TYPE Systematic Review PUBLISHED 12 September 2022 DOI 10.3389/fimmu.2022.932559 Ligand-based CAR-T cell: OPEN ACCESS Different strategies to drive T EDITED BY Catherine Sautes-Fridman, U1138 Centre de Recherche des cells in future new treatments Cordeliers (CRC) (INSERM), France REVIEWED BY Alejandro Ramı́rez-Chacón 1,2, Sergi Betriu-Méndez 3,4, Masakazu Kamata, University of Alabama at Birmingham, Ariadna Bartoló-Ibars 3,4, Azucena González 3,4,5, United States Kailin Xu, Mercè Martı́ 1,2 and Manel Juan 3,4,5* Xuzhou Medical University, China 1 Immunology Unit, Department of Cellular Biology, Physiology and Immunology, Universitat *CORRESPONDENCE Autònoma de Barcelona (UAB), Cerdanyola del Vallès, Spain, 2 Laboratory of Cellular Immunology, Manel Juan Institute of Biotechnology and Biomedicine (IBB), Cerdanyola del Vallès, Spain, 3 Immunology mjuan@clinic.cat Department, Hospital Clı´nic de Barcelona, Centre de Diagnòstic Biomèdic (CDB), Barcelona, Spain, 4 Immunology Department, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) – SPECIALTY SECTION Fundació Clínic per a la Recerca Biomèdica (FCRB) Universitat de Barcelona (UB), Barcelona, Spain, This article was submitted to 5 Immunology Department, Hospital Sant Joan de Déu, Barcelona, Spain Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology RECEIVED 29 April 2022 Chimeric antigen receptor (CAR)-based therapies are presented as innovative ACCEPTED 22 August 2022 treatments for multiple malignancies. Despite their clinical success, there is PUBLISHED 12 September 2022 scientific evidence of the limitations of these therapies mainly due to CITATION immunogenicity issues, toxicities associated with the infusion of the product, Ramı´rez-Chacón A, Betriu-Méndez S, and relapses of the tumor. As a result, novel approaches are appearing aiming to Bartoló-Ibars A, González A, Martı´ M and Juan M (2022) Ligand-based CAR- solve and/or mitigate the harmful effects of CAR-T therapies. These include T cell: Different strategies to drive T strategies based on the use of ligands as binding moieties or ligand-based CAR- cells in future new treatments. Front. Immunol. 13:932559. T cells. Several proposals are currently under development, with some undergoing doi: 10.3389/fimmu.2022.932559 clinical trials to assess their potential benefits. In addition to these, therapies such as COPYRIGHT chimeric autoantibody receptor (CAAR), B-cell receptor antigen for reverse © 2022 Ramı´rez-Chacón, Betriu- targeting (BAR), and even chimeric human leukocyte antigen (HLA) antibody Méndez, Bartoló-Ibars, González, Martı´ and Juan. This is an open-access article receptor (CHAR) have emerged, benefiting from the advantages of antigenic distributed under the terms of the ligands as antibody-binding motifs. This review focuses on the potential role that Creative Commons Attribution License ligands can play in current and future antitumor treatments and in other types of (CC BY). The use, distribution or reproduction in other forums is diseases, such as autoimmune diseases or problems associated permitted, provided the original with transplantation. author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in KEYWORDS accordance with accepted academic T cells, chimeric antigen receptor (CAR), ligands, receptor, antigen, CAAR, BAR practice. No use, distribution or reproduction is permitted which does not comply with these terms. Introduction Immunotherapy using autologous genetically engineered chimeric antigen receptor (CAR) T (CAR-T) cells is widely emerging as one of the major breakthroughs for treating cancer. The aim of these therapies is focused on driving T-cell cytotoxicity specifically Frontiers in Immunology 01 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 against tumor antigens in cancer cells (1, 2). However, the Autologous T cells expressing CAR molecules are activated identification of suitable targeted tumor-associated antigens upon non-HLA-restricted ligand recognition, subsequent (TAAs) remains a challenge nowadays due to life-threatening posttranslational phosphorylation of CD3z is produced, and toxicity derived from off-tumor on-target antigen recognition the zeta-chain-associated protein kinase 70 (Zap-70) is (3–5). recruited, inducing the assembly of downstream proteins. Various approaches are being proposed for both Meanwhile, CAR-costimulatory regions can activate hematological and non-hematological malignancies (6). Phosphoinositide 3-kinase (PI3K)/Protein kinase B (AKT), tumor Remarkable antitumor responses have been achieved from necrosis factor (TNF) receptor-associated factor 2 (TRAF2)/ anti-CD19 CAR-T therapies against B-cell acute lymphoblastic Mitogen-activated protein kinase (p38MAPK), and c-Jun N- leukemia (B-ALL) and other refractory B-cell malignancies, terminal kinase (JNK) pathways. Finally, CAR functional demonstrated in pivotal clinical trials (7–11). This has led to domains enable the CAR to integrate all downstream signaling the approval by Food and Drug Administration (FDA) and/or pathways that end up with the expression and activation of European Medicines Agency (EMA) of several CD19-directed transcriptional modulators—Nuclear factor kappa B (NF-kB), products, such as tisagenlecleucel (KYMRIAH®, Novartis) (12), Nuclear factor of activated T-cells (NFAT), Signal transducer and axicabtagene ciloleucel (YESCARTA®, Kite Pharma-Gilead) activator of transcription 3 (STAT3), Activator protein 1 (AP-1)— (13), and lisocabtagene maraleucel (BREYANZI ® , Juno to drive the effector function of CAR-T cells (22, 23). Therapeutics-Celgene-BMS) (14) for treating large B-cell Largely, the extracellular antigen recognition domain is a lymphoma and brexucabtagene autoleucel (TECARTUS®, Kite single-chain variable fragment (scFv) composed of a variable Pharma-Gilead) (15) for treating relapsed/refractory mantle cell heavy chain (VH) and a variable light chain (VL), which are lymphoma. In this context, our group obtained the first usually derived from a murine monoclonal antibody (moAb), European-developed CAR-T approved by the Spanish Agency joined by a linker region (1) (Figure 2). Due to the scFv murine of Medicine [Agencia Española del Medicamento y Productos nature, immunogenicity is one of the major issues regarding in Sanitarios (AEMPS)] with the authorization of ARI-0001 (at vivo long-term expression of CAR-T cells (24, 25). Moreover, Hospital Clı ́nic de Barcelona) administration for relapsed or aggregation and instability can lead to poor persistence and refractory CD19+ B-ALL in adult patients (16). Another CAR-T loss of effectivene ss (26). Even though the moAb treatment recently approved by the FDA is idecabtagene characteristics of scFv allow potential benefits for the CAR-T vicleucel (ABECMA®, Celgene-BMS) based on the recognition therapy, cell persistence, antitumor response efficiency, and of B-cell maturation agents (BCMAs) for treating off-tumor on-target toxicity need to be improved, and multiple myeloma (MM) (17). Besides approved therapies, thus, innovative approaches can bring improvements in ciltacabtagene autoleucel (of CARTITUDE clinical this regard. trials, Janssen-Johnson&Johnson) (18) is also a BCMA- directed CAR-T product expected to be authorized for MM treatment. Changing the extracellular target- Despite this range of validated products, researchers are striving to broaden the clinical benefit of CAR-T cells while binding region, changing the “CAR” exploring new cutting-edge applications. Nevertheless, concept to other but resistance mechanisms, such as T-cell exhaustion, similar receptors immunosuppression, or antigen loss, carrying to relapse and therapy failure, have been reported during these trials (19, 20). In Optimized design of every region of a synthetic CAR has fact, an estimated 30% of relapses after anti-CD19 therapy been shown to be relevant to its clinical success (27). Different were related to antigen loss (20). This highlights the need to strategies are being developed to increase CAR-T cell response, improve or fine-tune CAR-T therapies to avoid tumor escape by one of which is proposing a new extracellular target-binding identifying novel tumor antigen targets, testing various region (28). Given this, non-antibody-based strategies are being CAR-expressing cells as CAR-Natural killer (NK), and, proposed as a promising improvement for CAR-T cell therapies most importantly, enhancing the CAR molecule itself. based on the interaction of surface receptors with their natural The minimal structure of a CAR consists of an extracellular ligands (29). Specifically, ligand-based CAR-T cells take benefit antigen recognition domain, tethered to a hinge domain from the receptor-binding domain of soluble molecules, i.e., followed by a transmembrane region, and an intracellular cytokines, growth factors, immunoglobulin superfamily signaling domain (CD3z). Additionally, different generations proteins, or chimeric peptides for targeting TAA to induce the can be found according to the number of modular intracellular antitumor response (26) (Figure 2). Similar to scFv-based CAR- costimulatory domains (mainly 4-1BB, CD28, or OX-40) (1, T cells, the ligand recognition is in an HLA-independent 21) (Figure 1). manner. Additionally, the downstream signaling T-cell Frontiers in Immunology 02 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 A B C D FIGURE 1 Chimeric antigen receptor (CAR) generations. (A) First-generation CAR includes a single-chain variable fragment (scFv) extracellular region and a T-cell activation domain. This minimal structure can recognize the antigen in an HLA-independent manner. By adding a costimulatory domain, (B) second-generation CAR is more able to expand and persist due to this second signal. (C) The third-generation CAR has an additional costimulatory signaling domain to increase proliferation, survival, and activity of engrafted T cells. Recently, (D) the fourth-generation CAR has been developed to include extra genes, such as recognition domains for transcription factors involved in mediating signal transduction. The idea is to modulate the effect of the CAR, facing an immunosuppressive tumor microenvironment by cytokine production or other additional effects. activation is maintained in the ligand-based CAR-Ts as all four Moreover, its target must be tumor specific or highly expressed generations can be found, but most approaches use the second- while having minimal presence in normal tissues, making it and third-generation CARs (Table 1). necessary to optimize the proposals as much as possible. One of the main constraints lies in finding the right ligand Worldwide, a considerable number of preclinical studies have for the CAR structure, as the pool of candidates is still limited. been published and clinical trials are currently ongoing, B A D C FIGURE 2 Schematic differences between conventional chimeric antigen receptor T (CAR-T) cell and ligand-based CAR-T cell. (A) Second-generation conventional CAR-T cell structure, including T-cell activation domain (signal 1) and costimulatory regions. The recognition domain of this CAR- T is composed of the single-chain variable fragment (scFv) that allows direct interaction with the tumor-associated antigen to trigger the antitumor response. (B) Schematic representation of the different chains that form an antibody and how the scFv domain is obtained from the variable heavy and light chains of a monoclonal antibody to be used in the CAR-T structure. (C) Structure of a second-generation ligand-based CAR-T cell that shares the same domains as