of solid

FIGURE 2
A detailed timeline of CAR-T therapy development from the laboratory bench to the approval of several products by the US FDA for medical use. B-
ALL, B-cell acute lymphoblastic leukemia; DLBCL, diffuse large B-cell lymphoma; FDA, Food and Drug Administration; FL, follicular lymphoma; MCL,
mantel cell lymphoma; MM, multiple myeloma; MSKCC, Memorial Sloan Kettering Cancer Center; NCI, National Cancer Institute; UPenn, University
of Pennsylvania.

Frontiers in Immunology 03 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

FIGURE 3
A summary of CAR-T therapy of different ovarian cancer-associated antigens assessed in different investigational stages.

tumors including ovarian cancer. In this regard, various antigens clinical trial (NCT00562640) investigating the safety and feasibility
have been investigated in the field of mAb-based therapy for of autologous WT1-specific T lymphocytes in patients with
ovarian cancer (Table 1). Among these target antigens, VEGF, recurrent ovarian cancer (25). The enrolled patients included
EGFR, EpCAM, FRa, CA125, MUC1, PD-1, PD-L1, and CTLA-4 twelve patients aged between 23 to 72 years old and who had at
are the ones comprehensively investigated. Alongside mAb-based least 4 lines of prior failed therapies. According to the results, no
therapies, other types of novel immunotherapeutic approaches are dose-limiting toxicities (DLTs) were documented during the course
being investigated against ovarian cancer, which including T-cell- of the study. Median PFS and median OS were reported as 1.8 and
based therapies. T-cell receptors (TCRs) are antigen receptors 11.0 months, respectively. Moreover, 1-year PFS and 1-year OS
expressed on the surface of T cells. a/b T cells are a group of T were reported to be 8.3% and 41.7%, respectively. Of note, stable
lymphocytes in which the TCR is composed of an a and a b chain disease was only observed in one patient; whereas eleven others
that function together and recognize antigenic peptide fragments experienced progressive disease (25). It was reported that there was
that are presented by major histocompatibility complexes (MHCs) a rise in the level of WT1-specific cytotoxic T lymphocyte
(22). T cells have been used for the aim of cancer therapy in precursors in 9 out of 12 patients after the treatment course.
different methods including TILs, TCR-engineered T cells, and Overall, such studies can conclude that TCR-engineered T cells
CAR-Ts (23, 24). TCR-engineered T cells are T lymphocytes that with antigen-specific TCRs are well-tolerated in patients and can
have been genetically engineered to recognize a specific peptide mediate mild therapeutic benefits in patients with recurrent ovarian
antigen presented by a particular type of MHC. This process entails cancer (25). Of note, more clinical trials with broader patient
the genetic engineering of T cells for the expression of TCR a and b populations are required to elucidate the safety and clinical
chains which are specific for a certain antigen. These a and b chains efficacy of TCR-engineered T-cell therapy in ovarian cancer.
come from the genes of an activated T cell (24). Using this method, CAR-Ts are T cells genetically engineered to express CARs on
TCR-engineered T cells demonstrate enhanced affinity and their surface by the means of which they are capable of targeting cells
specificity to desired cancer antigens. Dissimilar to CAR-Ts which proficient in the expression of TAAs or TSAs of interest in a fashion
are only capable of interacting with cell surface-expressed antigens, similar to that of mAbs and without the engagement of MHC. The
TCR-engineered T cells can interact with MHC-presented genetic manipulation of CAR-Ts usually entails the application of
intracellular and surface proteins (24). TCR-engineered T-cell viral gene introduction techniques (such as by the means of lentiviral
therapy has been investigated in a wide range of malignancies or retroviral particles) or non-viral gene introduction methods
including ovarian cancer. Wilms’ tumor protein 1 (WT1), (including transposons, mRNA electroporation, etc.) (26). In
melanoma-associated antigen 4 (MAGE-A4), and New York reference to the structural characteristics of CAR molecules, CARs
esophageal-1 (NY-ESO-1) are among the targets investigated in are composed of three topological domains; extracellular domain,
this regard. For instance, Kyi et al. reported the results Phase I transmembrane domain, and intracellular domains (Figure 4). The

Frontiers in Immunology 04 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

TABLE 1 Monoclonal antibodies FDA-approved or under investigation for the treatment of patients with ovarian cancer (from 2015 to August 2023).

Generic Trade name/investi- Target Format Notes

name gational name
Bevacizumab Avastin® VEGF Whole antibody FDA-approved in 2018 for
ovarian cancer

Mirvetuximab Elahere® FR-a Whole antibody FDA-approved in 2022 for

soravtansine conjugated to a drug ovarian cancer

Catumaxomab Removab® EpCAM/CD3 Trifunctional antibody NCT00822809

Cetuximab Erbitux® EGF receptor Whole antibody NCT00086892

Panitumumab Vectibix® EGFR Whole antibody NCT01388621

Farletuzumab MORAb-003 FR-a Whole antibody NCT00849667

Oregovomab OvaRex® MAb-B43.13 CA125 Whole antibody NCT04498117

Amatuximab MORAb-009 Mesothelin Whole antibody NCT00325494

Atezolizumab Tecentriq® PD-L1 Whole antibody NCT03038100

Tisotumab Tivdak® Tissue factor (TF) Whole antibody NCT03657043

vedotin conjugated to a drug

Durvalumab Imfinzi® PD-L1 Whole antibody NCT04742075,

NCT03899610

Nivolumab Opdivo® PD-1 Whole antibody NCT05601752

Pembrolizumab Keytruda® PD-1 Whole antibody NCT02674061,

NCT02865811

Ipilimumab Yervoy® CTLA-4 Whole antibody NCT02498600

Sabatolimab MBG453 Mucin domain-3 (TIM-3) Whole antibody NCT02608268

Spartalizumab PDR001 PD-1 Whole antibody NCT02608268

Avelumab Bavencio® PD-L1 Whole antibody NCT02580058

Magrolimab Hu5F9-G4 CD47 Whole antibody NCT03558139,

NCT02216409

- hu3S193 Lewis-Y – NCT01137071,

NCT00617773

Anetumab BAY 94–9343 Mesothelin Whole antibody NCT02751918

ravtansine conjugated to a drug

Navicixizumab – Vascular endothelial growth factor (VEGF) and Bispecific NCT03030287

delta-like ligand 4 (DDL4)

Lifastuzumab LIFA NaPi2b Whole antibody NCT01911598

Vedotin conjugated to a drug

- TQB2450 PD-L1 Whole antibody NCT04236362

- INCAGN01949 OX40 Whole antibody NCT02923349

- DMUC5754A MUC16 Whole antibody NCT01335958

conjugated to a drug

Abagovomab – CA125 Whole antibody NCT00418574

Tocilizumab Actemra® IL-6 receptor Whole antibody NCT01637532

Trastuzumab Herceptin® HER2/neu Whole antibody NCT00189579

Ganitumab AMG 479 Type 1 insulin-like growth factor receptor Whole antibody NCT00719212
(IGF-1R)

Tremelimumab Imjudo® CTLA-4 Whole antibody NCT03899610

Olaratumab Lartruvo® Platelet-derived growth factor receptor-a Whole antibody NCT00913835

(PDGFR-a)

(Continued)

Frontiers in Immunology 05 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

TABLE 1 Continued

Generic Trade name/investi- Target Format Notes

name gational name
- DMUC4064A MUC16 Whole antibody NCT02146313
conjugated to a drug

Pertuzumab Perjeta® HER2 Whole antibody NCT01684878

Camrelizumab SHR-1210 PD1 Whole antibody NCT03827837

- RO5323441 Placental growth factor (PlGF) Whole antibody NCT01148758

Dostarlimab Jemperli® PDCD1 Whole antibody NCT04679064

- HuMax-IL8 IL-8 Whole antibody NCT02536469

- MOv18 FR-a Whole antibody NCT02546921

Gatipotuzumab PankoMab-GEX TA-MUC1 Whole antibody NCT01899599,

NCT01222624

- LY3022855 CSF-1R Whole antibody NCT02718911

Enoticumab REGN421 DDL4 Whole antibody NCT00871559

Seribantumab MM-121 ErbB3 Whole antibody NCT01447706

- DKN-01 DKK1 Whole antibody NCT03395080

Monalizumab IPH2201 CD94/NKG2 Whole antibody NCT02671435

Conatumumab AMG-655 TRAIL-R2 (CD262) Whole antibody NCT00819169

- PF-06647263 EFNA4 Whole antibody NCT02078752

conjugated to a drug

IMGN901 CD56 Whole antibody NCT00346385

Telisotuzumab ABT-700 c-Met Whole antibody NCT01472016

Figitumumab CP-751871 IGF-1 receptor Whole antibody –

Imalumab BAX69 Macrophage inhibitory factor (MIF) Whole antibody NCT01765790

extracellular domain harbors the antigen-recognition domain and a indicated target antigen(s). CAR-T products are conventionally
fragment that bridges this domain to the transmembrane domain, administered through the intravenous route. In the context of blood-
known as the spacer. Researchers have used single-chain variable based oncological indications, these engineered cells can easily
fragments (scFvs) or single domains on a heavy chain (VHH) as the encounter the cells proficient in the expression of the indicated target
antigen-recognition moieties of CARs; however, other applicable antigen, and efficiently eliminate them (30). This phenomenon has
fragments have also been incorporated into CARs for this aim (29). rendered CAR-T therapy an applicable frontline treatment for certain
The signaling domains that initiate the downstream signaling patients with hematologic cancers. However, CAR-T therapy has
cascades of CAR-Ts necessary for efficient activation and encountered serious limitations in the context of solid tumors and
antitumor responses are located at the intracellular portion of these researchers have put tremendous effort into addressing these
chimeric receptors. Depending on the generation of the CAR hindrances in recent years (20, 30). Lack of qualified target antigens,
construct, CARs could have no, one, and two co-stimulatory the immunosuppression and metabolic shortcomings of the tumor
domains (1st-, 2nd-, and 3rd-generation CARs, respectively). Other microenvironment (TME) impinging on the infused T cells, and
generations of CARs have also been devised, and they are structurally ineffective trafficking into tumor sites are all reasons for the failure of
tailored versions of the 2nd-generation CARs as 4th- and 5th- CAR-T therapy in the treatment of solid tumors (20, 30).
generation CARs are endowed with an inducer for the production
and secretion of cytokines of interest or the intracellular receptor
fragment that responds to cytokine stimuli, respectively. Over the 3 CAR-T therapy obstacles in
past years, researchers have further engineered different platforms of solid tumors
CAR-Ts, each of which developed to be capable of overcoming
certain limitations entwined with this treatment modality (Figure 4). At first, CAR-T therapy was proposed as a novel treatment
CAR-Ts mediate cytolytic reactions (by means of secreting perforin modality against solid tumors; however, this novel treatment
and granzyme B, as well as various proinflammatory cytokines) against managed to mediate more prominent remissions in patients with
target cells upon the engagement of their chimeric receptors with the certain blood-based malignancies over the course of the past years.

Frontiers in Immunology 06 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

FIGURE 4
The details of a CAR molecule and the various strategic twists proposed by scientists to overcome the obstacles of CAR-T therapies. From the early
days of CAR-T development, various components have been incorporated into CAR constructs. Since such components could substantially
influence the functionality of the CAR-Ts, their selection has been a subject of paramount importance. For instance, the length of the spacer
fragment can influence the cytokine production profile of the CAR-Ts, or the choice of the co-stimulatory domain(s) can have substantial impacts
on the expansion, persistence, or even the phenotypic features of the CAR-Ts. Researchers have engineered CAR-Ts to secrete TRBAs (for example
against a different TAA, rather than the one targeted by CAR-Ts, to augment antitumor responses) or other forms of mAbs for neutralizing the
mediators of cytokine release syndrome (CRS) or to counteract immunosuppression. CAR-Ts can also be manipulated to produce factors, such as
ILs, that boost their effector function, or to express enzymes that help modify the components of the tumor microenvironment for facilitating
intratumoral trafficking of the immune effector cells. In regard to increasing CAR-T accumulation in tumor sites, investigators have also engineered
CAR-Ts to express the cognate receptors of the molecules that are overexpressed by tumor cells, and they have reported encouraging results (27,
28). Ultimately, CAR-T therapies can also benefit from the novel gene-modifying techniques in a way that they can be utilized for the generation of
immunosuppression-resistant or off-the-shelf CAR-Ts through the disruption of immunosuppressive genes or TCR ab chains, respectively. AD,
activation domain; CsD, costimulatory domain; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICOS, inducible T-cell costimulator; IL,
interleukin; mAb, monoclonal antibody; scFv, single-chain variable fragment; TAA, tumor-associated antigen; TALEN, transcription activator-like
effector nucleases; TCR, T-cell receptor; TM, transmembrane domain; TRBA, T-cell-redirecting bispecific antibodies.

Such unexpected outcomes accentuated the bold differences that immunosuppressive environment whilst surrounded by elements
exist between the CAR-T therapy of solid tumors and hematologic that support their immune escape, progression, and invasion (30).
malignancies (20, 30). Researchers have proposed and evaluated Such tumor islets are often populated with tumor-associated blood
various counterstrategies for bypassing these hindrances; however, vessels that are morphologically different from the healthy tissue
tremendous effort and time need to be put into better identifying the vessels (30). The endothelial cells of these abnormal vessels usually
behavior and characteristics of solid tumors beforehand. Some of undergo modifications (induced by tumor-secreted factors) that
these differences are briefly outlined in this section. result in impaired expression of molecules (such as VCAM-1,
ICAM-1, ICAM-2, etc.) that correspond to those expressed by T
cells and necessary for extravasation (32, 33). Moreover, pericytes
3.1 Tumor accessibility offer structural integrity to the mentioned blood vessels which leads
to further tumor progression (34). Another important role in this
CAR-Ts are often administered through the intravenous route. scenario is played by cancer-associated fibroblasts (CAFs) (35, 36).
In the bloodstream and lymph nodes, CAR-Ts encounter and These fibroblasts produce a formidable web of the extracellular
consequently engage with their target cells to initiate cytolytic matrix (ECM) that remarkably restricts the migration of
reactions against them (which is basically what happens in the lymphocytes toward target tumor cells (35, 36). Another factor
case of lymphomas and leukemias) (31). However, this storyline is that limits the accessibility of CAR-Ts to tumor islets is the presence
not as straight in the context of solid tumors as it is in hematologic of tumor-associated macrophages (TAMs) as it has been evident
cancers. Solid tumor cells tend to aggregate in isolated territories that the depletion of these macrophages correlates with an amplified
(termed tumor islets) that allow them to establish an number of T cells in tumor sites (37). Moreover, the metabolic

Frontiers in Immunology 07 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

characteristics of the TME could also impinge on the targeting results in cytotoxic reactions against healthy tissues, which
transmigration capacity of CAR-Ts as findings have indicated is clinically recognized as “on-target off-tumor” effects (43). In the
that non-restricted outgrowth of tumor cells results in hypoxic context of CD19-redirected CAR-T products, such unfavorable
conditions in the TME, impairing T cell motility (38). Moreover, effects lead to the depletion of normal B cells (regarded as B cell
various distinct signaling pathways also have been known to aplasia); thereby increasing the risk of bacterial and/or viral
contribute to the diminished expression of certain chemotactic infections in the respective patients (31). Such adverse events are
factors (CCL4, CCL5, etc.) by the cellular elements of the TME manageable through different strategies such as immunoglobulin
which leads to the exclusion of T lymphocytes from tumor islets replacement (31). However, in the context of solid tumors, such
(39–41). unintended effects lead to serious, and sometimes detrimental and
irreversible, damage to the vital organs of the recipients (44).
Therefore, the research for qualified target antigens in solid
3.2 Immunosuppressive TME tumor CAR-T therapy is a subject of current investigation, and it
must continue.
There are various cellular and molecular elements in the TME
that act in favor of tumor cells in terms of progression and
invasiveness (42, 43). The cellular elements include CAFs, TAMs, 4 Target antigens for ovarian
Tregs, and myeloid-derived suppressor cells (MDSCs) that impose cancer CAR-T therapy
harsh conditions on T cells restricting or diminishing their
antitumor efficacy (42, 43). Moreover, various immunoinhibitory 4.1 Müllerian inhibiting substance
molecules are also secreted and/or surface-displayed by such type 2 receptor (MISIIR)
cellular elements or tumor cells themselves that further impair
the functionality of CAR-T/Ts in a pro-tumor fashion within the The overexpression of the TGF-b family member MISIIR is
TME (42, 43). reported in a high rate of ovarian cancer cases (45). Owing to its
scarcity in healthy tissues and its vital role in apoptosis induction in
tumors, MISIIR has been considered an interesting target antigen in
3.3 Tumor heterogeneity ovarian cancer immunotherapy, as well as other endometrial
malignancies (45). In 2020, Rodriguez-Garcia and colleagues
Solid tumor bulks are often populated with different types of reported the development and assessment of MISIIR-redirected
malignant cells that exhibit distinct characteristics in terms of CAR-Ts (using a fully human scFv as the targeting domain of the
behavior (drug resistance patterns) and target antigen expression CAR construct) and reported that these therapeutics mediated
(43). For instance, in the context of ovarian cancer, certain MISIIR-dependent cytotoxicity in vitro and in preclinical animal
malignant cells might be proficient in the expression of models (45). In the in vitro experiments, MISIIR-redirected CAR-
mesothelin, MISIIR, and EpCAM, whereas there might be tumor Ts were capable of mediating cytolytic reactions against a variety of
cells within a given tumor site that express neither. This occurrence MISIIR-positive cell lines as well as patient-derived cancer cells,
renders it impossible to target all the tumor cells within a tumor site while sparing healthy human cells (45). Conclusively, Rodriguez-
with a single CAR-T product and necessitates precise screening for Garcia and colleagues suggested that MISIIR might be a suitable
profiling the antigen expression patterns of the tumor cells prior to cancer immunotherapy target antigen for the development of
targeted immunotherapy. Moreover, the number of antigen MISIIR-redirected CAR-Ts against MISIIR-positive gynecologic
molecules displayed over the surface of a tumor cell is an disorders (45). However, broader investigations on the targeting
important factor that determines whether a CAR-T can establish of MISIIR via CAR-Ts are warranted in the context of
productive CAR-antigen engagement with that cell to initiate ovarian cancer.
cytolytic reactions (20). Solid tumor cells might exhibit different
rates of expression for a certain target antigen within a TME (20).
Low expression rates give tumor cells protection against CAR- 4.2 Olfactory receptor family 5
T recognition. subfamily V member 1 (OR5V1)

In 2022, Martin and colleagues reported that OR5V1 is

3.4 Lack of qualified target antigens expressed in a variety of ovarian cancer histological samples,
while its expression in normal tissues is restricted to the testis
The principal concept of CAR-T therapy was developed upon (46). Owing to these findings, Martin et al. reported the
the redirection of modified T cells against malignant cells proficient development of an scFv-based 2nd-generation CAR-T redirected
in the expression of TSAs of interest; however, the practicality of against the OR5V1 antigen (OR5V1-redirected CAR-Ts) and
this concept is restricted by a lack of known TSAs. To compensate, eventually introduced this construct into human T cells via
researchers considered targeting TAAs. Due to the expression of retroviral transduction (46). The results of the in vitro assays
such TAAs by the cells of healthy tissues, their CAR-T-mediated demonstrated that OR5V1-redirected CAR-Ts mediated specific

