Advertisement
Review

Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors

Aurore Morello, Michel Sadelain and Prasad S. Adusumilli
DOI: 10.1158/2159-8290.CD-15-0583 Published February 2016

Abstract

Chimeric antigen receptors (CAR) are synthetic receptors that target T cells to cell-surface antigens and augment T-cell function and persistence. Mesothelin is a cell-surface antigen implicated in tumor invasion, which is highly expressed in mesothelioma and lung, pancreas, breast, ovarian, and other cancers. Its low-level expression in mesothelia, however, commands thoughtful therapeutic interventions. Encouragingly, recent clinical trials evaluating active immunization or immunoconjugates in patients with pancreatic adenocarcinoma or mesothelioma have shown responses without toxicity. Altogether, these findings and preclinical CAR therapy models using either systemic or regional T-cell delivery argue favorably for mesothelin CAR therapy in multiple solid tumors.

Significance: Recent success obtained with adoptive transfer of CAR T cells targeting CD19 in patients with refractory hematologic malignancies has generated much enthusiasm for T-cell engineering and raises the prospect of implementing similar strategies for solid tumors. Mesothelin is expressed in a wide range and a high percentage of solid tumors, which we review here in detail. Mesothelin CAR therapy has the potential to treat multiple solid malignancies. Cancer Discov; 6(2); 133–46. ©2015 AACR.

Introduction

Adoptive cell therapy using engineered T cells is emerging as a promising strategy to rapidly establish tumor immunity and eradicate small or large tumor burdens. T cells may be engineered to target a tumor antigen through a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) (1, 2). In contrast to TCRs, which are restricted to human leukocyte antigen, CARs provide direct binding to cell-surface proteins, carbohydrates, or glycolipids. CARs intrinsically mediate T-cell activation as well as co-stimulation in the case of second-generation CARs (3). The use of CARs specific for CD19, a B-cell activation receptor, has recently been shown to induce durable remissions in patients with relapsed, chemo-refractory B-cell malignancies, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma, in multiple clinical trials (4–10). Second-generation CARs achieve these outcomes through both targeted tumor killing and functional T-cell enhancement (3, 11). Given the potential high efficiency of CAR therapy, it is critical to identify appropriate antigens to tackle solid tumors, in order to achieve tumor eradication with minimal or tolerable on-target/off-tumor toxicity to healthy tissues.

Searching For CAR Targets in Solid Tumors

Whereas CD19 provides a nearly ideal target for B-cell malignancies, no single antigen with equivalent characteristics has yet been identified for solid tumors. CD19 is highly and relatively homogenously expressed in most B-cell malignancies, possibly including their putative tumor-initiating cells. CD19 is functionally involved in B-cell activation, and may contribute to tumor survival and thus increase the likelihood of its expression in most tumor cells. Finally, in normal cells, CD19 expression is confined to the hematopoietic B-cell lineage, nonvital cells that can be dispensed (the B-cell aplasia induced by CD19 CAR T cells is not lethal and is clinically manageable). An ideal solid tumor CAR target would thus be overexpressed in all cancer cells, absent or with very low expression in nonvital normal tissue, and found in many patients. Furthermore, if expression of the target antigen is associated with tumor invasion or metastasis formation, CAR therapy may be directed to the more aggressive cancer cells and be less vulnerable to tumor relapse. Solid tumor CAR targets under investigation are altered gene products arising from genetic mutations or altered splicing (EGFRvIII), altered glycosylation patterns (MUC1), cancer-testis antigen-derived peptides (MAGE), overexpressed differentiation antigens [CEA, PSMA, GD2, MUC16, HER2/ERBB2, and mesothelin (MSLN)], or tumor-associated stroma (FAP and VEGFR; see Supplementary Table S1). Examples of solid-tumor antigens currently being investigated in CAR T-cell clinical trials are shown in Fig. 1.

Figure 1.
Figure 1.

Antigens targeted in solid tumor CAR T-cell therapy clinical trials. Antigens listed in the figure were compiled by search of the active clinical trials in the clincialtrials.govdatabase. CEA, carcinoembryonic antigen; FAP, fibroblast-associated protein; FR, folate receptor; GD2, Ganglioside; GPC3, Glypican 3; L1-CAM, L1 cell adhesion molecule; MUC16, Mucin 16; PSMA, prostate-specific membrane antigen.

Although overexpressed antigens are numerous and relatively frequent, they raise concerns about “on-target/off-tumor” side effects due to the high sensitivity of T cells for low-level antigen expression, which can be greater than that of monoclonal antibodies (12). For instance, the use of ERBB2 CAR T cells, administered at a high cell dose, has resulted in a fatal adverse event, attributed in part to low-level ERBB2 expression in healthy lung epithelial and cardiovascular cells (13). Thus, an optimal solid-tumor antigen target is one whose expression either is restricted to tumor cells or occurs only at very low levels in expendable normal tissues. EGFRvIII and chondroitin sulfate proteoglycan-4 (CSPG4) have been suggested to be examples for such favorable scenarios (14, 15).

MSLN is emerging as an attractive target for cancer immunotherapy, considering its low expression on normal mesothelial cells and high expression in a broad spectrum of solid tumors. The MSLN-targeted immunotherapies reported to date support a favorable safety profile (16, 17). MSLN is a potential CAR target in a number of common solid tumors (Fig. 2; Supplementary Table S2).

Figure 2.
Figure 2.

Frequency and distribution pattern of the MSLN protein in solid malignancies. MSLN is expressed in a wide range of solid tumors. For most cancers, MSLN expression is homogeneously distributed on the cell surface, with low cytoplasmic expression. For stomach and lung cancers, MSLN is frequently expressed in the cytoplasm, as well as on the cell surface. The distribution of MSLN in cytoplasm and/or cell surface is represented in the figure, where data were available. The frequency and distribution of MSLN were estimated from the published literature (Supplementary Table S2).

Discovery and Early Characterization of MSLN

In searching for targets for monoclonal antibody therapy for solid tumors, Chang and colleagues discovered the MSLN protein, which they found to be specifically expressed on ovarian cancer cells but not on normal human tissues, with the exception of mesothelial cells (18). MSLN is a glycoprotein anchored to the plasma membrane by a glycophosphatidyl inositol (GPI) domain. It is initially synthesized as a 69 kDa cell-surface protein. After cleavage of the amino terminus by the furin protease, a 40-kDa C-terminal fragment remains attached to the membrane and a soluble 32-kDa N-terminal fragment, named megakaryotic-potentiating factor (MPF), is released (17). A soluble form of MSLN has also been detected in the sera of patients with solid tumors, which is referred to as soluble MSLN-related protein (SMRP). SMRP is generated either by alternative splicing or by proteolytic cleavage of the MSLN mature form, induced by the TNFα-converting enzyme ADAM17 (19).

MSLN Function

The biologic function of MSLN seems to be nonessential in normal tissues, given that MSLN knockout mice exhibit normal development, reproduction, and blood cell count (20). In contrast, preclinical and clinical studies increasingly show that aberrant MSLN expression plays an active role in both malignant transformation of tumors and tumor aggressiveness by promoting cancer cell proliferation, contributing to local invasion and metastasis, and conferring resistance to apoptosis induced by cytotoxic agents (21–24). MSLN can act bidirectionally, either by directly activating intracellular pathways via its GPI domain or by interacting with its receptor, CA125/MUC16. Overexpression of MSLN alone is sufficient to constitutively activate the NFκB, MAPK, and PI3K intracellular pathways promoting cell proliferation and resistance to apoptosis (25). Several preclinical studies, including ours, support the finding that MSLN overexpression promotes cell migration and invasion by inducing activation and expression of the matrix metalloproteases MMP7 (26) and MMP9 (21). In addition, the high-affinity interaction between MSLN and CA125 leads to heterotypic cell adhesion, which facilitates metastasis of ovarian cancer cell lines (27). These observations correlate with clinicalobservations showing that MSLN expression, as well as elevated serum SMRP levels, is associated with progressing tumor burden, increasing stage, and poor overall survival (22–24, 28, 29). Cancer cells that possess an invasive phenotype express high amounts of membranous MSLN, rather than the cytoplasmic form (30, 31). Our group (22) and others (29) have reported that in patients with lung adenocarcinoma, MSLN is expressed at metastatic sites and correlates with tumor aggressiveness and KRAS mutation. These discoveries strengthen the rationale for targeting MSLN-expressing cancer cells with CARs.