mentioned with scFv-based CAR-T cells but incorporating a ligand as a target recognition domain. (D) Immune cytokines, growth factors, immunoglobulin superfamily proteins, and chimeric peptides, among others, are listed as potential molecules to be used as ligand-based CAR-T cell recognition domains (see some abbreviations in Table 1). Frontiers in Immunology 03 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 validating suitable ligands and elucidating their clinical Because of its orphan disease condition, IL-11Ra was proposed potential (Table 1). as a suitable candidate for CAR-T therapy (45). A second-generation CAR-T was designed using IL-11 peptide (CGRRAGGSC) as the extracellular domain (46). In Ligand-based Chimeric Antigen vitro, IL-11Ra-CAR-T cells were cytotoxic to four different OS Receptor (CAR)-T cell cell lines compared with control T cells. After in vitro injection in OS mouse models, engineered T cells accumulated in lung preclinical studies metastasis nodules that resulted in selective tumor cell lysis and Over the recent years, efforts have been focused on proving tumor regression, with no visible lung metastases in three of the CAR-T success against solid tumors, but several difficulties have five mice treated compared with controls (30). arisen such as finding a specific TAA, insufficient cell expansion after recognition, tumor penetration, and evasion mechanisms. These obstacles for CAR-T therapies need to be overcome (39– Adnectin-based CAR-T cell 41). Indeed, different approaches are being evaluated, including ligand-based strategies. Adnectin is derived from the 10th type III domain of human fibronectin (10Fn3) (47). The 10Fn3 domain interacts with integrins and belongs to the immunoglobulin superfamily. Its structure is IL-11-based CAR-T cell close to the antibody variable domain but with better stability and no dependence on disulfide bonds (48). This feature allows the The IL-11/IL-11Ra signaling pathway is involved in several manipulation of this domain to generate mutants with different biological activities, and it is supposed to induce an antiapoptotic interactions. Hence, a similar scFv structure, increased stability, and effect via STAT3 activation (42, 43). It has been shown that human nature make adnectin an interesting candidate for ligand- human IL-11Ra is overexpressed in several types of cancer, based CAR-T therapies (31). including osteosarcoma (OS) and lung-associated metastases. One of the target membrane surface receptors to direct Immunohistochemistry results from Huang et al. (30) showed adnectin-CAR-T cells is the epithelial growth factor receptor that four different OS cell lines overexpress IL-11Ra within (EGFR). This receptor has tyrosine kinase activity that governs 20%–60% and 14 of 16 patients were positive for IL-11Ra in fundamental cellular processes, including proliferation, cell their OS lung metastasis samples. In contrast, IL-11Ra was not migration, metabolism, and survival (49). Moreover, it is one expressed in the surrounding normal lung tissue or other of the most suitable candidates targeted in cancer therapies, essential tissues (30). OS treatment has been stagnant during since it is overexpressed in several tumors such as breast, lung, these years, and finding new treatments is still needed (44). and head and neck (50). TABLE 1 List of preclinical studies and current clinical trials using ligand-based CAR-T cells in course. References Ligand Target CAR structure Disease Preclinical studies (30) IL-11 IL11-Ra IL11 - CD28 -CD3 z OS and lung metastases (31) Adnectin EGFR Adnectin- CD28 - 4-1BB - CD3 z Lung cancer (32) FLT3L FLT3 FLT3L - 4-1BB - CD3 z AML (33) GM-CSF GMR GM-CSF - CD28 - CD3 z AML, JMML (34) EPHRIN B2 EPHB4 EPHRIN B2 -CD28 - CD3 z RMS (35) Tri-APRIL BCMA/TACI Tri-APRIL - 4-1BB - CD3 z MM (36) TPO MPL R TPO – CD28 - CD3 z AML (37) IL-10 IL-10R IL-10 - 4-1BB - CD3 z AML (38) CD27 CD70 CD27- CD3 z Diffuse large B-cell lymphoma, follicular lymphoma, AML Clinical trials NCT02208362 IL-13 IL-13Ra2 IL-13(E13Y) - 4-1BB - CD3 z Glioma NCT01818323 T1E ErbB 1-4 T1E - CD28 - CD3 z HNSCC NCT03287804 APRIL BCMA/TACI APRIL - CD28 - OX40 - CD3 z MM NCT04661384 IL-13 IL-13Ra2 IL-13(E13Y) - 4-1BB - CD3 z Leptomeningeal glioblastoma, Ependymoma or medulloblastoma OS, osteosarcoma; EGFR, epidermal growth factor receptor; FLT3L, FMS-like tyrosine kinase 3 ligand; FLT3, FMS-like tyrosine kinase 3; AML, acute myeloid leukemia; GM-CSF, granulocyte-monocyte colony-stimulating factor; GMR, granulocyte-monocyte colony-stimulating factor receptor; JMML, juvenile myelomonocytic leukemia; EPHB4, ephrin type-B receptor 4; RMS, rhabdomyosarcoma; APRIL, a proliferation-inducing ligand; BCMA, B-cell maturation antigen; TACI, transmembrane activator and calcium-modulator and cyclophilin ligand interactor; TPO, thrombopoietin; MPLR, myeloproliferative leukemia receptor; MM, multiple myeloma; HNSCC, head and neck squamous cell carcinoma. The clinical trials are collected from clinicaltrials.gov. Frontiers in Immunology 04 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 Han et al. (31) designed CETUX-CAR (scFv derived from FLT3L CAR-T cell cetuximab) and adnectin-CAR-T cells targeting EGFR (both third- generation CAR-T) to compare their activity (31). Ligand-based Acute myeloid leukemia (AML) is still a rare malignancy but CAR-T therapy was developed after revising a previous work by represents a third of all diagnosed leukemias. Two ligand-based Emanuel et al. (51) to generate adnectin clones for this aim. Four CAR-T cells have been proposed against AML, FLT3L CAR-T adnectin clones were evaluated (E1, E2, E3, E4) with different and granulocyte-monocyte colony-stimulating factor receptor binding affinities. E3 was considered the most eligible (51). In (GMR) CAR-T (32, 33). Approximately 30% of AML cases have comparison to CETUX-CAR-T therapy, E3 CAR-T cell displayed mutated the FMS-like tyrosine kinase-3 (FLT3), mainly internal relatively lower binding affinity toward EGFR but higher selectivity tandem duplication (ITD) mutations that lead to constitutive against EGFR-overexpressing cancer cells. Nevertheless, it has activity of tyrosine kinase domain (TKD) and promote, via comparable reactivity, cytotoxicity, and hence antitumor different signaling pathways, the progression of AML with response when incubated with lung carcinoma H292 cells (31). poor prognosis (57, 58). As scFv-based CAR-T targeting FLT3 These characteristics expect new broad opportunities to selectively has no optimal results, Wang et al. (32) developed a second- target EGFR-positive tumor cells, avoiding classical issues of classic generation ligand-based CAR-T cell with the FLT3 ligand CAR-T therapies, which will be discussed later. (FLT3L) as the recognizing domain (32, 59). FLT3L CAR-T cell can specifically recognize FLT3-positive cells, and in vitro studies have proven their cytotoxic efficacy EPHB4-based CAR-T cell against 10 different primary AML cell lines, five with FLT3-ITD and five with wild-type (WT) FLT3 expression. Moreover, Ephrin type-B receptor 4 (EPHB4), a member of the family of treated mice showed longer survival, but results also revealed receptor tyrosine kinases (RTKs), is ubiquitously expressed in that recognition seemed to be independent of FLT3 levels on distinct types of malignancies as rhabdomyosarcoma (RMS). cells, relying on the FLT3 genotype (32). EPHB4 expression is negligible in vital tissues except of a weak Cytotoxicity efficacy was proven in vitro against 10 different expression in normal placenta cells (52, 53). Differences in ligand- primary AML cell lines, five with FLT3-ITD and five with WT dependent or ligand-independent activation of EPHB4 have been FLT3 expression. FLT3L CAR-T cell can specifically recognize reported, being stimulation without ligand binding the one that FLT3-positive cells and display cytotoxicity. In vivo experiments leads to cell growth and transformation. In RMS and other verify this idea, since treated mice showed longer survival. In malignancies, EPHRIN B2 interaction with EPHB4 may induce fact, results revealed that recognition seemed to be independent apoptosis and lack of proliferation (54). Based on the fact that of FLT3 levels in cells but relied on the FLT3 genotype (32). EPHRIN B2 is a unique ephrin ligand that interacts with EPHB4, FLT3L-FLT3 interaction allows dimerization and a second-generation CAR-T cell with an extracellular portion of phosphorylation of FLT3 and activation of downstream EPHRIN B2 can be considered for RMS treatment (55). signaling pathways that end up in cell growth and survival (60, In vitro robust and sustained killing activity against RMS, 61). Since FLT3L CAR-T allows ligand-dependent activation, it OS, and triple-negative breast cancer (TNBC) cells was assessed can stimulate this phosphorylation and may promote cell growth by Kubo et al. (34), even following multiple tumor rechallenges, in FLT3 WT. Thus, FLTL3 WT is less sensitive to CAR-T indicating no reduction of antitumor effect. Even though the cytotoxicity. Otherwise, FLT3-ITD is constitutively activated interaction with EPHRIN B2 should induce weak proliferation, (ligand-independent) developing different phosphorylation in vitro results refuse this idea and do not promote proliferation profiles that are more sensitive to CAR-T therapy when FLT3 in RMS cells (55). Another considered point was the possible CAR-T interacts. This may allow the CAR-T therapy the ability effect off immunomodulatory effect of the P3F fusion gene, to distinguish between both types of cells, being more lethal for which undergoes some RMS variants, on CAR-T activity. mutated FLT3. So, distinguishing the receptor by genotype can Nevertheless, this translocation product did not modulate the be a novel strategy with potential benefit in such types of EPHB4-CAR-T activity (56). tumors (32). After substantiating that EPHRIN B2 could bind EPHB4 mouse receptors, the antitumor effect and off-tumor on-target toxicity were in vivo verified with RMS tumor xenograft models. GMR CAR-T cell The results showed decreased tumor growth rates and prolonged survival in treated animals with EPHB4-CAR-T compared with Another different approach for AML treatment is GMR CAR-T anti-CD19 CAR-T control without any sign of adverse effects cells. The granulocyte-macrophage colony-stimulating factor (GM- (55). These promising results have led to the generation of novel CSF) is an immunomodulatory cytokine capable of tuning the studies, and future clinical trials are being proposed. phenotype of myeloid cells but also T cells through myeloid Frontiers in Immunology 05 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 intermediaries (62). Its main target is GM-CSF receptor (GMR), against MPL+ AML cell lines in vitro but also satisfactory results composed of two subunits: a subunit (CD116) that is present in in murine AML xenograft models. Notably, on-target off-tumor normal and AML and juvenile myelomonocytic leukemia (JMML) toxicities were detected in the bone marrow compartment myeloid cells and b subunit (CD131) shared with IL-3 and IL-5 during the trials. The authors justify that bone marrow toxicity receptors. Recent studies revealed that GMR can be found as could be advantageous for the model, as most patients with AML complexes of two a subunits (low-affinity receptors) or both a receive a bone marrow transplant and treatment with TPO- and b subunits (high-affinity receptors) (33). based CAR-T cells may be helpful to replace the adverse effects AML expresses both complexes, and