Frontiers in Immunology 08 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

tumoricidal effects against HeLa cells in a dose-dependent fashion 4.4 Chondroitin sulfate
while managing to spare a panel of normal human cells (46). proteoglycan 4 (CSPG4)
Moreover, Martin and colleagues established HeLa-bearing
preclinical mouse models and reported that the treatment subjects The expression of CSPG4 has been reported in different
experienced delayed tumor outgrowth under OR5V1-redirected oncological indications, including ovarian cancer, breast cancer,
CAR-T treatment (46). Conclusively, these researchers glioblastoma, etc., which renders this antigen as an interesting target
demonstrated the antitumor potential of OR5V1-redirected CAR- antigen in targeted immunotherapy (50). This antigen can also be
T against OR5V1-positive malignancies with negligible toxicities leveraged for targeting tumor-associated vasculature, owing to its
(46). To further elucidate the suitability of OR5V1 as an expression on the blood vessel cells of malignant tissues (51). In
immunotherapy target antigen, in-depth preclinical and clinical recent years, researchers have attributed a number of roles to
assessments must be taken into consideration. CSPG4, which might further validate its importance as a target
antigen; such roles include contribution to the formation of
metastatic lesions, contribution to tumor aggressiveness, and
4.3 Annexin A2 arranging tumor progression signals (52, 53). Based on these
findings that implicate the importance of CSPG4 for tumors
In 2018, Cua and colleagues reported the development of a proficient in its expression, researchers have proposed that it is
variety of mAbs that target embryonic stem cells of human origin very unlikely for tumor cells to undertake mechanisms for CSPG4
(47). These researchers reported that such mAbs could be applied down-regulation and/or loss following its targeting via CAR-Ts
for or engineered into different mAb-based platforms for (54). In 2022, Harrer et al. reported the results of a study
therapeutic purposes (47). Among these mAbs, 2448 was reported investigating the drug-induced expression of CSPG4 on the
to recognize a unique antigen on a variety of tumor cells (47). Cua SKOV-3 ovarian cancer cell line, and targeting these cells using
et al. demonstrated that 2448 can potentially bind a specific site on CSPG-4-redirected CAR-Ts (54). In detail, these researchers used
annexin A2, expressed in breast cancer and ovarian cancer (47). decitabine (5-aza-2-deoxycytidine), a US FDA-approved
Further engineering techniques revealed that 2448 could also medication for the treatment of myelodysplastic syndrome (MS),
mediate antigen-dependent cytotoxicity while applied as an ADC, to induce the expression of CSPG4 on the surface of SKOV-3 cells
alongside mediating antibody-dependent cellular cytotoxicity in in (54). Decitabine is a DNA methylation inhibitor that has been
vitro and animal-based experiments (47). Ultimately, Cua and known to have upregulatory effects on the expression level of
colleagues suggested that annexin A2 might be taken into CSPG4 in a number of melanoma cell lines (55). Generally,
consideration as a qualified cancer immunotherapy target antigen decitabine and its structural relatives such as azacitidine have
and that 2448 could be further applied for therapeutic advantage been reported to induce unregulated expression of various TAAs
(47). Annexin A2 has been known to arrange different molecular on cancer cells, rendering CAR-T-mediated targeting of these target
mechanisms in cancer progression, as its expression fluctuates in antigens more feasible (56). First, these researchers demonstrated
different oncological indications (48). In 2020, Leong and colleagues that decitabine mediates CSPG4 upregulation in SKOV-3 cells in a
derived a CAR targeting domain from 2448 and developed annexin dose-dependent manner (54). Of note, SKOV-3 cells are deficient in
A2-redirected CAR-Ts (49). In detail, Leong et al. evaluated the the expression of CSPG4 (57). Next, Harrer and colleagues co-
effects of different CAR spacer domains (short, intermediate, and cultivated CSPG4-positive SKOV-3 cells with mRNA-
long; with 12, 122, 229 amino acids, respectively) on the cytotoxic electroporated 2nd-generation CSPG4-redirected CAR-Ts (54).
activity and proinflammatory cytokine secretion ability of the The results indicated that CSPG4-redirected CAR-Ts mediated
developed annexin A2-redirected CAR-Ts, and reported that effective target antigen-specific antitumor activity against ovarian
CAR-Ts with the longer spacer fragment outperformed those cancer cells induced to express CSPG4, in four different effector-to-
with the short or intermediate spacer fragments in mediating target ratios (54). Additionally, CSPG4-redirected CAR-Ts showed
cytolytic reactions against the annexin A2-positive cell line remarkable IFN-g production and secretion following being
IGROV-1 (49). Moreover, it was demonstrated that the subjected to decitabine-treated CSPG4-expressing SKOV-3 cells
tumoricidal effects mediated by the annexin A2-redirected CAR- (54). However, it is worth mentioning that CSPG4 is not
Ts (with the long spacer) were annexin A2-dependent, as they were generally expressed by ovarian cancer cells; therefore, it cannot be
only against the IGROV-1 and SKOV-3 cell lines, but not the considered a target antigen for CAR-T therapy of ovarian cancer,
IMR90 and HFF-1 cell lines (49). Leong et al. also reported more but the results of this study can highlight the potential of this cell
encouraging results as their annexin A2-redirected CAR-Ts surface antigen as a secondary inducible target antigen for CAR-T
prolonged the survival of ovarian cancer xenograft animals and therapy of various types of solid tumors including ovarian cancer.
shrunk tumors by almost 75% (49). Such novel findings accentuate Moreover, the effects of decitabine for inducing CSGP4 expression
the fact that annexin A2 might be considered a therapeutic target in ovarian cancer cells should be more broadly investigated since
antigen for ovarian cancer, as well as breast cancer; however, the ovarian cancer cells used in the mentioned study are not
broader preclinical and substantiated clinical evidence is required patient-derived primary samples. Also, preclinical data using
before confidently classifying this antigen as a potential target. mouse models are critically required for evaluating the safety and

Frontiers in Immunology 09 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

efficacy of the methods described above. Above all this, CSPG4 has IV administration of CNA3103 (5 × 106) into mouse models
been employed as a CAR-T therapy target in various types of mediated remission and prolonged their survival remarkably (63).
malignancies including glioblastoma and B-cell precursor leukemia, According to a recent report, Bandara and colleagues developed a
which highlights the importance of this target antigen for different panel of six different LGR5-redirected CAR-T products and
types of immunotherapies (58–60). demonstrated that four of these products were capable of
mediating meaningful tumoricidal effects in vitro against cell lines
of colorectal cancer (64). Furthermore, the investigators also
4.5 Leucine-rich repeat-containing G evaluated these four products in xenograft models of human
protein-coupled receptor 5 (LGR5) colorectal cancer and reported that three CAR-T products
mediated significant tumor suppression (64). Such studies might
Wang and colleagues have reported the expression of LGR5 in a highlight the potential suitability of LGR5 as a target antigen for the
variety of ovarian cancer cell lines (including OAW28, COV318, CAR-T therapy of ovarian cancer, as well as colorectal cancer;
and COV362), and have also reported its elevated expression level however, such CAR-T products need to be thoroughly and strictly
in ovarian cancer tissue samples in patients who had relapsed evaluated in clinical settings to substantiate such claims.
tumors (61). It has been demonstrated that this antigen has
distinctive roles in cancer emergence and metastasis, and its
expression has also been reported in a variety of malignancies 4.6 CD44v6
including colon cancer and ovarian cancer (62). In this regard,
Wang et al. generated LGR5-redirected CAR-Ts and set out to CD44v6 has been recognized as a variant of CD44 whose role in
evaluate their tumoricidal effects on different cell lines of ovarian tumor proliferation, aggressiveness, and migration in a variety of
cancer origin and patient-derived tumor cells in monolayer and 3D malignancies (including breast cancer, ovarian cancer, head and
culture conditions (61). In the monolayer model, Wang et al. neck epithelia, colorectal cancer, etc.) has been evident, according to
reported that their LGR5-redirected CAR-Ts mediated experimental findings (65–68). Based on the findings achieved in
tumoricidal effects in an antigen density-dependent fashion, as preclinical experiments, targeting CD44v6 has been correlated with
they remarkably suppressed the outgrowth of the ovarian cancer tumor outgrowth suppression in multiple myeloma (MM) and
cell lines COV318 and COV362 (which overexpress LGR5), while acute myeloid leukemia (AML) (65). Recently, Porcellini and
not enforcing the same reactions against SKOV3 and OV90 cell colleagues developed CD44v6-redirected CAR-Ts, which were
lines that display LGR5 on their surface at lower levels (61). also equipped with an at-will depletion switch, and assessed their
Moreover, LGR5-redirected CAR-Ts also mediated pronounced antitumor efficacy against CD44v6-positive cell lines (IGROV-1 of
cytolytic reactions against patient-derived ovarian cancer tumor ovarian cancer origin and MR232 of lung cancer origin) and in
cells (61). In reference to the 3D culture condition experiments, preclinical mouse models (65). Porcellini et al. reported that their
LGR5-redirected CAR-Ts mediated tumoricidal effects against CD44v6-redirected CAR-Ts mainly expressed the markers of T
patient-derived ovarian tumor cells and the OAW28, COV318, memory stem cells (Tscm) and T central memory cells (Tcm) subsets,
and COV362 cell lines (61). According to another investigation, and they exhibited CD44v6-dependent expansion and tumoricidal
Thompson and colleagues developed a real-time cytotoxicity assay effects upon co-cultivation with the MR232 and IGROV-1 cell lines
that could be applied in laboratory settings to efficiently compare (65). Moreover, the researchers established IGROV-1-bearing
the tumoricidal effects of different CAR-T platforms (to reach a immunodeficient NSG mouse models and reported that CD44v6-
candidate therapeutic for future clinical assessments) (62). Using redirected CAR-Ts infiltrated and proliferated at tumor foci upon
this technique, Thompson et al. were able to establish a measure intravenous administration, which eventually culminated in
(which was defined as the time CAR-Ts needed to enforce meaningful tumor outgrowth suppression (65). Conclusively,
tumoricidal reactions against 50% of the target tumor cells) to opt Porcellini and colleagues asserted that CD44v6-redirected CAR-
for a certain LGR5-redirected CAR-T product as their therapeutic Ts could be of therapeutic advantage for the treatment of CD44v6-
candidate for clinical assessment (called CNA3103) (62). Moreover, positive ovarian cancer, as well as other related malignancies;
these researchers reported that their therapeutic candidate was able however, carefully conducted clinical evaluations are to be taken
to remarkably exhibit tumoricidal reactions against different cell into consideration before drawing such conclusions (65). Other
lines of ovarian cancer and colon cancer origin (62). Ultimately, researchers have also evaluated the antitumor efficacy of CD44v6-
Thompson et al. suggested that this technique might also be applied redirected CAR-Ts in the context of other malignancies (69). For
in Phase I dose-escalation clinical trials for dose optimization instance, Haist and colleagues developed CD44v6-redirected CAR-
purposes (62). Aside from the expression of LGR5 in ovarian Ts and demonstrated a direct pattern between the expression level
cancer, its expression in colorectal cancer might also support its of CD44v6 by primary blasts of human head and neck squamous
suitability as a target antigen in the CAR-T therapy of colorectal cell carcinoma and the tumoricidal effects of CD44v6-redirected
cancer (63). According to a 2022 report by McPeake and colleagues, CAR-Ts (69). In the context of AML, Tang and colleagues reported
LGR5-redirected CAR-Ts might be promising therapeutics for the that individuals with AML, or SKM-1 and K562 cell lines, with the
treatment of colorectal cancer as administration of CNA3103 into FLT3 or DNMT3A mutations had higher expression levels of
preclinical mouse models mediated complete remission in all of the CD44v6 (in comparison with the patients without the mentioned
subjects (100%) (63). Moreover, these researchers also reported that mutations) (70). Furthermore, these researchers developed

Frontiers in Immunology 10 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

CD44v6-redirected CAR-Ts and demonstrated that these effector investigators asserted that PD-L1 plays an important role in
cells secreted proinflammatory cytokines and exhibited CD44v6- promoting tumor metastasis to the peritoneum, as achieved
dependent tumoricidal effects upon co-cultivation with CD44v6- through exhausting functional cytotoxic T lymphocytes, and they
positive cells (while sparing CD44v6-negative cells), which were also proposed that targeted therapy of this antigen is a potential
also consistent with the outcomes of their in vivo experiments (70). strategy for preventing this occurrence (73). Such studies accentuate
Ultimately, Tang and colleagues asserted that CAR-T-mediated the importance of PD-L1 in solid tumors, especially ovarian cancer.
targeting of CD44v6 in patients with FLT3 or DNMT3A In 2022, Ma and colleagues reported that the tumoricidal efficacy of
mutations might serve as a qualified therapeutic option; however, 2nd generation HER2-redirected CAR-Ts was hampered by the
clinical scrutiny is warranted in this matter (70). According to component of malignant pleural effusion (MPE) or malignant
another study, Casucci and colleagues also generated CD44v6- ascites (MA), leading to a compromised expansion and cytokine
redirected CAR-Ts and demonstrated that these effector cells production ability of the CAR-Ts (74). These researchers
were capable of enforcing tumoricidal effects against primary investigated the reason for this occurrence and identified a high-
blasts of AML and MM patients while managing not to attack level expression of PD-L1 by the cells of MPE/MA for this negative
hematopoietic stem cells (71). Recently, Stornaiuolo and colleagues impact (74). Of note, MPE/MA is often reported in individuals with
published the results of an investigation that aimed to optimize the advanced non-hematologic malignancies, such as ovarian cancer,
tumoricidal efficacy of CD44v6-redirected CAR-Ts by focusing on which also coincides with tumor metastasis to the peritoneum (74).
the structure of the CAR spacer domain, which was derived from a Such characteristics have been identified as potential elements that
fragment of the human low-affinity nerve growth factor receptor hamper the therapeutic effects of CAR-T treatments, through the
(LNGFR) (72). The investigators attributed the varied phenotypes formation of a highly immunosuppressive TME (74). To overcome
of the generated CAR-Ts to the length of the different spacer this limitation, Ma and colleagues developed CAR-Ts engineered to
fragments incorporated into the CAR constructs of each of the co-express two distinct CAR constructs; one of them was a 2nd
developed CAR-T products, which might in their own way affect the generation chimeric receptor redirected against HER2, whereas the
level of spontaneous antigen-independent signaling and the rate of other one was composed of a PD-L1-specific scFv fused to the 4-
CAR surface presentation (72). The researchers reported that one of 1BB co-stimulatory domain (referred to as [Link] CSR) (74). It
the CAR-T products (designated as [Link]-Ts) was reported that co-expression and subsequent engagement of the
exhibited pronounced tumoricidal effects in vitro and in vivo, PD-L1-specific CAR molecules on the surface of the HER2-
whose T cell populations were mainly composed of Tcm (72). redirected CAR-Ts enabled them to outperform conventional
Such investigations accentuate the importance of how CAR CAR-Ts in terms of expansion rate and counteract the
design might remarkably affect the antitumor efficacy of the suppressive effects of MPE/MA upon their co-cultivation with
developed CAR-Ts (60, 72). irradiated SKOV3 cell line (proficient in the expression of PD-L1)
(74). Moreover, Ma and colleagues further evaluated the efficacy of
[Link] CSR-positive HER2-redirected CAR-Ts in mediating
4.7 PD-L1 prolonged survival in xenograft models of pleural and peritoneal
metastasis (74). Briefly, the investigators established pleural and
Peritoneal metastasis of ovarian tumors has often been reported peritoneal metastasis NSG mouse models (using the PD-L1-positive
in patients with ovarian tumors, and these malignant cells somehow cell line SKOV3, or the lung adenocarcinoma cell line A549), ten
manage to escape immunosurveillance; however, the exact days following which, the animal models underwent CAR-T
underlying mechanism for this occurrence is not completely treatment via the intrapleural or intraperitoneal route (74).
deciphered (73). To this aim, Abiko and colleagues conducted an According to the results, [Link] CSR-positive HER2-
investigation focused on assessing the correlation between PD-L1 redirected CAR-Ts were able to mediate prolonged elimination of
expression and tumor metastasis to the peritoneum (73). According the established tumors (74). The investigators asserted that both
to the findings, these researchers reported a direct relationship components of the PD-L1-specific chimeric receptor (scFv and the
between PD-L1 expression by human ovarian tumors and 4-1BB signaling domain) were responsible for the enhanced efficacy
metastasis to the peritoneum, as achieved through of the CAR-Ts (74). To further investigate this concept, in March
immunohistochemistry and microarray techniques (73). 2021, a clinical investigation was initiated with eighteen individuals
Moreover, it was reported that the overexpression of PD-L1 had diagnosed with HER2-positive malignancies with pleural or
debilitating impacts on the cytolytic reactions of cytotoxic T peritoneal metastatic tumors. No findings have yet been published.
lymphocytes, as well as their degranulation, whereas PD-L1
deficiency improved the antitumor effects exerted by such
lymphocytes (73). Furthermore, these researchers demonstrated 4.8 5T4
that cytotoxic T lymphocytes exhibited an exhausted gene
expression profile which was attributed to PD-L1 overexpression 5T4 is a 72 kDa oncofetal antigen (which is also recognized as
by ovarian tumors (73). Abiko et al. also conducted preclinical trophoblast glycoprotein) that might be considered a qualified
experiments and reported that PD-L1 deficiency culminated in cancer immunotherapy target antigen as it exhibits a restricted
suppressed peritoneal tumor outgrowth correlating with expression profile in healthy cells, but its expression is remarkably
protracted survival in the animal subjects (73). Ultimately, these elevated in different stages of cancer progression of different types of

Frontiers in Immunology 11 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

tumors, including ovarian cancer (75). In 2018, Owens et al. epitope of 5T4 (77). Moreover, the researchers also reported that
generated two distinct sets of CAR-Ts redirected against 5T4 the engineered T cells were capable of recognizing the surface-
using two different mAb derivatives (named H8 and 2E4) and presented peptide of 5T4 in T2 cells (77). Ultimately, Xu and
evaluated the antitumor activity of these effector cells both in vitro colleagues suggested that T cells engineered with novel TCRs
and in vivo (76). Of note, it was reported that these two mAbs capable of recognizing the mentioned 5T4 epitope might be of
exhibited different affinities for their target antigen, 5T4 (76). therapeutic advantage for the treatment of 5T4-positive individuals
Briefly, in vitro assessments of these researchers included co- with HLA-A2-positive indications; however, following careful and
cultivation of 5T4-redirected CAR-Ts with autologous cancer cells sufficient clinical examinations (77).
as well as two ovarian cancer cell lines, including SKOV-3 and
OVCAR-3 (76). Both 5T4-redirected CAR-T products produced
high levels of INF-g upon encountering the ovarian cancer cell lines 4.9 TAG72
and autologous cell samples (76). Moreover, these CAR-Ts also
managed to produce and secrete moderate and minimum levels of Investigators have proven that abnormally glycosylated forms
IL-2 upon co-cultivation with the ovarian cancer cell lines and of target antigens could be of interest in targeted cancer
autologous tumor cells, respectively (76). In addition to these immunotherapy (20, 43). Among such glycoforms, TAG72
outcomes, Owens et al. used preclinical mouse models established exhibits an elevated expression profile in various oncological
using luciferase-expressing SKOV-3 cells (76). According to the indications, including ovarian cancer (20, 43). In detail, TAG72 is
results, 5T4-redirected CAR-T treatment culminated in the a sialyl Tn hapten found in various glycosylated proteins (20). In
therapeutic benefit of the preclinical animals against the 2018, Murad and colleagues incorporated a humanized TAG72-
established tumors (76). Ultimately, Owens and colleagues specific targeting fragment into the construct of a 4-1BB/CD3z-
asserted that the isolation of T cells from ovarian cancer patients based CAR and developed TAG72-redirected CAR-Ts (78). The
and their reprogramming for the expression of 5T4-redirected CAR investigators reported that their TAG72-redirected CAR-Ts
molecules can lead to the targeted responses of the engineered T successfully mediated tumoricidal effects and proinflammatory
cells to autologous cancer cells proficient in the expression of the cytokine secretion in response to TAG72-positive cell lines of
targeted antigen (76). Recently, Guo and colleagues developed 2nd- ovarian cancer origin (OVCAR3 and OV90) and ascites of
generation 5T4-redirected CAR-Ts and aimed to assess their corresponding patients (78). The researchers further reported that
antitumor functionality and proinflammatory cytokine secretion intraperitoneal administration of TAG72-redirected CAR-Ts into
ability upon their co-cultivation with a variety of 5T4-positive preclinical xenografts of ovarian cancer culminated in suppressed
ovarian cancer cell lines, including SKOV3, A2780, and ES2 cell tumor outgrowth and prolonged survival in the treatment subjects,
(76). These researchers reported that their 5T4-redirected CAR-Ts which was more pronounced as repeated administrations were
mediated remarkable tumoricidal effects against the mentioned cell taken into consideration (78). Of note, Murad and colleagues also
lines in vitro, alongside secreting high levels of GM-CSF, IFN-g, and reported a decline in the expression level of the target antigen,
IL-2 (76). Moreover, Guo et al. also established xenograft models of which was parallel with low CAR-T persistence in the treatment
peritoneal ovarian cancer by infusing SKOV3-luc cells into B-NDG subjects (78). Ultimately, the researchers concluded that TAG72-
mouse models (76). Following localized administration of the 5T4- redirected CAR-Ts might be therapeutically valuable for the
redirected CAR-Ts into the animal subjects, it was reported that the treatment of TAG72-positive ovarian cancer cases; however,
treatment managed to hinder tumor outgrowth; therefore, translation of such preclinical findings into clinical assumptions
prolonging the survival of the animal subjects (76). Ultimately, requires in-depth clinical evaluations (78). Other researchers have
the investigators suggested that this study provided a groundwork also assessed the applicability of TAG72-redirected CAR-Ts for the
for the future clinical assessment of 5T4-redirected CAR-Ts in 5T4- treatment of patients with other solid tumors, such as those with
positive ovarian cancer patients (76). Other researchers have also colorectal cancer (79). In a study by Hege and colleagues, the
assessed the antitumor efficacy of T cell-based immunotherapies in researchers reported the outcomes of two Phase I clinical trials
other 5T4-positive malignancies, including renal cell carcinoma (designated as C-9701 and C-9702) in which metastatic colorectal
(RCC) (77). For instance, an investigational team previously cancer patients were admitted to undergo first-generation TAG72-
reported that they identified high-avidity T-cell clones that could redirected CAR-T treatment (79). In the former trial, intravenous
specifically target tumor cells that display a certain epitope of 5T4 CAR-T administration was considered for the subjects (ten
(amino acids 17 to 25) via HLA-A2 (77). Briefly, single-cell RNA patients), whereas in the latter (six patients), metastatic colorectal
sequencing was employed to identify the TCRa and TCRb cancer patients with liver involvement underwent CAR-T
sequences of these T cell clones, and eventually whole TCRa and administration directly into their hepatic artery (79). According
TCRb sequences were introduced into T cells via lentiviral to the results, mild CRS without evident bystander effects against
transduction (77). Of note, seven different pairs of TCRa/b were healthy tissues was reported (79). Moreover, the presence of
identified over the course of this study (77). The results of the in neutralizing antibodies was confirmed in some patients, which
vitro experiments demonstrated that CD8+ TCR-engineered T cells were mounted against the humanized targeting domain of the
secreted proinflammatory cytokines and mediated targeted TAG72-redirected CAR-Ts (known as CC49) (79). Since the
tumoricidal effects upon encountering target cells (solid tumor investigators reported no objective tumor responses to the
cell lines and primary RCC cells) displaying the mentioned treatment, various points were elucidated over the course of these