MSLN Expression in Solid Tumors

Physiologically, MSLN is expressed on mesothelial cells of the peritoneal and pleural cavities and pericardium; it is expressed minimally on the epithelial cell surface of the trachea, ovaries, rete testis, tonsils, and fallopian tubes (32). Overexpression of MSLN was initially observed in mesothelioma and ovarian cancer, and subsequently in lung, esophageal, pancreatic, gastric, biliary, endometrial, thymic, colon, and breast cancers (17, 21–24). MSLN overexpression thus has an estimated incidence of 340,000 patients and prevalence of 2 million patients a year (Supplementary Tables S3 and S4) in the United States alone. The frequency and distribution patterns of MSLN expression differ for each tumor subtype, as summarized in Fig. 2 and Supplementary Table S2. Using the 5B2 MSLN-specific antibody, we developed an MSLN expression score integrating MSLN intensity and distribution (21). In our series, MSLN expression was found in 90% of epithelioid malignant pleural mesothelioma (n = 139; ref. 21), 69% of lung adenocarcinoma (n = 1,209; ref. 22), 60% of breast cancers (n = 314; ref. 24), and 46% of esophageal cancers (n = 125; ref. 23). Furthermore, we observed that MSLN expression is more prevalent in aggressive histologic subtypes of lung (KRAS+ tumors; ref. 22), breast (triple-negative; ref. 24), and esophageal cancers (high-grade dysplasia and adenocarcinoma; ref. 23). These findings have been corroborated in other studies (29, 33). Within the cancer cell, MSLN expression may be luminal/membranous or cytoplasmic. In mesothelioma tumors, MSLN expression is homogeneously distributed on the cell surface (21). In lung adenocarcinoma, we and others have found that the MSLN expression pattern is heterogeneous, with expression in the cytoplasm and on the cell surface (22, 29). In gastric cancer, cytoplasmic expression was found to be more prevalent than membranous expression (30). In addition to the studies characterizing MSLN expression by IHC analysis, functional genomic mRNA profiling studies in a large cancer database (n = 19,746) have reported MSLN expression in other solid tumors, such as thyroid, renal, and synovial sarcoma tumors, which were not previously reported (34).

MSLN Vaccines and Immunoconjugates

Given MSLN's distribution, protumorigenic functions, and immunogenicity (see below), various immunotherapeutic strategies have been devised, some of which have shown encouraging results in early-phase clinical trials (Table 1). These strategies include the use of (1) tumor vaccines, (2) antibody-based therapies, and (3) adoptive T-cell therapy (CAR T cells; Fig. 3).

Figure 3.
Figure 3.

MSLN-targeted immunotherapy strategies. Several therapeutic strategies have been designed for targeting MSLN on tumor cells: (1) tumor vaccine strategy; (2–4) antibody-based therapies; and (5) adoptive CAR T-cell therapy. These therapies are being evaluated in phase I and/or phase II clinical trials.

View this table:
Table 1.

Phase I/II clinical trials for MSLN-targeted immunotherapies

Phase I and II clinical studies have been conducted at Johns Hopkins University with CRS-207, an attenuated form of Listeria monocytogenes vector that overexpresses human MSLN, either alone (35) or in combination with cyclophosphamide and GVAX (irradiated allogeneic cell line–secreting GM-CSF; refs. 36, 37). Although no clinical responses were reported, MSLN-specific CD8 T-cell responses were induced following cyclophosphamide, GVAX, and CRS-207 administration, along with a modest increase in survival (36). Significantly, no toxicities were observed in the patients. In addition to CD4+ and CD8+ T-cell responses (38), MSLN-specific antibody immune responses (39) were observed in patients with ovarian and pancreatic cancers, confirming the immunogenicity of MSLN and further supporting the safety of its immunotherapeutic targeting.

Phase I studies with SS1P, an anti-MSLN immunotoxin engineered by fusing a murine anti-MSLN variable antibody fragment to the PE38 portion of Pseudomonas exotoxin, enrolled patients with advanced mesothelioma, ovarian cancer, or pancreatic cancer (40). As a single agent, SS1P exhibits moderate antitumor efficacy. Impressive antitumor responses were seen in patients with mesothelioma who received SS1P, together with pentostatin and cyclophosphamide, to deplete T and B cells (41). Leveraging the knowledge that chemotherapies act in concert by disrupting the tumor structure, thereby allowing better penetration of the SS1P molecule, SS1P, in combination with cisplatin and pemetrexed, has resulted in partial responses in 77% of 13 patients with mesothelioma (42). A limitation of the strategies that use SS1P immunotoxin is the development of neutralizing antibodies specific for the toxin portion of the construct and possibly the chimeric SS1 antibody as well. Fully human anti-MSLN monoclonal antibodies have been evaluated in the preclinical setting (43, 44), with the goal of identifying agents with a lower immunogenicity profile—an important concern in the development of CARs as well.

Another therapeutic strategy based on the MSLN antibody uses amatuximab (also called MORAb-009; ref. 45). Amatuximab binds to MSLN, thereby inhibiting adhesion between cell lines expressing CA125, and it elicits antibody-dependent cell-mediated cytotoxicity. Phase II clinical trials have been conducted with amatuximab treatment alone or in combination with pemetrexed and cisplatin. Combination treatment is well tolerated; objective tumor response and stable disease were achieved in 40% and 51% of patients with nonresectable pleural mesothelioma, respectively (n = 89; ref. 45).

Therapeutic agents have been linked to anti-MSLN antibody, with the idea that the drugs will be released into the cytoplasm following internalization of the antibody: (1) duocarmycin, a DNA alkylating agent, which led to the development of MDX-1382 MED2460 (Medarex); and (2) DM4, a tubulin polymerase inhibitor, which led to the development of BAY 94-9343 (46). Interestingly, in vitro, BAY 94-9343 is able to induce a bystander-killing effect on neighboring MSLN-negative cancer cells without affecting nonproliferating cells, an observation that is of particular interest in the context of heterogeneous antigen-expressing tumors (46). A phase I clinical trial investigating the safety of BAY 94-9343 is currently under way. Taken together, these reports demonstrate the safety and feasibility of MSLN as a target for CAR T-cell immunotherapy.

MSLN CAR Design and Preclinical Evaluation

CARs consist of an ectodomain [commonly derived from a single-chain variable fragment (scFv)], a hinge, a transmembrane domain, and an endodomain (typically comprising signaling domains derived from CD3ζ and costimulatory receptors; Fig. 4A). Several MSLN-specific scFvs have been reported, including the murine SS1 scFv (47–50) and two fully human scFvs (51, 52), spanning all three generations of CAR design based on their signaling domains (Fig. 4B). First-generation CARs contain the CD3ζ cytoplasmic domain, which is sufficient to initiate T-cell activation and enable T-cell–mediated cytotoxicity (3, 52, 53). Second-generation CARs further enhance T-cell function and persistence through the incorporation of signaling domains that rescue and amplify the activation signal provided by the CD3ζ cytoplasmic domain. Co-stimulatory elements may be derived from receptors such as 4-1BB (33, 48, 54, 55), CD28 (47, 51, 52), or ICOS (50). Dual signaling prevents T-cell anergy and increases persistence and function by augmenting T-cell proliferation and cytokine production (IFNγ and IL2) and reducing activation-induced cell death through the recruitment of the PI3K, TRAF, and/or other pathways (3, 47, 52). Third-generation CARs comprise three signaling domains, typically encompassing those of CD3ζ and two co-stimulatory domains, for example CD28 and 4-1BB or CD28 and OX40 (47, 56). Compared with second-generation CARs, third-generation CARs have shown inconsistent antitumor activity in vivo (47, 57). A recently described MSLN CAR construct was generated to provide the DAP12 killer immunoglobulin-like receptor activation (58). Preclinical experiments demonstrated that T cells engineered with this CAR displayed increased potency in vivo, compared with second-generation MSLN CARs comprising either CD28 or 4-1BB signaling domains (58). Other co-stimulatory domains have been tested in combination with other antigens, including FcRγ, OX40, DAP10, and CD27 (3, 56, 59). Choosing an appropriate co-stimulation domain is essential to sustain CAR T-cell activity and calibrate T-cell persistence. However, the ideal co-stimulation domain may depend on context, as CAR function depends on multiple extraneous factors, such as antigen density (60, 61), CAR stoichiometry (61), CAR affinity (62–64), and the immunologic features of the tumor microenvironment (65–68).

Figure 4.
Figure 4.