since CD116 is of pretransplant conditioning regimens. overexpressed in more than 60% of AML, mainly in those with poor prognosis, Saito et al. (33) proposed a second- generation ligand-based CAR-T cell targeting GMR (33, 63) IL-10-based CAR-T cell after they demonstrated antiproliferative effects of the same construct against JMML (63). Different CAR-Ts were built and Among the diverse ways of drawing an antitumor response evaluated to enhance this effect against AML. Referring to a in AML is that based on the IL-10 receptor (IL-10R). IL-10R is a previous work by Ló pez et al. (64), they used GM-CSF as a receptor composed of four members, two alpha (IL-10RA) and binding region (33) mutated in residue 21 that plays a key role in two beta (IL-10RB) molecules, being hematopoietic-specific and the functionality of the cytokine but not affecting the binding ubiquitous, respectively. Published data infer that the IL-10/IL- (64). After screening analysis of several mutated GM-CSFs, 10R pathway, when with aberrant function, is involved in E21K and E21R, both had increased antitumor response. In promoting the stemness of AML cells (37). For this reason, it vitro and in vivo results revealed E21K mutation as the one with seems reasonable to validate the CAR-T response against IL- durable in vitro cytotoxicity and complete suppression of the 10R. To this end, Chen et al. (37) designed a second-generation progression of CD116+ AML cells in vivo, correlating strongly ligand-based CAR-T cell using IL-10 as a binding motif and with the CD116 levels in tumor cells. These may appear to assessed the degranulation and cytokine secretion from T cells conflict with other reported data in which E21K-mutated GM- and killing of the AML-targeted cells in culture. Following good CSF had a reduced binding capacity to high-affinity receptors in vitro results, they assessed the product in a murine AML but maintained the binding capacity to low-affinity receptors, xenograft model and obtained prolonged survival in treated leading to less AML interaction than scFv-CARs (33, 64). models compared with those that did not undergo CAR- Although the mechanism has not been identified yet, it seems T treatment. that the reduction of time interaction with receptors would enhance T-cell stimulation (65). Despite all of these important results, off-tumor adverse effects were not tested and this would CD27 CAR-T cell be necessary to evaluate for further applications (33). CD70 is the membrane-bound ligand of the CD27 receptor, which belongs to the TNF receptor superfamily. This interaction Thrombopoietin-based CAR-T cell is considered a potential target to address CD70-positive malignancies, such as diffuse large B-cell lymphoma and Thrombopoietin (TPO) is a hematopoietic growth factor follicular lymphoma, as well as AML, since CD70 is expressed produced not only by the liver but also in the bone marrow and on most of its leukemic blasts, while its expression is low or kidney niches. TPO is defined as a natural ligand to the absent in normal bone marrow samples (66). Importantly, CD70 myeloproliferative leukemia (MPL) receptor, also known as expression is transient and restricted to a subset of highly CD110. Overexpression of MPL has been characterized as a activated T, B, and dendritic cells under physiological negative prognosis factor for AML progression due to the effects conditions, playing a role in T-cell activation. However, it is of the associated signaling, such as Janus Kinase 2 (JAK2)/ not essential for the development and maintenance of a STAT5, Phosphoinositide 3-kinase (PI3K)/Protein kinase B functional immune system. (AKT), and proto-oncogene, serine/threonine kinase (Raf1)/ In this context, Sauer et al. (38) developed a first-generation Mitogen-activated protein kinase (MAPK) (36). CAR-T cell based on a ligand, CD27z-CAR, which uses the full- The TPO/MPL pathway is essential for the survival and self- length CD27 cDNA as a recognition domain. Additionally, their renewal of leukemia stem cells (LSCs) and hematopoietic stem research incorporated the design of several CAR-T sequences cells (HSCs) and is therefore involved in the progression of with the CD70-specific scFv to compare reactivity against AML. For this reason, Zoine et al. (36) proposed a second- the target. generation ligand-based CAR-T cell using the biologically active All CAR-T cell populations mediated cytotoxicity against region of the TPO protein to target the MPL receptor. The CD70-positive tumor cell lines but not CD70-negative cells in in outcomes showed not only a significantly specific cytotoxicity vitro assays (38). However, because the efficacy of CAR-T cells is Frontiers in Immunology 06 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 determined not only by their cytolytic activity but also by their worse prognosis, hypothetically owing to IL-13/IL13Ra2 ability to proliferate after the tumor challenge, they were interaction (73–75). IL-13 is an immunomodulatory cytokine subjected to successive cocultures. The results showed that promotes apoptosis and transforming growth factor alpha differences in their ability to kill and proliferate during (TGF-b) secretion when it interacts with IL13Ra1/IL-4Ra high- successive cocultures, with CD27z-CAR able to kill tumor cells affinity heterodimer. Alternatively, IL-13 has a higher affinity to during five consecutive cocultures in at least two of the four IL13Ra2 but does not induce intracellular signaling (76). donors in contrast to other CAR-T cell populations, as well as Therefore, overexpression of IL13Ra2 in GBM may reduce the highest production of T helper 1 (TH1)-type cytokines, such proapoptotic signaling and promote cell survival (75). as Interferon (IFN)-g and TNF-a (38). Considering these facts, IL13Ra2 is a suitable candidate for Following these results, the same research evaluated the different treatments as is IL-13-zetakine (77, 78). This product is CD27z-CAR in vivo effects in CD70-positive AML murine an adoptive T-cell therapy engineered with a CAR structure xenograft models. Their results demonstrated an efficient whose recognition domain is IL-13 cytokine, which contains the ability to control leukemic growth, leading to complete E13Y mutation, for targeting IL13Ra2. The importance of this leukemia remission in all mice by day 21 (38). Furthermore, mutation relies on reducing the affinity to IL13Ra1/IL-4Ra they could demonstrate a significant expansion of the heterodimer but increasing IL13Ra2 binding compared with transduced T cells in the in vivo models, thus corroborating WT IL-13 (77). The aim is to specifically redirect the cytotoxic the relationship between the administered therapy and the activity of T cells to GBM cells that overexpress this TAA remission of pathology. compared with normal CNS cells. During the last few years, a first-generation IL-13 CAR-T was developed and tested, obtaining a sustained cytotoxic Ligand-based CAR-T cell response to both cancer-initiating cells and differentiated GBM clinical trials cells in vitro. Also, there was evidence of antitumor activity and limitation of the progression of established IL13Ra2-positive Although anti-CD19 scFv-based CAR-T therapies have tumors in xenograft mice without clear collateral damage on clinically succeeded, as several products have already been healthy tissue (77, 78). With these results, Brown et al. (79) approved (12, 13), the reality is that limited clinical conducted a first-in-human pilot clinical trial (NCT00730613) information is still available for other strategies, as could be to assess the activity and safety of IL-13-zetakine after for ligand-based CAR-T cells. However, a few phase I/II clinical intracranial delivery in three patients with recurrent GBM. trials are currently trying to elucidate the safety and bioactivity Indeed, two out of three showed transient antitumor activity of different approaches. in the absence of severe adverse events. Although the survival rate was 11 months, the small cohort denied the capacity to establish the therapy survival benefit (67). IL-13-zetakine CAR-T cell Aware that the CAR-T response needs to be improved, Brown et al. (80) started to tune the IL-13-zetakine structure. One of the most hopeful proposals is focused on treating Thus, the second-generation CAR-T cell was developed using 4- central nervous system (CNS) solid tumors, such as glioblastoma 1BB as the costimulatory domain and CD3z as the intracellular multiforme (GBM) (67–69). GBM is one of the most lethal signaling domain (80). Preclinical results showed an enhanced primary brain tumors, and its outcome remains poor. High- response. As the first-generation IL-13 CAR-T activity was grade glioblastoma does not respond to standard treatments transient, and its persistence was limited, one of the aims was such as surgery or chemotherapy mainly because of tumor to analyze the antitumor response and cell persistence of the heterogeneity (70). Diverse differentiation status has been second-generation one. For this reason, a current phase I clinical found in GBM cell populations: stem-like cancer-initiating trial (NCT02208362) (81) studies the activity, adverse effects, cells (GSCs), expressing stem cell markers and maintaining and best dose of these CAR-T cells. In fact, one patient has certain self-renewal capacity, and differentiated glioblastoma reported a transient complete response after complete CAR-T cells (71). It has been proposed that GSCs are responsible for dose administration with important improvements in the quality this lack of response because of their natural resistance to of life for up to 7.5 months (68). The trial is still ongoing, but this conventional treatments (72). case appears to be a great hope. Another clinical trial has just IL-13 receptor a2 (IL13Ra2) is demonstrated to be started (NCT04661384) to test this CAR-T therapy in patients expressed within 50%–80% of GBM cells, independently of with leptomeningeal disease from glioblastoma, ependymoma, differentiation status, but not significantly expressed in normal or medulloblastoma, but the results are not expected to be CNS tissue. IL13Ra2-positive tumors are associated with a analyzed until December 2022 (69). Frontiers in Immunology 07 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 Pan-ErbB CAR-T cell and selective stimulation, allowing better expansion. Preclinical studies have revealed that T4 immunotherapy (CAR-T The ErbB receptor family comprises a synergistic dynamic combining T1E28z and 4ab chimeric receptors) achieves a signaling network composed of four members, EGFR/ErbB-1, relevant antitumor response in HNSCC, EOC, and malignant ErbB-2/NEU/HER2, ErbB-3/HER3, and ErbB-4/HER4 (49). mesothelioma in vitro (83–85). After ligand-dependent stimulation, diverse homodimer or Considering that T1E polypeptide can efficiently bind to heterodimer combinations may occur (50). ErbB-2 is ErbB mouse receptors, in vivo efficiency and toxicity were tested demonstrated to be the preferred member for dimer in diverse immunocompromised xenograft mice, including all formation, while ErbB-3 pairing is essential, because of its lack three malignancies mentioned above (89). T cells elicit of intrinsic tyrosine kinase (TK) activity. Upon dimer activation, antitumor activity in the absence of relevant toxicity when tissue development, proliferation, and differentiation are delivered intratumorally or intraperitoneally at a moderate promoted (50). dose. Nevertheless, after high-dose intraperitoneal delivery, Several studies have revealed