Frontiers in Immunology 12 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

trials (79). To overcome the issue of anaphylaxis, CAR-Ts could be this study entailed preclinical mouse models of ovarian cancer
equipped with fully human or humanized targeting domains, and to established using subcutaneously administered SKOV3 cells (83).
tackle the issue of limited CAR-T persistence, the effector cells must According to the results, the administration of EpCAM-redirected
be genetically engineered to express CAR molecules that harbor CAR-Ts into mouse models reduced the size of the established
auxiliary stimulatory domains (such as 2nd- and 3rd-generation tumors and remarkably slowed down tumor outgrowth in
CARs) (60, 80). Antigen-dependent tumor escape is one of the most comparison with the control group (83). Overall, these
frequently undertaken mechanisms by which tumor cells evade the researchers suggested that EpCAM might act as a potent target
immune system (30, 42). Targeting more than one antigen might antigen for CAR-T therapy of ovarian cancer; even though more in-
serve as a potential counterstrategy in targeted cancer depth assessments are required (83). In addition to this study,
immunotherapy (60). In the context of ovarian cancer, Shu and Herbel and colleagues reported the results of an ongoing study
colleagues devised a dual CAR-T platform capable of recognizing investigating the expression of THY1 and EpCAM on the surface of
TAG72 and CD47 on the surface of tumor cells of interest (81). ovarian cancer cells and the suitability of these target antigens for
Despite the constitutive expression of CD47 (which acts as a “don’t ovarian cancer CAR-T therapy with the aim of addressing the “on-
eat me” signal to macrophages) on the surface of a vast variety of target off-tumor” toxicity associated with this platform of cancer
tumor cells, it is also displayed on the surface of healthy cells; immunotherapy (84). In detail, THY1 is a cell surface protein
therefore, to prevent dual CAR-Ts from targeting undesired cells, present in fibroblasts and hematopoietic stem cells (84). First,
Shu and colleagues designed a CD47-specific CAR (with an anti- Herbel et al. assessed the expression of THY1 and EpCAM on a
CD47 scFv as the targeting domain) that does not harbor any number of primary ovarian cancer patient-derived samples using
intracellular signaling domains (81). Upon co-binding of dual CAR- high-content imaging (84). The results of this investigation
Ts to CD47 and TAG72 on the surface of tumor cells, CAR-Ts indicated that THY1-EpCAM-redirected CAR-Ts mediated
could start eradicating TAG72-positive malignant cells (81). tumoricidal functionality against target cells in vitro (84). Overall,
Ultimately, Shu and colleagues suggested that this proof-of- these researchers added that they are planning to assess the efficacy
concept might also serve as a potential strategy for the treatment and safety of these multi-targeting THY1-EpCAM-redirected CAR-
of other relatable malignancies (81). To date, a limited number of Ts in animal models of ovarian cancer. Until then, more
studies have investigated the safety and tumoricidal efficacy of substantiated data in regard to the efficacy of these CAR-Ts
TAG72-redirected CAR-Ts, which are only in preclinical stages. remains to be obtained (84). In 2020, Qin et al. reported that
To further elucidate the suitability of this target antigen and the murine EpCAM-redirected CAR-Ts can mediate lung attack and
applicability of TAG72-redirected CAR-Ts for the treatment of lethality in immunocompetent preclinical mouse models of breast
ovarian cancer, as well as other TAG72-positive malignancies, cancer established using the breast cancer cell line 4T1 (85). In
profound preclinical and clinical assessments are warranted. detail, these CAR-Ts mediated effective antitumor activity and
produced INF-g when co-cultured with different EpCAM-
expressing cancer cell lines, including 4T1 and MC38; however,
4.10 Epithelial cell adhesion they did not demonstrate cytotoxicity against the EpCAM-deficient
molecule (EpCAM) normal fibroblast control cells NIH-3T3, indicating the antigen-
specific tumoricidal activity of these effector cells (85). These results
EpCAM is a membrane-anchored glycosylated protein with were consistent with those of the previously published studies
physiological expression by epithelial cells and elevated expression investigating EpCAM-redirected CAR-Ts in several types of solid
levels by malignant cells of various advanced carcinomas (82). This tumors including peritoneal carcinomas, prostate cancer, and
characteristic renders EpCAM a favorable target antigen for the colorectal cancer (86–88). In reference to the in vivo assays,
CAR-T therapy of solid tumors, including ovarian cancer. In 2021, EpCAM-redirected CAR-Ts significantly reduced tumor burden
Fu et al. generated EpCAM-redirected CAR-Ts and evaluated the in preclinical mouse models in comparison with control T cells (85).
antitumor efficacy of these cells in vitro and in vivo (83). Briefly, It is worth mentioning that these researchers also evaluated the
these researchers first assessed the expression of EpCAM on ovarian antitumor response of these CAR-Ts in mouse models of colon
cancer cell lines and patient-derived samples (83). According to the cancer established using the MC38 colon cancer cell line, and
results of the immunohistochemistry analysis, the expression level demonstrated that these CAR-Ts reduced tumor outgrowth and
of EpCAM in ovarian cancer tissue is remarkably higher than its extended the survival of the tumor models (85). However, these
expression in ovarian para-cancerous tissues (83). Moreover, researchers indicated that their murine EpCAM-redirected CAR-Ts
SKOV3 cells exhibited a high level of EpCAM expression (83). In recognized and became activated by EpCAM expressed on non-
the next step, Fu et al. co-cultured EpCAM-redirected CAR-Ts with malignant tissues leading to on-target off-tumor toxicities (85).
SKOV3 cells and evaluated the CAR-T mediated cytotoxicity and Moreover, Qin et al. demonstrated an expression pattern of EpCAM
the level of INF-g secretion (83). According to the result of the real- in the lung bronchioles (85). It was also demonstrated that alveolar
time cell analysis assay, EpCAM-redirected CAR-Ts mediated EpCAM expression in normal lung tissues results in the recruitment
effective tumoricidal activity against SKOV3 cells in vitro (83). of EpCAM-redirected CAR-Ts and their activation in the
Moreover, these effector cells produced significant levels of INF-g mentioned sites leading to CAR-T-mediated lung inflammation
upon co-cultivation with the target cells (83). In vivo assessments of and tissue damage (85). Such data highlight the importance of

Frontiers in Immunology 13 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

broader research, both in the preclinical and clinical stages, in this THP1 cell line (90). Upon administration of FRb-redirected CAR-
field to prevent the occurrence of such unwanted adverse Ts into the preclinical subjects, suppression of tumor progression
events (85). was evident (90). One of the main concerns of selecting FRb as a
target antigen is its possible shared expression in CD34-positive
hematopoietic stem and progenitor cells (HSCs). Lynn and
4.11 Folate receptor b (FRb) colleagues investigated this matter and elucidated that FRb-
redirected CAR-Ts did not mediate cytolytic reactions against
Lynn and colleagues reported the applicability of FRb- HSCs (90). These researchers also evaluated a strategy to improve
redirected CAR-Ts for the elimination of tumor-associated the recognition of FRb-positive cells by FRb-redirected CAR-Ts
macrophages in ovarian cancer (89). In the TME, TAMs have and demonstrated that all-trans retinoic acid could elevate FRb
been recognized as elements supportive of tumor progression (30, expression levels in AML cell lines (THP1 and MV411) (90).
89). In the context of ovarian cancer, a dismal prognosis has been According to another study, Lynn et al. elucidated how the
attributed to the presence of TAMs (89). FRb is known as a surface- affinity of the CAR target antigen recognition domain could be a
expressed molecule that plays an important role in folic acid uptake factor of paramount importance in the context of CAR-T therapy,
(89). In a variety of individuals with cancer and preclinical cancer and how transient CAR expression policies could minimize the
animal models, the expression of FRb has been documented (89). health risks associated with stable CAR expression in T cells (91).
Moreover, Lynn and colleagues have also reported the expression of Briefly, these researchers reported the isolation of a high-affinity
this GPI-linked molecule in M2-polarized macrophages (89). In this scFv (2.48 nM) specific for FRb and demonstrated that CAR-Ts
regard, these researchers developed FRb-redirected CAR-Ts using a equipped with this scFv showed pronounced tumoricidal capacity
high-affinity targeting domain specific for FRb (89). It was against FRb-positive AML cells in comparison with FRb-redirected
demonstrated that FRb-redirected CAR-Ts were capable of CAR-Ts that harbored a low-affinity scFv as their targeting domain,
mediating cytolytic effects and secreting INF-g against M2 in preclinical experiments (91). Since the FRb-redirected CAR-Ts
polarized macrophages, and upon co-cultivation of FRb- exhibited cytolytic activities against mature CD14-positive
redirected CAR-Ts with the SKOV3 cell line (deficient in the monocytes and to minimize the risk of CAR-T-mediated cytolysis
expression of FRb), the engineered T cells mediated tumoricidal of mature myeloid lineage, Lynn et al. proposed the strategy of
effects while M2 polarized macrophages were present (proficient in developing mRNA-based FRb-redirected CAR-Ts and
the expression of FRb) (89). Based on the findings by Lynn and demonstrated that these engineered T cells managed to maintain
colleagues that FRb exhibits an elevated expression level in TAMs their tumoricidal efficacy in preclinical experimental conditions
isolated from individuals with ovarian cancer, these researchers (91). Studies such as those discussed herein highlight the
suggested that FRb-redirected CAR-Ts could be of therapeutic importance of CAR-T-mediated targeting of FRb in solid tumors,
benefit for the elimination of such TAMs (89). To further validate such as ovarian cancer, as well as myeloid-derived malignancies;
these findings, these researchers established ID8-based ovarian however, clinical validation of these findings could shed more light
cancer mouse models and set out to assess the tumoricidal on the suitability of FRb as a cancer immunotherapy target
efficacy of FRb-redirected CAR-Ts (referred to as CL10) in vivo antigen (91).
(89). Of note, ID8 is a murine cell line of ovarian epithelial that is
biologically similar to human ovarian cancer (89). Upon
administration of CL10 into the animal models, the researchers 4.12 Folate receptor a (FRa)
reported CAR-T-mediated elimination of FRb-positive TAMs and
suppression in the outgrowth of the established tumors (89). FRa (alternatively known as FOLR1) could be considered a
Moreover, the researchers proposed that future investigations favorable target antigen in the CAR-T therapy of ovarian cancer
could focus on the synergistic effects of CL10 coupled with CAR- owing to its elevated level of expression in a high percentage of
Ts redirected against a TAA or TSA of interest since it has been ovarian cancer cases (92, 93). Alongside this oncological indication,
experimentally evident that CAR-T-mediated elimination of TAMs FRa overexpression has been documented in a variety of solid
could lead to better tumor control in solid tumors (89). Lynn and tumors, inclusive of lung cancer, mesothelioma, and breast cancer,
colleagues also published a paper on FRb-redirected CAR-T- as well as other carcinomas (92, 94, 95). Moreover, its localized
mediated targeting of AML blasts, based on the findings that FRb expression in epithelial cells renders it unreachable to FRa-specific
expression has been evident in a high percentage of AML samples therapeutics, and its negligible expression in normal tissues further
(90). Briefly, these researchers genetically modified the FRb- validates its possible suitability as a CAR-T therapy target antigen
negative cell line C3023 (of ovarian cancer origin) to stably (96). One of the earliest studies on this topic was conducted by
express FRb (90). Upon co-cultivation of the FRb-redirected Kershaw and colleagues as they genetically manipulated autologous
CAR-Ts (equipped with an scFv called m909 as the targeting T cells for the expression of chimeric receptors redirected against
domain) with the engineered C3023 cell line alongside other FRb- FRa using an scFv fused to the intracellular domain of the Fc
positive AML cell lines, it was demonstrated that the CAR-Ts were gamma receptor (97). Individuals with ovarian cancer were divided
capable of mediating antigen-dependent tumoricidal effects against into two cohorts as eight of them underwent T cell therapy with
the target cells (90). Moreover, the investigators also established high-dose IL-2 (cohort I) and six underwent pre-treatment with
xenograft mouse models of FRb-positive human AML based on the dual-specific T cells scheduled to be followed by allogeneic

Frontiers in Immunology 14 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

peripheral blood mononuclear cells (PBMCs) for immunization CAR-Ts and evaluated their characteristics in the presence of a
(cohort II) (97). The findings indicated no tumor shrinkage, as five panel of cytokines (IL-2, -7, -15, -18, and -21) in preclinical
individuals in the first cohort showed attention-requiring toxicities conditions (101). The first three cytokines supported the
(grade 3/4; attributable to high-dose IL-2) whereas those in the expansion of CAR-Ts ex vivo as compared to IL-18, IL-21, or the
second cohort exhibited milder signs of toxicities (grade 1/2) (97). absence of any cytokine treatment (101). Moreover, the highest
Further examination of the administered T cells demonstrated poor degree of CAR-T differentiation was observed in the IL-2 treatment
tumor-site trafficking in most of the patients in cohort I (97). group whereas CAR-Ts in the IL-7 and IL-21 treatment groups
Moreover, Kershaw and colleagues reported a sharp decline in the more shifted towards stem cell-like memory T cells and less
number of administered T cells in most patients, which was differentiated populations, respectively (101). In terms of
attributed to the emergence of an inhibitory element in some tumoricidal effects and cytokines secretion, CAR-Ts in the IL-2
patients (97). Our speculation for this occurrence is the formation and IL-15 treatment groups were superior to others in vitro;
of neutralizing antibodies against the scFv incorporated into the however, CAR-Ts in the former cytokine treatment group
CAR construct of these engineered T cells (97). This scFv was exhibited the weakest level of tumoricidal effects in vivo, as an
derived from the murine mAb MOv18 (97, 98). Examination of the incremental pattern was observed in the antitumor efficacy of CAR-
treated patients’ sera for the presence of such neutralizing Ts in IL-15 and IL-21 cytokine groups (101). Ultimately, Xu and
antibodies would have possibly elucidated the reason for such colleagues concluded that IL-7 and IL-15 are the more suitable
poor in vivo CAR-T persistence. options for supporting the expansion of CAR-Ts ex vivo whereas
It has been evident that the FRa-specific humanized mAb IL-15 and IL-21 are the preferred cytokines for in vivo
farletuzumab has strong tumor growth suppression in animal- administration following adoptive transfer of CAR-Ts (101).
based models of human solid tumors, and Lin and colleagues One of the strategies proposed by researchers for overcoming
reported that such tumoricidal effects are exerted through the limitations of CAR-T-mediated on-target off-tumor toxicities is
antibody-dependent cellular cytotoxicity (ADCC) by conducting the development of CAR-Ts equipped with AND or OR gates (42,
an investigation using mouse models of ovarian cancer (99). 102). However, the practicality of this approach is somewhat
Moreover, farletuzumab has also been assessed in various clinical questioned by the set of target antigens such strategies are
settings for therapeutic purposes in individuals with ovarian cancer developed upon (42, 102). In the context of HGSC, Banville and
(NCT03386942, NCT02289950, etc.) (99). An scFv derived from colleagues conducted an investigation to identify antigens that
such mAbs could be of interest for the development of FRa- could be targeted via combinatorial CAR-T platforms by
redirected CAR-Ts to overcome the issues of murine scFv investigating the simultaneous expression of FRa, mesothelin,
immunogenicity. In contrast with the MOv18-derived scFv, Song and CA125 in different HGSC tumor samples (103). Briefly, all of
and colleagues incorporated a fully human FRa-specific scFv the mentioned target antigens exhibited more than 90% expression
(named C4) into a CAR construct to develop FRa-redirected in the tumor samples whereas the first two were also substantially
CAR-Ts (100). In vitro assays indicated that C4-based CAR-Ts expressed by normal tissues at the transcript level (103). CA125,
were able to mediate tumoricidal effects against FRa-positive mesothelin, and FRa had the highest to lowest expression rates,
ovarian cancer cell lines, SKOV3 and OVCAR5, upon co- respectively, in tumor samples and percentages of malignant cells at
cultivation, alongside secreting elevated levels of INF-g (100). the protein level (103). These researchers reported that an OR gate
Moreover, Song and colleagues established ovarian cancer mouse CAR-T platform based on CA125 and mesothelin would be the
models by intraperitoneal injection of SKOV3 cells into NGC mice most applicable choice as a high percentage of malignant cells in
and reported that intravenous administration of C4-based CAR-Ts around 60% of the assessed tumor samples were proficient in the
resulted in significant tumor volume reduction (100). Furthermore, expression of either antigen (103). Conclusively, Banville and
a comparison of C4-based CAR-Ts with MOv19-based CAR-Ts colleagues asserted that an OR gate CAR-T platform based on
(whose targeting domain is based on a MOv19-derived scFv; a mAb mesothelin and CA125 would be capable of targeting a high
similar to MOv18) showed that these two products mediated proportion of malignant cells in most clinical cases (103).
comparable cytolytic reactions against SKOV3 and A1847 cell However, a personalized AND or OR gate must be developed for
lines alongside secreting comparable levels of INF-g (100). each patient undergoing CAR-T therapy since the expression
Moreover, these two CAR-T products also mediated comparable pattern of each of these antigens could differ in patients with
therapeutic effects in mouse models with established tumors (100). different stages of advanced ovarian cancer.
Upon the co-cultivation of each of these CAR-T products with
HEK293T and IOSE6 (with minimal levels of FRa expression), it
was demonstrated that C4-based CAR-Ts secreted lower levels of 4.13 MUC16
INF-g and TNF-a in comparison to those secreted by MOv19-based
CAR-Ts (100). This lower antigen reactivity of C4-based CAR-Ts MUC16 (alternatively known as mucin 16) is a heavily
could be attributed to the lower affinity of their scFv to FRa, which glycosylated protein with important roles in cellular maintenance
could be a factor of paramount importance in reducing on-target and epithelial protection; however, its tumor-associated expression
off-tumor effects of CAR-Ts towards healthy tissues with has been correlated with tumor cell progression and migration in
physiological levels of target antigen expression (100). According numerous oncological indications (104). The expression of MUC16
to another study, Xu and colleagues developed FRa-redirected has been documented in a high percentage of ovarian cancer cases

