CAR T-cell therapy for pleural mesothelioma: rationale, preclinical development, and clinical trials

Abstract

The aim of adoptive T-cell therapy is to promote tumor-infiltrating immune cells following the transfer of either tumor-harvested or genetically engineered T lymphocytes. A new chapter in adoptive T-cell therapy began with the success of chimeric antigen receptor (CAR) T-cell therapy. T cells harvested from peripheral blood are transduced with genetically engineered CARs that render the ability to recognize cancer cell-surface antigen and lyse cancer cells. The successes in CAR T-cell therapy for B-cell leukemia and lymphoma have led to efforts to expand this therapy to solid tumors. Herein, we discuss the rationale behind the preclinical development and clinical trials of T-cell therapies in patients with malignant pleural mesothelioma. Furthermore, we highlight the ongoing investigation of combination immunotherapy strategies to synergistically potentiate endogenous as well as adoptively transferred immunity.

1. Introduction

Malignant pleural mesothelioma (MPM) is an aggressive malignancy, with an overall median survival of 8 months [1]. In patients receiving trimodality therapy for resectable MPM, consisting of chemotherapy, surgery, and radiation, median survival is 20 months [2]. Translational studies provided initial rationale for applying immunotherapy to improve outcomes for patients with MPM. Increased tumor-infiltrating lymphocytes (TILs) have been associated with improved survival in patients with MPM [3–4]. By analyzing a large cohort of MPM tumors, we previously observed that an increased inflammatory response in the tumor and stroma [5] and the ratio of immunosuppressive macrophages to immune effector lymphocytes [6] were associated with improved survival in patients with MPM [7]. Many recent investigations have built towards understanding how best to harness the immune response to MPM to improve prognosis.

Immune checkpoint blockade therapy has shown some promise for improving disease responses however the demonstrated clinical impact in trials to date have been limited. Single-agent programmed cell death protein 1 (PD-1) blockade (pembrolizumab) [8] or cytotoxic T lymphocyte antigen 4 blockade (tremelimumab) [9] was not superior to chemotherapy in patients with MPM. In the phase III CheckMate 743 trial, dual-agent immunotherapy with ipilimumab and nivolumab was associated with a 4-month survival benefit in all comers compared with chemotherapy (18.1 vs. 14.1 months), in patients with unresectable MPM [10] with the most clinically significant benefit in those with non-epithelioid histologies.

Adoptive cell therapy is a form of immunotherapy with an evolving role in cancer care. Adoptive cell therapy promotes tumor-infiltrating immune cells by redirecting the patient’s own immune cells, such as T cells, to target cancer cells. Adoptive transfer of ex vivo cultured and expanded TILs isolated from a resected tumor specimen has led to reliable antitumor activity only in melanomas, thus far limiting the applicability of this approach to other tumors [11]. Alternatively, T cells can be genetically engineered to express a chimeric antigen receptor (CAR) to recognize a cell-surface antigen and kill cancer cells. CAR T-cell therapy with a CD19-targeted approach has produced remission rates of >80% in patients with adult [12] and pediatric [13] acute lymphoblastic leukemia. CD19-targeted therapy has also achieved sustained remissions in patients with B-cell lymphoma [14–16] and mantle cell lymphoma [17]. In addition, CD22 CAR T-cell therapy was successful in patients with B-cell acute lymphoblastic leukemia [18], as was B-cell maturation antigen CAR T-cell therapy in patients with multiple myeloma [19]. Overall, at present, four CD19 CAR T-cell therapies have been approved by the US Food and Drug Administration (FDA) [20].

Despite the success of CAR T-cell therapy in hematologic malignancies, translating this to solid tumors, such as MPM, remains challenging owing to a variety of obstacles. These obstacles include heterogeneity in tumor antigen expression, an immunosuppressive tumor microenvironment, and inhibition of immune cell trafficking, all of which can limit CAR T-cell function. Building on studies of the tumor immune microenvironment and immunotherapy, we developed and translated mesothelin (MSLN)–targeted CAR T-cell therapy for MPM, non-small cell lung cancer, and triple-negative breast cancer to phase I/II clinical trials (NCT02414269, NCT02792114). Mesothelin is a cell-surface antigen expressed on solid-tumor cells. Recognizing the importance of overcoming tumor-mediated immune inhibition, we developed and translated a next-generation CAR T-cell therapy that combats immunosuppression by the use of a PD-1 dominant negative receptor (DNR)—this approach is currently being evaluated in an ongoing phase 1 clinical trial (NCT04577326). Herein, we discuss the rationale behind and the preclinical development of adoptive T-cell therapies and describe ongoing clinical trials that specifically focus on CAR T-cell therapies for patients with MPM.