aberrant expression or function cytokine release syndrome (CRS) appeared, providing evidence of some ErbB receptors, mainly ErbB-2 dimers with ErbB-1 or that intratumor administration seems to be the safest route for ErbB-3, as a determinant of the pathogenesis of many solid tumors and that CRS dose-dependently appeared (83, 89). malignancies, such as mesothelioma, epithelial ovarian To build on this, van Schalkwyk et al. (90) designed a phase I carcinoma (EOC), or head and neck carcinoma [head and clinical trial (NCT01818323) to assess the safety of T4 neck squamous cell (HNSCC)] (82–85). Consequently, there is immunotherapy to treat HNSCC that is not suitable for a considerable interest in targeting ErbB family members, but conventional active therapy. Primary results are expected to be problems of selective pressure and tumor resistance have been published in April 2022. If results are robust, other clinical emerging due to the overexpression of non-targeted approaches should be initiated to evaluate CD4+ CAR-T therapy receptors (82). against other malignancies, such as EOC or malignant To circumvent this, diverse approaches are currently trying mesothelioma (91). to redirect their mechanism toward two or more ErbB dimers to prevent the signaling network from escaping and continuing tumor progression. This idea includes T1E28z or pan-ErbB AUTO2: APRIL-based CAR-T cell CAR-T cell, a second-generation CAR-T therapy that includes T1E as a binding moiety for treating many epithelial MM represents 13% of all hematologic cancers, and it is malignancies (83). characterized by extreme growth of malignant plasma cells (PCs) T1E is a chimeric polypeptide that takes benefit from in the bone marrow, aberrant production of monoclonal different ErbB ligand properties: epidermal growth factor immunoglobulin, and immunosuppression, among others (92, (EGF) and TGF-a selectively bind to ErbB-1 with high affinity 93). Over the past decade, autologous stem cell transplants, but weaker or no affinity for ErbB-2/ErbB-3 heterodimers, proteasome inhibitors (PIs), and immunomodulatory drugs respectively. Structural analysis of both revealed that EGF is (IMiDs) have significantly raised survival rates, and moAbs unable to bind ErbB-2/ErbB-3 with high affinity because of the further improved relapsed/refractory multiple myeloma lack of essential amino acids in the N-terminal region, whereas (RRMM) outcomes (93–95). Nonetheless, overall survival is TGF-a cannot bind despite having these crucial residues (86). extremely reduced in patients with RRMM after IMiDs or IP Thus, a chimera was developed introducing N-terminal linear treatments. Therefore, there remains a need for new approaches region of TGF-a into the EGF C-terminal sequence, resulting in that could lead to durable remissions in MM patients, especially high affinity for ErbB-2/ErbB-3 maintaining ErbB-1 specificity in RRMM (94). (87). ErbB-4 heterodimer binding was also reported. This made Although the CARTITUDE-1 trial seems to have recently T1E a promiscuous ligand ideal for multitargeting ErbB dimers, promising results (18), treatment of MM still involves many preventing antigen loss and signaling compensation. challenges, focusing on TAA detection. Since CD19 has a Davies et al. (83) engineered T cells with T1E28z and reduced expression in malignant cells, some RRMMs appear to evaluated its binding capacity, resulting in eight of nine be CD19-negative and other well-defined antigens (CD56 or possible ErbB homo and heterodimers, with most affinity CD38) have expression levels in other tissues, then other detected against cells that coexpressed ErbB-1 and ErbB-2 antigens need to be validated (95). (83). One of the main challenges of CAR-T therapies is the BCMA is another suitable candidate for CAR-T therapies enrichment and expansion of T cells. For this reason, they also because it is absent on hematopoietic stem cells but selectively introduced a chimeric cytokine receptor named 4ab, in which expressed on PCs, and BCMA is almost present in MM cells. IL-4 receptor-a ectodomain has been coupled to the shared b After the first anti-BCMA CAR-T cell trial, remission was chain used by IL-2/15 (88). With IL-4, T cells receive a potent reported in four of 12 patients, but high doses were required Frontiers in Immunology 08 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 for persistent remission due to the low expression of BCMA in other approaches in which ligands are incorporated as the MM cells. Moreover, similar to CD19 therapies, tumor escape by binding moiety of the CAR structure. In this regard, we found downregulation of targeted BCMA was reported (96, 97). the so-called chimeric autoantibody receptor (CAAR) (103), B- Considering this, Lee et al. (98) attempted overcoming low cell receptor antigen for reverse targeting (BAR) (104), and target density and antigen escape targeting two TNF-receptor chimeric HLA antibody receptor (CHAR) (Figure 3). superfamily members, BCMA and a transmembrane activator Treatments for autoimmune diseases specifically eliminate and calcium-modulator and cyclophilin ligand interactor self-reactive cells while preserving protective immunity (103). (TACI) (98). Both are coexpressed on the majority of PC and However, this premise is proving difficult to implement, as both MM cells and may play a similar role in providing PC with autoimmunity and cancer are closely related, requiring optimal survival signaling (35). For this reason, a bispecific third- management of autoimmune therapies to prevent cancer generation CAR was constructed using a murine truncated development due to general immunosuppression (105). For version of a proliferation-inducing ligand (APRIL), a natural this reason, novel approaches try to avoid the classic issues ligand for both BCMA and TACI with nanomolar affinity (99, associated with autoimmunity treatments. 100). The final product, AUTO-2, is retrovirally transduced to In this sense, CAAR molecules consist of a chimeric produce CAR-T cells expressing APRIL-CAR-T and the RQR8 immunoreceptor that includes an autoantigen as the switch system that acts as a marker but also as a suicide molecule extracellular domain. This technology directs the modified T- when adverse effects of the therapy occur (101, 102). cell response toward autoreactive memory B cells expressing The preclinical evaluation demonstrated cytolysis at low autoantibodies as their surface immunoglobulins (sIg-BCR), or levels of target antigen, even when the BCMA expression was autoantibody-producing PCs, which are autoantibody-secreting downregulated or lost. These data were confirmed in vivo, where cells (103). This would generate selective therapy against reactive an improved disease control compared with scFv-based CAR immunity, thus avoiding a general suppression of the was observed (101), but the problem still was the need for abovementioned protective immunity. This strategy has been considerable T-cell doses to achieve relevant responses with a used by Ellebrecht et al. (103), who have constructed a CAAR T short follow-up duration. A phase I/II clinical trial cell using the different forms of the Dsg3 autoantigen target of (NCT03287804) (102) was initiated to test the safety and pathological autoantibodies present in a significant percentage of efficacy of AUTO2 in RRMM patients. Phase I showed that patients with pemphigus vulgaris (PV). Their results expect eight of 11 (81.9%) treated individuals achieved expansion and potential benefits that these CAARs could bring to the persistence of CAR-T cells in peripheral blood, while four of 11 treatment of autoimmune diseases. (36.4%) had a complete/very good/partial response up to 2 years. BCR signaling has been identified as an important pathway However, the average death during the trial was eight of 11 (72, in B-cell lymphomagenesis, and there is increasing evidence that 73), so the duration of response could not be quantified, and antigenic stimulation of the BCR is a trigger for proliferation. thus, phase II was not initiated. Several autoantigens, such as ARS2 and LRPAP1, have been Even though the results of the clinical trial seemed to show a proposed as stimulatory ligands of the BCR and its pathway in glimmer of hope, APRIL-based CAR-T cells required better one quarter of diffuse large B-cell lymphomas (DLBCLs) and optimization. After evaluation of all of these studies, Schmidts almost half of the mantle cell lymphomas (MCLs), respectively et al. (35) generated a second-generation CAR where they (104–106). changed the extracellular domain of AUTO2 for a tri-APRIL From the study of these BCR antigens arise structures binding moiety (TriPRIL) (35). The study hypothesized that defined as BAR. BAR-bodies were initially designed with the preserving the trimeric form of the natural ligand would increase idea of conjugating toxins to these BCR antigens. One example is the binding affinity and efficacy against MM cells. Also, they the research led by Thurner et al. (107), where it is shown that used human APRIL so it would reduce immunogenicity. Indeed, LRPAP1-based BARs conjugated to Pseudomonas aeruginosa data revealed that antitumor activity was enhanced in vitro exotoxin A toxin are internalized and specifically kill MCL cells against BCMA+ and BCMA- cells and in vivo with xenograft with LRPAP1-reactive BCRs by inducing apoptosis. models compared with monomeric APRIL-based CAR-T cells Further research has led to the construction of an antibody- (35). Therefore, TriPRIL CAR-T therapy holds promise for like structure that incorporates the sequence of these identified treating MM, including the absence of BCMA. Further clinical BCR antigens, or at least their BCR-binding epitope, replacing trials will be required to elucidate its potential clinical benefits. the variable fragments of the scFv heavy and light chains, with the aim of transducing T cells and targeting malignant B-clones with unique specificity for these BCRs responsible for tumor Beyond ligand-based CAR-T expansion (104, 106). Since approaches such as that of Bewarder et al. (106) that uses the BCR antigen to target MCL cells have Although this review focuses mainly on what the literature exclusive specificity for cells with the specific surface BCR, they refers to as ligand-based CAR-T cells, it is necessary to mention do not only represent a strictly tumor-specific approach but can Frontiers in Immunology 09 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 A B C FIGURE 3 Role of ligands in other antibody-mediated diseases. Recently, new approaches have emerged in which different ligands linked to antibody-like structures can be used to address specific pathologies. The sequence of these products shares similar domains to CAR-Ts but changes the extracellular domain. In panel (A), a chimeric autoantibody receptor (CAAR) can be seen in which an autoantigen is incorporated as a recognition domain of the CAAR to redirect it toward autoantibodies on the autoreactive B-cell surface, thus facing autoimmune responses. Panel (B) shows the structure of a B-cell receptor antigen for reverse targeting (BAR). The recognition domain includes an antigen specific to the single B-cell receptor (BCR) clonally specific of each B-cell tumor. Finally, panel (C) represents an example of chimeric HLA antibody receptor (CHAR), where molecules of the class I HLA system replace the conventional recognition domain to direct the transduced T cell toward anti-HLA antibody-producing B cells to cope with posttransplant immune rejection. also be expected