Frontiers in Immunology 15 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

(105). The full-length protein of MUC16 is cleaved as a fragment is ectoMUC16-redirected CAR-Ts were developed, which were
released into the bloodstream (known as CA125; which has been further engineered to secrete IL-12 (108). Aside from favorable in
leveraged for diagnostic purposes) and the remainder is left vitro outcomes, IL-12-secreting ectoMUC16-redirected CAR-Ts
displayed over the cell surface (known as ectoMUC16; which has mediated strong tumoricidal responses in xenograft mouse
been exploited for therapeutic purposes by researchers) (104, 105). models of ovarian cancer (based on SCID-Beige mice) which led
CAR-T-mediated targeting of MUC16 has been investigated in to extended survival of the treatment subjects and persistence of the
multiple studies which are briefly discussed in this section. For effector cells alongside elevated levels of INF-g (108). Based on these
instance, Chekmasova and colleagues derived an scFv from the favorable findings, a Phase I clinical investigation was initiated to
mAb 4H11 by fusing VH and VL by means of a flexible linker evaluate the safety and therapeutic efficacy of these IL-12-secreting
peptide (106). This scFv was applied as the targeting domain of 2nd- CAR-Ts (which were also equipped with a safety switch for their at-
generation CAR-Ts (whose CAR construct was based on the CD28 will elimination following administration) in individuals with
co-stimulatory and CD3z activation domains) redirected against ectoMUC16-proficient ovarian carcinoma for the first time (108,
ectoMUC16 (106). Upon co-cultivation of the generated CAR-Ts 109). Yeku and colleagues suggested that a potential counterstrategy
with ectoMUC16-proficient cell lines, SKOV3 and OVCAR3, it was against antigen-dependent tumor relapse (antigen loss or
demonstrated that the CAR-Ts were capable of mediating specific downregulation) would be to engage other parties of the immune
tumoricidal reactions in a dose-dependent fashion, alongside system to augment the antitumor responses, aside from those
exhibiting antigen-dependent proliferation (106). Moreover, these mediated by CAR-Ts (110). These researchers investigated
CAR-Ts secreted significantly elevated levels of IL-2 and INF-g over whether genetic manipulation of ectoMUC16-redirected CAR-Ts
the course of a two-day co-cultivation (106). To further validate for the secretion of IL-12 could have a therapeutic advantage against
these findings, Chekmasova and colleagues developed allogeneic peritoneal ovarian tumors with high or low levels of ectoMUC16
(from a healthy donor) and autologous ectoMUC16-redirected expression in vitro and in preclinical animal models (110). The
CAR-Ts and demonstrated that these engineered T cells managed researchers reported that these CAR-Ts were able to mediate
to enforce cytolytic reactions against primary patient-derived tumor tumoricidal responses against ID8 cells with high or low levels of
cells proficient in the expression of ectoMUC16 (106). These ectoMUC16 expression, with IL-12-secreting ectoMUC16-
researchers also established ovarian cancer SCID-Beige mouse redirected CAR-Ts exhibiting more effective tumoricidal reactivity
models (based on the OVCAR3 cell line) and demonstrated that in comparison with that of conventional ectoMUC16-redirected
intravenous or intraperitoneal administration of the ectoMUC16- CAR-Ts (110). Moreover, peritoneal tumor mouse models were
redirected CAR-Ts resulted in tumor regression and prolonged established by intraperitoneal injection of tumor cells into C57BL/6
survival of the animal models, without any significant difference mice, and it was demonstrated that 12-secreting ectoMUC16-
between CAR-T delivery routes (106). Ultimately, Chekmasova and redirected CAR-Ts prolonged the survival of the treated mice (for
colleagues asserted that such encouraging outcomes could be the whose tumor transplantation, a ratio of 1:1 of high ectoMUC16-
foundation of future clinical investigations with patients diagnosed expressing tumor cells: low ectoMUC16-expressing tumor cells
with MUC16-positive ovarian cancer (106). Chekmasova and co- were used) upon adoption transfer (110). Furthermore, it was
researchers conducted another study to investigate the antitumor elucidated that treatment with 12-secreting ectoMUC16-
efficacy of ectoMUC16-redirected CAR-Ts engineered to secrete IL- redirected CAR-Ts culminated in the amplification of mature
12 in preclinical mouse models of ovarian cancer (107). Briefly, T dendritic cells of the treated subjects’ peritoneum, as an
lymphocytes were engineered to express ectoMUC16-redirected incremental pattern was also observed in the TCR clonality of
CARs (based on the 4H11 scFv) and to secrete IL-12 (107). The this experimental group (110). Ultimately, Yeku and colleagues
researchers established ectoMUC16-positive ID8-based mouse asserted that the application of 12-secreting CAR-Ts could be a
models and demonstrated that adoptive transfer of IL-12- potential strategy to counteract the heterogeneity of solid
secreting ectoMUC16-redirected CAR-Ts resulted in complete tumors (110).
elimination of the peritoneal established tumor lesions, in According to another investigation, Yeku and colleagues
comparison with conventional ectoMUC16-redirected CAR-Ts attempted to evaluate strategies aimed at overcoming the
(107). Moreover, it was elucidated that there were higher immunosuppressive nature of the ovarian cancer TME (111).
numbers of IL-12-secreting ectoMUC16-redirected CAR-Ts in the Briefly, these researchers further modified ectoMUC16-redirected
peritoneum of the treatment subjects which also correlated with CAR-Ts to secrete IL-18 and reported that these CAR-Ts were able
elevated rates of endogenous T lymphocyte recruitment to the to significantly prolong the survival of ovarian cancer syngeneic
tumor lesions, in comparison with the other treated groups (107). mouse models, with high and low tumor burdens, upon adoptive
The investigators also asserted that such antitumor effects were not transfer (111). Another counterstrategy evaluated by Yeku et al. was
dependent on pre-treatment lymphodepletion and that treatment to engineer CAR-Ts for the secretion of PD-1-specific scFvs which
was regarded as well-tolerated (107). Ultimately, the researchers culminated in augmented antitumor efficacy in mouse models of
concluded that ectoMUC16-redirected CAR-Ts could successfully ovarian cancer alongside prolonging the persistence of the CAR-Ts
eliminate transplanted ovarian tumors in mouse models, as such in a way that they resisted tumor rechallenge (111). Moreover, it
favorable therapeutic effects could also be further improved while was demonstrated that this approach resulted in an increase in the
the CAR-Ts were engineered to secrete IL-12 (107). Koneru and engagement of the endogenous immune elements (111). Yeku et al.
colleagues conducted a similar study in which 4H11-based also developed ectoMUC16-specific TRBAs and reported that they

Frontiers in Immunology 16 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

mediated significant tumor shrinkage in xenograft mouse models of negligible (118–121). Therefore, researchers have suggested that
ovarian cancer (111). Clinical assessment of such CAR-Ts and NKG2DLs could be leveraged for therapeutic purposes without
TRBAs could further validate their therapeutic efficacy in patients causing serious risks of toxicities towards healthy tissues (122).
with ectoMUC16-positive ovarian cancer (111). According to According to a study, Barber and colleagues devised a chimeric
another preclinical investigation, Li and colleagues generated a receptor by fusing NKG2D to the CD3z of the TCR, and genetically
lentivirally transduced dual CAR-T product redirected against engineered T cells to express this receptor, and demonstrated that
PD-L1 and ectoMUC16 (PD-L1-ectoMUC16-redirected CAR-Ts) the engineered T cells exhibited tumoricidal reactivity and secreted
and demonstrated that these engineered T lymphocytes were able to proinflammatory cytokines upon engagement of their engineered
enforce pronounced tumoricidal effects against OVCAR3 cells receptors with NKG2DLs (which were surface-expressed by more
alongside secreting elevated levels of IL-2, IFN-g, and TNF-a than 80% of human ovarian cancer biological samples, and the
upon co-cultivation with the mentioned cell line (112). Moreover, ovarian cancer cell lines A2780 and A2008) (122). Barber et al.
these researchers also developed OVCAR3-based mouse models of attempted to further validate their engineered T cells as these
ovarian cancer and reported that adoptive transfer of these dual researchers established GFP-positive ID8 cell line-based mouse
CAR-Ts resulted in the extended survival of the animal models, models of ovarian cancer, and treated the animal subjects with
which were two- to four-fold more pronounced than those induced their engineered T cells (5 × 106 cells) a week following tumor
by CAR-Ts redirected against either PD-L1 or ectoMUC16 (112). establishment (122). 2 months later, the animal models were
Such findings necessitate the evaluation of the safety profile and sacrificed to measure tumor burden and assess the antitumor
therapeutic efficacy of PD-L1-ectoMUC16-redirected CAR-Ts in efficacy of the engineered T cells (122). According to the results,
clinical settings. Recently, O’Cearbhaill and colleagues initiated a the engineered T cells were capable of mediating ID8-based tumor
Phase I clinical investigation (NCT02498912) to assess the safety outgrowth suppression, as measured by a decline in the percentage
and therapeutic efficacy of IL-12-secreting ectoMUC16-redirected of GFP-positive cells and the number of tumor lesions in the animal
CAR-Ts (equipped with a truncated EGFR-based safety switch) peritoneum (122). Ultimately, Barber and colleagues suggested that
administered through the intravenous or intraperitoneal route into their findings indicate that T cells endowed with NKG2D-based
eighteen individuals with ectoMUC16-proficient serous carcinoma chimeric receptors are potential therapeutic options for ovarian
of the ovary with prior lines of therapy (113). Briefly, patients were cancer; however, broader investigation at the clinical level is
put into five cohorts (I-V); as patients in cohorts I to IV underwent warranted for such assertions (122). Barber and colleagues
CAR-T treatment with four different doses (ranging from 3 × 105 to conducted another investigation to further elaborate on the
1 × 107 CAR-Ts/kg), and those in cohort V were lymphodepleted applicability of T cells engineered with NKG2D-based chimeric
with Cy/Flu before CAR-T treatment (with the 3 × 106 CAR-Ts/kg receptors (designed in the previous study) by determining if
dose) (113). According to the findings, no significant clinical signs adoptive transfer of these T cells could prolong the survival of
of on-target off-tumor toxicities nor any DLTs were documented in ovarian cancer animal models, and whether the immune system of
the evaluated patients of the first four cohorts, as two out of three the treatment subjects could mount responses to antigens associated
patients treated in cohort V experienced DLTs (113). Moreover, with ovarian cancer (123). Briefly, B6 mouse models of the GFP-
CRS was reported to occur at all investigated doses (113). positive ID8 cell line were established, and then treated with 5 × 106
Ultimately, According to the Response Evaluation Criteria in Solid engineered T cells (or control T cells) via intraperitoneal
Tumors (RECIST) criteria, the best documented response to the administration (123). According to the results, treatment with the
CAR-T treatment was stable disease (113). Conclusively, the engineered T cells significantly prolonged the survival of the animal
researchers asserted that they aim to increase the in vivo models in comparison with the control group (123). Moreover, the
persistence of these CAR-Ts by combining them with PD-1- investigators further elucidated that the tumor-free surviving
specific therapy (113). Such findings highlight the importance and treatment subjects managed to develop protective immune
possible suitability of MUC16 as a CAR-T therapy target antigen; reactions against ovarian cancer (which include memory CD8-
however, hurdles must be recognized and arduously overcome to positive T cell and CD4-positive T cell reactions) since they
better the therapeutic responses achieved in ovarian cancer patients. rejected another ovarian tumor development with the ovarian
cancer cells, 225 days following the initial time of tumor
establishment (123). Furthermore, the investigators asserted that
4.14 NKG2D ligands (NKG2DLs) the complete tumoricidal capacity of the engineered T cells was also
dependent on the secretion of perforin and IFN-g, as well as GM-
NKG2D is surface-expressed by a variety of immune cells, CSF (123).
including cytotoxic T lymphocytes, gd T lymphocytes, and According to another study, Song and colleagues generated
Natural Killer (NK) cells, in humans (114). In T lymphocytes, NKG2DL-redirected CAR-Ts by incorporating the extracellular
NKG2D engages with DAP10 only to trigger co-stimulatory signals domain of NKG2D into a CAR construct (based on 4-1BB and
for TCRs, whereas, in NK cells, it provides main activation signals CD3z costimulatory and activation domains, respectively), and set
(115–117). In various types of solid tumors, such as ovarian cancer, out to evaluate the feasibility of augmenting the sensitivity of
NKG2DLs (such as MICA/B, ULBP, Letal, etc.) are frequently NKG2DL-positive tumor cells by pharmacologically increasing
expressed; fortunately, mRNAs for their ligands are reported to the expression rate of NKG2DL (124). However, these researchers
exist in healthy tissues, but their surface expression is restricted or reported that the expansion of the NKG2DL-redirected CAR-Ts

Frontiers in Immunology 17 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

was hindered, in comparison with that of the control T cell groups, induced increase in the population of tumor-resident endogenous
mainly due to the expression of NKG2DLs by the NKG2DL- CD4-positive T cells and CD8-positive T cells in a fashion
redirected CAR-Ts, which would consequently result in their dependent on CXCR3, as well as expansion in the population of
fratricide (124, 125). To overcome this issue, the investigators tumor- and lymph-resident tumor-reactive endogenous CD4-
increased the duration of in vitro expansion (124). According to positive T cells (127). Moreover, the researchers reported a CAR-
the results of the in vitro experiments, upon the co-cultivation of the T-induced increase in antigen presentation to CD4-positive T cells
NKG2DL-redirected CAR-Ts with NKG2DL-positive ovarian which was dependent on CAR-T-mediated secretion of INF-g and
cancer cell lines, A1847 and OVCAR5, the effector cells mediated GM-CSF (127). Ultimately, the researchers asserted that efficient
strong cytolytic reactions against the target cells (at the effector: tumor eradication mediated by NKG2DL-redirected CAR-Ts relied
target ratio of 3, 1, and 0.3), while they managed to spare the AE17 on the presence of endogenous CD8-positive T cells (127).
mesothelioma cell line deficient in the expression of NKG2DLs Toxicities associated with the activation and expansion of CAR-
(124). Next, Song et al. treated the ovarian cancer cell lines A2780, Ts following administration are a result of cytokine release (128). In
PEO-1, and OVCAR5 (which expressed low to moderate levels of the context of NKG2DL-redirected CAR-Ts, Ng and colleagues
NKG2DLs) with 2 mM of sodium valproate (which is a histone attempted to overcome this clinical hindrance by designing a 2nd
deacetylase inhibitor), and demonstrated that this treatment generation CAR construct based on the extracellular domain of
increases the surface expression of NKG2DLs on these cells, NKG2D, as the targeting domain, and the 4-1BB co-stimulatory
which consequently culminates in their increased susceptibility to domain and the DAP12 activation domain (128). In a comparative
NKG2DL-redirected CAR-T treatment (as was evident from the view, DAP12-based NKG2DL-redirected CAR-Ts secreted lower
elevated levels of secreted IFN-g in their co-culture) (124). levels of INF-g, TNF-a, and IL-2, and their proliferation capacity
Ultimately, Song et al. concluded that the application of sodium was lower in response to repeated antigen encounter while
valproate with NKG2DL-redirected CAR-T treatment could have mediating tumoricidal effects in vitro, without any observable
therapeutic benefits for patients with ovarian cancer; however difference between DAP12-based NKG2DL-redirected CAR-Ts
clinical assessment would have to validate the safety and clinical and CD3z-based NKG2DL-redirected CAR-Ts in the context of
applicability of this strategy. According to another investigation, mediating cytolytic reactions (128). Ng and colleagues further
Spear and colleagues developed T cells engineered to express established NSG mouse models based on the HCT116 colorectal
NKG2D-based CAR constructs (referred to as chNKG2D-CAR- cancer cell line and reported that the administration of DAP12-
Ts) and demonstrated how adoptive transfer of these cells resulted based NKG2DL-redirected CAR-Ts and CD3z-based NKG2DL-
in sufficient antitumor effects against NKG2DL-positive and redirected CAR-Ts mediated similar tumoricidal effects that led to
NKG2DL-negative ovarian cancer tumors and how chNKG2D- the elimination of the established tumors (128). Of note, high
CAR-T treatment conferred antitumor immunity against mortality rates were reported only in the xenograft group treated
NKG2DL-deficient ovarian cancer cells (126). Briefly, these with the CD3z-based NKG2DL-redirected CAR-Ts which was a
researchers established ID8 cell line-based ovarian cancer mouse result of graft-versus-host disease (GvHD), as higher levels of serum
models and reported remarkable chNKG2D-CAR-T-mediated cytokines were documented in this experimental group (128).
tumoricidal effects against populations of ovarian tumors that Ultimately, the investigators concluded that designing CARs
expressed fluctuating levels of NKG2DL (from 7 to 50%) (126). based on the DAP12 activation domain might be a feasible
However, the researchers asserted that these antitumor responses strategy for minimizing the adverse events associated with CAR-
were strongly dependent on the expression rate of NKG2DL and the T-mediated CRS (128).
percentage of NKG2DL-positive tumor cells within a tumor bulk Despite the fact that various studies reported the expression of
(126). Moreover, Spear and colleagues evaluated whether successful NKG2DL or PD-L1 in numerous types of oncological indications,
treatment with chNKG2D-CAR-T could provide host immunity to whether the simultaneous expression of these two antigens is
animal models against an ID8-based ovarian cancer rechallenge present in different tumors is less explored (129). In this regard,
(126). To this aim, first, GFP-positive ID8 cells were modified with a Jiang and colleagues investigated the expression of NKG2DL and
Rae1 shRNA (ID8/GFP-shRae1) to render them NKG2DL-deficient PD-L1 in human ovarian cancer tissue samples and demonstrated
(of note, this cell line is deficient in the expression of Mult1 or H-60 that almost 80% of the samples exhibited the co-expression of the
NKG2DLs) (126). Next, chNKG2D-CAR-Ts were intraperitoneally mentioned antigens (129). Briefly, these researchers designed a
administered into the same animal models free of the ID8-based unique dual CAR circuit that was based on two separate CAR
ovarian tumors, and, eight weeks following treatment, it was constructs; one based on the extracellular domain of NKG2D fused
demonstrated that the formation of ovarian tumor lesions as well to the DAP12 activation domain (which provides the principal
as tumor progression were suppressed in these mouse models, activation signals), and the other based on a high-affinity PD-L1-
compared with the control group (126). According to another specific scFv fused to the co-stimulatory domain of 4-1BB (which
study, Spear and colleagues demonstrated that adoptive T cell provides auxiliary signals necessary for efficient activation of CAR-
therapy results in conferring host T cell-based immunity that Ts following antigen encounter) (129). The appliance of this high-
cooperates in the eradication of tumor lesions, and the formation affinity scFv was taken into consideration as a potential strategy to
of tumor-reactive immune reactions (127). Briefly, upon the enable dual CAR-Ts to recognize tumor cells with low-level
administration of NKG2DL-redirected CAR-Ts into ID8-based expression of PD-L1 (129). According to the results of the in vivo
preclinical mouse models, the researchers reported a CAR-T- experiments, adoptive transfer of the dual CAR-Ts resulted in the