CAR T-cell design. A, structure of the CAR. The CAR contains an scFv binding domain specific for MSLN fused to a transmembrane domain and intracellular signaling domain (CD3ζ). The CAR expressed in the patient's own T cells after transduction provides both specificity and effector function activation. B, different generations of the MSLN CAR. Three generations of CAR T cells differing by their signaling domains (CD28 and/or 4-1BB) have been designed to increase the activation strength of T cells. Combinational antigen recognition with balanced signaling has been described. CCR, chemokine receptor.

A particular concern regarding MSLN CARs is interference from soluble MSLN, which in principle could occupy and block the scFv portion. Reassuringly, MSLN CAR T-cell activation (cytokine secretion and cytotoxic activity) is dependent on MSLN expression on the cell surface (47, 52). Significantly, the presence of serum SMRP does not alter MSLN CAR T-cell efficiency in vitro or in vivo, even at high levels (51, 52). Similar findings have been reported with carcinoembryonic antigen (CEA; ref. 69) and HER2-targeted CARs (70). The lack of CAR blockade by serum protein may be explained by the avidity of CAR T cells for membranous target antigen, which may be increased by interactions between adhesion molecules and other accessory molecules present on the surface of the T cell and tumor cells (71).

The efficiency of MSLN CAR T-cell therapy has been investigated in subcutaneous or orthotopic mouse models of mesothelioma, ovarian cancer, and lung cancer (47, 51, 52). We established a clinically relevant orthotopic mouse model of malignant pleural mesothelioma in which the tumor is aggressive loco-regionally with extensive lymphangiogenesis and mediastinal lymph node metastases mimicking human pleural mesothelioma (21, 72, 73). In this model, we administered MSLN CAR T cells systemically or intrapleurally (52). Intrapleural delivery resulted in greater T-cell proliferation, T-cell redistribution to extra-thoracic metastatic sites, tumor eradication, and survival, than a 30-fold higher T-cell dose administered systemically (52). Systemically administered CAR T cells are sequestered in the lungs prior to tumor infiltration. Regional administration of MSLN CAR T cells facilitated earlier antigen encounter and T-cell activation, cytokine secretion, and effector function of CD4+ CAR T cells, which was associated with increased CD8+ CAR T-cell proliferation and function. Furthermore, intrapleurally administered MSLN CAR T cells persisted long-term and eliminated a tumor rechallenge 200 days after the initial tumor eradication. On the basis of these findings, we are now initiating a phase I study to investigate MSLN CAR T cells administered regionally to subjects with mesothelioma, lung cancer, or breast cancer with pleural metastases (NCT02414269, Table 1).

MSLN CARs in Clinical Trials

With more than 150,000 individuals diagnosed with primary and metastatic pleural disease each year in the United States alone (mostly from lung and breast cancers; ref. 52), a treatment that is effective against these diseases has the potential to make a significant impact. Multiple phase I clinical trials are currently being initiated to determine the safety and the maximum tolerated dose of MSLN CAR T cell therapy. The risk of on-target/off-tumor toxicity has led to different strategies to address this safety concern.

One such strategy is based on the transfection of mRNA that encodes the MSLN CAR, which results in transient CAR expression for only a few days. In preclinical models, this approach has shown promise; multiple infusions of mRNA CAR T cells have produced a robust antitumor effect in vivo (49). However, transient expression of the CAR may limit the long-term efficacy of the therapy. A clinical trial conducted at the University of Pennsylvania administered autologous T cells electroporated with the mRNA encoding for a second-generation MSLN CAR (SS1–4-1BB CAR; refs. 74, 75). In this study, 4 patients with advanced mesothelioma or pancreatic tumors were treated with MSLN CAR T cells infused intravenously or intratumorally. Multiple MSLN CAR T-cell infusions were well tolerated, with no off-target toxicities (pleuritis, pericarditis, or peritonitis) observed. Encouragingly, moderate clinical responses were observed in this phase I study, and MSLN CAR T cells were detected in the tumor. The antitumoral activity of MSLN CAR T cells in vivo was established by the transient elevation of inflammatory cytokines in the sera, including IL12, IL6, G-CSF, MIP1β, MCP1, IL1RA, and RANTES (74, 75). No severe cytokine release syndrome (CRS) was reported with MSLN CARs (74, 75). Interestingly, the clinical evidence also highlights the capacity of CAR T-cell therapy to elicit a systemic antitumor cellular and humoral immune response by favoring epitope spreading (74). The detection of a polyclonal IgG antibody response is consistent with the classic process of epitope spreading, where tumor lysis and inflammation induced by MSLN CAR T cells lead to the release of multiple antigens that are cross-presented on dendritic cells and activate the host immune response. This observation underscores the indirect mechanism present in MSLN CAR T-cell therapy to potentiate a broad antitumor immune response. A serious adverse event was subsequently reported by Maus and colleagues, as one study subject developed severe anaphylaxis and cardiac arrest after the third infusion of MSLN CAR T cells. This reaction was related to the high production of IgE antibodies directed against the MSLN CAR (75), which included the murine SS1 scFv. This effect may be attributable to the multiple injections of the CAR T cells, which may have resulted in an effective prime/boost regimen to stimulate the host humoral immune response. The use of a fully human MSLN CAR (51, 52) will hopefully abrogate or at least reduce the risk of developing such an anti-CAR antibody response.

Another approach to increase T-cell safety is to utilize a suicide gene to eliminate T cells in the event of an emerging adverse event. CAR T cells can be eliminated by drug-induced activation of a suicide gene, such as the herpes simplex thymidine kinase (HSV-TK) gene (76), inducible caspase-9 (iCaspase-9; ref. 77), or EGFRΔ gene (78). Unlike HSV-TK, the latter two are human proteins with a minimal immunogenic potential. These “safety-switch systems” have been successful in clinical investigation (77) and can rapidly deplete CAR T cells if required. The clinical-grade construct that incorporates an iCaspase-9 safety switch—which we will use in an upcoming clinical trial of intrapleural MSLN-targeted CAR T-cell therapy (NCT02414269, Table 1)—has been shown to be safe in preclinical experiments wherein a single dose of the AP1903 small molecule eliminated intrapleurally administered MSLN CAR T cells at the peak of their proliferation within 4 hours.

Future CARs and their Paths

The solid-tumor microenvironment poses several obstacles for MSLN CAR T cells that may limit their antitumor efficacy (79). To optimize the efficiency of CAR T cells, numerous approaches are under evaluation to tame the host tumor microenvironment or generate “armored” CAR T cells that can overcome immune barriers. Such strategies include (i) promoting CAR T-cell infiltration, (ii) augmenting the functional persistence of CAR T cells, (iii) enhancing CAR T cells to overcome inhibitory signals encountered in the tumor microenvironment, and (iv) improving safety by preventing on-target/off-tumor toxicity (Table 2A).

View this table:
Table 2.

Genetic engineering strategies and combinational therapies potentiating CAR T-cell efficacy

To enhance trafficking to solid tumors, MSLN CAR T cells have been engineered to overexpress the chemokine receptor CCR2B, because mesothelioma cells highly express its chemokine ligand, CCL2, and T cells express a minimal amount of CCR2B (54). CCR2B overexpression significantly improved specific homing of MSLN CAR T cells to the tumor microenvironment, as well as the efficiency of the therapy following systemic administration. Potentiation of trafficking by cotransduction of chemokine receptors, such as CCR2 or CCR4, has been demonstrated previously in other preclinical models for T-cell therapy engineered with CARs (80) as well as TCRs (81). This strategy is particularly relevant for MSLN CAR T-cell therapy because solid tumors overexpress CCR2 and CCR4 chemokine ligands.

Upon T-cell activation following tumor infiltration, multiple intracellular factors, such as diacylglycerol kinase (DGK), impair T-cell effector functions and promote T-cell anergy. Riese and colleagues demonstrated that genetic deletion of DGKζ significantly increased the antitumor activity of MSLN CAR T cells, as shown by the enhancement of effector cytokine secretion, FASL, and TRAIL expression, and the cytotoxic functions in vitro (55). In addition to the strategies that investigated the CAR T-cell intracellular pathways such as DGKζ or AKT (82), other genetic engineering strategies to enhance CAR T-cell effector function have been described (IL12, IL7, and IL15 secretion) in other models, with examples provided in Table 2A.