to be more effective but less toxic than the cells, resulting in potent responses that can lead to the currently available CAR-T cells with specificity for CD19, as they elimination of tumor cells expressing the target antigen (1). As should not affect other cells and should work equally well in the mentioned above, those approaches are being considered as one presence of autoantibodies against the antigen in question. of the further progress in the field of antitumor therapies, even In the same line as CAARs and BARs, T cells that express including several commercial products available for clinical use CHAR with the ability to kill B cells that produce donor-specific (12–16). Specifically, it is the conventional CAR, with the scFv class I HLA antibodies are being developed to treat antibody- fragment as the recognition domain, that is mostly proposed, mediated rejection in the field of solid organ transplantation. since they were the first to appear and to get results. In any case, One of the main problems in solid organ transplantation is the scFv-based CAR is not exempt from limitations and concerns, presence, or de novo generation, of donor-specific antibody anti- such as immunogenicity or toxicity (5, 24). For this reason, HLA molecules (anti-HLA-DSA), which is associated with a ligand-based CAR-T cells are now emerging as a suitable high risk of antibody-mediated rejection (108). Our thinking has alternative to address them (110). They are presented as an changed from considering rejection as a primarily T cell- alternative therapy because most of the preliminary and clinical mediated process. Insufficient control of the humoral arm of a studies conducted to date show similar results in terms of recipient’s immune system by current immunosuppressive activity and efficacy (110). regimens is now the pathogenic factor primarily responsible It is relevant to highlight the commonalities between the two for allograft dysfunction and loss (109). This new CHAR could technologies. The proof of concept involves engineering of be a therapeutic approach for personalized desensitization of autologous T cells, allowing the CAR expression on the cell HLA-sensitized recipients and even for antibody-mediated surface, so that the modified cells can acquire tumor specificity. rejection in solid organ transplantation. Since the same intracellular signaling domains are used, upon recognition of the target molecule, the internal signaling Discussion necessary to enable T-cell activation and expansion will be triggered (22, 23). CAR-T cell-based therapeutic strategies allow the Both approaches share HLA-independent target recognition, production of significant numbers of tumor-specific reactive T which allows for less restriction in the recognition of what they Frontiers in Immunology 10 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 are meant to act against. The main difference lies in the viral vector peptides will still exist, but ligand-based CAR-T cells interaction on which target recognition depends. While scFv- may be less likely to prompt immune responses (35). based CAR-T cells rely on moAb–target binding, which is Despite that fact, it requires further investigation to elucidate defined as a higher-affinity interaction, ligand-based CAR-Ts the possible benefit of using ligand-based CAR-T cells to reduce use ligand–receptor binding, which is presumably a lower- immunogenicity, as there is a lack of results to prove it. affinity interaction (28). Using the properties of this modification in the CAR structure, the aim is to mitigate the issues detected after administration of conventional CAR- Tonic CAR signaling and scFv instability T therapies. Throughout the design process of scFv molecules, many studies have highlighted problems of oligomerization either as Immunogenicity part of a CAR structure or in a soluble form. Oligomerization occurs mainly through a process of domain swapping, where the CAR-T cells have the potential to trigger both cellular and VH region of one scFv is incorrectly associated with the VL humoral immune responses against non-self-components of the region of another scFv (26, 111). This causes the aggregation of CAR structure, but its clinical implication remains poorly CAR structures, which will result in dysfunctionalities leading to investigated and exhibits great variability depending on the tonic signaling through constitutive activation via the signaling CAR-T and type of tumor (24). described above. Not to mention that these oligomerizations Human anti-mouse antibodies (HAMAs) against mouse- could lead to problems in target recognition by the CAR (111). derived scFvs have been detected in a subset of patients. Anti- CAR-transduced T-cell tonic signaling is widely described in idiotype antibodies are also reported, directed toward specific many investigations. In addition to aggregations of scFv, high regions of scFv binding sequence, since the hypervariable region levels of cell surface CAR expression, the addition of endogenous (the idiotype and the allotype determinants) of the scFv are TCR-associated signaling, and the incorporation of certain highly immunogenic. Additionally, antibodies against CAR intracellular signaling domains into the CAR sequence can peptides originated from the fusion of the domains that make trigger constitutive cytokine release, prolonged and excessive up its structure can also be found (24, 25). Regarding the cellular expansion, and thus further T-cell exhaustion (26, 111). immune response, specific cytotoxic T cells could arise from the However, natural ligands are probably more stable and have a processing and cross-presentation of foreign peptides of the lower risk of dimerization and domain swapping if no CAR structure. Finally, immune response could also be multimeric ligands appear, and these will require further triggered by residual elements from gene transfer viral vectors, study. Thus, ligand-based approaches would reduce potential which are inevitably immunogenic (24). tonic signaling that prevents early exhaustion of CAR-T therapy, Despite the lack of conclusive evidence, it is inferred that thus prolonging its functionality and thus improving the these elements may interfere negatively with CAR-T cell activity probability of success of the intervention when administered by either neutralizing their recognition capacity, causing loss of in in vivo models. CAR membrane expression, or directly increasing CAR-T cell apoptosis (24, 25). Immunogenicity arising from the non- human origin of scFv could be reversed using human moAbs, Affinity-tuning and toxicity management but the full ability and a sufficient human moAb library to obtain them are not currently available. Also, humanized scFv can be an Another potential advantage of ligand-based CAR-T cells is option, but problems exist with cell surface stability, their capacity to multitarget (110). The native forms of ligands dimerization, and aggregation that limit the desired cytotoxic that are proposed for CAR structure often can bind to different activity (26, 110). receptors, being bispecific or trispecific. Recent publications Ligand-based CAR-T cells have been proposed to dodge consider that one of the main drawbacks encountered is the these adverse effects considering that natural human ligands are relapsed/refractory state of some malignancies after CAR-T used to replace the scFv region, as non-human sequences are infusion mainly due to downregulation of the specific antigen eliminated, and the immunogenicity of the product is reduced against which the cytotoxic activity is directed (antigen loss) (21, (both HAMA and cytotoxic T cells). It is worth mentioning that 31). Given that scFv has a single specificity for a particular the reduction in immunogenicity will be greater the more peptide, ligand-based CAR-T cells may provide a safeguard original ligand sequence is included (full-length sequence) in against antigen loss, anticipating one of the possible the final recombinant molecule (26, 31, 35). Antibodies directed mechanisms of tumor evasion (112). For instance, the APRIL- against the region of the fused domains and immunogenicity of CAR proposed by Lee et al. (98) has bispecificity for BCMA and Frontiers in Immunology 11 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 TACI, leading to increased recognition and lysis of MM cells, that are less affine but induce greater cytotoxic activity on either BCMA+ or BCMA-, compared to an scFv-based CAR-T the tumor. cell directed against BCMA that does not prevent the Ultimately, if the potential benefits associated with the ability proliferation of BCMA- tumor cells (35, 98). to prevent and/or reduce tumor evasion of the immune response This potential benefit from ligand-based CAR-T cells must can be balanced during in vitro development and testing in in be balanced against the toxicity problems that have already been vivo models with a thorough analysis of CAR-T interactions with described in some patients when treated with scFv-based CAR-T known and undescribed targets, if, in addition, possible cells (5). Specifically, these off-tumor on-target toxicities arise modifications of the ligand used to modulate the cytotoxicity because target molecules can be constitutively expressed in of the CAR-T product can be described, and if immunogenicity healthy tissues, causing harmful activity by altering normal issues are resolved, we may be talking about a therapeutic tissue functionality (3). If a product with bispecificity or alternative that will potentially discriminate tumor from trispecificity is infused, the potential number of off-target sites healthy tissue, be tumor-specific, and reduce the risk of at which it can act increases significantly, thereby increasing the adverse effects (31, 110). risk of adverse effects (4, 21, 35). Therefore, this promiscuous binding could be also analyzed as a disadvantage of the ligand- based design if other known or unknown binding partners exist Challenges ahead for ligand-based in healthy tissues, leading to off-tumor on-target toxicities. For CAR-T cells this reason, these potential toxic effects of ligand-based CAR-T cells need to be carefully explored in animal models. Although the use of natural ligand-based CARs presumably However, this could be countered by considering the has many advantages, these alternative CAR designs have their sensitivity of the CAR interaction toward the recognized own limitations. These include the potential for off-tumor molecule. Normally, the process of production of scFv regions toxicity, unwanted target-associated signaling, and possible depends on somatic hypermutation mechanisms, in which interference with the physiological interaction between the molecules with the highest affinity for the target are selected endogenous ligand and the target. (113). Thus, at low concentrations of the target molecule in the As has already been mentioned in this review, the possibility tumor, the CAR-T cell is able to reach the activation threshold of ligand binding to different targets may lead, on the one hand, and trigger the cytotoxic response (31, 114). However, this to a reduction in the ability to evade the antitumor response, at characteristic of high-affinity CARs makes them poorly able to the same time, it may trigger on-target off-tumor toxicities by discriminate between target cells with various levels of antigen increasing the range of possible interactions outside the tumor expression. Considering that tumors tend to overexpress certain (4, 21). Although