2. Modes of tumor killing by antigen-targeting immunotherapies

Multiple tumor-associated antigen-targeting immunotherapy approaches are in development for both hematological and solid malignancies (Figure 1). Antibody based therapy is one such approach. Antibody-mediated cellular cytotoxicity is primarily achieved by natural killer cells, in addition to other myeloid cells (Figure 1A). The antibody Fc domain binds to Fc receptors on the effector cell leading to the release of cytokines that kill the target cells. An additional mechanism of antibody-mediated killing is antibody-dependent phagocytosis, in which phagocytes recognize target cells that are opsonized by antibodies. Antibody-dependent phagocytosis has the potential to contribute to antigen presentation of tumor antigens, leading to further T cell–mediated cytotoxicity [21]. Rituximab, an antibody-based therapy targeted against CD20, has shown success in patients with non-Hodgkin lymphoma [22].

Fig 1.

Modes of tumor killing by antigen-targeting immunotherapies. A, Antibody-mediated cytotoxicity. Plasma cells produce tumor antigen–specific IgG antibodies, which bind to the tumor cells. An Fc-receptor (FcR) on a natural killer cell recognizes the Fc portion of the antibody, leading to release of cytolytic granules and killing of tumor cells. B, Bispecific T-cell engager (BiTE)–mediated cytotoxicity. A BiTE is a synthetic molecule with two single-chain variable fragments (scFvs) with different specificities that are linked together. One scFv is specific to CD3 on a T cell, and the other scFv binds specifically to an antigen on a tumor cell. A BiTE engages both the tumor and the T cell, leading to T-cell activation, release of cytolytic granules, and killing of tumor. C, Chimeric antigen receptor (CAR) T cell–mediated cytotoxicity. The CAR T cell recognizes a cell surface–expressed antigen, leading to its activation, release of cytolytic granules, and killing of tumor. D, T cell–mediated cytotoxicity. An antigen-presenting cell (dendritic cell or macrophage) engulfs a tumor-associated antigen from an apoptotic tumor cell and presents it to a naive T cell. The peptide–major histocompatibility complex (MHC) molecule binds to a specific T-cell receptor (TCR), leading to T-cell activation. In addition, the antigen-presenting cell provides a costimulatory signal through its B7 molecule to the T cell through a CD28 molecule. This leads to activation and proliferation of the T cell, which recognizes a peptide-MHC on the tumor, leading to killing of tumor cells.

A bispecific T-cell engager (BiTE) is a synthetic antibody designed to induce T cell–mediated killing of an antigen target (Figure 1B). A BiTE consists of two binding regions linked by a flexible peptide: a single-chain variable fragment (scFv) domain targeting a tumor antigen and a second scFv recognizing CD3 on a T cell. Linking the T cell and the tumor cell by the use of a BiTE leads to T cell–mediated killing [23]. At present, blinatumomab, an anti-CD19 BiTE, has been approved by the FDA for acute lymphoblastic leukemia [24]. In addition, HPN536, a tri-specific T-cell activating construct (TriTAC), consisting of three binding domains; mesothelin, CD3ε on T cells, and a binding domain for serum albumin (to extend half-life), has demonstrated antitumor efficacy in in vivo models of pancreatic, ovarian, and non-small cell lung cancer [25].

CAR T cells can be engineered to produce BiTEs; in a study by Choi et al., the production of EGFR-BiTEs by EGFRvIII specific CAR T cells improved efficacy against heterogeneous tumor by recruiting endogenous T-cell activity against tumor cells [26].

T-cell mediated cytotoxicity, and CAR T-cell mediated cytotoxicity are other mechanisms employed by antigen-targeting immunotherapies. In T cell–mediated cytotoxicity, the T-cell receptor (TCR) binds to a tumor peptide–major histocompatibility complex (MHC). MHC class I is expressed on every nucleated cell, and MHC class II is expressed on antigen-presenting cells (APCs). The activation of a naïve T cell requires antigen presentation by an APC (such as a dendritic cell or a macrophage) [27]. A costimulatory signal provided by CD28 on the T cell and by a B7 family ligand on the APC is also required for activation [28]. Once activated, the TCR complex mediates T-cell cytotoxicity against both surface and intracellular antigens, unlike CAR T cells, which can only recognize antigens expressed on the cell surface (Figure 1C, 1D). However, TCR-mediated cytotoxicity requires antigen processing, presentation, and peptide-loaded MHC expression by the target cell, and loss of MHC class I expression is a known mechanism of tumor escape from TCR-mediated cytotoxicity [29]. To overcome this barrier, T cells can be genetically engineered to express either an engineered TCR targeted against a tumor-associated MHC peptide or a CAR targeting tumor antigen expressed on the tumor cell surface [11]. Whereas engineered TCR T-cell therapies require that a patient’s tumor expresses the target antigen in the specific MHC haplotype that matches the engineered TCR, CAR T-cell therapies do not have this restriction and can be designed to target any antigen that is expressed on a tumor cell surface.