Within the solid tumor, CAR T cells are confronted with a tumor-induced immunosuppressive microenvironment that can limit CAR T-cell potency. Tumor cells and associated-stroma cells, including Tregs and myeloid-derived suppressor cells, express inhibitory molecules, such as TGFβ, IDO, and PD-L1, which limit CAR T-cell efficacy (48, 55, 65, 67, 68). Although second-generation CARs are relatively efficient in the immunosuppressive microenvironment, as shown by us and other investigators (83), co-stimulation alone may not be sufficient (66). To potentiate CAR T cells in an immunosuppressive environment, multiple approaches have been investigated, including antibody-based therapy and genetic approaches, such as engineering T cells to express a dominant-negative TGFβ receptor that restores T-cell effector functions in an immunocompetent mouse melanoma model (84). This strategy, which is currently being investigated in a clinical trial utilizing CAR T cells targeting the HER2 antigen (clinicalTrials.gov, NCT00889954), could readily be adapted for use with MSLN CAR T-cell therapy, as TGFβ is an immunosuppressive factor in lung cancer, ovarian cancer, and mesothelioma. Overexpression of PD-L1 by tumor cells has been shown to induce CAR T-cell exhaustion (48, 65). The immunosuppressive effect of the PD-L1/PD-1 pathway can be reverted by the addition of PD-1/PD-L1–blocking antibody (48), a PD1/CD28 converter (85, 86), or a PD-1 dominant-negative receptor. Our results demonstrate that coexpression of a PD-1 dominant-negative receptor together with an MSLN CAR potentiates long-term eradication of mesothelioma tumors (87).

To improve the specificity and safety of CAR T cells, a trans-signaling strategy was developed where CD3ζ signaling is physically dissociated to the co-stimulatory signal through the transduction of two CARs specific for different antigens (Fig. 4B); these dual-CAR T cells eliminate only cancer cells that coexpress the two targeted antigens (53, 88). In one such strategy, T cells are engineered to express an MSLN-specific CAR containing a CD3ζ domain and a folate receptor–specific CAR containing a CD28 co-stimulation domain (Fig. 4B). Cotransduced T cells possess superior antitumor activity against cancer cells expressing both antigens, compared with first-generation CAR T cells, and equivalent activity compared with second-generation CAR T cells. Thus, this study demonstrates the ability to manage on-target toxicity on normal tissue, as well as the ability to combine MSLN CARs with another tumor antigen to improve the safety, specificity, and efficacy of MSLN CAR T-cell therapy.

Potential Combination Therapies

In addition to the genetic engineering strategies described above, rational combinatorial approaches with therapeutic agents that are already in clinical practice are being investigated to enhance therapy response by improving T-cell engraftment, sensitizing tumor cells to apoptosis, and stimulating the host immune system. Examples of such combinations investigated in preclinical studies potentiating CAR T-cell efficacy are shown in Table 2B. Preconditioning to achieve host lymphodepletion by use of cyclophosphamide, fludarabine, or radiotherapy is commonly used to promote engraftment of adoptively transferred T cells. Other promising approaches include combining CAR T-cell therapy with small-molecule inhibitors, monoclonal antibodies, oncolytic viruses, or whole-cell vaccines. Although these studies are conducted in tumor models, such as melanoma or leukemia, some of these may be applicable to solid tumors, including MSLN-expressing solid tumors.

Conclusion

CAR therapy using second-generation CARs has rapidly translated to clinical impact in CD19+ malignancies, paving the way for unprecedented enthusiasm for adoptive cell therapy and engineered T cells. Having such a powerful technology at hand, one important future direction for CAR research is the identification of suitable targets for tackling solid tumors. MSLN offers exciting prospects based on its high expression in a variety of cancers and low-level expression in normal tissues. The latter commands a thoughtful targeting strategy, noting that MSLN-targeted immunotherapies have been very well tolerated. These clinical outcomes, combined with the preclinical data obtained with MSLN CARs, argue favorably for clinical trials targeting mesothelioma and breast, lung, ovarian, and pancreatic cancers, which will soon be performed at multiple centers (NCT01355965, NCT01897415, NCT011583686, NCT02159716, NCT02414269, and NCT02465983).

Disclosure of Potential Conflicts of Interest

M. Sadelain is a consultant/advisory board member for Juno Therapeutics. No potential conflicts of interest were disclosed by the other authors.

Grant Support

This work is supported by grants from the NIH (R21 CA164568-01A1, R21 CA164585-01A1, U54 CA137788, P50 CA086438-13, and P30 CA008748), the U.S. Department of Defense (LC110202 and BC132124), the Lake Road Foundation, and Stand Up To Cancer—Cancer Research Institute Cancer Immunology Translational Cancer Research Grant (SU2C-AACR-DT1012). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research.

Acknowledgments

The authors thank David Sewell and Alex Torres of the Memorial Sloan Kettering Thoracic Surgery Service for their editorial assistance.

Footnotes

  • Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).

  • Received May 13, 2015.
  • Revision received August 31, 2015.
  • Accepted September 8, 2015.