this can be contrasted with the modulation of molecules above basal levels present in other tissues (82), this the interaction affinity, making the therapies more selective, this means that the CAR-T cells activate the cytotoxic response not aspect must be widely considered in the testing phases in animal only toward the tumor but also toward other healthy tissues, models. It is worth mentioning that toxic effects are also present increasing the risk of off-tumor on-target toxicities. Finally, in conventional CAR-T therapies, and therefore, it is a pending scFv-CAR-T cells have constitutive basal activation of CAR task for all CAR-T cell therapies. signaling by their extracellular domain, increasing the off- Another aspect to consider is the possible unwanted target effects of the therapies and the earlier exhaustion of the signaling that may be generated in the target cell because of T cell (31). the interaction with the ligand fused to the CAR. For example, if By using ligands and not antibody chains as the target- the ligand plays a role in cell proliferation and survival, their binding molecule, a lower-affinity interaction will be achieved interaction could increase tumor growth. This is a very (110). Additionally, the ability to modify native ligand sequences preliminary approach that requires further study, but a also offers some flexibility in CAR binding. An example is the possible alternative would be to introduce modifications in the GM-CSF-CAR, where mutations are introduced into the target- ligands that prevent signal transduction in the target, as binding domain so that the product has less affinity toward its proposed by Saito et al. (33), who introduced mutations in target (33). By reducing the affinity, a high TAA expression level GM-CSF at residue 21, a key to the functionality of the ligand will be needed to activate the T cell, increasing the selectivity of but maintaining the binding capacity. ligand-based CAR-T therapy against tumor cells. However, Finally, possible interference of endogenous ligands on the previous studies have shown that the affinity of a CAR toward interaction of the ligand that is used as the CAR binding moiety its target is inversely proportional to the activation threshold of should also be considered, generating a competition between the T cell, although the mechanism is not yet fully described both for binding to the target (35). This could result in reduced (114). Thus, by modifying the ligands, we can generate CARs functionality of the CAR-T cell. Although this is presumed to be Frontiers in Immunology 12 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 a mild effect, it should be considered individually as a reason for Data availability statement the study depending on the type of ligand. The original contributions presented in the study are included in the article/Supplementary Material. Further Conclusion inquiries can be directed to the corresponding author. As described in this review, ligand-based CAR-T cells offer several advantages over CARs containing an scFv domain as a binding moiety. Although they should generate greater toxicity Author contributions problems due to their ability to recognize multiple off-tumor MJ define the main aspects of the review. AR-C, AG, and targets, clear advantages are described: 1) they are less likely to MM contributed to the conception and design of the review. provoke an immune response, as the ligands are derived from AR-C organized the database and wrote the first draft of the natural human sequences and therefore fewer murine regions manuscript. SB-M and AB-I wrote sections of the manuscript. will be present in the CAR structure; 2) somatic hypermutation All authors contributed to manuscript revision, read, and phenomena are not necessary, thus reducing anti-idiotype approved the submitted version. antibodies; 3) ligand-based CARs are often able to bind to multiple targets, thus reducing the potential for tumor escape; 4) the nature of the ligands and their binding to the receptor allow for a certain tuning capacity that reduces their sensitivity Acknowledgments and enables therapies with a greater ability to discriminate between tumor tissues, which tend to overexpress the target, Thanks to Immunology department (Hospital Clı ́nic de and healthy tissues; and 5) less tonic signaling and longer lasting Barcelona) and Institute of Biotechnology and Biomedicine functionality should be detected associated with a reduction in (IBB-UAB) for their help during the writing of this article. the probability of ligand aggregation. This work has been partially supported by grants from the Ideally, ligand-based CAR-T therapies appear to be Instituto de Salud Carlos III, Spanish Ministry of Health (FIS proposals that would improve the safety profile of CAR-T cells PI18/00775, PICI18/00012 and complementary grant for and increase cell persistence, maintaining similar levels of CONCORD-023), Fondo Europeo de Desarrollo Regional response to those achieved with scFv-based CAR-T cells in (FEDER) “una manera de hacer Europa”, and “La Caixa” hematological malignancies and translate these to solid Foundation (CP042702/LCF/PR/GN18/50310007). tumors. They should therefore be presented as a significant advance in cancer immunotherapy. Nevertheless, there is still a lack of data and much research to be done to truly elucidate their Conflict of interest potential benefit and corroborate their safety profile. Therefore, public and private institutions should invest in the development The authors declare that the research was conducted in the and testing of these products and technologies. However, there is absence of any commercial or financial relationships that could a conflict of interest, as native ligand sequences cannot be be construed as a potential conflict of interest. patented like scFv; at the basic research level, it would facilitate the production of therapies, but, for the time being, there is a lack of investment to bring them to clinical trials and to Publisher’s note be able to analyze the issues mentioned in this review. It is also particularly important for the scientific community to engage All claims expressed in this article are solely those of the with these types of therapies, as more information is still needed. authors and do not necessarily represent those of their affiliated In conclusion, what emerges from this work is that ligands are organizations, or those of the publisher, the editors and the intended to offer a future alternative for developing new reviewers. Any product that may be evaluated in this article, or therapies, but more support and effort will be needed to claim that may be made by its manufacturer, is not guaranteed get results. or endorsed by the publisher. References 1. June CH, Sadelain M. Chimeric antigen receptor therapy. N Engl J Med 2. Sermer D, Brentjens R. CAR T-cell therapy: Full speed ahead. Hematol Oncol (2018) 4379(1):64–73. doi: 10.1056/NEJMra1706169 (2019) 37 Suppl 1(S1):95–100. doi: 10.1002/hon.2591 Frontiers in Immunology 13 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 3. Neelapu SS, Tummala S, Kebriaei P, Wierda W, Gutierrez C, Locke FL, et al. expressed by tisagenlecleucel. J Immunol Methods (2020) 476:112692. doi: Chimeric antigen receptor T-cell therapy - assessment and management of 10.1016/j.jim.2019.112692 toxicities. Nat Rev Clin Oncol (2018) 15(1):47–62. doi: 10.1038/nrclinonc.2017.148 26. Rafiq S, Hackett CS, Brentjens RJEngineering strategies to overcome the 4. Abreu TR, Fonseca NA, Gonçalves N, Moreira JN. Current challenges and current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol (2020) 17(3):147–67. emerging opportunities of CAR-T cell therapies. J Control Release (2020) 319:246– doi: 10.1038/s41571-019-0297-y 61. doi: 10.1016/j.jconrel.2019.12.047 27. Fujiwara K, Tsunei A, Kusabuka H, Ogaki E, Tachibana M, Okada N. Hinge 5. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. and transmembrane domains of chimeric antigen receptor regulate receptor Case report of a serious adverse event following the administration of T cells expression and signaling threshold. Cells (2020) 9(5):1182. doi: 10.3390/ transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther (2010) cells9051182 18(4):843–51. doi: 10.1038/mt.2010.24 28. Hanssens H, Meeus F, De Veirman K, Breckpot K, Devoogdt N. The 6. Atrash S, Bano K, Harrison B, Abdallah AO. CAR-T treatment for antigen-binding moiety in the driver’s seat of CARs. Med Res Rev (2021) 42(1): hematological malignancies. J Investig Med (2020) 68(5):956–64. doi: 10.1136/ 306–42. doi: 10.1002/med.21818 jim-2020-001290 29. Fabian F, Maucher M, Riester Z, Hudecek M. New targets and technologies 7. Ying Z, Huang XF, Xiang X, Liu Y, Kang X, Song Y, et al. A safe and potent for CAR-T cells. Curr Opin Oncol (2020) 32(5):510–7. doi: 10.1097/ anti-CD19 CAR T cell therapy. Nat Med (2019) 256. 201925(6):947–53. doi: CCO.0000000000000653 10.1038/s41591-019-0421-7 30. Huang G, Yu L, Cooper LJ, Hollomon M, Huls H, Kleinerman ES. 8. Grupp SA, Maude SL, Shaw PA, Aplenc R, Barrett DM, Callahan C, et al. Genetically modified T cells targeting interleukin-11 receptor a-chain kill Durable remissions in children with Relapsed/Refractory ALL treated with T cells human osteosarcoma cells and induce the regression of established osteosarcoma engineered with a CD19-targeted chimeric antigen receptor (CTL019). Blood lung metastases. Cancer Res (2012) 72(1):271–81. doi: 10.1158/0008-5472.CAN- (2015) 126(23):681–1. doi: 10.1182/blood.V126.23.681.681 11-2778 9. Neelapu SS, Locke FL, Bartlett NL, Lekakis LJ, Miklos DB, Jacobson CA, et al. 31. Han X, Cinay GE, Zhao Y, Guo Y, Xiaoyang Z, Wang P. Adnectin-based Axicabtagene ciloleucel CAR T-cell therapy in refractory Large b-cell lymphoma. N design of chimeric antigen receptor for T cell engineering. Mol Ther (2017) 25 Engl J Med (2017) 377(26):2531–44. doi: 10.1056/NEJMoa1707447 (11):2466–76. doi: 10.1016/j.ymthe.2017.07.009 10. Maude SL, Laetsch TW, Buechner J, Rives S, Boyer M, Bittencourt H, et al. 32. Wang Y, Xu Y, Li S, Liu J, Xing Y, Xing H, et al. Targeting FLT3 in acute Tisagenlecleucel in children and young adults with b-cell lymphoblastic leukemia. myeloid leukemia using ligand-based chimeric antigen receptor-engineered T cells. N Engl J Med (2018) 378(5):439–48. doi: 10.1056/NEJMoa1709866 J Hematol Oncol (2018) 11(1): 60. doi: 10.1186/s13045-018-0603-7 11. Catellà M, Caballero-Baños M, Ortı́z-Maldonado V, Gonzá lez-Navarro EA, 33. Saito S, Hasegawa A, Nagai M, Inada Y, Morokawa H, Nakashima I, et al. Suñé G, Antoñana-Vidó sola A, et al. Point-Of-Care CAR T-cell production (ARI- Mutated GM-CSF-Based CAR T-cells targeting CD116/CD131 complexes exhibit 0001) using a closed semi-automatic bioreactor: Experience from an academic enhanced anti-tumor effects against acute myeloid leukemia. Blood (2020) 136 phase I clinical trial. Front Immunol (2020) 11. doi: 10.3389/fimmu.2020.00482 (Supplement 1):36–7. doi: 10.1182/blood-2020-134395 12. O'Learly MC, Lu X, Huang Y, Lin X, Mahmood I, Przepiorka D, et al. FDA 34. Kubo H, Yagyu S, Nakamura K, Yamashima K, Tomida A, Kikuchi K, et al. Approval summary: Tisagenlecleucel for treatment of patients with relapsed or Development of non-viral, ligand-dependent, EPHB4-specific chimeric antigen refractory b-cell precursor acute lymphoblastic leukemia. Clin Cancer Res (2019) 25 receptor T cells for treatment of rhabdomyosarcoma. Mol Ther - Oncolytics (2021) (4):1142–6. doi: 10.1158/1078-0432.CCR-18-2035 20:646–58. doi: 10.1016/j.omto.2021.03.001 