Frontiers in Immunology 18 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

elimination of ovarian cancer-established tumors with metastatic and reported that intratumoral administration of mesothelin-
peritoneal lesions in preclinical animal models (129). A dual CAR-T redirected CAR-Ts culminated in tumor outgrowth suppression
platform, such as the one developed by Jiang and colleagues, might while the animals were under soluble mesothelin treatment (136).
offer therapeutic benefits for the treatment of metastatic peritoneal Ultimately, Lanitis et al. concluded that mesothelin-redirected
tumor lesions proficient in the expression of NKG2DL and PD-L1 CAR-Ts, whose targeting domains are based on the mentioned
(129). According to another study, Wang and colleagues fully human scFv, can potentially eliminate mesothelin-positive
investigated whether treatment of the ovarian cancer cell line tumors in preclinical conditions while being capable of
SKOV3 with romidepsin (an anticancer agent utilized in the overcoming the issue of anaphylaxis induced by the immunogenic
treatment of T-cell lymphomas) could increase their susceptibility targeting domains of CAR constructs (136). In 2016, Tanyi and
to NKG2DL-redirected CAR-T treatment through increasing colleagues reported the results of a Phase I clinical investigation
NKG2DL expression (130). The results of the in vitro (NCT02159716) assessing the tumoricidal efficacy of 2nd-generation
experiments implicated increased surface expression of NKG2DL mesothelin-redirected CAR-Ts intravenously administered to six
in the SKOV3 cells which consequently resulted in the increased individuals with ovarian cancer (137). Briefly, these autologous
tumoricidal effects of the NKG2DL-redirected CAR-Ts against CAR-Ts benefited from a mesothelin-specific scFv derived from the
these cells (accompanied by elevated levels of secreted INF-g) SS1 mAb (of murine origin), the 4-1BB co-stimulatory domain, and
(130). Ultimately, Wang and colleagues concluded that increasing the CD3z main activation domain (137). Moreover, four
the expression rate of the CAR-T-targeted antigens could enhance individuals underwent a single round of CAR-T therapy with the
the antitumor efficacy of this platform of immunotherapy; however 3 × 107/m2 dosage whereas two other individuals received a single
in-depth clinical evaluations of such strategies are warranted for round with a higher dose (3 × 108/m2) (137). Of note, this treatment
further elucidation (130). scheme entailed CAR-T treatment with or without the use of
lymphodepleting chemotherapy (137). According to the results,
no CRS nor serious adverse events related to the administration of
4.15 Mesothelin the CAR-Ts were observed, as only two patients experienced grade
3 adverse events (such as abdominal discomfort) and one
Mature mesothelin is a 40 kDa membrane-expressed protein experienced grade 3 to 4 adverse events (namely, pleural effusion
which is the result of a 71 kDa Furin-cleaved protein known as the and rapid and shallow breathing, as well as shortness of breath)
precursor mesothelin (131). Primarily introduced in the 1990s, this (137). Moreover, the investigators reported higher in vivo
protein exhibits negligible expression levels in the cells of the expansion rates for the CAR-Ts of the patients treated with the
peritoneum and pleura, whereas its elevated expression in higher dosage and lymphodepletion (137). Examination of tumor
malignant mesothelioma patients has been confirmed in a high samples of three out of four patients (75%) revealed the presence of
percentage of tumor samples (131, 132). Despite its undeciphered, the mesothelin-redirected CAR-Ts, which confirmed their sufficient
and probably inessential, physiological function in normal cells, its infiltration, with their tumoricidal efficacy being evident by the
elevated tumor-associated expression has been correlated with elimination of pleural tumor cells three weeks following treatment
tumor aggressiveness and progression (133–135). Ever since its without lymphodepleting regimens (137). Ultimately, based on the
discovery, mesothelin has been an interesting target antigen in RECIST criteria, the investigators reported that all six patients
investigations relating to cancer immunotherapy. In the context of achieved stable disease (137). The authors asserted that such
ovarian cancer CAR-T therapy, mesothelin can be named as the findings highlight the safety and applicability of mesothelin-
most researched target antigen, which has been the subject of redirected CAR-Ts in individuals with serous carcinoma of the
various CAR-T-based preclinical and clinical investigations, ovary and further clinical evaluations might benefit from these
which are briefly discussed in this section. outcomes (137).
In 2012, Lanitis and colleagues conducted an investigation to According to another investigation, Tanyi and colleagues, for
address the issue of poor CAR-T persistence in vivo induced by the the first time, reported the development of CRS in the pleural
formation of neutralizing antibodies against the targeting domain of cavities of a middle-aged female individual with serous ovarian
CAR-Ts derived from animal-based mAbs (136). To this aim, these carcinoma who had undergone mesothelin-redirected CAR-T
researchers proposed that using fully human targeting domains can therapy (3 × 107/m2) without any lymphodepleting regimen
be taken into consideration in the construction of CAR molecules; (138). This occurrence was characterized by elevated levels of IL-
therefore, they applied a fully human scFv, called P4, specific for 6 and a high population of mesothelin-redirected CAR-Ts in the
human mesothelin as the antigen-recognition domain of their CAR patient’s pleural fluid; three weeks following CAR-T administration,
construct (136). According to the results of the in vitro experiments, this severe toxicity was resolved with the application of the IL-6-
these mesothelin-redirected CAR-Ts secreted proinflammatory specific mAb tocilizumab (138). These researchers suggested that
cytokines and mediated strong tumoricidal reactions upon their the formation of an environment by the patient’s pleural fluid in
co-cultivation with mesothelin-positive cells, as this functionality which CAR-Ts and cancer cells could interact could be the
was not suppressed by the presence of recombinant mesothelin or underlying mechanism for this event (138). According to another
its cancer cell-secreted form (136). Moreover, Lanitis and colleagues study, Gruzdyn and colleagues lentivirally transduced primary T
developed human ovarian cancer xenograft mouse models by cells to develop scFv-based mesothelin-redirected CAR-Ts, and
subcutaneous inoculation of the A1847 cell line into NSG mice they reported that the CAR-Ts significantly secreted elevated

Frontiers in Immunology 19 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

levels of granzyme B and INF-g upon their co-cultivation with the other relatable malignancies. According to another study, Haas
mesothelin-positive SKOV3 ovarian cancer cell line (139). and colleagues reported the findings of a Phase I clinical
Moreover, these researchers demonstrated that increasing the investigation that evaluated the safety and efficacy of mesothelin-
effector:target cell ratio from 10:1 to 20:1 resulted in higher redirected CAR-Ts in fifteen individuals with ovarian cancer,
percentages of CAR-T-mediated SKOV3 cell lysis (from ~40 to pancreatic adenocarcinoma, and pleural mesothelioma (five
61%) (139). Experimental evidence has suggested that ovarian patients in each oncological indication group; who were also
tumor cells evade the immune system by means of secreting refractory to chemotherapy) (142). Briefly, lentivirally transduced
inhibitory cytokines, including IL-10 (140). In line with the CAR-Ts were generated by incorporating a mesothelin-specific scFv
previously discussed study [by Gruzdyn et al. (139)] and to (derived from the murine mAb, SS1) into the construct of a 2nd-
further investigate the inhibitory impact of TME-derived IL-10 on generation CAR (based on 4-1BB and CD3z as the co-stimulatory
CAR-T therapy, Batchu and colleagues conducted a study and domain and activation domain, respectively), and then the patients
reported that blockade of IL-10 in the tumor milieu remarkably underwent a single round of CAR-T administration (with 1-3 × 107
reverses its pro-tumor effects and enables mesothelin-redirected or 1-3 × 108 engineered effector cells/m2) with cyclophosphamide-
CAR-Ts to more efficiently exert their tumoricidal effects (140). induced lymphodepletion (1.5 g/m2) or without lymphodepletion
Briefly, these researchers developed mesothelin-redirected CAR-Ts (142). Conclusively, it was reported that the CAR-Ts were well-
and prepared a conditioned medium from two-day cultivation of tolerated, as only one case of toxicity was reported (grade 4) in a
SKOV3 cells without serum in which IL-10 was present or depleted non-lymphodepleted patient in the low-dose CAR-T group (142).
via antibodies (referred to as IL-10-proficient or IL-10-deficient Eleven out of fifteen (~ 73%) patients achieved stable disease, as
medium, respectively) (140). Cultivation of mesothelin-redirected CAR-T persistence was reported to be transient with their
CAR-Ts in the IL-10-proficient media resulted in significantly expansion peak between day 6 to 14 (of note, prior
suppressed secretion of granzyme B and INF-g, as their secretion lymphodepletion improved effector cell expansion, but had no
levels were not completely returned to the co-cultivation levels even positive effects on their persistence after 28 days) (142).
in the presence of IL-10-deficient media (140). Moreover, a sharp According to the blood examination results of fourteen patients,
decline in the cytolytic reactions of mesothelin-redirected CAR-Ts neutralizing antibodies against the CAR constructs of the CAR-Ts
was reported (to 19% at the E:T ratio of 10:1 and 32% at the E:T were found in eight patients (~ 57%), which necessitates the
ratio of 20:1) in the presence of IL-10-proficient media (140). application of fully human or humanized antigen recognition
Simultaneous expression of CAR-T-targeted TAAs by healthy domains (scFvs or VHHs) in the design of CAR constructs for
tissues results in off-tumor toxicities against unintended tissues, to clinical applications (60, 80, 142, 143).
overcome which, some researchers have suggested the development According to another study, Zhang and colleagues focused on
of mRNA-based CAR-Ts that transiently express CARs redirected targeting two distinct epitopes of mesothelin (its membrane-distal
against TAAs of interest (60, 141). According to a study by Hung region and membrane proximal region; hereinafter referred to as
and colleagues, the researchers generated an automated and efficient meso-I and meso-III, respectively) by developing two different
platform for the large-scale development (~ 2 × 1010) of mRNA- mesothelin-redirected CAR-T products (144). For in vitro analysis
based human mesothelin-redirected CAR-Ts that enables the of the antitumor reactivities of the developed CAR-T products,
generation of vast populations of CAR-Ts from a single round of meso-I- or meso-III-redirected CAR-Ts were co-cultivated with the
leukapheresis to be used for multiple infusions into treatment human gastric carcinoma cell line HGC-27 or human ovarian
subjects (141). According to in vitro killing assays, the developed cancer cell line SKOV3, and it was demonstrated that meso-III-
CAR-Ts mediated strong tumoricidal effects against the murine redirected CAR-Ts exhibited more pronounced tumoricidal effects
ovarian cancer cell line Defb29 engineered to express human against the target cells, as they also secreted higher levels of IL-2,
mesothelin, as it was reported that CAR expression rate grew low INF-g, and TNF-a, and demonstrated a higher expression rate of
within a week following in vitro cultivation with a possible CD107a in comparison with those of meso-I-redirected CAR-Ts
correlation with CAR-T expansion (141). Moreover, the (144). Furthermore, Zhang et al. investigated the antitumor efficacy
investigators established human mesothelin-positive ovarian of the developed CAR-Ts in more realistic tumor models of the
tumor mouse models (using 3 × 105 human mesothelin-positive HGC-27 and SKOV3 cell lines by developing 3D tumor spheroids
Defb29 cells), and reported that a single round intraperitoneal and reported that meso-III-redirected CAR-Ts cytolyzed higher
administration of mesothelin-redirected CAR-Ts resulted in percentages of the target cells over a period of 24 hours (144). In
tumor outgrowth suppression and prolonged survival of the mouse models of HGC-27-based gastric cancer, meso-III-redirected
animal models in a CAR-T dose-dependent fashion (141). The CAR-Ts, which were administered intravenously ten days following
researchers also reported that repeated weekly intraperitoneal tumor establishment, enforced more effective tumoricidal effects
injection of an optimal mesothelin-redirected CAR-T dose which resulted in more pronounced tumor volume shrinkage in the
resulted in better tumor regression and more protracted survival animal models, as compared to those of meso-I-redirected CAR-Ts
rates in the mouse models (141). Of note, no serious CAR-T- (144). Moreover, in mouse models of SKOV3-based ovarian cancer,
mediated off-tumor toxicities were reported by the investigators meso-III-redirected CAR-Ts were administered intravenously
(141). Such findings highlight the potential applicability of mRNA- seven days or fourteen days following tumor establishment, and it
based mesothelin-redirected CAR-Ts for clinical evaluations in was demonstrated that the CAR-Ts were able to mediate similar
individuals with mesothelin-positive ovarian tumors, as well as survival rates (over the course of 40 days) and similar tumor volume

Frontiers in Immunology 20 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

shrinkage in the treatment subjects (144). Based on these findings, it antigens, as only a small percentage (~ 10%) of the sample were
can be concluded that meticulous selection of the target antigen deficient in their co-expression (147). FOLR1-redirected CAR-Ts,
epitope could be a factor of paramount importance in the context of mesothelin-redirected CAR-Ts, and tandem CAR-Ts were co-
CAR-T therapy development, which could potentially lead to better cultivated with the SNU119 and SKOV3 ovarian cancer cell lines,
therapeutic effects. and it was demonstrated that tandem CAR-Ts mediated higher
According to a 2021 report, Liu and colleagues conducted an percentages of target cell lysis in comparison with those of the single
investigation to assess the inhibitory effects of PD1-PD-L1 antigen-redirected CAR-Ts or control T cells (147). Moreover,
interaction on the tumoricidal effects of mesothelin-redirected significantly higher levels of IL-2, IL-12, INF-g, and TNF-a were
CAR-Ts, using the SKOV3 and HCT116 (of colorectal cancer secreted by tandem CAR-Ts upon their co-cultivation with the
origin) cell lines and a shRNA-based silencing strategy for PD-1 SNU119 cells, in comparison with those of other CAR-Ts or T cell
silencing (145). According to the results of the in vitro experiments, groups (147). These researchers further developed ovarian cancer
PD-1-silenced mesothelin-redirected CAR-Ts exhibited stronger xenograft models based on B-NDG mice and SNU119 cells and
tumoricidal effects against the SKOV3 and HCT116 cell lines treated the animal models with intravenous administration of the
upon co-cultivation, alongside secreting higher levels of INF-g, in three different CAR-T products or control T cells (1 × 107 cells) ten
comparison with those of wild-type mesothelin-redirected CAR-Ts days following tumor transplantation (147). Tandem CAR-T
(145). Such findings highlight the importance of PD1-PD-L1 axis significantly outperformed other treatment groups in terms of
disruption in the context of solid tumor CAR-T therapy which mediating prolonged survival and eliminating large established
warrants meticulous in-depth preclinical and clinical evaluations ovarian tumor lesions; however, tandem CAR-Ts exhibited a
(145). Moreover, other gene silencing techniques (such as CRISPR- comparable persistence rate and infiltration capacity only to
Cas or TALEN) alongside other immunoinhibitory genes can also FOLR1-redirected CAR-Ts, which was higher than those of the
be taken into consideration for future assessments (24, 102, 145). other CAR-T/T cell groups (147). Ultimately, Liang and colleagues
Another investigation further investigated how the concluded that FOLR1/mesothelin tandem CAR-Ts engineered to
immunosuppressive elements of ovarian tumors impede the secrete IL-12 could minimize the risks of disease recurrence through
functionality of CAR-Ts and demonstrated that the co- the elimination of antigen escape variants while maximizing
stimulatory domains of CAR constructs could play important targeted antitumor responses (147). However, broad clinical
roles in this matter (146). Briefly, Schoutrop and colleagues investigation must elucidate the safety and tolerability of such
developed two different mesothelin-redirected CAR-T products, CAR-T platforms, since the selection of two co-expressed target
one of which harbored the CD28 co-stimulatory domain whereas antigens absent from healthy tissues could be a rather challenging
the other had the 4-1BB co-stimulatory domain, and evaluated the task (102).
in vivo functionality of these CAR-Ts in SKOV3-based xenograft According to a 2021 report by Liu and colleagues, it was reported
mouse models of ovarian cancer (146). Briefly, adoptive transfer of that disruption of adenosine 2a receptor (A2aR) culminates in
CD28-based CAR-Ts resulted in significant protracted survival of improved tumoricidal efficacy of mesothelin-redirected CAR-Ts in
the animal models whereas 4-1BB-based CAR-T treatment preclinical conditions (148). Accumulating evidence demonstrates
mediated persistent remission in several treatment subjects (146). that tumor cell-derived adenosine, within the TME, binds its cognate
Moreover, examination of tumor-in filtrating CA R-Ts receptor, A2aR, on the surface of T cells leading to their impaired
demonstrated that both CAR-T products exhibited elevated levels tumoricidal reactivity through triggering downstream signaling
of immunoinhibitory receptor (LAG3 and PD-1) expression which cascades (148). To evaluate the effects of A2aR disruption on the
coincided with the expression of relative ligands (HLA-DR and PD- tumoricidal efficacy of mesothelin-redirected CAR-Ts, Liu et al. used
L1) by mesothelin-positive ovarian tumors (146). However, it was a specific shRNA for A2aR disruption (148). Briefly, it was
demonstrated that tumor-infiltrating CD28-based CAR-Ts demonstrated that A2aR-disrupted mesothelin-redirected CAR-Ts
exhibited more pronounced characteristics of exhaustion in outperformed conventional mesothelin-redirected CAR-Ts in terms
comparison with 4-1BB-based CAR-Ts (146). Such findings of mediating cytolytic reactions and secreting proinflammatory
substantiated the proposition that immunoinhibitory axes impair cytokines upon their co-cultivation with the SKOV3 and HCT116
the persistence of CAR-Ts in the TME of ovarian cancer (146). cell lines (148). Moreover, adoptive transfer of both mesothelin-
Most patients with solid tumors, such as ovarian cancer, often redirected CAR-T products into SKOV3-based xenograft mouse
suffer from tumor recurrence which is somehow a result of antigen models resulted in remarkable tumor regression, as compared with
heterogeneity within the TME (43, 147). In the context of the control T cell group, with the A2aR-disrupted CAR-Ts mediating
mesothelin-redirected CAR-T therapy for ovarian cancer, a recent more pronounced tumoricidal reactions (148). Conclusively, these
study by Liang and colleagues attempted to address this issue researchers asserted that shRNA-based gene disruption might hold
through the development of tandem CAR-Ts that target FOLR1 therapeutic promise for augmenting the antitumor efficacy of CAR-
(by the means of the MOv19 scFv) and mesothelin (by the means of Ts within harsh TME conditions (148). The therapeutic benefit and
the P4 scFv) while engineered to secrete IL-12 for supportive clinical feasibility of this strategy need to be meticulously assessed in
immunomodulatory effects (147). Briefly, these researchers put clinical investigations with ovarian cancer patients.
together a panel of twenty co-expressed genes with elevated Aside from the encouraging results of 2 nd -generation
expression levels using the Gene Expression Omnibus, and then mesothelin-redirected CAR-Ts, their 3rd-generation counterparts
selected FOLR1 and mesothelin as two co-expressed potential target could also be therapeutically valuable in the context of ovarian