References

  1. 1.↵
    1. Ho WY,
    2. Blattman JN,
    3. Dossett ML,
    4. Yee C,
    5. Greenberg PD
    . Adoptive immunotherapy: engineering T cell responses as biologic weapons for tumor mass destruction. Cancer Cell 2003;3:431–7.
    OpenUrlCrossRefPubMed
  2. 2.↵
    1. Sadelain M,
    2. Riviere I,
    3. Brentjens R
    . Targeting tumours with genetically enhanced T lymphocytes. Nat Rev Cancer 2003;3:35–45.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. van der Stegen SJ,
    2. Hamieh M,
    3. Sadelain M
    . The pharmacology of second-generation chimeric antigen receptors. Nat Rev Drug Discov 2015;14:499–509.
    OpenUrlCrossRefPubMed
  4. 4.↵
    1. Davila ML,
    2. Riviere I,
    3. Wang X,
    4. Bartido S,
    5. Park J,
    6. Curran K,
    7. et al.
    Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 2014;6:224ra25.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. Kochenderfer JN,
    2. Dudley ME,
    3. Kassim SH,
    4. Somerville RP,
    5. Carpenter RO,
    6. Stetler-Stevenson M,
    7. et al.
    Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol 2015;33:540–9.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    1. Grupp SA,
    2. Kalos M,
    3. Barrett D,
    4. Aplenc R,
    5. Porter DL,
    6. Rheingold SR,
    7. et al.
    Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368:1509–18.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Porter DL,
    2. Hwang WT,
    3. Frey NV,
    4. Lacey SF,
    5. Shaw PA,
    6. Loren AW,
    7. et al.
    Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia. Sci Transl Med 2015;7:303ra139.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Kochenderfer JN1,
    2. Rosenberg SA
    . Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors. Nat Rev Clin Oncol 2012;10:267–76.
    OpenUrl
  9. 9.↵
    1. Jensen MC,
    2. Riddell SR
    . Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunol Rev 2014;257:127–44.
    OpenUrlCrossRefPubMed
  10. 10.↵
    1. Sadelain M
    . CAR therapy: the CD19 paradigm. J Clin Invest 2015;125:3392–400.
    OpenUrlCrossRefPubMed
  11. 11.↵
    1. Zhao Z,
    2. Condomines M,
    3. van der Stegen SJ,
    4. Perna F,
    5. Kloss CC,
    6. Gunset G,
    7. et al.
    Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T Cells. Cancer Cell 2015;28:415–28.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Ahmed N,
    2. Salsman VS,
    3. Yvon E,
    4. Louis CU,
    5. Perlaky L,
    6. Wels WS,
    7. et al.
    Immunotherapy for osteosarcoma: genetic modification of T cells overcomes low levels of tumor antigen expression. Mol Ther 2009;17:1779–87.
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Morgan RA,
    2. Yang JC,
    3. Kitano M,
    4. Dudley ME,
    5. Laurencot CM,
    6. Rosenberg SA
    . Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 2010;18:843–51.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Beard RE,
    2. Zheng Z,
    3. Lagisetty KH,
    4. Burns WR,
    5. Tran E,
    6. Hewitt SM,
    7. et al.
    Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells. J Immunother Cancer 2014;2:25.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Johnson LA,
    2. Scholler J,
    3. Ohkuri T,
    4. Kosaka A,
    5. Patel PR,
    6. McGettigan SE,
    7. et al.
    Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma. Sci Transl Med 2015;7:275ra22.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    1. Villena-Vargas J,
    2. Adusumilli PS
    . Mesothelin-targeted immunotherapies for malignant pleural mesothelioma. Ann Cardiothorac Surg 2012;1:466–71.
    OpenUrlPubMed
  17. 17.↵
    1. Pastan I,
    2. Hassan R
    . Discovery of mesothelin and exploiting it as a target for immunotherapy. Cancer Res 2014;74:2907–12.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Chang K,
    2. Pai LH,
    3. Batra JK,
    4. Pastan I,
    5. Willingham MC
    . Characterization of the antigen (CAK1) recognized by monoclonal antibody K1 present on ovarian cancers and normal mesothelium. Cancer Res 1992;52:181–6.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Sapede C,
    2. Gauvrit A,
    3. Barbieux I,
    4. Padieu M,
    5. Cellerin L,
    6. Sagan C,
    7. et al.
    Aberrant splicing and protease involvement in mesothelin release from epithelioid mesothelioma cells. Cancer Sci 2008;99:590–4.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Bera TK,
    2. Pastan I
    . Mesothelin is not required for normal mouse development or reproduction. Mol Cell Biol 2000;20:2902–6.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    1. Servais EL,
    2. Colovos C,
    3. Rodriguez L,
    4. Bograd AJ,
    5. Nitadori J,
    6. Sima C,
    7. et al.
    Mesothelin overexpression promotes mesothelioma cell invasion and MMP-9 secretion in an orthotopic mouse model and in epithelioid pleural mesothelioma patients. Clin Cancer Res 2012;18:2478–89.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Kachala SS,
    2. Bograd AJ,
    3. Villena-Vargas J,
    4. Suzuki K,
    5. Servais EL,
    6. Kadota K,
    7. et al.
    Mesothelin overexpression is a marker of tumor aggressiveness and is associated with reduced recurrence-free and overall survival in early-stage lung adenocarcinoma. Clin Cancer Res 2014;20:1020–8.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    1. Rizk NP,
    2. Servais EL,
    3. Tang LH,
    4. Sima CS,
    5. Gerdes H,
    6. Fleisher M,
    7. et al.
    Tissue and serum mesothelin are potential markers of neoplastic progression in Barrett's associated esophageal adenocarcinoma. Cancer Epidemiol Biomarkers Prev 2012;21:482–6.
    OpenUrlAbstract/FREE Full Text
  24. 24.↵
    1. Tozbikian G,
    2. Brogi E,
    3. Kadota K,
    4. Catalano J,
    5. Akram M,
    6. Patil S,
    7. et al.
    Mesothelin expression in triple negative breast carcinomas correlates significantly with basal-like phenotype, distant metastases and decreased survival. PLoS One 2014;9:e114900.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Bharadwaj U,
    2. Marin-Muller C,
    3. Li M,
    4. Chen C,
    5. Yao Q
    . Mesothelin confers pancreatic cancer cell resistance to TNF-alpha-induced apoptosis through Akt/PI3K/NF-kappaB activation and IL-6/Mcl-1 overexpression. Mol Cancer 2011;10:106.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Chen SH,
    2. Hung WC,
    3. Wang P,
    4. Paul C,
    5. Konstantopoulos K
    . Mesothelin binding to CA125/MUC16 promotes pancreatic cancer cell motility and invasion via MMP-7 activation. Sci Rep 2013;3:1870.
    OpenUrlPubMed
  27. 27.↵
    1. Rump A,
    2. Morikawa Y,
    3. Tanaka M,
    4. Minami S,
    5. Umesaki N,
    6. Takeuchi M,
    7. et al.
    Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion. J Biol Chem 2004;279:9190–8.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Robinson BW,
    2. Creaney J,
    3. Lake R,
    4. Nowak A,
    5. Musk AW,
    6. de Klerk N,
    7. et al.
    Soluble mesothelin-related protein–a blood test for mesothelioma. Lung Cancer 2005;49:S109–11.
    OpenUrlPubMed
  29. 29.↵
    1. Thomas A,
    2. Chen Y,
    3. Steinberg SM,
    4. Luo J,
    5. Pack S,
    6. Raffeld M,
    7. et al.
    High mesothelin expression in advanced lung adenocarcinoma is associated with KRAS mutations and a poor prognosis. Oncotarget 2015;6:11694–703.
    OpenUrlCrossRefPubMed
  30. 30.↵
    1. Einama T,
    2. Homma S,
    3. Kamachi H,
    4. Kawamata F,
    5. Takahashi K,
    6. Takahashi N,
    7. et al.
    Luminal membrane expression of mesothelin is a prominent poor prognostic factor for gastric cancer. Br J Cancer 2012;107:137–42.
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Kawamata F,
    2. Homma S,
    3. Kamachi H,
    4. Einama T,
    5. Kato Y,
    6. Tsuda M,
    7. et al.
    C-ERC/mesothelin provokes lymphatic invasion of colorectal adenocarcinoma. J Gastroenterol 2014;49:81–92.
    OpenUrlPubMed
  32. 32.↵
    1. Ordonez NG
    . Application of mesothelin immunostaining in tumor diagnosis. Am J Surg Pathol 2003;27:1418–28.
    OpenUrlCrossRefPubMed
  33. 33.↵