13. Bouchkouj N, Kasamon YL, de CRA, George B, Lin X, Lee S, et al. FDA 35. Schmidts A, Ormhøj M, Choi BD, Taylor AO, Bouffard AA, Scarfò I, et al. Approval summary: Axicabtagene ciloleucel for relapsed or refractory Large b-cell Rational design of a trimeric April-based CAR-binding domain enables efficient lymphoma. Clin Cancer Res (2019) 25(6):1702–8. doi: 10.1158/1078-0432.CCR-18- targeting of multiple myeloma. Blood Adv (2019) 3(21):3248–60. doi: 10.1182/ 2743 bloodadvances.2019000703 14. BREYANZI (lisocabtagene maraleucel). FDA. Available at: https://www.fda. 36. Zoine JT, Prince C, Story JY, Branella GM, Lytle AM, Fedanov A, et al. gov/vaccines-blood-biologics/cellular-gene-therapy-products/breyanzi- Thrombopoietin-based CAR-T cells demonstrate in vitro and in vivo cytotoxicity lisocabtagene-maraleucel. to MPL positive acute myelogenous leukemia and hematopoietic stem cells. Gene 15. TECARTUS (brexucabtagene autoleucel). FDA. Available at: https://www. Ther (2021) 29(5), 1–12. doi: 10.1038/s41434-021-00283-5 fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/tecartus- 37. Chen N, Xu Y, Mou J, Rao Q, Xing H, Tian Z, et al. Targeting of IL-10R on brexucabtagene-autoleuce. acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells. 16. Ortı́z-Maldonado V, Rives S, Castellà M, Alonso-Saladrigues A, Benı́tez- Blood Cancer J (2021) 11(8):144. doi: 10.1038/s41408-021-00536-x Ribas D, Caballero-Baños M, et al. CART19-BE-01: A multicenter trial of ARI- 38. Sauer T, Parikh K, Sharma S, Omer B, Sedloev D, Chen Q, et al. CD70- 0001 cell therapy in patients with CD19 + Relapsed/Refractory malignancies. Mol specific CAR T cells have potent activity against acute myeloid leukemia without Ther (2021) 29(2):636–44. doi: 10.1016/j.ymthe.2020.09.027 HSC toxicity. Blood (2021) 138(4):318–30. doi: 10.1182/blood.2020008221 17. ABECMA (idecabtagene vicleucel). FDA. Available at: https://www.fda.gov/ 39. Bagley SJ, O’Rourke DM. Clinical investigation of CAR T cells for solid vaccines-blood-biologics/abecma-idecabtagene-vicleucel. tumors: Lessons learned and future directions. Pharmacol Ther (2020) 205:107419. 18. Madduri D, Berdeja JG, Usmani SZ, Jakubowiak A, Agha M, Cohen AD, doi: 10.1016/j.pharmthera.2019.107419 et al. CARTITUDE-1: Phase 1b/2 study of ciltacabtagene autoleucel, a b-cell 40. Ma S, Li X, Wang X, Cheng L, Li Z, Zhang C, et al. Current progress in CAR- maturation antigen-directed chimeric antigen receptor T cell therapy, in T cell therapy for solid tumors. Int J Biol Sci (2019) 15(12):2548–60. doi: 10.7150/ Relapsed/Refractory multiple myeloma. Blood (2020) 136(Supplement 1):22–25. ijbs.34213 doi: 10.1182/blood-2020-136307 41. Zhang B-L, Qin D-Y, Mo Z-M, Li Y, Wei W, Wang Y-S, et al. Hurdles of 19. Shah NN, Fry TJ. Mechanisms of resistance to CAR T cell therapy. Nat Rev CAR-T cell-based cancer immunotherapy directed against solid tumors. Sci China Clin Oncol (2019) 16(6):372–85. doi: 10.1038/s41571-019-0184-6 Life Sci (2016) 59(4):340–8.39. doi: 10.1007/s11427-016-5027-4 20. Ruella M, Barrett DM, Kenderian SS, Shestova O, Hofmann TJ, Perazzelli J, 42. Du X, Williams DA. Interleukin-11: Review of molecular, cell biology, and et al. Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19- clinical use. Blood (1997) 89(11):3897–908. doi: 10.1182/blood.V89.11.3897 directed immunotherapies. J Clin Invest (2016) 126(10):3814–26. doi: 10.1172/ 43. Putoczki TL, Ernst M. IL-11 signaling as a therapeutic target for cancer. JCI87366 Immunotherapy (2015) 7(4):441–53. doi: 10.2217/imt.15.17 21. Petersen CT, Krenciute G. Next generation CAR T cells for the 44. Bishop MW, Janeway KA, Gorlick R. Future directions in the treatment of immunotherapy of high-grade glioma. Front Oncol (2019) 69. doi: 10.3389/ osteosarcoma. Curr Opin Pediatr (2016) 28(1):26–33. doi: 10.1097/ fonc.2019.00069 MOP.0000000000000298 22. CAR Signaling Networks. Available at: https://www.cellsignal.com/ 45. Lewis VO, Ozawa MG, Deavers MT, Wang G, Shintani T, Arap W, et al. The pathways/car-signaling-networks. interleukin-11 receptor a as a candidate ligand-directed target in osteosarcoma: 23. Karlsson H, Svensson E, Gigg C, Jarvius M, Olsson- Strömberg U, Savoldo Consistent data from cell lines, orthotopic models, and human tumor samples. B, et al. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Cancer Res (2009) 69(5):1995–9. doi: 10.1158/0008-5472.CAN-08-4845 Receptors. PLoS One (2015) 10(12):e0144787. doi: 10.1371/journal.pone.0144787 46. Zurita AJ, Troncoso P, Cardó -Vila M, Logothetis CJ, Pasqualini R, Arap W. 24. Wagner DL, Fritsche E, Pulsipher MA, Ahmed N, Hamieh M, Hedge M, Combinatorial screenings in patients: the interleukin-11 receptor alpha as a et al. Immunogenicity of CAR T cells in cancer therapy. Nat Rev Clin Oncol (2021) candidate target in the progression of human prostate cancer. Cancer Res (2004) 18(6):379–93. doi: 10.1038/s41571-021-00476-2 64(2):435–9. doi: 10.1158/0008-5472.CAN-03-2675 25. Potthoff B, McBlane F, Spindeldreher S, Sickert D. A cell-based 47. Sachdev E, Gong J, Rimel B, Mita M. Adnectin-targeted inhibitors: rationale immunogenicity assay to detect antibodies against chimeric antigen receptor and results. Curr Oncol Rep (2015) 17(8):35. doi: 10.1007/s11912-015-0459-8 Frontiers in Immunology 14 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 48. Simeon R, Chen Z. In vitro-engineered non-antibody protein therapeutics. 69. Feldman LA. Brain tumor-specific immune cells (IL13Ralpha2-CAR T cells) Protein Cell (2018) 9(1):3–14. doi: 10.1007/s13238-017-0386-6 for the treatment of leptomeningeal glioblastoma, ependymoma, or 49. Wang Z. ErbB receptors and cancer. Methods Mol Biol (2017) 1652:3–35. medulloblastoma. In: ClinicalTrials.gov 2022 (City of Hope Medical Center doi: 10.1007/978-1-4939-7219-7_1 Duarte, California, United States) 50. Olayioye MA, Neve RM, Lane HA, Hynes NE. The ErbB signaling network: 70. Shergalis A, Bankhead 3A, Luesakul U, Muangsin N, Neamati N. Current receptor heterodimerization in development and cancer. EMBO J (2000) 19 challenges and opportunities in treating glioblastoma. Pharmacol Rev (2018) 70 (13):3159–67. doi: 10.1093/emboj/19.13.3159 (3):412–45. doi: 10.1124/pr.117.014944 51. Emanuel SL, Engle LJ, Chao G, Zhu R-R, Cao C, Lin Z, et al. A fibronectin 71. Gao L, Wang Y, Ma W, Xing B. Glioblastoma cell differentiation trajectory scaffold approach to bispecific inhibitors of epidermal growth factor receptor and predicts the immunotherapy response and overall survival of patients. Aging (2020) insulin-like growth factor-I receptor. mAbs (2011) 3(1):38–48. doi: 10.4161/ 12(18):18297–321. doi: 10.18632/aging.103695 mabs.3.1.14168 72. Brown CE, Warden CD, Starr R, Deng X, Badie B, Yuan YC, et al. Glioma 52. Randolph ME, Cleary MM, Bajwa Z, Svalina MN, Young MC, Mansoor A, IL13Ra2 is associated with mesenchymal signature gene expression and poor et al. EphB4/EphrinB2 therapeutics in rhabdomyosarcoma. PLoS One (2017) 12(8): p a t ie n t p ro g n o s i s. P l oS O n e ( 2 0 13 ) 8 ( 1 0 ) :e 7 7 76 9 . d o i: 1 0 .1 3 71 / e0183161. doi: 10.1371/journal.pone.0183161 journal.pone.0077769 53. Berardi AC, Marsilio S, Rofani C, Salvucci O, Altavista P, Perla FM, et al. 73. Debinski W, Gibo DM, Hulet SW, Connor JR, Gillespie GY. Receptor for Up-regulation of EphB and ephrin-b expression in rhabdomyosarcoma. Anticancer interleukin 13 is a marker and therapeutic target for human high-grade gliomas. Res (2008) 28(2 A):763–9. Clin Cancer Res (1999) 5(5):985–90. 54. Noren NK, Lu M, Freeman AL, Koolpe M, Pasquale EB. Interplay between 74. Thaci B, Brown CE, Binello E, Werbaneth K, Sampath P, Sengupta S. EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth. Proc Natl Significance of interleukin-13 receptor alpha 2–targeted glioblastoma therapy. Acad Sci USA (2004) 101(15):5583. doi: 10.1073/pnas.0401381101 Neuro Oncol (2014) 16(10):1304–12. doi: 10.1093/neuonc/nou045 55. Kubo H, Yagyu S, Nakamura K, Yamashima K, Tomida A, Kikuchi K, et al. 75. Hsi LC, Kundu S, Palomo J, Xu B, Ficco R, Vogelbaum MA, et al. Silencing Development of non-viral, ligand-dependent, EPHB4-specific chimeric antigen IL-13Ra2 promotes glioblastoma cell death via via endogenous signaling. Mol receptor T cells for treatment of rhabdomyosarcoma. Mol Ther - Oncolytics (2021) Cancer Ther (2011) 10(7):1149–60. doi: 10.1158/1535-7163.MCT-10-1064 20:646–58. doi: 10.1016/j.omto.2021.03.001 76. Hershey GK. IL-13 receptors and signaling pathways: an evolving web. J 56. Kikuchi K, Tsuchiya K, Otabe O, Gotoh T, Tamura S, Katsumi Y, et al. Allergy Clin Immunol (2003) 111(4):677–90. doi: 10.1067/mai.2003.1333 Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma. 77. Kahlon KS, Brown C, Cooper LJN, Raubitschek A, Forman SJ, Jensen MC. Biochem Biophys Res Commun (2008) 365(3):568–74. doi: 10.1016/ Specific recognition and killing of glioblastoma multiforme by interleukin 13- j.bbrc.2007.11.017 zetakine redirected cytolytic T cells. Cancer Res (2004) 64(24):9160–6. doi: 10.1158/ 57. Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA, 0008-5472.CAN-04-0454 et al. The presence of a FLT3 internal tandem duplication in patients with acute 78. Brown CE, Starr R, Aguilar B, Shami AF, Martinez C, D'Apuzzo M, et al. myeloid leukemia (AML) adds important prognostic information to cytogenetic Stem-like tumor-initiating cells isolated from IL13Ra2 expressing gliomas are risk group and response to the first cycle of chemotherapy: analysis of 854 patients targeted and killed by IL13-zetakine-redirected T cells. Clin Cancer Res (2012) 18 from the united kingdom medical research council AML 10 and 12 trials. Blood (8):2199–209. doi: 10.1158/1078-0432.CCR-11-1669 (2001) 98(6):1752–9. doi: 10.1182/blood.V98.6.1752 79. Forman S. Cellular adoptive immunotherapy using genetically modified T- 58. Riccioni R, Diverio D, Riti V, Buffolino S, Mariani G, Boe A, et al. lymphocytes in treating patients with recurrent or refractory high-grade malignant Interleukin (IL)-3/granulocyte macrophage-colony stimulating factor/IL-5 glioma. In: ClinicalTrials.gov. (2011) (City of Hope Comprehensive Cancer Center) receptor alpha and beta chains are preferentially expressed in acute myeloid 80. Brown CE, Aguilar B, Starr R, Yang X, Chang W-C, Weng L, et al. leukaemias with mutated FMS-related tyrosine kinase 3 receptor. Br J Haematol Optimization of IL13Ra2-targeted chimeric antigen receptor T cells for (2009) 144(3):376–87. doi: 10.1111/j.1365-2141.2008.07491.x improved anti-tumor efficacy against glioblastoma. Mol Ther (2018) 26(1):31. 59. Chen L, Mao H, Zhang J, Chu J, Devine S, Caligiuri MA, et al. Targeting doi: 10.1016/j.ymthe.2017.10.002 FLT3 by chimeric antigen receptor T cells for the treatment of acute myeloid 81. Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, et al leukemia. Leukemia (2017) 31(8):1830. doi: 10.1038/leu.2017.147 Genetically modified T-cells in treating patients with recurrent or refractory 60. Kazi JU, Ronnstrand L. FMS-like tyrosine kinase 3/FLT3: From basic malignant glioma. In: ClinicalTrials.gov (2014) (City of Hope Medical Center) science to clinical implications. Physiol Rev (2019) 99(3):1433–66. doi: 10.1152/ 82. Garrett JT, Olivares G, Rinehart C, Granja-Ingram ND, Sá nchez V, physrev.00029.2018 Chakrabarty A, et al. Transcriptional and posttranslational up-regulation of 61. Grafone T, Palmisano M, Nicci C, Storti S. An overview on the role of FLT3- HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase. PNAS tyrosine kinase receptor in acute myeloid leukemia: biology and treatment. Oncol (2011) 108(12):5021–6. doi: 10.1073/pnas.1016140108 Rev (2012) 6(1):64–74. doi: 10.4081/oncol.2012.e8 83. Davies DM, Foster J, van der Stegen S, Parente-Pereria A, Chiapero-Stanke 62. Bhattacharya P, Thiruppathi M, Elshabrawy H, Alharshawi K, Kumar P, L, Delinassios GJ, et al. Flexible targeting of ErbB dimers that drive tumorigenesis Prabhakar BS. GM-CSF: An immune modulatory cytokine that can suppress by using genetically engineered T cells. Mol Med (2012) 18(1):565–76. doi: 10.2119/ autoimmunity. Cytokine (2015) 75(2):261–71. doi: 10.1016/j.cyto.2015.05.030 molmed.2011.00493 63. Nakazawa Y, Matsuda K, Kurata T, Sueki A, Tanaka M, Sakashita K, et al. 84. Klampatsa A, Achkova DY, Davies DM, Parente-Pereira AC, Woodman N, Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen Rosekilly J, et al. Intracavitary ‘T4 immunotherapy’ of malignant mesothelioma receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia. J using pan-ErbB re-targeted CAR T-cells. Cancer Lett (2017) 393:52–9. doi: Hematol Oncol (2016) 9(1):27. doi: 10.1186/s13045-016-0256-3 10.1016/j.canlet.2017.02.015 64. Ló pez AF, Shannon MF, Hercus T, Nicola NA, Cambareri B, Dottore M, 85. Parente-Pereira AC, Whilding LM, Brewig N, Stegen SJC, Davies DM, et al. Residue 21 of human granulocyte-macrophage colony-stimulating factor is Wilkie S, et al. Synergistic chemoimmunotherapy of epithelial ovarian cancer using critical for biological activity and for high but not low affinity binding - EMBO J. ErbB-retargeted T cells combined with carboplatin. J Immunol (2013) 191(5):2437– (1992) 11(3):909–16. doi: 10.1002/j.1460-2075.1992.tb05129.x 45. doi: 10.4049/jimmunol.1301119 65. Ghorashian S, Kramer AM, Onuoha S, Wright G, Bartram J, Richardson R, 86. Wingens M, Walma T, Van Ingen H, Stortelers C, Van Leeuwen JEM, Van et al. Enhanced CAR T cell expansion and prolonged persistence in pediatric Zoelen EJJ, et al. Structural analysis of an epidermal growth factor/transforming patients with ALL treated with a low-affinity CD19 CAR. Nat Med (2019) 25 growth factor-a chimera with unique ErbB binding specificity. J Biol Chem (2003) (9):1408–14. doi: 10.1038/s41591-019-0549-5 278(40):39114–23. doi: 10.1074/jbc.M305603200 87. Stortelers C, Van de Poll MLM, Lenferink AEG, Gadellaa MM, Van Zoelen 66. Shaffer DR, Savoldo B, Yi Z, Chow KKH, Kakarla S, Spencer DM, et al. T C, Van Zoelen EJJ. Epidermal growth factor contains both positive and negative Cells redirected against CD70 for the immunotherapy of CD70- determinants for interaction with ErbB-2/ErbB-3 heterodimers. Biochemistry positive malignancies. Blood (2011) 117(16):4304–14. doi: 10.1182/blood-2010- (2002) 41(13):4292–301. doi: 10.1021/bi012016n 04-278218 88. Wilkie S, Burbridge SE, Chiapero-Stanke L, Pereira ACP, Cleary S, van der 67. Brown CE, Badie B, Barish ME, Weng L, Ostberg JR, Chang WC, et al. Stegen SJC, et al. Selective expansion of chimeric antigen receptor-targeted T-cells Bioactivity and safety of IL13Ra2-redirected chimeric antigen receptor CD8+ T with potent effector function using interleukin-4. J Biol Chem (2010) 285 cells in patients with recurrent glioblastoma. Clin Cancer Res (2015) 21(18):4062– (33):25538–44. doi: 10.1074/jbc.M110.127951 72. doi: 10.1158/1078-0432.CCR-15-0428 89. Van Der Stegen SJ, Davies DM, Wilkie S, Foster J, Sosabowski JK, Burnet J, 68. Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, et al. et al. Preclinical in vivo modeling of cytokine release syndrome induced by ErbB- Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J retargeted human T cells: identifying a window of therapeutic opportunity? J Med (2016) 375(26):2561–9. doi: 10.1056/NEJMoa1610497 Immunol (2013) 191(9):4589–98. doi: 10.4049/jimmunol.1301523 Frontiers in Immunology 15 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 90. Van Schalkwyk MC, Papa SE, Jeannon JP, Urbano TG, Spicer JF, Maher J. therapy of autoimmune disease. Sci (2016) 353(6295):179–84. doi: 10.1126/ Design of a phase i clinical trial to evaluate intratumoral delivery of ErbB-targeted science.aaf6756 chimeric antigen receptor T-cells in locally advanced or recurrent head and neck 104. Bewarder M, Thurner L, Neumann F, Fadle N, Regitz E, Kemele M, et al. cancer. Hum Gene Ther Clin Dev (2013) 24(3):134–42. doi: 10.1089/humc.2013.144 BAR-bodies: B-cell receptor antigens as the targeting moiety of antibodies in 91. Papa S, van Schalkwyk M, Maher J. Clinical evaluation of ErbB-targeted substitution for the variable region of heavy and light chains. Blood (2018) 132 CAR T-cells, following intracavity delivery in patients with ErbB-expressing solid (Supplement 1):2940–0. doi: 10.1182/blood-2018-99-115348 tumors. Methods Mol Biol (2015) 1317:365–82. doi: 10.1007/978-1-4939-2727- 105. Yasunaga M. Antibody therapeutics and immunoregulation in cancer and 2_21 autoimmune disease. Semin Cancer Biol (2020) 64:1–12. doi: 10.1016/ 92. Palumbo A, Anderson K. Multiple myeloma. N Engl J Med (2011) 364 j.semcancer.2019.06.001 (11):1046–60. doi: 10.1056/NEJMra1011442 106. Bewarder M, Kiefer M, Will H, Olesch K, Moelle C, Stilgenbauer S, et al. 93. Cho SF, Lin L, Xing L, Li Y, Yu T, Andersin KC, et al. BCMA-targeting The b-cell receptor autoantigen LRPAP1 can replace variable antibody regions to therapy: Driving a new era of immunotherapy in multiple myeloma. Cancers target mantle cell lymphoma cells. HemaSphere (2021) 5(8):E620. doi: 10.1097/ (Basel) (2020) 12(6):1–29. doi: 10.3390/cancers12061473 HS9.0000000000000620 94. Kazandjian D. Multiple myeloma epidemiology and survival: A unique 107. Thurner L, Hartmann S, Fadle N, Kemele M, Bock T, Bewarder M, et al. malignancy. Semin Oncol (2016) 43(6):676–81. doi: 10.1053/j.seminoncol. LRPAP1 is a frequent proliferation-inducing antigen of BCRs of mantle cell 2016.11.004 lymphomas and can be used for specific therapeutic targeting. Leuk (2018) 331. 95. Kumar SK, Rajkumar SV, Dispenzieri A, Lacy MQ, Hayman SR, Buadi FK, 201833(1):148–58. doi: 10.1038/s41375-018-0182-1 et al. Improved survival in multiple myeloma and the impact of novel therapies. 108. Lionaki S, Panagiotellis K, Iniotaki A, Boletis JN. Incidence and Blood (2008) 111(5):2516–20. doi: 10.1182/blood-2007-10-116129 clinical significance of de novode novo donor specific antibodies after 96. Ali SA, Shi V, Maric I, Wang M, Stroncek DF, Rose JJ, et al. T Cells kidney transplantation. Clin Dev Immunol (2013) 2013:849835. doi: 10.1155/ expressing an anti-b-cell maturation antigen chimeric antigen receptor cause 2013/849835 remissions of multiple myeloma. Blood (2016) 128(13):1688–700. doi: 10.1182/ 109. Loupy A, Hill GS, Jordan SC. The impact of donor-specific anti-HLA blood-2016-04-711903 antibodies on late kidney allograft failure. Nat Rev Nephrol (2012) 8(6):348–57. doi: 97. Raje N, Berdeja J, Lin Y, Siegel D, Jagannath S, Deepu M, et al. Anti-BCMA 10.1038/nrneph.2012.81 CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma. N Engl J 110. Murad JM, Graber DJ, Sentman CL. Advances in the use of natural Med (2019) 380(18):1726–37. doi: 10.1056/NEJMoa1817226 receptor- or ligand-based chimeric antigen receptors (CARs) in haematologic 98. Lee L, Draper B, Philip B, Chin M, Galas-Filipowicz D, et al. An malignancies. Best Pract Res Clin Haematol (2018) 31(2):176–83. doi: 10.1016/ APRIL-based chimeric antigen receptor for dual targeting of BCMA and TACI j.beha.2018.03.003 in multiple myeloma. Blood (2018) 131(7):746–58. doi: 10.1182/blood-2017-05- 111. Nieba L, Honegger A, Krebber C, Plückthun A. Disrupting the 781351 hydrophobic patches at the antibody variable/constant domain interface: 99. Patel DR, Wallweber HJA, Yin J, Shriver SK, Marsters SA, Gordon NC, et al. improved in vivoin vivo folding and physical characterization of an engineered Engineering an APRIL-specific b cell maturation antigen *. J Biol Chem (2004) 279 scFv fragment. Protein Eng Des Sel (1997) 10(4):435–44. doi: 10.1093/protein/ (16):16727–35. doi: 10.1074/jbc.M312316200 10.4.435 100. Hymowitz SG, Patel DR, Wallweber HJA, Runyon S, Yan M, Yin JP, et al. 112. Zah E, Lin M-Y, Silva-Benedict A, Jensen MC. Chen YY. T cells expressing Structures of APRIL-receptor complexes: Like bcma, taci employs only a single CD19/CD20 bispecific chimeric antigen receptors prevent antigen escape by cysteine-rich domain for high affinity ligand binding. J Biol Chem (2005) 280 malignant b cells. Cancer Immunol Res (2016) 4(6):498–508. doi: 10.1158/2326- (8):7218–27. doi: 10.1074/jbc.M411714200 6066.CIR-15-0231 101. Philip B, Kokalaki E, Mekkaoui L, Thomas S, Straathof K, Flutter B, et al. A 113. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M. scFv highly compact epitope-based marker/suicide gene for easier and safer T-cell antibody: Principles and clinical application. Clin Dev Immunol (2012) therapy. Blood (2014) 124(8):1277–87. doi: 10.1182/blood-2014-01-545020 2012:980250. doi: 10.1155/2012/980250 102. Philip B, Kokalaki E, Mekkaoui L, Thomas S, Straathof K, Flutter B, et al. A 114. Chmielewski M, Hombach A, Heuser C, Adams GP, Abken H. highly compact epitope-based marker/suicide gene for easier and T Cell activation by antibody-like immunoreceptors: Increase in affinity safer T-cell therapy. Blood (2014) 124(8):1277–87. doi: 10.1182/blood-2014-01- of the single-chain fragment domain above threshold does not increase 545020 T cell activation against antigen-positive target cells but 103. Ellebrecht CT, Bhoj VG, Nace A, Choi EJ, Mao X, Cho MJ, et al. decreases selectivity. J Immunol (2004) 173(12):7647–53. doi: 10.4049/ Reengineering chimeric antigen receptor T cells for targeted jimmunol.173.12.7647 Frontiers in Immunology 16 frontiersin.org Ramı́rez-Chacón et al. 10.3389/fimmu.2022.932559 Continued Glossary BAR B-cell receptor antigen for reverse targeting sIg surface immunoglobulin BCR B-cell receptor CAR chimeric antigen receptor PV pemphigus vulgaris TA tumor antigen DLBCL diffuse large B-cell lymphoma TAA tumor-associated antigen MCL mantle cell lymphoma BALL B-cell acute lymphoblastic leukemia CHAR chimeric HLA antibody receptor FDA Food and Drug Administration DSA donor-specific antibody EMA European Medicines Agency HAMA human anti-mouse antibody BCMA B-cell maturation antigen VH variable heavy chain AEMPS Agencia Española del Medicamento y Productos Sanitarios (Spanish VL variable light chain drug agency) scFv single-chain variable fragment moAb monoclonal antibody OS osteosarcoma EGFR epidermal growth factor receptor FLT3L FMS-like tyrosine kinase 3 ligand FLT3 FMS-like tyrosine kinase 3 AML acute myeloid leukemia GM- granulocyte-monocyte colony-stimulating factor CSF GMR granulocyte-monocyte colony-stimulating factor receptor JMML juvenile myelomonocytic leukemia TPO thrombopoietin MPLR myeloproliferative leukemia receptor EPHB4 ephrin type-B receptor 4 RMS rhabdomyosarcoma APRIL a proliferation-inducing ligand TACI transmembrane activator and calcium-modulator and cyclophilin ligand interactor MM multiple myeloma HNSCC head and neck squamous cell carcinoma 10Fn3 10th type III domain of human fibronectin TNBC triple-negative breast cancer ITD internal tandem duplication TKD tyrosine kinase domain WT wild type CNS central nervous system GBM glioblastoma multiforme GSC stem-like cancer-initiating cell IL13Ra2 interleukin-13 receptor a2 TK tyrosine kinase EOC epithelial ovarian cancer EGF epidermal growth factor TGF-a transforming growth factor a CRS cytokine release syndrome PC plasma cell PI proteasome inhibitor IMiD immunomodulatory drug RRMM relapsed/refractory multiple myeloma TNF tumor necrosis factor CAAR chimeric autoantibody receptor (Continued) Frontiers in Immunology 17 frontiersin.org