Frontiers in Immunology 21 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

cancer. According to a 2021 report, Zhang and colleagues models of liver cancer and pancreatic cancer (151). Relying on these
incorporated a mesothelin-specific scFv into a 3rd-generation findings, Pang et al. initiated a Phase I clinical investigation to
CAR construct based on the CD28/4-1BB co-stimulatory domains evaluate the safety and applicability of these CAR-Ts in individuals
and the CD3z activation domain only to develop mesothelin- with ovarian cancer (proficient in the expression of glypican 3 or
redirected CAR-Ts (149). Upon co-cultivation of these CAR-Ts mesothelin), as well as other advanced solid tumors
with the SKOV3 and OVCAR3 ovarian cancer cell lines, the effector (NCT03198546) (151). The results of this trial relating to the
cells mediated mesothelin-dependent tumoricidal effects against the treatment of ovarian cancer patients are yet to be obtained;
target cells (149). Moreover, the CAR-Ts secreted significantly however, these researchers reported that intratumoral
elevated levels of INF-g and TNF-a upon co-cultivation with the administration of the IL-7/CCL19-engineered glypican 3-
SKOV3, OVCAR3, and HCT116 cell lines (149). To further redirected CAR-Ts led to complete tumor eradication a month
evaluate the antitumor efficacy of these mesothelin-redirected following the treatment in a patient with hepatocellular carcinoma
CAR-Ts, Zhang et al. established mouse models of ovarian (151). In reference to IL-7/CCL19-engineered mesothelin-
cancer, breast cancer, and colorectal cancer (by subcutaneous redirected CAR-Ts, it was reported that the adoptive transfer of
injection of SKOV3, MCF7, and HCT116 cells into the animal these CAR-Ts to a pancreatic cancer patient led to the complete
models, respectively), and reported that intravenous administration elimination of tumors almost 8 months following CAR-T treatment
of the mesothelin-redirected CAR-Ts (2.5 × 105 effector cells) (151). Ultimately, it can be concluded that engineering CAR-Ts for
resulted in significant shrinkage of the established tumor lesions the secretion of IL-7 and CCL19 could have a therapeutic advantage
(149). Moreover, the adoptive transfer of the mesothelin-redirected against solid tumors by increasing the tumor-homing capacity of
CAR-Ts into mesothelin-positive patient-derived xenograft mouse CAR-Ts; however, in the context of ovarian cancer, clinical
models of colorectal cancer or gastric cancer resulted in the investigations with large patient populations must be conducted
eradication of the tumor bulks and prolonged survival of the for more elucidation (151). In 2022, Chen and colleagues isolated a
animal models (149). Such findings highlighted the fact that 3rd- mesothelin-specific scFv without any cross-reactivity using the
generation mesothelin-redirected CAR-Ts could also be choices of phage-display technology and then developed 2nd-generation
therapeutic value against ovarian cancer in preclinical settings; CAR-Ts (153). Following successful in vitro and in vivo
however, clinical studies must be conducted to directly assess the antitumor functionality of these mesothelin-redirected CAR-Ts
safety profile and tumoricidal efficacy of 2nd-generation and 3rd- against ovarian cancer cell lines and xenograft mouse models,
generation mesothelin-redirected CAR-Ts in patients with respectively, Chen and colleagues evaluated the safety profile and
mesothelin-positive ovarian cancer (149). In 2022, Li and therapeutic efficacy of autologous mesothelin-redirected CAR-Ts in
colleagues incorporated the humanized version of a mesothelin- three individuals with ovarian carcinoma (153). No CRS nor
specific single-domain antibody (known as F3M) into a 2nd- neurotoxicity (greater than grade 2) was reported in any of the
generation CAR construct based on the 4-1BB and CD3z co- three patients, as two of them experienced stable disease with a PFS
stimulatory domain and activation domain, respectively, and of 4.6 and 5.8 months (153).
developed mesothelin-redirected CAR-Ts that exhibited effective In 2022, Tanyi and colleagues reported the results of a Phase I
tumoricidal efficacy in preclinical settings (150). As an attempt to clinical investigation in which fourteen patients with mesothelin-
counteract the immunosuppressive effects of TME-derived TGF-b, positive solid tumors, inclusive of ovarian cancer, underwent
Li and colleagues engineered T cells to co-express the mentioned treatment with mesothelin-redirected CAR-Ts harboring a
CAR alongside a dominant-negative TGF-b receptor type II (150). humanized scFv as the targeting domain incorporated into a 4-
Li et al. demonstrated that the developed CAR-Ts exhibited 1BB/CD3z-based CAR construct (154). The patients were put into
ameliorated efficacy in the presence of TGF-b and in preclinical four different cohorts (1 to 4); three patients were treated with a
mouse models, in a way that these CAR-Ts were capable of resisting single intravenous administration of 3 × 107 CAR-Ts/m2, three
TGF-b immunosuppressive effects (150). In October 2020, a Phase I underwent pre-treatment lymphodepletion and a single
clinical investigation was initiated to investigate the safety and intravenous administration of 3 × 10 7 CAR-Ts/m 2 , two
tumoricidal efficacy of these CAR-Ts in fifteen patients with underwent a single intravenous administration of 3 × 108 CAR-
ovarian cancer; however, in 2022, the collaborators and sponsors Ts/m 2 , and six underwent lymphodepletion and an initial
were considering bringing closure to the investigation. intravenous administration of 3 × 107 CAR-Ts/m2 scheduled to
To address one of the most important obstacles of solid tumor be followed by a maximum of two other intravenous infusions
CAR-T therapy, which is poor CAR-T trafficking into the tumor (cohort 1, 2, 3, and 4, respectively) (154). Briefly, the treatment was
sites, Pang and colleagues developed glypican 3- or mesothelin- reported to be well-tolerated, as CAR-T expansion directly
redirected CAR-Ts engineered to express and secrete IL-7 and correlated with lymphodepletion and higher infusion doses (154).
CCL19 and demonstrated that these CAR-Ts exhibited enhanced Moreover, the administered CAR-Ts exhibited tumor infiltration in
migration capacity and expansion rate in vitro (151). Previous nine out of fourteen patients (~ 64%) (154). In accordance with the
studies have demonstrated that IL-7 and CCL19 expression RECIST criteria, eight patients experienced stable disease (~ 57%);
correlates with an enhanced infiltration rate of T cells in which lasted for three and nine months in two of the patients (154).
preclinical experiments (42, 151, 152). Moreover, these In reference to the adverse events and toxicities, CRS (grades 3 and
researchers reported that adoptive transfer of their IL-7/CCL19- 4) was documented in four patients, as multiple adverse events were
engineered CAR-Ts mediated strong tumor regression in xenograft also observed (which included hypotension, fatigue, and hypoxia)

Frontiers in Immunology 22 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

(154). Serious pulmonary-related toxicities were only documented contrast, while CAR-Ts engage with their target cells upon antigen
in the patients of cohort 3; one of whom experienced failure of the encounter, they mediate cytolytic reactions against that cell, which
respiratory system, which was related to the high dosage of the might result in serious irreversible organ damage in the cases of off-
administered CAR-Ts (154). The instigators declared 3 × 107 CAR- tumor effects. Therefore, it is reasonable to assume that CAR-Ts have
Ts/m2 as the maximum tolerated dose based on the findings of this different toxicity profiles in comparison with those of mAbs, or any
trial (154). Ultimately, Tanyi and colleagues asserted that these other targeted treatment modality, and antigens considered suitable for
findings could pave the way for future clinical trials assessing the mAb therapies might not necessarily be suitable for CAR-T therapy as
safety and therapeutic applicability of CAR-Ts in mesothelin- well. Another justification might be based on the lack of qualified
positive tumors, including ovarian cancer, and that meticulous targeting domains for the construction of potent CARs against such
strategies could be taken into consideration for increasing the targets (which might arise from proprietary rights). Ultimately, since
safety profile and tumor-homing capacity of such CAR-Ts (such CAR-T therapy of ovarian is still a progressing field, it is believed that
as localized CAR-Ts administration) (154). Studies such as those the suitability of these antigens for CAR-T therapy of ovarian cancer will
discussed in this section highlight the importance of mesothelin as a be a subject of investigation in the upcoming years (as some of these
potential target antigen in ovarian cancer CAR-T therapy, and the antigens have already been assessed in the case of other solid tumors).
fact that researchers have put a tremendous deal of effort into Out of the target antigens discussed, three mAbs against PD-L1 have
assessing CAR-T therapy in the treatment of mesothelin-positive been approved by the US FDA which include durvalumab (IMFINZI®;
ovarian cancer; however, the findings of future clinical trials with approved for the treatment of bladder cancer in 2017), avelumab
larger patient populations could shed more light on the downsides (Bavencio®; approved for the treatment of Merkel cell carcinoma in
and upsides of ovarian cancer CAR-T therapy, and propose 2017), atezolizumab (Tecentriq®; approved for the treatment of bladder
applicable strategies for overcoming the limitations. cancer in 2016) (156–158). These approvals might somehow
corroborate the suitability of PD-L1 as a target antigen of cancer
immunotherapy; however, each immunotherapy platform warrants
5 Conclusion further in-depth assessments of its own.
Aside from selecting the ideal target antigen, successful CAR-T
Reaching the destination is worth having a long and twisted way to therapy in solid tumors including ovarian cancer depends on various
go; this might be the case with CAR-T therapy in the treatment of factors that are critical for tackling the roadblocks of this type of
ovarian cancer. Researchers must keep in mind that numerous dots treatment. The future of this type of therapy relies on the engineering of
must be connected to successfully fight this advanced indication with next-generation CAR-Ts and their successful employment. For
CAR-Ts. Over the past years, tremendous effort has been put into instance, since most of the targeted antigens in the CAR-T therapy
assessing the safety and therapeutic potential of CAR-T therapy of ovarian cancer are TAAs, researchers need to develop strategies to
redirected against various ovarian cancer-associated target antigens overcome the issue of on-target off-tumor toxicities. One of the most
both in preclinical and clinical investigations. To fully validate the applicable strategies in this matter is the use of CAR targeting domains
suitability of each of these target antigens in the context of ovarian with a moderate affinity, rather than high-affinity ones (159).
cancer CAR-based therapies, broad and in-depth clinical findings are Experimental findings have demonstrated that this strategy is feasible
required. However, other platforms of cancer immunotherapy have in minimizing the off-tumor effects of CAR-Ts on healthy tissues (159).
been developed against some of these antigens, as well as other target Other strategies could be based on the transient expression of CAR
antigens whose targeted therapy might hold therapeutic promise. For molecules in CAR-Ts developed using mRNAs or the use of suicide
instance, ImmunoGen developed a FRa-specific ADC named switches for the elimination of the infused T cells (160, 161). In the
mirvetuximab soravtansine (under the trade name Elahere®) which context of ovarian cancer CAR-T therapy, various researchers reported
was approved by the US FDA in November 2022 for the treatment of the presence of neutralizing antibodies against the targeting domain of
patients with epithelial ovarian cancer resistant to platinum or CARs derived from animal-based targeting moieties. Such antigen-
peritoneal cancer with three or fewer lines of prior therapies (155). recognition domains must be replaced with humanized or fully human
The approval of this therapeutic was based on the ultimate findings of a targeting domains to overcome the issue of anaphylaxis (60, 80). As
clinical trial (NCT04296890) in which 106 patients with the mentioned discussed, the design of the CAR molecules (the spacer fragment, the
indications were required to undergo bevacizumab. The objective targeting domain, and the signaling domains) could have substantial
response rate (ORR) and duration of response (DOR) were reported effects on the phenotype and antitumor efficacy of the developed CAR-
to be ~ 32% and 6.9 months, respectively. Moreover, this therapeutic Ts; therefore, precise designing alongside meticulous selection of the
entered the market with a safety label for ocular toxicity; meaning that CAR components should be taken into consideration before the
despite encouraging clinical outcomes, there are still risks of off-tumor development of a CAR-T product. Researchers also used an
toxicities. The same assumption could be attributed to all of the antigens intelligent strategy to overcome the issue of low antigen density by
discussed throughout this review, as CAR-T therapies and mAbs exert ovarian cancer cells. As detailed throughout the text, various
their antitumor function through distinct mechanisms. In brief, mAbs therapeutics (such as chemotherapeutics) were used to positively
can play an antagonistic role by blocking ligands from binding their influence the expression of a certain antigen by tumor cells; thereby
cognate receptors, induce signaling in a cell upon antigen engagement, increasing their susceptibility to CAR-T-mediated cytolytic reactions.
or trigger target cell lysis through complement-dependent cytotoxicity To overcome the immunosuppressive nature of the TME of ovarian
(CDC) or antibody-dependent cellular cytotoxicity (ADCC). In cancer, CAR-Ts could be genetically engineered to be

Frontiers in Immunology 23 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

immunosuppression-resistant (using CRISPR-Cas, TALEN, shRNAs, targeting of each of these antigens will be in patients with ovarian
etc.) or secrete anti-immunoinhibitory molecules (such as anti-PD-1 cancer is the subject of future investigations, as limitations currently
scFvs or VHHs) (26). To overcome the limitation of poor CAR-T known or unknowns will have to be overcome. The future success in
infiltration in the context of ovarian cancer, localized administration of this field is highly dependent on the specificity and safety of the
CAR-Ts could be a potent solution instead of systemic administration. target antigens as well as the counterstrategies for tackling the
Moreover, CAR-Ts could also be genetically modified for the roadblocks of this type of solid tumor. In a similar fashion to CAR-
expression of chemokine receptors that correspond to those secreted T therapy in most types of solid tumors, the application of this type
by tumor cells for amplifying the presence of effector immune cells in of living drug immunotherapy in ovarian cancer requires far-
tumor sites. For instance, it has been demonstrated that CAR-Ts can be reaching detailed investigations, especially in clinical settings with
genetically engineered for the expression of IL-8 receptors (CXCR1 or larger patient populations.
CXCR2) (27, 162). Such CAR-Ts can respond to IL-8 secreted by
tumor cells in tumor islets which results in boosted CAR-T infiltration
rate and improved antitumor efficacy (27, 162). To solve the issue of Author contributions
tumor escape variants or tumor heterogeneity, the modification of
CAR-Ts for the expression of molecules that engage the endogenous FN: Investigation, Validation, Writing – original draft. KF:
elements of the immune system (such as IL-12) or the co-expression of Validation, Writing – original draft. PouSK: Conceptualization,
distinct CAR molecules that target two (or more) sets of target antigens Investigation, Supervision, Validation, Visualization, Writing –
associated with ovarian cancer could be taken into consideration. Aside original draft, Writing – review and editing. MMK: Writing –
from all of the discussed engineering novelties, CAR-Ts can be original draft. SDS: Investigation, Writing – original draft. PooSK:
designed to trigger bystander antitumor activity by other Conceptualization, Investigation, Supervision, Validation,
components of the immune system. For instance, CAR-Ts can be Visualization, Writing – original draft, Writing – review and editing.
engineered to secret TRBAs which can help them circumvent antigen
escape and induce paracrine immune responses by bystander T cells
against the tumor cells (163). Similarly, CAR-T can also be engineered Funding
to express CD40 ligand (CD40L) on their surface which can act
through two mechanisms. In detail, CD40L-expressing CAR-Ts can The author(s) declare that no financial support was received for
interact with CD40-expressing tumor cells which results in a direct the research, authorship, and/or publication of this article.
antitumor effect. Also, CD40L-expressing CAR-Ts have the ability to
license antigen-presenting cells (APCs) that can mediate the
recruitment and cytokine production of other endogenous immune Conflict of interest
effector cells (164, 165). Moreover, allogeneic CAR-Ts have been used
in clinical settings owing to their various advantages (166). However, The authors declare that the research was conducted in the
the limitation of alloreactivity of these products should be addressed to absence of any commercial or financial relationships that could be
avoid compromised antitumor responses in patients. To address this, construed as a potential conflict of interest.
researchers have used genetic manipulation methods to knock out the
TCR a or b chain which renders the produced T-cell product devoid of
TCR surface presentation resulting in a significantly lower rate of Publisher’s note
alloreactivity (167–171). Moreover, similar methods have also been
applied for the knockout of human leukocyte antigens (HLA) All claims expressed in this article are solely those of the authors
expression in T-cell-based products to minimize their alloreactivity and do not necessarily represent those of their affiliated
and expand their safer clinical application (172, 173). organizations, or those of the publisher, the editors and the
In the long run, it can cautiously be concluded that considerable reviewers. Any product that may be evaluated in this article, or
preclinical and clinical efforts have been made in the field of ovarian claim that may be made by its manufacturer, is not guaranteed or
cancer CAR-T therapy. However, how well-tolerated CAR-T endorsed by the publisher.

References
1. Jayson GC, Kohn EC, Kitchener HC, Ledermann JA. Ovarian cancer. Lancet 5. Chien J, Poole EM. Ovarian cancer prevention, screening, and early detection:
(2014) 384:1376–88. doi: 10.1016/s0140-6736(13)62146-7 report from the 11th biennial ovarian cancer research symposium. Int J Gynecol Cancer
2. Mallen A, Soong TR, Townsend MK, Wenham RM, Crum CP, Tworoger SS. Off J Int Gynecol Cancer Soc (2017) 27:S20–2. doi: 10.1097/igc.0000000000001118
Surgical prevention strategies in ovarian cancer. Gynecol Oncol (2018) 151:166–75. 6. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA: Cancer J
doi: 10.1016/[Link].2018.08.005 Clin (2022) 72:7–33. doi: 10.3322/caac.21708
3. Slatnik CL, Duff E. Ovarian cancer: Ensuring early diagnosis. Nurse Practitioner 7. Stewart C, Ralyea C, Lockwood S. Ovarian cancer: an integrated review. Semin
(2015) 40:47–54. doi: 10.1097/[Link].0000450742.00077.a2 Oncol Nurs (2019) 35:151–6. doi: 10.1016/[Link].2019.02.001
4. Jasen P. From the “Silent killer” to the “Whispering disease”: ovarian cancer and the 8. Ozols RF. Challenges for chemotherapy in ovarian cancer. Ann Oncol Off J Eur Soc
uses of metaphor. Med History (2009) 53:489–512. doi: 10.1017/S0025727300000521 Med Oncol (2006) 17 Suppl 5:v181–7. doi: 10.1093/annonc/mdj978

Frontiers in Immunology 24 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

9. Schorge JO, McCann C, Del Carmen MG. Surgical debulking of ovarian cancer: 33. Griffioen AW, Damen CA, Blijham GH, Groenewegen G. Tumor angiogenesis is
what difference does it make? Rev Obstet Gynecol (2010) 3:111–7. accompanied by a decreased inflammatory response of tumor-associated endothelium.
10. Iorio GC, Martini S, Arcadipane F, Ricardi U, Franco P. The role of radiotherapy Blood (1996) 88:667–73. doi: 10.1182/blood.V88.2.667.bloodjournal882667
in epithelial ovarian cancer: a literature overview. Med Oncol (Northwood London 34. Bergers G, Song S. The role of pericytes in blood-vessel formation and
England) (2019) 36:64. doi: 10.1007/s12032-019-1287-8 maintenance. Neuro Oncol (2005) 7:452–64. doi: 10.1215/s1152851705000232
11. Maibach F, Sadozai H, Seyed Jafari SM, Hunger RE, Schenk M. Tumor- 35. Salmon H, Franciszkiewicz K, Damotte D, Dieu-Nosjean MC, Validire P,
infiltrating lymphocytes and their prognostic value in cutaneous melanoma. Front Trautmann A, et al. Matrix architecture defines the preferential localization and
Immunol (2020) 11:2105. doi: 10.3389/fimmu.2020.02105 migration of T cells into the stroma of human lung tumors. J Clin Invest (2012)
12. Stanton SE, Disis ML. Clinical significance of tumor-infiltrating lymphocytes in 122:899–910. doi: 10.1172/jci45817
breast cancer. J Immunother Cancer (2016) 4:59. doi: 10.1186/s40425-016-0165-6 36. Yamauchi M, Barker TH, Gibbons DL, Kurie JM. The fibrotic tumor stroma. J
13. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C, et al. Clin Invest (2018) 128:16–25. doi: 10.1172/jci93554
Type, density, and location of immune cells within human colorectal tumors predict 37. Peranzoni E, Lemoine J, Vimeux L, Feuillet V, Barrin S, Kantari-Mimoun C, et al.
clinical outcome. Sci (New York NY) (2006) 313:1960–4. doi: 10.1126/science.1129139 Macrophages impede CD8 T cells from reaching tumor cells and limit the efficacy of anti-
14. Arigami T, Uenosono Y, Ishigami S, Matsushita D, Hirahara T, Yanagita S, et al. PD-1 treatment. Proc Natl Acad Sci USA (2018) 115:E4041–e50. doi: 10.1073/
Decreased density of CD3+ tumor-infiltrating lymphocytes during gastric cancer pnas.1720948115
progression. J Gastroenterol Hepatol (2014) 29:1435–41. doi: 10.1111/jgh.12551 38. Sugiura A, Rathmell JC. Metabolic barriers to T cell function in tumors. J
15. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G, Immunol (2018) 200:400–7. doi: 10.4049/jimmunol.1701041
et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J 39. Dangaj D, Bruand M, Grimm AJ, Ronet C, Barras D, Duttagupta PA, et al.
Med (2003) 348:203–13. doi: 10.1056/NEJMoa020177 Cooperation between constitutive and inducible chemokines enables T cell engraftment
16. Smith SL. Ten years of Orthoclone OKT3 (muromonab-CD3): a review. J and immune attack in solid tumors. Cancer Cell (2019) 35:885–900.e10.
Transpl Coord (1996) 6:109–19. doi: 10.7182/prtr.1.6.3.8145l3u185493182 doi: 10.1016/[Link].2019.05.004