    1. Tchou J,
    2. Wang LC,
    3. Selven B,
    4. Zhang H,
    5. Conejo-Garcia J,
    6. Borghaei H,
    7. et al.
    Mesothelin, a novel immunotherapy target for triple negative breast cancer. Breast Cancer Res Treat 2012;133:799–804.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Lamberts LE,
    2. de Groot DJ,
    3. Bense RD,
    4. de Vries EG,
    5. Fehrmann RS
    . Functional genomic mRNA Profiling of a large cancer data base demonstrates mesothelin overexpression in a broad range of tumor types. Oncotarget 2015 Jul 1. [Epub ahead of print].
  35. 35.↵
    1. Le DT,
    2. Brockstedt DG,
    3. Nir-Paz R,
    4. Hampl J,
    5. Mathur S,
    6. Nemunaitis J,
    7. et al.
    A live-attenuated Listeria vaccine (ANZ-100) and a live-attenuated Listeria vaccine expressing mesothelin (CRS-207) for advanced cancers: phase I studies of safety and immune induction. Clin Cancer Res 2012;18:858–68.
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Le DT,
    2. Wang-Gillam A,
    3. Picozzi V,
    4. Greten TF,
    5. Crocenzi T,
    6. Springett G,
    7. et al.
    Safety and survival with GVAX pancreas prime and listeria monocytogenes-expressing mesothelin (CRS-207) boost vaccines for metastatic pancreatic cancer. J Clin Oncol 2015;33:1325–33.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Lutz ER,
    2. Wu AA,
    3. Bigelow E,
    4. Sharma R,
    5. Mo G,
    6. Soares K,
    7. et al.
    Immunotherapy converts nonimmunogenic pancreatic tumors into immunogenic foci of immune regulation. Cancer Immunol Res 2014;2:616–31.
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    1. Chen Y,
    2. Ayaru L,
    3. Mathew S,
    4. Morris E,
    5. Pereira SP,
    6. Behboudi S
    . Expansion of anti-mesothelin specific CD4+ and CD8+ T cell responses in patients with pancreatic carcinoma. PLoS One 2014;9:e88133.
    OpenUrlPubMed
  39. 39.↵
    1. Ho M,
    2. Hassan R,
    3. Zhang J,
    4. Wang QC,
    5. Onda M,
    6. Bera T,
    7. et al.
    Humoral immune response to mesothelin in mesothelioma and ovarian cancer patients. Clin Cancer Res 2005;11:3814–20.
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    1. Hassan R,
    2. Bullock S,
    3. Premkumar A,
    4. Kreitman RJ,
    5. Kindler H,
    6. Willingham MC,
    7. et al.
    Phase I study of SS1P, a recombinant anti-mesothelin immunotoxin given as a bolus I.V. infusion to patients with mesothelin-expressing mesothelioma, ovarian, and pancreatic cancers. Clin Cancer Res 2007;13:5144–9.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Hassan R,
    2. Miller AC,
    3. Sharon E,
    4. Thomas A,
    5. Reynolds JC,
    6. Ling A,
    7. et al.
    Major cancer regressions in mesothelioma after treatment with an anti-mesothelin immunotoxin and immune suppression. Sci Transl Med 2013;5:208ra147.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    1. Hassan R,
    2. Sharon E,
    3. Thomas A,
    4. Zhang J,
    5. Ling A,
    6. Miettinen M,
    7. et al.
    Phase 1 study of the antimesothelin immunotoxin SS1P in combination with pemetrexed and cisplatin for front-line therapy of pleural mesothelioma and correlation of tumor response with serum mesothelin, megakaryocyte potentiating factor, and cancer antigen 125. Cancer 2014;120:3311–9.
    OpenUrlCrossRefPubMed
  43. 43.↵
    1. Ho M,
    2. Feng M,
    3. Fisher RJ,
    4. Rader C,
    5. Pastan I
    . A novel high-affinity human monoclonal antibody to mesothelin. Int J Cancer 2011;128:2020–30.
    OpenUrlCrossRefPubMed
  44. 44.↵
    1. Feng Y,
    2. Xiao X,
    3. Zhu Z,
    4. Streaker E,
    5. Ho M,
    6. Pastan I,
    7. et al.
    A novel human monoclonal antibody that binds with high affinity to mesothelin-expressing cells and kills them by antibody-dependent cell-mediated cytotoxicity. Mol Cancer Ther 2009;8:1113–8.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    1. Hassan R,
    2. Kindler HL,
    3. Jahan T,
    4. Bazhenova L,
    5. Reck M,
    6. Thomas A,
    7. et al.
    Phase II clinical trial of amatuximab, a chimeric anti-mesothelin antibody with pemetrexed and cisplatin in advanced unresectable pleural mesothelioma. Clin Cancer Res 2014;20:5927–36.
    OpenUrlAbstract/FREE Full Text
  46. 46.↵
    1. Golfier S,
    2. Kopitz C,
    3. Kahnert A,
    4. Heisler I,
    5. Schatz CA,
    6. Stelte-Ludwig B,
    7. et al.
    Anetumab ravtansine: a novel mesothelin-targeting antibody-drug conjugate cures tumors with heterogeneous target expression favored by bystander effect. Mol Cancer Ther 2014;13:1537–48.
    OpenUrlAbstract/FREE Full Text
  47. 47.↵
    1. Carpenito C,
    2. Milone MC,
    3. Hassan R,
    4. Simonet JC,
    5. Lakhal M,
    6. Suhoski MM,
    7. et al.
    Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc Natl Acad Sci U S A 2009;106:3360–5.
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    1. Moon EK,
    2. Wang LC,
    3. Dolfi DV,
    4. Wilson CB,
    5. Ranganathan R,
    6. Sun J,
    7. et al.
    Multifactorial T-cell hypofunction that is reversible can limit the efficacy of chimeric antigen receptor-transduced human T cells in solid tumors. Clin Cancer Res 2014;20:4262–73.
    OpenUrlAbstract/FREE Full Text
  49. 49.↵
    1. Zhao Y,
    2. Moon E,
    3. Carpenito C,
    4. Paulos CM,
    5. Liu X,
    6. Brennan AL,
    7. et al.
    Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 2010;70:9053–61.
    OpenUrlAbstract/FREE Full Text
  50. 50.↵
    1. Guedan S,
    2. Chen X,
    3. Madar A,
    4. Carpenito C,
    5. McGettigan SE,
    6. Frigault MJ,
    7. et al.
    ICOS-based chimeric antigen receptors program bipolar TH17/TH1 cells. Blood 2014;124:1070–80.
    OpenUrlAbstract/FREE Full Text
  51. 51.↵
    1. Lanitis E,
    2. Poussin M,
    3. Hagemann IS,
    4. Coukos G,
    5. Sandaltzopoulos R,
    6. Scholler N,
    7. et al.
    Redirected antitumor activity of primary human lymphocytes transduced with a fully human anti-mesothelin chimeric receptor. Mol Ther 2012;20:633–43.
    OpenUrlCrossRefPubMed
  52. 52.↵
    1. Adusumilli PS,
    2. Cherkassky L,
    3. Villena-Vargas J,
    4. Colovos C,
    5. Servais E,
    6. Plotkin J,
    7. et al.
    Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity. Sci Transl Med 2014;6:261ra151.
    OpenUrlAbstract/FREE Full Text
  53. 53.↵
    1. Lanitis E,
    2. Poussin M,
    3. Klattenhoff AW,
    4. Song D,
    5. Sandaltzopoulos R,
    6. June CH,
    7. et al.
    Chimeric antigen receptor T cells with dissociated signaling domains exhibit focused anti-tumor activity with reduced potential for toxicity. Cancer Immunol Res 2013;1:43–53.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    1. Moon EK,
    2. Carpenito C,
    3. Sun J,
    4. Wang LC,
    5. Kapoor V,
    6. Predina J,
    7. et al.
    Expression of a functional CCR2 receptor enhances tumor localization and tumor eradication by retargeted human T cells expressing a mesothelin-specific chimeric antibody receptor. Clin Cancer Res 2011;17:4719–30.
    OpenUrlAbstract/FREE Full Text
  55. 55.↵
    1. Riese MJ,
    2. Wang LC,
    3. Moon EK,
    4. Joshi RP,
    5. Ranganathan A,
    6. June CH,
    7. et al.
    Enhanced effector responses in activated CD8+ T cells deficient in diacylglycerol kinases. Cancer Res 2013;73:3566–77.
    OpenUrlAbstract/FREE Full Text
  56. 56.↵
    1. Hombach AA,
    2. Abken H
    . Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28-OX40 signalling. Int J Cancer 2011;129:2935–44.
    OpenUrlPubMed
  57. 57.↵
    1. Zhong XS,
    2. Matsushita M,
    3. Plotkin J,
    4. Riviere I,
    5. Sadelain M
    . Chimeric antigen receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+ T cell-mediated tumor eradication. Mol Ther 2010;18:413–20.
    OpenUrlCrossRefPubMed
  58. 58.↵
    1. Wang E,
    2. Wang LC,
    3. Tsai CY,
    4. Bhoj V,
    5. Gershenson Z,
    6. Moon EK,
    7. et al.
    Generation of potent T cell immunotherapy for cancer using DAP12-based, multichain, chimeric immunoreceptors. Cancer Immunol Res 2015;3:815–26.
    OpenUrlAbstract/FREE Full Text
  59. 59.↵