3. CARs

CARs are synthetic membrane receptors that redirect T lymphocytes to recognize and bind to a target ligand. The CAR consists of an scFv composed of the antigen-specific variable heavy and light chains with a flexible linker [30]. The CAR also includes a transmembrane domain and an intracellular signaling domain. The design of a CAR begins with the selection of a tumor-associated antigen target, a costimulatory domain(s), and additional engineering strategies to overcome anticipated barriers posed by the tumor microenvironment (Figure 2).

Fig 2.

Types of chimeric antigen receptors (CARs). A, Antigen targets for malignant pleural mesothelioma (MPM) currently in clinical trials are mesothelin and fibroblast activating protein. Targets in preclinical investigation include ErbB family receptors, chondroitin sulfate proteoglycan 4, and the oncofetal glycoprotein 5T4. B, The evolution of CAR designs, showing a first-generation CAR with a single-chain variable fragment (scFv) and a CD3ζ signaling domain, followed by a second-generation CAR with an scFv and a costimulatory domain along with a CD3ζ signaling domain, and a third-generation CAR with an scFv and two costimulatory domains along with a CD3ζ signaling domain. C, Cytokine-secreting CAR T cells. The CAR T cells are designed to produce IL-12 upon their activation in the tumor microenvironment, leading to recruitment of endogenous antitumor immune cells. D, Chemokine receptor–expressing CAR T cells. Chemokine receptor–expressing CAR T cells traffic to the tumor microenvironment, guided by the chemokine gradient created by the tumor cells. E, Dominant negative receptor (DNR). A programmed cell death protein 1 (PD-1) DNR binds to the programed death ligand 1 (PD-L1) inhibitory ligand on the tumor cell. The PD-1 DNR lacks an intracellular signaling domain, preventing transmission of an inhibitory signal to the T cell.

3.1. Cell-surface antigen targets for CARs

The success of CAR T-cell therapy requires the selection of a candidate tumor-associated antigen target that is overexpressed on cancer cells in a high percentage of patient tumors, with limited or no expression on normal tissue. In multiple B-cell malignancies, CD19 is homogenously expressed, and its expression is limited to the hematopoietic B-cell lineage that can result in B-cell aplasia, which is a manageable side effect treated with immunoglobulin replacement [31, 32]. Potential targets for CAR T-cell therapy in solid tumors include overexpressed differentiation antigens such as MSLN, human epidermal growth factor receptor 2 (HER2/ERBB2), tumor-associated stroma-expressed fibroblast activating protein (FAP), cancer-testis antigen-derived peptides (MAGE), and altered gene products arising from genetic mutations or altered splicing (e.g. EGFRvIII) [33, 34].

Antigen targeting with CARs is subject to limitations due to unintended targeting of normal tissue. In “on-target/off-tumor” CAR activity, the expression of the target-antigen on healthy tissue may lead to CAR mediated toxicity as has been observed in clinical trials of CAR T-cell therapy in solid tumors. This includes lethal pulmonary toxicity and cytokine storm in an ERBB2-targeted CAR, which were attributed to target-antigen expression on healthy lung tissue [35]; hepatic toxicity in a carbonic anhydrase IX–targeted CAR, which was attributed to antigen expression on healthy bile duct epithelial cells [36]; and pulmonary toxicity in a CEACAM5-targeted CAR T-cell trial, which was attributed to antigen expression on healthy lung epithelium [37]. Along with on-target/off-tumor toxicity, recognition of structurally related but “off-target” peptides on normal tissues may occur. For example, T cells engineered with a MAGE-A3 TCR caused fatal cardiac toxicity owing to recognition of the unrelated peptide titin [38]. As such, a CAR T-cell target for solid tumors must be carefully chosen to optimize efficacy and avoid on-target/off-tumor, and off-target toxicity.

3.1.1. Mesothelin

MSLN is a glycoprotein attached to the plasma membrane by phosphatidyl inositol [39] and is expressed only at low levels in mesothelial cells of the pleura, peritoneum, and pericardium [40]. MSLN-knockout mice demonstrate no anatomic or histologic abnormalities, indicating that MSLN is not essential for normal development [41].

MSLN is expressed in 85% to 90% of malignant mesothelioma tumors, as well as in additional cancers, including lung and esophageal cancer [33]. We have shown that in vitro MSLN expression promotes matrix metalloproteinase secretion, leading to increased invasion and migration of tumor cells and decreased survival in vivo [42]. MSLN binds to CA125/MUC16 [43], which may contribute to metastasis of MSLN-expressing tumors. We have reported that MSLN expression is associated with increased tumor stage in MPM [42] and decreased survival in lung adenocarcinoma [44, 45] and is a marker of neoplastic transformation in Barrett’s esophagus and esophageal adenocarcinoma [46].

The selective overexpression of MSLN on multiple cancers and its association with an aggressive phenotype led us to investigate MSLN as a target of CAR T-cell therapy in patients with MPM (Figure 2A). In an orthotopic mouse model of MPM, MSLN-targeted CAR T cells induced long-term remissions, an effect that was enhanced by regional delivery [47] and checkpoint blockade [48].

3.1.2. FAP

FAP is expressed in the tumor stroma of multiple epithelial tumors, including all histologic subtypes of mesothelioma [49], with limited expression in normal adult tissue [50]. In two in vivo studies, FAP-directed CAR T cells inhibited the growth of solid tumors [51, 52]; one mechanism of action was the stimulation of antitumor responses by native T cells [51]. A single in vivo study found lethal bone toxicity and cachexia with the use of FAP-directed CAR T cells [53]; however, this finding was not replicated in two subsequent studies [51, 52].

3.1.3. Additional antigen targets

Additional targets investigated in preclinical studies include ErbB family receptors, the oncofetal glycoprotein 5T4, and chondroitin sulfate proteoglycan 4 (CSPG4). In a study of MPM histologic samples, at least one ErbB family receptor was expressed on 88% of tumors, prompting the investigation of a CAR T-cell construct directed at multiple ErbB family receptors. This CAR T-cell construct inhibited tumor growth in vivo [54]. 5T4 has also been proposed as a target for CAR T-cell therapy for mesothelioma on the basis of its expression in multiple cancer types, including MPM [55], and its low expression on normal adult tissue [56]. Finally, CSPG4 has been proposed as a potential target for CAR T cells in MPM [57] on the basis of its expression in tumors [58] and the findings of in vitro cytotoxicity of CSPG4 CAR T cells against an MPM cell line [59].

3.2. Reengineering CD3ζ and costimulatory domains

The first generation of CAR T cells included a cytoplasmic CD3-ζ signaling domain but no costimulatory domains, which resulted in poor proliferative response [60] and limited clinical efficacy [37]. In contrast, second-generation CAR T cells contain one costimulatory domain and third-generation CAR T cells contain two costimulatory domains each, along with a CD3-ζ signaling domain (Figure 2B). The addition of an intracellular costimulatory domain improved the proliferation and persistence of CAR T cells [60]. All FDA-approved CAR T-cell therapies include either a CD28 [61] or a 4-1BB [32] costimulatory domain. Additional costimulatory domains tested in preclinical trials include MyD88/CD40 [62], OX40 [63], ICOS [64], and KIR2DS2/DAP12 [65]. CD28 costimulation differentiates CAR T cells into an effector memory phenotype, and 4-1BB costimulation differentiates them into a central memory phenotype [66]. Hence, the selection of an ideal costimulatory domain is context dependent.

In an additional effort to improve the efficacy of CAR T cells, it was hypothesized that redundant CD28 and CD3ζ may increase T-cell exhaustion. The use of a single immunoreceptor tyrosine-based activation motif improved tumor eradication—compared with CAR T cells with all three immunoreceptor tyrosine-based activation motif residues in CD3ζ—and resulted in increased central memory cells and decreased terminally differentiated cells and markers of exhaustion [67]. We have advanced the 1XX CD3ζ strategy into a phase I clinical trial with an MSLN-targeted CAR for patients with MPM [68]. In a similar approach, investigators from the University of Pennsylvania showed that a single amino acid residue in CD28 drove T-cell exhaustion and hindered the persistence of CD28-based CAR T cells. Changing this asparagine to phenylalanine (CD28-YMFM) promoted durable antitumor control, reduced T-cell differentiation and exhaustion, and increased skewing toward Th17 cells [69].

3.3. Armored CARs

In the development of next-generation CAR T cells, investigators “armored” CARs with cytokines, chemokine receptors, or DNRs for checkpoint inhibition, which empowered them to better infiltrate solid tumors and resist the immunosuppressive microenvironment.

3.3.1. Cytokine-secreting CARs

Armored CAR T cells engineered to produce IL-12 upon activation (Figure 2C) improved the efficacy of CEA-targeting CAR T cells [70], MUC16-targeting CAR T cells [71], and CAR T cells targeted toward VEGFR-2 on the tumor vasculature [72]. This effect was mediated by autocrine signaling of armored CAR T cells [71] and IL-12–mediated stimulation of an antitumor response by endogenous macrophages [70]. However, in a clinical trial of TILs with inducible IL-12, dose-dependent toxicity (including hepatic and hemodynamic instability) was observed, indicating the need for further investigation of the promoter sequence and other aspects of IL-12 expression [73]. Armored CAR T cells engineered to produce IL-18 were shown to improve antitumor efficacy in syngeneic solid-tumor models (through both autocrine signaling and stimulation of an endogenous immune response) [74] and reduce the number of immunoinhibitory M2 macrophages and T-regulatory cells in the tumor [75]. Armored CARs producing IL-15 have been shown to improve antitumor activity in vivo against glioblastoma [76] and neuroblastoma [77].

3.3.2. Chemokine receptor expression on CAR T cells

One barrier to CAR T-cell trafficking to the tumor site is a chemokine–chemokine receptor mismatch, with CAR T cells not expressing receptors to the chemokines produced by tumors [78]. On the basis of the observation that MPM cells produce the cytokine CCL2, MSLN-targeted CAR T cells were transduced with the CCL2 receptor (CCR2). CCR2-expressing cells had increased trafficking to the tumor and increased efficacy [79] (Figure 2D). Increased CAR T-cell homing and antitumor efficacy were also seen in GD2-targeted CAR T cells in an in vivo model of neuroblastoma [80], as well as in TCR-transduced T cells in an in vivo model of lung cancer [81]. In addition, the administration of an oncolytic virus engineered to induce the cytokine CXCL11 on tumor cells also caused an increase in CAR T-cell trafficking to tumor and increased antitumor efficacy [78].

3.3.3. T-cell intrinsic checkpoint blockade

Despite impressive responses with immune checkpoint inhibitor (ICI) agents, the majority of solid tumor patients who received checkpoint blockade did not have an objective response [82]. One possibility was that patients with a nonresponse lacked a preexisting immune infiltrate that could be reactivated by ICI agents. CAR T-cell therapy has the potential to provide such an infiltrate. In addition, we have shown that CAR T cells were subject to exhaustion after repeated antigen exposure and were inhibited by programed death ligand 1 (PD-L1)–expressing tumor cells [48]. In a preclinical orthotopic model of MPM, our laboratory showed that administration of a PD-1–blocking antibody was able to “rescue” exhausted CAR T cells and induce tumor regressions. To avoid repeat administration of anti–PD-1 antibody therapy and associated off-target effects, we implemented a cell-intrinsic strategy and designed a PD-1 DNR consisting of a PD-1 receptor without an intracellular signaling domain, designed to bind PD-L1 ligands and avoid inhibitory signaling by the endogenous receptor (Figure 2E). CAR T cells with a PD-1 DNR were able to induce long-term tumor suppression without repeat dosing [48]—this CAR construct is under investigation in a phase 1 clinical trial for patients with MPM (NCT04577326) [68]. Other methods to augment CAR T function through intrinsic immune checkpoint resistance include CAR T cells engineered to secrete anti–PD-L1 antibodies [83] and an anti–PD-1 scFv [84] and a CAR T-cell switch receptor construct containing the extracellular domain of PD-1 and the cytoplasmic costimulatory domain of CD28 [85]; these have improved antitumor efficacy in vivo.

Genome-editing approaches have also been adopted. In particular, CAR T cells in which the PD-1 [86] and TGF-β receptor II [87] genes were disrupted with CRISPR/Cas9 gene editing have demonstrated improved efficacy in vivo.

4. Regional delivery as a strategy to overcome barriers in solid-tumor cell therapy

Multiple other barriers to CAR T cell efficacy exist including inefficient tumor infiltration following systemic delivery due to pulmonary sequestration of CAR T cells, checkpoint inhibition, tumor antigen heterogeneity, and immunosuppressive cells, including tumor-associated macrophages (Figure 3A, 3B).

Fig 3.

Immunosuppressive tumor microenvironment of malignant pleural mesothelioma. Multiplex immunofluorescence imaging of malignant pleural mesothelioma, showing [A] heterogeneous mesothelin antigen expression in tumor and [B] an immunosuppressive tumor microenvironment, including inhibitory tumor-associated macrophages interacting with T cells.

One strategy to improve CAR T-cell trafficking to and infiltration of, the tumor is regional administration of CAR T cells. Using an orthotopic model of MPM, we demonstrated that intrapleural delivery of CAR T cells was more effective than intravenous administration, leading to eradication of the tumor and inducing long-term remissions. Regionally administered CAR T cells efficiently infiltrate tumors and circulate systemically within 24 h; antigen-activated CD4 CAR T cells provide enhanced helper function to CD8 CAR T cells and achieve better proliferation, even with a 30-fold lower dose of cells [47]. Intrapleural delivery was adopted in our clinical trial of MSLN-targeted CAR T cells [88, 89] and in a trial of FAP-directed CAR T cells in patients with MPM [90]. Regional intraperitoneal administration of CAR T cells has also demonstrated increased efficacy in in vivo models of peritoneal ovarian cancer [91] and peritoneal metastasis from colorectal cancer [92], as has regional portal vein delivery in a model of intrahepatic metastasis [93]. Similarly, intratumoral delivery in subcutaneous models of squamous cell carcinoma [87] and melanoma [94] and intracerebroventricular administration in a model of breast cancer metastatic to the brain [95] resulted in improved efficacy of CAR T cells. CAR T cells administered in an implanted polymer scaffold had improved proliferation, persistence, and antitumor efficacy, compared with both intravenous and intratumoral injection, in an in vivo model of pancreatic cancer [96]. Finally, in a phase I clinical trial, intracavitary and intraventricular administration of IL13Rα2-targeted CAR T cells in a patient with glioblastoma resulted in a transient complete response [97].

5. CAR T-cell therapy trials for patients with MPM

A summary of CAR T-cell therapy clinical trials for patients with MPM is provided in Table 1. In a University of Pennsylvania clinical trial, T cells were engineered to transiently express a CAR TCR with an anti-MSLN murine scFv, resulting in a transient partial response in 1 patient with mesothelioma and stable disease in 1 patient with pancreatic cancer. In addition, the researchers found evidence that the CAR T-cell therapy induced an endogenous humoral antitumor response, which was indicated by an immunoblot assay that showed antitumor antibodies [98]. However, 1 of 4 patients experienced anaphylactic shock requiring cardiopulmonary resuscitation during their third infusion of mRNA-transduced T cells, which was attributed to a reaction against repeated infusions of the murine scFv [99]. However, the trial did not show evidence of on-target/off-tumor toxicity [98]. A subsequent University of Pennsylvania clinical trial evaluating lentiviral-transduced, MSLN-targeted CAR T cells in patients with MPM, pancreatic cancer, and ovarian cancer demonstrated a best response of stable disease in 11 of 15 patients, without on-target/off-tumor toxicity [100]. A trial of MSLN-targeted CAR T cells with a human scFv, administered through intravenous or intrapleural administration, is currently ongoing at the University of Pennsylvania (NCT03054298).

Table 1.

CAR T-cell therapy clinical trials to treat malignant pleural mesothelioma

Target Antigen	CAR T-Cell Product	Costimulatory Domain	NCT Identifier, Phase	Clinical Site	Combination Therapy	Delivery	Patients, No.	Reference(s)
Mesothelin	mRNA transduced, mouse scFv	4-1BB	NCT01355965, phase I	University of Pennsylvania		Intravenous	4	[98], [99]
	Lentiviral transduced, mouse scFv	4-1BB	NCT02159716, phase I	University of Pennsylvania	With and without cyclophosphamide pretreatment	Intravenous	15	[100]
	Lentiviral transduced, human scFv		NCT03054298, phase I	University of Pennsylvania		Intravenous and intrapleural
	iCasp9M28z	CD28	NCT02414269, phase I/II	Memorial Sloan Kettering Cancer Center	With and without cyclophosphamide preconditioning, with and without pembrolizumab	Intrapleural	19	[88], [89]
	M28z-1XX-PD1DNR	CD28	NCT04577326, phase I	Memorial Sloan Kettering Cancer Center	Cyclophosphamide	Intrapleural	1	[68]
	Anti-mesothelin CAR	CD28	NCT01583686, phase I/II	National Cancer Institute	Fludarabine, cyclophosphamide, aldesleukin	Intravenous	15
	mRNA transduced PBMC		NCT03608618, phase I	MaxCyte	Cyclophosphamide	Intraperitoneal	11	[102]
	TRuC		NCT03907852, phase I/II	TCR2 Therapeutics	With and without cyclophosphamide and fludarabine pretreatment, with and without pembrolizumab	Intravenous	5	[103]
	Anti–PD-1 nanobodies		NCT04489862, phase I	Wuhan Union Hospital	Cyclophosphamide	Intravenous
	Anti–PD-1 antibody		NCT03615313, phase I/II	Shanghai Cell Therapy Research Institute	Fludarabine, cyclophosphamide	Intravenous
			NCT02580747, phase I	Chinese PLA General Hospital
			NCT02930993, phase I	China Meitan General Hospital	Cyclophosphamide	Intravenous
			NCT03638206, phase I/II	The First Affiliated Hospital of Zhengzhou University	Fludarabine, cyclophosphamide	Intravenous
FAP	FAP-specific redirected T cells		NCT01722149, phase I	University Hospital of Zurich	Neoadjuvant chemotherapy	Intrapleural		[90], [101]

Abbreviations: CAR, chimeric antigen receptor; FAP, fibroblast activating protein; NCT, National Clinical Trial; PBMC, peripheral blood mononuclear cell; PD-1, programmed cell death protein 1; PD1DNR, PD-1 dominant negative receptor; scFv, single-chain variable fragment; TRuC, T-cell receptor fusion construct.

A phase I clinical trial of 3 patients who received FAP-targeted CAR T cells through intrapleural injection found that the treatment was safe, without treatment-related toxicity. [90, 101].

A National Cancer Institute study of MSLN-targeted CAR T cells in MSLN-expressing cancers (NCT01583686), including MPM, was stopped because of slow accrual and low efficacy. An ongoing trial of mRNA-transduced unexpanded peripheral blood mononuclear cells that expressed an anti-MSLN CAR administered via intraperitoneal injection for peritoneal mesothelioma and ovarian cancer has demonstrated a best response of stable disease, with no CAR T cell–related toxicities grade >2 [102]. In addition, an anti-MSLN TCR fusion construct consisting of an antigen-binding domain fused to a CD3-ε subunit that incorporates into the native TCR is being investigated in an ongoing clinical trial. In results reported for 5 patients (4 with mesothelioma and 1 with ovarian cancer), the TCR fusion construct was associated with an unconfirmed partial response or stable disease, with 1 instance of grade 3 toxicity attributed to cytokine release syndrome [103]. Additional clinical trials in progress for MSLN-targeted CAR T cells in mesothelioma include CAR T cells secreting anti–PD-1 antibodies (NCT03615313) and nanobodies (NCT04489862).

At Memorial Sloan Kettering Cancer Center, we are conducting a clinical trial of MSLN-targeted CAR T-cell therapy administered intrapleurally for patients (N=21) with MSLN-expressing malignant pleural disease from MPM (19 patients), lung cancer (1 patient), and breast cancer (1 patient). In this study, 14 patients received anti–PD-1 therapy after administration of CAR T cells. By the interpretation of the investigator, this study showed complete metabolic response by positron emission tomography in 2 patients, with no CAR T cell–related toxicities grade >2 observed [88, 89].

6. Future directions of combination therapies

6.1. Checkpoint blockade and CAR T-cell therapy

Checkpoint blockade treatments involve administration of ICI agents against PD-1, PD-L1, or cytotoxic T lymphocyte antigen 4. The successes with ICI therapy come from reinvigorating T cells that are exhausted. Activation induces expression and accumulation of checkpoint molecules on the CAR T-cell surface; when these checkpoint molecules interact with their ligands present in the microenvironment, the T cells become exhausted. Attenuating this interaction between a checkpoint molecule and its ligands by the use of ICI agents helps to improve the persistence and efficacy of CAR T-cell therapies (Figure 4A). Correlative studies from our clinical trial [88, 89] raised the possibility that combination therapy induced an endogenous immune response against the tumor—and further suggested that therapy consisting of a combination of intrinsic and extrinsic checkpoint blockade may represent an optimal approach [104].

Fig 4.

Future directions. A, Checkpoint blockade, i) Chimeric antigen receptor (CAR) T cells with extrinsically administered anti–programmed cell death protein 1 (PD-1) and/or anti–programed death ligand 1 (PD-L1) antibodies block inhibitory signals, allowing CAR T cells to function effectively. ii) Gene-edited CAR T cells with disrupted PD-1 gene, thereby blocking the receipt of inhibitory signals. iii) CAR T cells expressing PD-1 dominant negative receptor (DNR) combats PD-L1–mediated immunosuppression in the tumor microenvironment, allowing for the efficient functioning of CAR T cells. scFv, single-chain variable fragment; TAA, tumor-associated antigen. B, Radiation- and chemotherapy-mediated cytotoxicity. i) CAR T cells target antigen-positive tumor cells (red). ii) Subsequent chemotherapy and/or radiation kills antigen-negative tumor cells (blue), leading to the release of tumor neoantigens. iii) Dendritic cells engulf and transport antigens to the lymph nodes. iv) Major histocompatibility complex–peptide–T cell receptor binding in the lymph nodes activates endogenous naïve T cells, iv) which proliferate and migrate to attack antigen-negative tumor. C, Surgery and CAR T-cell therapy. i) Neoadjuvant CAR T-cell therapy. Regional delivery of CAR T cells with response, allowing for curative resection of the remaining tumor. ii) Adjuvant CAR T-cell therapy. The tumor is surgically resected, followed by adjuvant systemic CAR T-cell administration targeting circulating tumor cells, leading to a sustained remission.

6.2. Radiation therapy or chemotherapy and CAR T-cell therapy

An additional strategy to overcome high tumor burden and antigen heterogeneity using CAR T-cell therapy is combining CAR T-cell therapy with radiation or chemotherapy (Figure 4B). Radiation or chemotherapy has the potential to induce an endogenous immune response against antigen-negative tumor, acting in a synergistic fashion while CAR T cells target antigen-positive tumors. In an in vivo study of adoptive T cells, radiation therapy increased antitumor efficacy through increased trafficking to the tumor, increased release of tumor-associated antigens, and priming of T cells by APCs [105]. An additional mechanism through which radiation and CAR T cells exert a synergistic effect is the sensitization of antigen-negative tumor cells to TRAIL-mediated cytotoxicity by CAR T cells [106]. In addition, radiation also exerts a synergistic effect with CAR T cells through increased CAR T-cell trafficking to the tumor, which has been demonstrated in in vivo glioblastoma models of NKG2D-targeted [107] and GD2-targeted [108] CAR T cells.

6.3. Dendritic cells and CAR T-cell therapy

An endogenous immune response against antigen-negative tumors can also be affected by interventions that stimulate endogenous APCs. One strategy is to induce expression of CD40L on CAR T cells, which stimulates APCs and induces an endogenous antitumor response [109]. A STING (stimulator of interferon genes) agonist also has been demonstrated to stimulate endogenous APCs. In in vivo models of pancreatic cancer and melanoma, a STING agonist co-delivered with CAR T cells in a biomaterial implant stimulated APCs and induced an endogenous immune response that resulted in eradication of heterogeneous tumor [96]. The use of a dendritic cell vaccine consisting of dendritic cells pulsed in vitro with the antigen target has been shown to increase the efficacy of CAR T cells targeted toward a human leukocyte antigen–restricted intracellular target [110]. The use of a dendritic cell vaccine has also been tested in a pilot clinical trial of patients with glioblastoma treated with cytomegalovirus-specific T cells [111], as well as in a clinical trial of patients with hepatocellular carcinoma treated with activated T cells after tumor resection [112].

6.4. Surgery and CAR T-cell therapy

The expansion of CAR T-cell therapy for the treatment of solid tumors introduces the possibility of CAR T-cell therapy as a neoadjuvant or an adjuvant therapy combined with surgical tumor resection (Figure 4C). In the neoadjuvant setting, CAR T-cell therapy has the potential to reduce tumor burden, but most importantly generate neoantigen responses from the lysed tumor that can be effective for long-term immunity. In a clinical trial of human epidermal growth factor receptor 2–targeted CAR T cells in patients with sarcoma, 3 of 19 patients had stable disease after CAR T-cell therapy and underwent surgical resection [113]. In the adjuvant setting, CAR T cells provide the potential to eradicate micro-metastases and persist in the body as a “living drug,” controlling tumor recurrences. This possibility was hinted at by the ability of CAR T cells to control tumor after re-challenge in in vivo studies, including after up to 10 tumor rechallenges conducted in a preclinical model in our laboratory [68].

7. Conclusion

Future investigations involving CAR T-cell therapy for MPM and other solid tumors should encompass combination immunotherapies to synergistically potentiate endogenous as well as adoptively transferred immunity.

Highlights.

• Tumor antigen-targeted CAR T-cell therapy is effective for leukemia and lymphoma

• CAR T-cell therapy is in phase I/II clinical trials for pleural mesothelioma

• Regional delivery of CAR T cells achieve systemic immunity

• Combination of CAR T cells and anti-PD1 strategies is under investigation

Abbreviations:

APC

antigen-presenting cell

BiTE

bispecific T-cell engager

CAR

chimeric antigen receptor

CSPG4

chondroitin sulfate proteoglycan 4

DNR

dominant negative receptor

FAP

fibroblast activation protein

MHC

major histocompatibility complex

MPM

malignant pleural mesothelioma

MSLN

mesothelin

PD-1

programmed cell death protein 1

PD-L1

programed death ligand 1

TAA

tumor-associated antigen

TCR

T-cell receptor

TIL

tumor-infiltrating lymphocyte

scFv

single-chain variable fragment

STING

stimulator of interferon genes