17. Jen EY, Xu Q, Schetter A, Przepiorka D, Shen YL, Roscoe D, et al. FDA approval: 40. Spranger S, Bao R, Gajewski TF. Melanoma-intrinsic b-catenin signalling
blinatumomab for patients with B-cell precursor acute lymphoblastic leukemia in prevents anti-tumour immunity. Nature (2015) 523:231–5. doi: 10.1038/nature14404
morphologic remission with minimal residual disease. Clin Cancer Res (2019) 25:473– 41. Li X, Xiang Y, Li F, Yin C, Li B, Ke X. WNT/b-catenin signaling pathway
7. doi: 10.1158/[Link]-18-2337 regulating T cell-inflammation in the tumor microenvironment. Front Immunol (2019)
18. Jen EY, Ko CW, Lee JE, Del Valle PL, Aydanian A, Jewell C, et al. FDA approval: 10:2293. doi: 10.3389/fimmu.2019.02293
gemtuzumab ozogamicin for the treatment of adults with newly diagnosed CD33- 42. Safarzadeh Kozani P, Safarzadeh Kozani P, Ahmadi Najafabadi M, Yousefi F,
positive acute myeloid leukemia. Clin Cancer Res (2018) 24:3242–6. doi: 10.1158/1078- Mirarefin SMJ, Rahbarizadeh F. Recent advances in solid tumor CAR-T cell therapy:
[Link]-17-3179 driving tumor cells from hero to zero? Front Immunol (2022) 13:795164.
19. O'Leary MC, Lu X, Huang Y, Lin X, Mahmood I, Przepiorka D, et al. FDA doi: 10.3389/fimmu.2022.795164
approval summary: tisagenlecleucel for treatment of patients with relapsed or 43. Nasiri F, Kazemi M, Mirarefin SMJ, Mahboubi Kancha M, Ahmadi Najafabadi
refractory B-cell precursor acute lymphoblastic leukemia. Clin Cancer Res (2019) M, Salem F, et al. CAR-T cell therapy in triple-negative breast cancer: Hunting the
25:1142–6. doi: 10.1158/[Link]-18-2035 invisible devil. Front Immunol (2022) 13:1018786. doi: 10.3389/fimmu.2022.1018786
20. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. CAR T cells 44. Brudno JN, Kochenderfer JN. Recent advances in CAR T-cell toxicity:
redirected against tumor-specific antigen glycoforms: can low-sugar antigens Mechanisms, manifestations and management. Blood Rev (2019) 34:45–55.
guarantee a sweet success? Front Med (2022) 16:322–38. doi: 10.1007/s11684-021- doi: 10.1016/[Link].2018.11.002
0901-2 45. Rodriguez-Garcia A, Sharma P, Poussin M, Boesteanu AC, Minutolo NG, Gitto SB,
21. Wang SS, Yan YS, Ho K. US FDA-approved therapeutic antibodies with high- et al. CAR T cells targeting MISIIR for the treatment of ovarian cancer and other
concentration formulation: summaries and perspectives. Antibody Ther (2021) 4:262– gynecologic Malignancies. Mol Ther (2020) 28:548–60. doi: 10.1016/[Link].2019.11.028
72. doi: 10.1093/abt/tbab027 46. Martin A, Galindo CMA, Biswas S, Mine J, Mandal G, Innamarato P, et al.
22. Porcelli S, Yockey CE, Brenner MB, Balk SP. Analysis of T cell antigen receptor Olfactory receptor OR5V1 is an effective target for CAR T cells in ovarian cancer (207).
(TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates Gynecol Oncol (2022) 166:S116. doi: 10.1016/S0090-8258(22)01433-0
preferential use of several V beta genes and an invariant TCR alpha chain. J Exp Med 47. Cua S, Tan HL, Fong WJ, Chin A, Lau A, Ding V, et al. Targeting of embryonic
(1993) 178:1–16. doi: 10.1084/jem.178.1.1 annexin A2 expressed on ovarian and breast cancer by the novel monoclonal antibody
23. Huang H, Nie CP, Liu XF, Song B, Yue JH, Xu JX, et al. Phase I study of adjuvant 2448. Oncotarget (2018) 9:13206–21. doi: 10.18632/oncotarget.24152
immunotherapy with autologous tumor-infiltrating lymphocytes in locally advanced 48. Wang CY, Lin CF. Annexin A2: its molecular regulation and cellular expression
cervical cancer. J Clin Invest (2022) 132. doi: 10.1172/jci157726 in cancer development. Dis Markers (2014) 2014:308976. doi: 10.1155/2014/308976
24. Safarzadeh Kozani P, Shokrgozar MA, Evazalipour M, Roudkenar MH. CRISPR/ 49. Leong L, Tan HL, Cua S, Yong KSM, Chen Q, Choo A. Preclinical activity of
Cas9-medaited knockout of endogenous T-cell receptor in Jurkat cells and generation embryonic annexin A2-specific chimeric antigen receptor T cells against ovarian
of NY-ESO-1-specific T cells: An in vitro study. Int Immunopharmacol (2022) cancer. Int J Mol Sci (2020) 21:381. doi: 10.3390/ijms21020381
110:109055. doi: 10.1016/[Link].2022.109055
50. Harrer DC, Dörrie J, Schaft N. CSPG4 as target for CAR-T-cell therapy of
25. Chrisann K, Ekaterina D, Qin Z, Sara K, Alexia I, Carol A, et al. Phase I dose various tumor entities-merits and challenges. Int J Mol Sci (2019) 20:5942.
escalation safety and feasibility study of autologous WT1-sensitized T cells for the doi: 10.3390/ijms20235942
treatment of patients with recurrent ovarian cancer. J Immunother Cancer (2021) 9:
e002752. doi: 10.1136/jitc-2021-002752 51. CheKenya M, Enger P, Thorsen F, Tysnes BB, Al-Sarraj S, Read TA, et al. The
glial precursor proteoglycan, NG2, is expressed on tumour neovasculature by vascular
26. Dabiri H, Safarzadeh Kozani P, Habibi Anbouhi M, Mirzaee Godarzee M, Haddadi pericytes in human Malignant brain tumours. Neuropathol Appl Neurobiol (2002)
MH, Basiri M, et al. Site-specific transgene integration in chimeric antigen receptor (CAR) 28:367–80. doi: 10.1046/j.1365-2990.2002.00412.x
T cell therapies. Biomark Res (2023) 11:67. doi: 10.1186/s40364-023-00509-1
52. Ilieva KM, Cheung A, Mele S, Chiaruttini G, Crescioli S, Griffin M, et al.
27. Jin L, Tao H, Karachi A, Long Y, Hou AY, Na M, et al. CXCR1- or CXCR2- Chondroitin sulfate proteoglycan 4 and its potential as an antibody immunotherapy
modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors. Nat tar g e t a c r o s s di ffe r e n t tu mo r t yp es . Front I mm unol ( 2017 ) 8 :191 1.
Commun (2019) 10:4016. doi: 10.1038/s41467-019-11869-4 doi: 10.3389/fimmu.2017.01911
28. Di Stasi A, De Angelis B, Rooney CM, Zhang L, Mahendravada A, Foster AE, 53. Price MA, Colvin Wanshura LE, Yang J, Carlson J, Xiang B, Li G, et al. CSPG4, a
et al. T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting potential therapeutic target, facilitates Malignant progression of melanoma. Pigment
CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood Cell Melanoma Res (2011) 24:1148–57. doi: 10.1111/j.1755-148X.2011.00929.x
(2009) 113:6392–402. doi: 10.1182/blood-2009-03-209650
54. Harrer DC, Schenkel C, Berking C, Herr W, Abken H, Dörrie J, et al. Decitabine-
29. Nasiri F, Safarzadeh Kozani P, Rahbarizadeh F. T-cells engineered with a novel mediated upregulation of CSPG4 in ovarian carcinoma cells enables targeting by
VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal CSPG4-specific CAR-T cells. Cancers (2022) 14:5033. doi: 10.3390/cancers14205033
efficacy to their FMC63-based counterparts. Front Immunol (2023) 14:1063838.
doi: 10.3389/fimmu.2023.1063838 55. Luo W, Wang X, Kageshita T, Wakasugi S, Karpf AR, Ferrone S. Regulation of
high molecular weight-melanoma associated antigen (HMW-MAA) gene expression
30. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. Addressing the by promoter DNA methylation in human melanoma cells. Oncogene (2006) 25:2873–
obstacles of CAR T cell migration in solid tumors: wishing a heavy traffic. Crit Rev 84. doi: 10.1038/[Link].1209319
Biotechnol (2022) 42:1079–98. doi: 10.1080/07388551.2021.1988509
56. Leick MB, Silva H, Scarfò I, Larson R, Choi BD, Bouffard AA, et al. Non-
31. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. Optimizing the clinical cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid
impact of CAR-T cell therapy in B-cell acute lymphoblastic leukemia: looking back while leukemia. Cancer Cell (2022) 40:494–508.e5. doi: 10.1016/[Link].2022.04.001
moving forward. Front Immunol (2021) 12:765097. doi: 10.3389/fimmu.2021.765097
57. Yang J, Liao Q, Price M, Moriarity B, Wolf N, Felices M, et al. Chondroitin
32. Piali L, Fichtel A, Terpe HJ, Imhof BA, Gisler RH. Endothelial vascular cell sulfate proteoglycan 4, a targetable oncoantigen that promotes ovarian cancer growth,
adhesion molecule 1 expression is suppressed by melanoma and carcinoma. J Exp Med invasion, cisplatin resistance and spheroid formation. Transl Oncol (2022) 16:101318.
(1995) 181:811–6. doi: 10.1084/jem.181.2.811 doi: 10.1016/[Link].2021.101318

Frontiers in Immunology 25 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

58. Harrer DC, Schuler G, Dörrie J, Schaft N. CSPG4-specific CAR T cells for high- 82. Trzpis M, McLaughlin PM, de Leij LM, Harmsen MC. Epithelial cell adhesion
risk childhood B cell precursor leukemia. Int J Mol Sci (2019) 20:2764. molecule: more than a carcinoma marker and adhesion molecule. Am J Pathol (2007)
doi: 10.3390/ijms20112764 171:386–95. doi: 10.2353/ajpath.2007.070152
59. CSPG4 shows promise for glioblastoma CAR T therapy. Cancer Discovery (2018) 83. Fu J, Shang Y, Qian Z, Hou J, Yan F, Liu G, et al. Chimeric Antigen receptor-T
8:524–5. doi: 10.1158/[Link]-nb2018-032 (CAR-T) cells targeting Epithelial cell adhesion molecule (EpCAM) can inhibit tumor
60. Safarzadeh Kozani P, Safarzadeh Kozani P, O'Connor RS. In like a lamb; out like growth in ovarian cancer mouse model. J Vet Med Sci (2021) 83:241–7. doi: 10.1292/
a lion: marching CAR T cells toward enhanced efficacy in B-ALL. Mol Cancer Ther jvms.20-0455
(2021) 20:1223–33. doi: 10.1158/[Link]-20-1089 84. Herbel C, Dittmer V, Martinez-Osuna M, Reiß S, Mallmann P, Ratiu D, et al.
61. Wang W, Bandara V, Lokman N, Napoli S, Gundsambuu B, Oehler M, et al. Abstract 2813: Identification of a novel tumor marker combination THY1-EPCAM for
Abstract 5183: LGR5 CAR-T cells: A novel potential treatment against high grade adaptor CAR T cell therapy in ovarian cancer. Cancer Res (2022) 82:2813–.
serous ovarian cancer. Cancer Res (2022) 82:5183–. doi: 10.1158/1538-7445.Am2022- doi: 10.1158/1538-7445.Am2022-2813
5183 85. Qin D, Li D, Zhang B, Chen Y, Liao X, Li X, et al. Potential lung attack and
62. Thompson EJ, Bandara V, Sadlon T, Gundsambuu B, Tan LY, Ricciardelli C, lethality generated by EpCAM-specific CAR-T cells in immunocompetent mouse
et al. Abstract 5184: Real-time cytotoxicity assays as a pre-clinical screening tool for models. OncoImmunology (2020) 9:1806009. doi: 10.1080/2162402X.2020.1806009
LGR5-targeting CAR-T cells for treatment of solid tumors. Cancer Res (2022) 82:5184. 86. Ang WX, Li Z, Chi Z, Du SH, Chen C, Tay JC, et al. Intraperitoneal
doi: 10.1158/1538-7445.Am2022-5184 immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in
63. McPeake DJ, Tyllis TS, Foeng J, Bandara V, Abbott CA, Gundsambuu B, et al. xenograft models of peritoneal carcinomatosis. Oncotarget (2017) 8:13545–59.
Abstract 5574: In vivo characterization of a novel CAR-T cell therapy directed towards doi: 10.18632/oncotarget.14592
LGR5 for the treatment of colorectal cancer. Cancer Res (2022) 82:5574–. doi: 10.1158/ 87. Deng Z, Wu Y, Ma W, Zhang S, Zhang YQ. Adoptive T-cell therapy of prostate
1538-7445.Am2022-5574 cancer targeting the cancer stem cell antigen EpCAM. BMC Immunol (2015) 16:1.
64. Bandara V, Mills S, Thompson E, Tan L, Gundsambuu B, Napoli S, et al. doi: 10.1186/s12865-014-0064-x
Abstract 5575: Pre-clinical validation of a LGR5-targeting CAR-T against colorectal 88. Zhang BL, Li D, Gong YL, Huang Y, Qin DY, Jiang L, et al. Preclinical evaluation
cancer. Cancer Res (2022) 82:5575–. doi: 10.1158/1538-7445.Am2022-5575 of chimeric antigen receptor-modified T cells specific to epithelial cell adhesion
65. Porcellini S, Asperti C, Corna S, Cicoria E, Valtolina V, Stornaiuolo A, et al. CAR molecule for treating colorectal cancer. Hum Gene Ther (2019) 30:402–12.
T cells redirected to CD44v6 control tumor growth in lung and ovary adenocarcinoma doi: 10.1089/hum.2018.229
bearing mice. Front Immunol (2020) 11:99. doi: 10.3389/fimmu.2020.00099 89. Lynn RC, Matsuyama T, Powell DJ. Targeting FRb+ tumor associated
66. Ma L, Dong L, Chang P. CD44v6 engages in colorectal cancer progression. Cell macrophages with car T cells in ovarian cancer. J Immunother Cancer (2015) 3:P32.
Death Dis (2019) 10:30. doi: 10.1038/s41419-018-1265-7 doi: 10.1186/2051-1426-3-S2-P32

67. Afify A, Purnell P, Nguyen L. Role of CD44s and CD44v6 on human breast 90. Lynn RC, Poussin M, Kalota A, Feng Y, Low PS, Dimitrov DS, et al. Targeting of
cancer cell adhesion, migration, and invasion. Exp Mol Pathol (2009) 86:95–100. folate receptor b on acute myeloid leukemia blasts with chimeric antigen receptor–
doi: 10.1016/[Link].2008.12.003 expressing T cells. Blood (2015) 125:3466–76. doi: 10.1182/blood-2014-11-612721

68. Mack B, Gires O. CD44s and CD44v6 expression in head and neck epithelia. PloS 91. Lynn RC, Feng Y, Schutsky K, Poussin M, Kalota A, Dimitrov DS, et al. High-
One (2008) 3:e3360. doi: 10.1371/[Link].0003360 affinity FRb-specific CAR T cells eradicate AML and normal myeloid lineage without
HSC toxicity. Leukemia (2016) 30:1355–64. doi: 10.1038/leu.2016.35
69. Haist C, Schulte E, Bartels N, Bister A, Poschinski Z, Ibach TC, et al. CD44v6-
targeted CAR T-cells specifically eliminate CD44 isoform 6 expressing head/neck 92. Knutson KL, Krco CJ, Erskine CL, Goodman K, Kelemen LE, Wettstein PJ, et al.
squamous cell carcinoma cells. Oral Oncol (2021) 116:105259. doi: 10.1016/ T-cell immunity to the folate receptor alpha is prevalent in women with breast or
[Link].2021.105259 ovarian cancer. J Clin Oncol (2006) 24:4254–61. doi: 10.1200/jco.2006.05.9311

70. Tang L, Huang H, Tang Y, Li Q, Wang J, Li D, et al. CD44v6 chimeric antigen 93. Kalli KR, Oberg AL, Keeney GL, Christianson TJ, Low PS, Knutson KL, et al.
receptor T cell specificity towards AML with FLT3 or DNMT3A mutations. Clin Transl Folate receptor alpha as a tumor target in epithelial ovarian cancer. Gynecol Oncol
Med (2022) 12:e1043. doi: 10.1002/ctm2.1043 (2008) 108:619–26. doi: 10.1016/[Link].2007.11.020

71. Casucci M, Nicolis di Robilant B, Falcone L, Camisa B, Norelli M, Genovese P, et al. 94. O'Shannessy DJ, Yu G, Smale R, Fu YS, Singhal S, Thiel RP, et al. Folate receptor
CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia alpha expression in lung cancer: diagnostic and prognostic significance. Oncotarget
and multiple myeloma. Blood (2013) 122:3461–72. doi: 10.1182/blood-2013-04-493361 (2012) 3:414–25. doi: 10.18632/oncotarget.489

72. Stornaiuolo A, Valentinis B, Sirini C, Scavullo C, Asperti C, Zhou D, et al. 95. Bueno R, Appasani K, Mercer H, Lester S, Sugarbaker D. The alpha folate
Characterization and functional analysis of [Link] T cells endowed with a new receptor is highly activated in Malignant pleural mesothelioma. J Thorac Cardiovasc
low-affinity nerve growth factor receptor-based spacer. Hum Gene Ther (2021) 32:744– Surg (2001) 121:225–33. doi: 10.1067/mtc.2001.111176
60. doi: 10.1089/hum.2020.216 96. Kelemen LE. The role of folate receptor alpha in cancer development,
73. Abiko K, Mandai M, Hamanishi J, Yoshioka Y, Matsumura N, Baba T, et al. PD- progression and treatment: cause, consequence or innocent bystander? Int J Cancer
L1 on tumor cells is induced in ascites and promotes peritoneal dissemination of (2006) 119:243–50. doi: 10.1002/ijc.21712
ovarian cancer through CTL dysfunction. Clin Cancer Res (2013) 19:1363–74. 97. Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z, Mavroukakis SA,
doi: 10.1158/[Link]-12-2199 et al. A phase I study on adoptive immunotherapy using gene-modified T cells for
74. Ma Q, He X, Zhang B, Guo F, Ou X, Yang Q, et al. A PD-L1-targeting chimeric ovarian cancer. Clin Cancer Res (2006) 12:6106–15. doi: 10.1158/[Link]-
switch receptor enhances efficacy of CAR-T cell for pleural and peritoneal metastasis. 06-1183
Signal Transduct Target Ther (2022) 7:380. doi: 10.1038/s41392-022-01198-2 98. Parker LL, Do MT, Westwood JA, Wunderlich JR, Dudley ME, Rosenberg SA, et al.
75. Guo C, Dong E, Lai Q, Zhou S, Zhang G, Wu M, et al. Effective antitumor activity Expansion and characterization of T cells transduced with a chimeric receptor against
of 5T4-specific CAR-T cells against ovarian cancer cells in vitro and xenotransplanted ovarian cancer. Hum Gene Ther (2000) 11:2377–87. doi: 10.1089/104303400750038480
tumors in vivo. MedComm (2020) 1:338–50. doi: 10.1002/mco2.34 99. Lin J, Spidel JL, Maddage CJ, Rybinski KA, Kennedy RP, Krauthauser CL, et al.
The antitumor activity of the human FOLR1-specific monoclonal antibody,
76. Owens GL, Sheard VE, Kalaitsidou M, Blount D, Lad Y, Cheadle EJ, et al.
farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent
Preclinical assessment of CAR T-cell therapy targeting the tumor antigen 5T4 in
cellular cytotoxicity. Cancer Biol Ther (2013) 14:1032–8. doi: 10.4161/cbt.26106
ovarian cancer. J Immunother (2018) 41:130–40. doi: 10.1097/cji.0000000000000203
100. Song DG, Ye Q, Poussin M, Liu L, Figini M, Powell DJ Jr. A fully human
77. Xu Y, Morales AJ, Cargill MJ, Towlerton AMH, Coffey DG, Warren EH, et al. chimeric antigen receptor with potent activity against cancer cells but reduced risk for
Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 off-tumor toxicity. Oncotarget (2015) 6:21533–46. doi: 10.18632/oncotarget.4071
tumor antigen on renal cell carcinoma. Cancer Immunol Immunother (2019) 68:1979–
93. doi: 10.1007/s00262-019-02419-4 101. Xu X-J, Song D-G, Poussin M, Ye Q, Sharma P, Rodrı́guez-Garcı́a A, et al.
Multiparameter comparative analysis reveals differential impacts of various cytokines
78. Murad JP, Kozlowska AK, Lee HJ, Ramamurthy M, Chang W-C, Yazaki P, et al. on CART cell phenotype and function ex vivo and in vivo. Oncotarget (2016) 7:82354.
Effective targeting of TAG72+ Peritoneal ovarian tumors via regional delivery of CAR- doi: 10.18632/oncotarget.10510
engineered T cells. Front Immunol (2018) 9:2268. doi: 10.3389/fimmu.2018.02268
102. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. Novel antigens of
79. Hege KM, Bergsland EK, Fisher GA, Nemunaitis JJ, Warren RS, McArthur JG, CAR T cell therapy: New roads; old destination. Transl Oncol (2021) 14:101079.
et al. Safety, tumor trafficking and immunogenicity of chimeric antigen receptor doi: 10.1016/[Link].2021.101079
(CAR)-T cells specific for TAG-72 in colorectal cancer. J Immunother Cancer (2017)
103. Banville AC, Wouters MCA, Oberg AL, Goergen KM, Maurer MJ, Milne K,
5:22. doi: 10.1186/s40425-017-0222-9
et al. Co-expression patterns of chimeric antigen receptor (CAR)-T cell target antigens
80. Safarzadeh Kozani P, Safarzadeh Kozani P, O'Connor RS. Humanized chimeric in primary and recurrent ovarian cancer. Gynecol Oncol (2021) 160:520–9.
antigen receptor (CAR) T cells. J Cancer Immunol (Wilmington) (2021) 3:183–7. doi: 10.1016/[Link].2020.12.005
81. Shu R, Evtimov VJ, Hammett MV, Nguyen NN, Zhuang J, Hudson PJ, et al. 104. Felder M, Kapur A, Gonzalez-Bosquet J, Horibata S, Heintz J, Albrecht R, et al.
Engineered CAR-T cells targeting TAG-72 and CD47 in ovarian cancer. Mol Ther MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress. Mol Cancer
Oncolytics (2021) 20:325–41. doi: 10.1016/[Link].2021.01.002 (2014) 13:129. doi: 10.1186/1476-4598-13-129

Frontiers in Immunology 26 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

105. Liu Q, Cheng Z, Luo L, Yang Y, Zhang Z, Ma H, et al. C-terminus of MUC16 128. Ng Y-Y, Tay JCK, Li Z, Wang J, Zhu J, Wang S. T cells expressing NKG2D CAR
activates Wnt signaling pathway through its interaction with b-catenin to promote with a DAP12 signaling domain stimulate lower cytokine production while effective in
tumorigenesis and metastasis. Oncotarget (2016) 7:36800–13. doi: 10.18632/ tumor eradication. Mol Ther (2021) 29:75–85. doi: 10.1016/[Link].2020.08.016
oncotarget.9191 129. Jiang G, Ng YY, Tay JCK, Du Z, Xiao L, Wang S, et al. Dual CAR-T cells to treat
106. Chekmasova AA, Rao TD, Nikhamin Y, Park KJ, Levine DA, Spriggs DR, et al. cancers co-expressing NKG2D and PD1 ligands in xenograft models of peritoneal
Successful eradication of established peritoneal ovarian tumors in SCID-Beige mice metastasis. Cancer Immunol Immunother (2023) 72:223–34. doi: 10.1007/s00262-022-
following adoptive transfer of T cells genetically targeted to the MUC16 antigen. Clin 03247-9
Cancer Res (2010) 16:3594–606. doi: 10.1158/[Link]-10-0192 130. Wang L, Lin X, Yu L, Sun P. Romidepsin Enhances the Killing Ability of
107. Chekmasova AA, Sandadi S, Spriggs DR, Brentjens RJ. Effect of modulation of NKG2D-CAR-T Cells through Enhanced Expression of NKG2DL against Ovarian
the hostile tumor microenvironment through adoptive transfer of IL-12 expressing Cancer Cells. CEOG (2022) 49. doi: 10.31083/j.ceog4910227
MUC-16 targeted T cells on ovarian tumors in vivo. J Clin Oncol (2012) 30:2586–. 131. Chang K, Pastan I, Willingham MC. Isolation and characterization of a
doi: 10.1200/jco.2012.30.15_suppl.2586 monoclonal antibody, K1, reactive with ovarian cancers and normal mesothelium.
108. Koneru M, Purdon TJ, Spriggs D, Koneru S, Brentjens RJ. IL-12 secreting Int J Cancer (1992) 50:373–81. doi: 10.1002/ijc.2910500308
tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo. 132. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen
Oncoimmunology (2015) 4:e994446. doi: 10.4161/2162402x.2014.994446 present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci
109. Koneru M, O'Cearbhaill R, Pendharkar S, Spriggs DR, Brentjens RJ. A phase I U.S.A. (1996) 93:136–40. doi: 10.1073/pnas.93.1.136
clinical trial of adoptive T cell therapy using IL-12 secreting MUC-16(ecto) directed 133. Servais EL, Colovos C, Rodriguez L, Bograd AJ, Nitadori J, Sima C, et al.
chimeric antigen receptors for recurrent ovarian cancer. J Transl Med (2015) 13:102. Mesothelin overexpression promotes mesothelioma cell invasion and MMP-9 secretion
doi: 10.1186/s12967-015-0460-x in an orthotopic mouse model and in epithelioid pleural mesothelioma patients. Clin
110. Yeku OO, Purdon T, Spriggs DR, Brentjens RJ. Interleukin-12 armored Cancer Res (2012) 18:2478–89. doi: 10.1158/[Link]-11-2614
chimeric antigen receptor (CAR) T cells for heterogeneous antigen-expressing 134. Bera TK, Pastan I. Mesothelin is not required for normal mouse development
ovarian cancer. J Clin Oncol (2018) 36:12–. doi: 10.1200/JCO.2018.36.5_suppl.12 or reproduction. Mol Cell Biol (2000) 20:2902–6. doi: 10.1128/mcb.20.8.2902-
111. Yeku OO, Rafiq S, Koneru M, Purdon TJ, Song M, Wang P, et al. Abstract IA21: 2906.2000
MUC16-directed immunotherapy for ovarian cancer. Clin Cancer Res (2020) 26:IA21– 135. Pastan I, Hassan R. Discovery of mesothelin and exploiting it as a target for
IA. doi: 10.1158/1557-3265.Ovca19-ia21 immunotherapy. Cancer Res (2014) 74:2907–12. doi: 10.1158/[Link]-14-0337
112. Li T, Wang J. Therapeutic effect of dual CAR-T targeting PDL1 and MUC16 136. Lanitis E, Poussin M, Hagemann IS, Coukos G, Sandaltzopoulos R, Scholler N,
antigens on ovarian cancer cells in mice. BMC Cancer (2020) 20:678. doi: 10.1186/ et al. Redirected antitumor activity of primary human lymphocytes transduced with a
s12885-020-07180-x fully human anti-mesothelin chimeric receptor. Mol Ther (2012) 20:633–43.
113. O'Cearbhaill RE, Park JH, Halton EF, Diamonte CR, Mead E, Lakhman Y, et al. doi: 10.1038/mt.2011.256
A phase I clinical trial of autologous chimeric antigen receptor (CAR) T cells genetically 137. Tanyi JL, Haas AR, Beatty GL, Stashwick CJ, O'Hara MH, Morgan MA, et al.
engineered to secrete IL-12 and to target the MUC16ecto antigen in patients (pts) with Anti-mesothelin chimeric antigen receptor T cells in patients with epithelial ovarian
MUC16ecto+ recurrent high-grade serous ovarian cancer (HGSOC). Gynecol Oncol cancer. J Clin Oncol (2016) 34:5511–. doi: 10.1200/JCO.2016.34.15_suppl.5511
(2020) 159:42. doi: 10.1016/[Link].2020.06.089
138. Tanyi JL, Stashwick C, Plesa G, Morgan MA, Porter D, Maus MV, et al. Possible
114. Sentman CL, Barber MA, Barber A, Zhang T. NK cell receptors as tools in compartmental cytokine release syndrome in a patient with recurrent ovarian cancer
cancer immunotherapy. Adv Cancer Res (2006) 95:249–92. doi: 10.1016/s0065-230x after treatment with mesothelin-targeted CAR-T cells. J Immunother (2017) 40:104–7.
(06)95007-6 doi: 10.1097/cji.0000000000000160
115. Groh V, Rhinehart R, Randolph-Habecker J, Topp MS, Riddell SR, Spies T. 139. Gruzdyn OV, Gruber SA, Batchu RB, Mahmud EM, Chukr FK, Dachepalli R,
Costimulation of CD8alphabeta T cells by NKG2D via engagement by MIC induced on et al. Mesothelin chimeric antigen receptor (CAR)-mediated therapy for ovarian
virus-infected cells. Nat Immunol (2001) 2:255–60. doi: 10.1038/85321 cancer. J Am Coll Surgeons (2017) 225:e47. doi: 10.1016/[Link].2017.07.645
116. Maasho K, Opoku-Anane J, Marusina AI, Coligan JE, Borrego F. NKG2D is a 140. Batchu RB, Gruzdyn OV, Mahmud EM, Rohondia S, Manmari SK, Tavva SP,
costimulatory receptor for human naive CD8+ T cells. J Immunol (2005) 174:4480–4. et al. Inhibition of IL-10 augments mesothelin chimeric antigen receptor T cell activity
doi: 10.4049/jimmunol.174.8.4480 in epithelial ovarian cancer. J Am Coll Surgeons (2018) 227:e215–6. doi: 10.1016/
117. Markiewicz MA, Carayannopoulos LN, Naidenko OV, Matsui K, Burack WR, [Link].2018.08.583
Wise EL, et al. Costimulation through NKG2D enhances murine CD8+ CTL function: 141. Hung CF, Xu X, Li L, Ma Y, Jin Q, Viley A, et al. Development of anti-human
similarities and differences between NKG2D and CD28 costimulation. J Immunol mesothelin-targeted chimeric antigen receptor messenger RNA-transfected peripheral
(2005) 175:2825–33. doi: 10.4049/jimmunol.175.5.2825 blood lymphocytes for ovarian cancer therapy. Hum Gene Ther (2018) 29:614–25.
118. Groh V, Rhinehart R, Secrist H, Bauer S, Grabstein KH, Spies T. Broad tumor- doi: 10.1089/hum.2017.080
associated expression and recognition by tumor-derived gamma delta T cells of MICA 142. Haas AR, Tanyi JL, O'Hara MH, Gladney WL, Lacey SF, Torigian DA, et al.
and MICB. Proc Natl Acad Sci USA (1999) 96:6879–84. doi: 10.1073/pnas.96.12.6879 Phase I study of lentiviral-transduced chimeric antigen receptor-modified T cells
119. Pende D, Rivera P, Marcenaro S, Chang CC, Biassoni R, Conte R, et al. Major recognizing mesothelin in advanced solid cancers. Mol Ther (2019) 27:1919–29.
histocompatibility complex class I-related chain A and UL16-binding protein doi: 10.1016/[Link].2019.07.015
expression on tumor cell lines of different histotypes: analysis of tumor susceptibility 143. Safarzadeh Kozani P, Naseri A, Mirarefin SMJ, Salem F, Nikbakht M, Evazi
to NKG2D-dependent natural killer cell cytotoxicity. Cancer Res (2002) 62:6178–86. Bakhshi S, et al. Nanobody-based CAR-T cells for cancer immunotherapy. biomark Res
120. Cerwenka A, Lanier LL. NKG2D ligands: unconventional MHC class I-like (2022) 10:24. doi: 10.1186/s40364-022-00371-7
molecules exploited by viruses and cancer. Tissue Antigens (2003) 61:335–43. 144. Zhang Z, Jiang D, Yang H, He Z, Liu X, Qin W, et al. Modified CAR T cells
doi: 10.1034/j.1399-0039.2003.00070.x targeting membrane-proximal epitope of mesothelin enhances the antitumor function
121. Raulet DH. Roles of the NKG2D immunoreceptor and its ligands. Nat Rev against large solid tumor. Cell Death Dis (2019) 10:476. doi: 10.1038/s41419-019-
Immunol (2003) 3:781–90. doi: 10.1038/nri1199 1711-1
122. Barber A, Zhang T, DeMars LR, Conejo-Garcia J, Roby KF, Sentman CL. 145. Liu G, Zhang Q, Li D, Zhang L, Gu Z, Liu J, et al. PD-1 silencing improves anti-
Chimeric NKG2D receptor–bearing T cells as immunotherapy for ovarian cancer. tumor activities of human mesothelin-targeted CAR T cells. Hum Immunol (2021)
Cancer Res (2007) 67:5003–8. doi: 10.1158/[Link]-06-4047 82:130–8. doi: 10.1016/[Link].2020.12.002
123. Barber A, Zhang T, Sentman CL. Immunotherapy with chimeric NKG2D receptors 146. Schoutrop E, El-Serafi I, Poiret T, Zhao Y, Gultekin O, He R, et al. Mesothelin-
leads to long-term tumor-free survival and development of host antitumor immunity in specific CAR T cells target ovarian cancer. Cancer Res (2021) 81:3022–35. doi: 10.1158/
murine ovarian cancer. J Immunol (2008) 180:72–8. doi: 10.4049/jimmunol.180.1.72 [Link]-20-2701
124. Song DG, Ye Q, Santoro S, Fang C, Best A, Powell DJ Jr. Chimeric NKG2D 147. Liang Z, Dong J, Yang N, Li SD, Yang ZY, Huang R, et al. Tandem CAR-T cells
CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by targeting FOLR1 and MSLN enhance the antitumor effects in ovarian cancer. Int J Biol
histone deacetylase inhibition. Hum Gene Ther (2013) 24:295–305. doi: 10.1089/ Sci (2021) 17:4365–76. doi: 10.7150/ijbs.63181
hum.2012.143 148. Liu G, Zhang Q, Liu G, Li D, Zhang L, Gu Z, et al. Disruption of adenosine 2A
125. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. CAR-T cell therapy receptor improves the anti-tumor function of anti-mesothelin CAR T cells both in vitro
in T-cell Malignancies: Is success a low-hanging fruit? Stem Cell Res Ther (2021) 12:527. and in vivo. Exp Cell Res (2021) 409:112886. doi: 10.1016/[Link].2021.112886
doi: 10.1186/s13287-021-02595-0 149. Zhang Q, Liu G, Liu J, Yang M, Fu J, Liu G, et al. The antitumor capacity of
126. Spear P, Barber A, Rynda-Apple A, Sentman CL. NKG2D CAR T-cell therapy mesothelin-CAR-T cells in targeting solid tumors in mice. Mol Ther Oncolytics (2021)
inhibits the growth of NKG2D ligand heterogeneous tumors. Immunol Cell Biol (2013) 20:556–68. doi: 10.1016/[Link].2021.02.013
91:435–40. doi: 10.1038/icb.2013.17 150. Li K, Xu J, Wang J, Lu C, Dai Y, Dai Q, et al. Dominant-negative transforming
127. Spear P, Barber A, Sentman CL. Collaboration of chimeric antigen receptor growth factor-b receptor-armoured mesothelin-targeted chimeric antigen receptor T
(CAR)-expressing T cells and host T cells for optimal elimination of established ovarian cells slow tumour growth in a mouse model of ovarian cancer. Cancer Immunol
tumors. OncoImmunology (2013) 2:e23564. doi: 10.4161/onci.23564 Immunother (2023) 72:917–28. doi: 10.1007/s00262-022-03290-6

Frontiers in Immunology 27 [Link]

Nasiri et al. 10.3389/fimmu.2023.1302307

151. Pang N, Shi J, Qin L, Chen A, Tang Y, Yang H, et al. IL-7 and CCL19-secreting 163. Choi BD, Yu X, Castano AP, Bouffard AA, Schmidts A, Larson RC, et al. CAR-
CAR-T cell therapy for tumors with positive glypican-3 or mesothelin. J Hematol Oncol T cells secreting BiTEs circumvent antigen escape without detectable toxicity. Nat
(2021) 14:118. doi: 10.1186/s13045-021-01128-9 Biotechnol (2019) 37:1049–58. doi: 10.1038/s41587-019-0192-1
152. Adachi K, Kano Y, Nagai T, Okuyama N, Sakoda Y, Tamada K. IL-7 and 164. Curran KJ, Seinstra BA, Nikhamin Y, Yeh R, Usachenko Y, van Leeuwen DG,
CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell et al. Enhancing antitumor efficacy of chimeric antigen receptor T cells through
survival in the tumor. Nat Biotechnol (2018) 36:346–51. doi: 10.1038/nbt.4086 constitutive CD40L expression. Mol Ther (2015) 23:769–78. doi: 10.1038/mt.2015.4
153. Chen J, Hu J, Gu L, Ji F, Zhang F, Zhang M, et al. Anti-mesothelin CAR-T 165. Kuhn NF, Purdon TJ, van Leeuwen DG, Lopez AV, Curran KJ, Daniyan AF,
immunotherapy in patients with ovarian cancer. Cancer Immunol Immunother (2023) et al. CD40 ligand-modified chimeric antigen receptor T cells enhance antitumor
72:409–25. doi: 10.1007/s00262-022-03238-w> function by eliciting an endogenous antitumor response. Cancer Cell (2019) 35:473–
88.e6. doi: 10.1016/[Link].2019.02.006
154. Tanyi J, Haas A, Aggarwal C, O’Hara M, Lacey S, Golden R, et al. Phase I study
of autologous T cells bearing fully-humanized chimeric antigen receptors targeting 166. Mailankody S, Matous JV, Chhabra S, Liedtke M, Sidana S, Oluwole OO, et al.
mesothelin in mesothelin- expressing cancers (314). Gynecol Oncol (2022) 166:S164–5. Allogeneic BCMA-targeting CAR T cells in relapsed/refractory multiple myeloma:
doi: 10.1016/S0090-8258(22)01537-2 phase 1 UNIVERSAL trial interim results. Nat Med (2023) 29:422–9. doi: 10.1038/
s41591-022-02182-7
155. Heo YA. Mirvetuximab soravtansine: first approval. Drugs (2023) 83:265–73.
doi: 10.1007/s40265-023-01834-3 167. Eyquem J, Mansilla-Soto J, Giavridis T, van der Stegen SJC, Hamieh M,
Cunanan KM, et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9
156. Syed YY. Durvalumab: first global approval. Drugs (2017) 77:1369–76.
enhances tumour rejection. Nature (2017) 543:113–7. doi: 10.1038/nature21405
doi: 10.1007/s40265-017-0782-5
168. Georgiadis C, Preece R, Nickolay L, Etuk A, Petrova A, Ladon D, et al. Long
157. Kim ES. Avelumab: first global approval. Drugs (2017) 77:929–37. doi: 10.1007/
terminal repeat CRISPR-CAR-coupled “Universal” T cells mediate potent anti-
s40265-017-0749-6>
leukemic effects. Mol Ther (2018) 26:1215–27. doi: 10.1016/[Link].2018.02.025
158. Markham A. Atezolizumab: first global approval. Drugs (2016) 76:1227–32. 169. Qasim W, Zhan H, Samarasinghe S, Adams S, Amrolia P, Stafford S, et al.
doi: 10.1007/s40265-016-0618-8 Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited
159. Liu X, Jiang S, Fang C, Yang S, Olalere D, Pequignot EC, et al. Affinity-tuned CAR T cells. Sci Trans Med (2017) 9:eaaj2013. doi: 10.1126/scitranslmed.aaj2013
ErbB2 or EGFR chimeric antigen receptor T cells exhibit an increased therapeutic index 170. Torikai H, Reik A, Liu P-Q, Zhou Y, Zhang L, Maiti S, et al. A foundation for
against tumors in mice. Cancer Res (2015) 75:3596–607. doi: 10.1158/[Link]- universal T-cell based immunotherapy: T cells engineered to express a CD19-specific
15-0159> chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood (2012)
160. Lin L, Cho SF, Xing L, Wen K, Li Y, Yu T, et al. Preclinical evaluation of CD8+ 119:5697–705. doi: 10.1182/blood-2012-01-405365
anti-BCMA mRNA CAR T cells for treatment of multiple myeloma. Leukemia (2021) 171. Valton J, Guyot V, Marechal A, Filhol JM, Juillerat A, Duclert A, et al. A
35:752–63. doi: 10.1038/s41375-020-0951-5> multidrug-resistant engineered CAR T cell for allogeneic combination
161. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F, Khoshtinat immunotherapy. Mol Ther (2015) 23:1507–18. doi: 10.1038/mt.2015.104
Nikkhoi S. Strategies for dodging the obstacles in CAR T cell therapy. Front Oncol 172. Torikai H, Reik A, Soldner F, Warren EH, Yuen C, Zhou Y, et al. Toward
(2021) 11:627549. doi: 10.3389/fonc.2021.627549 eliminating HLA class I expression to generate universal cells from allogeneic donors.
162. Whilding LM, Halim L, Draper B, Parente-Pereira AC, Zabinski T, Davies DM, Blood (2013) 122:1341–9. doi: 10.1182/blood-2013-03-478255
et al. CAR T-cells targeting the integrin avb6 and co-expressing the chemokine 173. Ren J, Zhang X, Liu X, Fang C, Jiang S, June CH, et al. A versatile system for
receptor CXCR2 demonstrate enhanced homing and efficacy against several solid rapid multiplex genome-edited CAR T cell generation. Oncotarget (2017) 8:17002–11.
Malignancies. Cancers (2019) 11:674. doi: 10.3390/cancers11050674 doi: 10.18632/oncotarget.15218

Frontiers in Immunology 28 [Link]