    1. Sentman CL,
    2. Meehan KR
    . NKG2D CARs as cell therapy for cancer. Cancer J 2014;20:156–9.
    OpenUrlCrossRefPubMed
  60. 60.↵
    1. Watanabe K,
    2. Terakura S,
    3. Martens AC,
    4. van Meerten T,
    5. Uchiyama S,
    6. Imai M,
    7. et al.
    Target antigen density governs the efficacy of anti-CD20-CD28-CD3 zeta chimeric antigen receptor-modified effector CD8+ T cells. J Immunol 2015;194:911–20.
    OpenUrlAbstract/FREE Full Text
  61. 61.↵
    1. Weijtens ME,
    2. Hart EH,
    3. Bolhuis RL
    . Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production. Gene Ther 2000;7:35–42.
    OpenUrlCrossRefPubMed
  62. 62.↵
    1. Song DG,
    2. Ye Q,
    3. Poussin M,
    4. Liu L,
    5. Figini M,
    6. Powell DJ Jr.
    . A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity. Oncotarget 2015 Jun 19. [Epub ahead of print].
  63. 63.↵
    1. Chmielewski M,
    2. Hombach A,
    3. Heuser C,
    4. Adams GP,
    5. Abken H
    . T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity. J Immunol 2004;173:7647–53.
    OpenUrlAbstract/FREE Full Text
  64. 64.↵
    1. Hudecek M,
    2. Lupo-Stanghellini MT,
    3. Kosasih PL,
    4. Sommermeyer D,
    5. Jensen MC,
    6. Rader C,
    7. et al.
    Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin Cancer Res 2013;19:3153–64.
    OpenUrlAbstract/FREE Full Text
  65. 65.↵
    1. Burga RA,
    2. Thorn M,
    3. Point GR,
    4. Guha P,
    5. Nguyen CT,
    6. Licata LA,
    7. et al.
    Liver myeloid-derived suppressor cells expand in response to liver metastases in mice and inhibit the anti-tumor efficacy of anti-CEA CAR-T. Cancer Immunol Immunother 2015;64:817–29.
    OpenUrlCrossRefPubMed
  66. 66.↵
    1. Kofler DM,
    2. Chmielewski M,
    3. Rappl G,
    4. Hombach A,
    5. Riet T,
    6. Schmidt A,
    7. et al.
    CD28 costimulation Impairs the efficacy of a redirected t-cell antitumor attack in the presence of regulatory t cells which can be overcome by preventing Lck activation. Mol Ther 2011;19:760–7.
    OpenUrlCrossRefPubMed
  67. 67.↵
    1. Lee JC,
    2. Hayman E,
    3. Pegram HJ,
    4. Santos E,
    5. Heller G,
    6. Sadelain M,
    7. et al.
    In vivo inhibition of human CD19-targeted effector T cells by natural T regulatory cells in a xenotransplant murine model of B cell malignancy. Cancer Res 2011;71:2871–81.
    OpenUrlAbstract/FREE Full Text
  68. 68.↵
    1. Ninomiya S,
    2. Narala N,
    3. Huye L,
    4. Yagyu S,
    5. Savoldo B,
    6. Dotti G,
    7. et al.
    Tumor indoleamine 2,3-dioxygenase (IDO) inhibits CD19-CAR T cells and is downregulated by lymphodepleting drugs. Blood 2015;125:3905–16.
    OpenUrlAbstract/FREE Full Text
  69. 69.↵
    1. Nolan KF,
    2. Yun CO,
    3. Akamatsu Y,
    4. Murphy JC,
    5. Leung SO,
    6. Beecham EJ,
    7. et al.
    Bypassing immunization: optimized design of “designer T cells” against carcinoembryonic antigen (CEA)-expressing tumors, and lack of suppression by soluble CEA. Clin Cancer Res 1999;5:3928–41.
    OpenUrlAbstract/FREE Full Text
  70. 70.↵
    1. Beecham EJ,
    2. Ortiz-Pujols S,
    3. Junghans RP
    . Dynamics of tumor cell killing by human T lymphocytes armed with an anti-carcinoembryonic antigen chimeric immunoglobulin T-cell receptor. J Immunother 2000;23:332–43.
    OpenUrl
  71. 71.↵
    1. Gonzalez PA,
    2. Carreno LJ,
    3. Cespedes PF,
    4. Bueno SM,
    5. Riedel CA,
    6. Kalergis AM
    . Modulation of tumor immunity by soluble and membrane-bound molecules at the immunological synapse. Clin Dev Immunol 2013;2013:450291.
    OpenUrlPubMed
  72. 72.↵
    1. Servais EL,
    2. Colovos C,
    3. Kachala SS,
    4. Adusumilli PS
    . Pre-clinical mouse models of primary and metastatic pleural cancers of the lung and breast and the use of bioluminescent imaging to monitor pleural tumor burden. Curr Protoc Pharmacol 2011;Chapter 14:Unit14 21.
  73. 73.↵
    1. Servais EL,
    2. Suzuki K,
    3. Colovos C,
    4. Rodriguez L,
    5. Sima C,
    6. Fleisher M,
    7. et al.
    An in vivo platform for tumor biomarker assessment. PLoS One 2011;6:e26722.
    OpenUrlCrossRefPubMed
  74. 74.↵
    1. Beatty GL,
    2. Haas AR,
    3. Maus MV,
    4. Torigian DA,
    5. Soulen MC,
    6. Plesa G,
    7. et al.
    Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies. Cancer Immunol Res 2014;2:112–20.
    OpenUrlAbstract/FREE Full Text
  75. 75.↵
    1. Maus MV,
    2. Haas AR,
    3. Beatty GL,
    4. Albelda SM,
    5. Levine BL,
    6. Liu X,
    7. et al.
    T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer Immunol Res 2013;1:26–31.
    OpenUrlAbstract/FREE Full Text
  76. 76.↵
    1. Cooper LJ,
    2. Ausubel L,
    3. Gutierrez M,
    4. Stephan S,
    5. Shakeley R,
    6. Olivares S,
    7. et al.
    Manufacturing of gene-modified cytotoxic T lymphocytes for autologous cellular therapy for lymphoma. Cytotherapy 2006;8:105–17.
    OpenUrlCrossRefPubMed
  77. 77.↵
    1. Di Stasi A,
    2. Tey SK,
    3. Dotti G,
    4. Fujita Y,
    5. Kennedy-Nasser A,
    6. Martinez C,
    7. et al.
    Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 2011;365:1673–83.
    OpenUrlCrossRefPubMed
  78. 78.↵
    1. Wang X,
    2. Chang WC,
    3. Wong CW,
    4. Colcher D,
    5. Sherman M,
    6. Ostberg JR,
    7. et al.
    A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 2011;118:1255–63.
    OpenUrlAbstract/FREE Full Text
  79. 79.↵
    1. Adusumilli PS
    . Translational immunotherapeutics: Chemoimmunotherapy for malignant pleural mesothelioma. Cancer 2014;120:3268–71.
    OpenUrlCrossRefPubMed
  80. 80.↵
    1. Craddock JA,
    2. Lu A,
    3. Bear A,
    4. Pule M,
    5. Brenner MK,
    6. Rooney CM,
    7. et al.
    Enhanced tumor trafficking of GD2 chimeric antigen receptor T cells by expression of the chemokine receptor CCR2b. J Immunother 2010;33:780–8.
    OpenUrlCrossRef
  81. 81.↵
    1. Asai H,
    2. Fujiwara H,
    3. An J,
    4. Ochi T,
    5. Miyazaki Y,
    6. Nagai K,
    7. et al.
    Co-introduced functional CCR2 potentiates in vivo anti-lung cancer functionality mediated by T cells double gene-modified to express WT1-specific T-cell receptor. PLoS One 2013;8:e56820.
    OpenUrlCrossRefPubMed
  82. 82.↵
    1. Sun J,
    2. Dotti G,
    3. Huye LE,
    4. Foster AE,
    5. Savoldo B,
    6. Gramatges MM,
    7. et al.
    T cells expressing constitutively active Akt resist multiple tumor-associated inhibitory mechanisms. Mol Ther 2010;18:2006–17.
    OpenUrlCrossRefPubMed
  83. 83.↵
    1. Loskog A,
    2. Giandomenico V,
    3. Rossig C,
    4. Pule M,
    5. Dotti G,
    6. Brenner MK
    . Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells. Leukemia 2006;20:1819–28.
    OpenUrlCrossRefPubMed
  84. 84.↵
    1. Zhang L,
    2. Yu Z,
    3. Muranski P,
    4. Palmer DC,
    5. Restifo NP,
    6. Rosenberg SA,
    7. et al.
    Inhibition of TGF-beta signaling in genetically engineered tumor antigen-reactive T cells significantly enhances tumor treatment efficacy. Gene Therapy 2013;20:575–80.
    OpenUrlCrossRefPubMed
  85. 85.↵
    1. Ankri C,
    2. Shamalov K,
    3. Horovitz-Fried M,
    4. Mauer S,
    5. Cohen CJ
    . Human T cells engineered to express a programmed death 1/28 costimulatory retargeting molecule display enhanced antitumor activity.J Immunol 2013;191:4121–9.
    OpenUrlAbstract/FREE Full Text
  86. 86.↵
    1. Prosser ME,
    2. Brown CE,
    3. Shami AF,
    4. Forman SJ,
    5. Jensen MC
    . Tumor PD-L1 co-stimulates primary human CD8(+) cytotoxic T cells modified to express a PD1:CD28 chimeric receptor. Mol Immunol 2012;51:263–72.
    OpenUrlCrossRefPubMed
  87. 87.↵
    1. Cherkassky L,
    2. Villena-Vargas J,
    3. Morello A,
    4. Rusch V,
    5. Sadelain M,
    6. Adusumilli P
    . Mesothelin-targeted T cells gene-engineered with 4-1BB costimulation overcome tumor-mediated immunoinhibition and eradicate established solid tumors. J Am Coll Surg 2014;219:S134.
    OpenUrl
  88. 88.↵
    1. Kloss CC,
    2. Condomines M,
    3. Cartellieri M,
    4. Bachmann M,
    5. Sadelain M
    . Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 2013;31:71–5.
    OpenUrlCrossRefPubMed
  89. 89.
    1. Kreitman RJ,
    2. Hassan R,
    3. Fitzgerald DJ,
    4. Pastan I
    . Phase I trial of continuous infusion anti-mesothelin recombinant immunotoxin SS1P. Clin Cancer Res 2009;15:5274–9.
    OpenUrlAbstract/FREE Full Text
  90. 90.
    1. Fujisaka Y,
    2. Kurata T,
    3. Tanaka K,
    4. Kudo T,
    5. Okamoto K,
    6. Tsurutani J,
    7. et al.
    Phase I study of amatuximab, a novel monoclonal antibody to mesothelin, in Japanese patients with advanced solid tumors. Invest New Drugs 2015;33:380–8.
    OpenUrlPubMed
  91. 91.
    1. Hassan R,
    2. Cohen SJ,
    3. Phillips M,
    4. Pastan I,
    5. Sharon E,
    6. Kelly RJ,
    7. et al.
    Phase I clinical trial of the chimeric anti-mesothelin monoclonal antibody MORAb-009 in patients with mesothelin-expressing cancers. Clin Cancer Res 2010;16:6132–8.
    OpenUrlAbstract/FREE Full Text
  92. 92.
    1. Di Stasi A,
    2. De Angelis B,
    3. Rooney CM,
    4. Zhang L,
    5. Mahendravada A,
    6. Foster AE,
    7. et al.
    T lymphocytes coexpressing CCR4 and a chimeric antigen receptor targeting CD30 have improved homing and antitumor activity in a Hodgkin tumor model. Blood 2009;113:6392–402.
    OpenUrlAbstract/FREE Full Text
  93. 93.
    1. Caruana I,
    2. Savoldo B,
    3. Hoyos V,
    4. Weber G,
    5. Liu H,
    6. Kim ES,
    7. et al.
    Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes. Nat Med 2015;21:524–9.
    OpenUrlCrossRefPubMed
  94. 94.
    1. Pegram HJ,
    2. Lee JC,
    3. Hayman EG,
    4. Imperato GH,
    5. Tedder TF,
    6. Sadelain M,
    7. et al.
    Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood 2012;119:4133–41.
    OpenUrlAbstract/FREE Full Text
  95. 95.
    1. Koneru M,
    2. Purdon TJ,
    3. Spriggs D,
    4. Koneru S,
    5. Brentjens RJ
    . IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors. Oncoimmunology 2015;4:e994446.
    OpenUrlPubMed
  96. 96.
    1. Chinnasamy D,
    2. Yu Z,
    3. Kerkar SP,
    4. Zhang L,
    5. Morgan RA,
    6. Restifo NP,
    7. et al.
    Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clin Cancer Res 2012;18:1672–83.
    OpenUrlAbstract/FREE Full Text
  97. 97.
    1. Chmielewski M,
    2. Kopecky C,
    3. Hombach AA,
    4. Abken H
    . IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Res 2011;71:5697–706.
    OpenUrlAbstract/FREE Full Text
  98. 98.
    1. Hoyos V,
    2. Savoldo B,
    3. Quintarelli C,
    4. Mahendravada A,
    5. Zhang M,
    6. Vera J,
    7. et al.
    Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 2010;24:1160–70.
    OpenUrlCrossRefPubMed
  99. 99.
    1. Markley JC,
    2. Sadelain M
    . IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell-mediated rejection of systemic lymphoma in immunodeficient mice. Blood 2010;115:3508–19.
    OpenUrlAbstract/FREE Full Text
  100. 100.
    1. Perna SK,
    2. Pagliara D,
    3. Mahendravada A,
    4. Liu H,
    5. Brenner MK,
    6. Savoldo B,
    7. et al.
    Interleukin-7 mediates selective expansion of tumor-redirected cytotoxic T lymphocytes (CTLs) without enhancement of regulatory T-cell inhibition. Clin Cancer Res 2014;20:131–9.
    OpenUrlAbstract/FREE Full Text
  101. 101.
    1. Singh H,
    2. Figliola MJ,
    3. Dawson MJ,
    4. Huls H,
    5. Olivares S,
    6. Switzer K,
    7. et al.
    Reprogramming CD19-specific T cells with IL-21 signaling can improve adoptive immunotherapy of B-lineage malignancies. Cancer Res 2011;71:3516–27.
    OpenUrlAbstract/FREE Full Text
  102. 102.
    1. Stephan MT,
    2. Ponomarev V,
    3. Brentjens RJ,
    4. Chang AH,
    5. Dobrenkov KV,
    6. Heller G,
    7. et al.
    T cell-encoded CD80 and 4–1BBL induce auto- and transcostimulation, resulting in potent tumor rejection. Nat Med 2007;13:1440–9.
    OpenUrlCrossRefPubMed
  103. 103.
    1. Curran KJ,
    2. Seinstra BA,
    3. Nikhamin Y,
    4. Yeh R,
    5. Usachenko Y,
    6. van Leeuwen DG,
    7. et al.
    Enhancing antitumor efficacy of chimeric antigen receptor T cells through constitutive CD40L expression. Mol Ther 2015;23:769–78.
    OpenUrlCrossRefPubMed
  104. 104.
    1. Wilkie S,
    2. Burbridge SE,
    3. Chiapero-Stanke L,
    4. Pereira AC,
    5. Cleary S,
    6. van der Stegen SJ,
    7. et al.
    Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4. J Biol Chem 2010;285:25538–44.
    OpenUrlAbstract/FREE Full Text
  105. 105.
    1. Condomines M,
    2. Arnason J,
    3. Benjamin R,
    4. Gunset G,
    5. Plotkin J,
    6. Sadelain M
    . Tumor-targeted human t cells expressing CD28-based chimeric antigen receptors circumvent CTLA-4 inhibition. PLoS One 2015;10:e0130518.
    OpenUrlCrossRefPubMed
  106. 106.
    1. Fedorov VD,
    2. Themeli M,
    3. Sadelain M
    . PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci Transl Med 2013;5:215ra172.
    OpenUrlAbstract/FREE Full Text
  107. 107.
    1. Sampson JH,
    2. Choi BD,
    3. Sanchez-Perez L,
    4. Suryadevara CM,
    5. Snyder DJ,
    6. Flores CT,
    7. et al.
    EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss. Clin Cancer Res 2014;20:972–84.
    OpenUrlAbstract/FREE Full Text
  108. 108.
    1. Sanchez C,
    2. Chan R,
    3. Bajgain P,
    4. Rambally S,
    5. Palapattu G,
    6. Mims M,
    7. et al.
    Combining T-cell immunotherapy and anti-androgen therapy for prostate cancer. Prostate Cancer Prostatic Dis 2013;16:123–31, S1.
    OpenUrlCrossRefPubMed
  109. 109.
    1. Song DG,
    2. Ye Q,
    3. Santoro S,
    4. Fang C,
    5. Best A,
    6. Powell DJ
    Jr. Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition. Hum Gene Ther 2013;24:295–305.
    OpenUrlCrossRefPubMed
  110. 110.
    1. John LB,
    2. Devaud C,
    3. Duong CP,
    4. Yong CS,
    5. Beavis PA,
    6. Haynes NM,
    7. et al.
    Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells. Clin Cancer Res 2013;19:5636–46.
    OpenUrlAbstract/FREE Full Text
  111. 111.
    1. Kobold S,
    2. Steffen J,
    3. Chaloupka M,
    4. Grassmann S,
    5. Henkel J,
    6. Castoldi R,
    7. et al.
    Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer. J Natl Cancer Inst 2015;107:364.
    OpenUrlPubMed
  112. 112.
    1. Karlsson H,
    2. Lindqvist AC,
    3. Fransson M,
    4. Paul-Wetterberg G,
    5. Nilsson B,
    6. Essand M,
    7. et al.
    Combining CAR T cells and the Bcl-2 family apoptosis inhibitor ABT-737 for treating B-cell malignancy. Cancer Gene Ther 2013;20:386–93.
    OpenUrlPubMed
  113. 113.
    1. Huye LE,
    2. Nakazawa Y,
    3. Patel MP,
    4. Yvon E,
    5. Sun J,
    6. Savoldo B,
    7. et al.
    Combining mTor inhibitors with rapamycin-resistant T cells: a two-pronged approach to tumor elimination. Mol Ther 2011;19:2239–48.
    OpenUrlCrossRefPubMed
  114. 114.
    1. Gargett T,
    2. Fraser CK,
    3. Dotti G,
    4. Yvon ES,
    5. Brown MP
    . BRAF and MEK inhibition variably affect GD2-specific chimeric antigen receptor (CAR) T-cell function in vitro. J Immunother 2015;38:12–23.
    OpenUrl
  115. 115.
    1. Nishio N,
    2. Diaconu I,
    3. Liu H,
    4. Cerullo V,
    5. Caruana I,
    6. Hoyos V,
    7. et al.
    Armed oncolytic virus enhances immune functions of chimeric antigen receptor-modified T cells in solid tumors. Cancer Res 2014;74:5195–205.
    OpenUrlAbstract/FREE Full Text
  116. 116.
    1. Caruana I,
    2. Weber G,
    3. Ballard BC,
    4. Wood MS,
    5. Savoldo B,
    6. Dotti G
    . K562-Derived whole-cell vaccine enhances antitumor responses of CAR-redirected virus-specific cytotoxic T lymphocytes in vivo. Clin Cancer Res 2015;21:2952–62.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top
Cancer Discovery: 6 (2)
February 2016
Volume 6, Issue 2

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Email Article
Citation Tools
Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors
Aurore Morello, Michel Sadelain and Prasad S. Adusumilli
Cancer Discov February 1 2016 (6) (2) 133-146; DOI: 10.1158/2159-8290.CD-15-0583
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement