Chimeric antigen receptor (CAR) T-cell therapy for thoracic malignancies

Abstract

Chimeric antigen receptor (CAR) T cells are patient T cells that are transduced with genetically engineered synthetic receptors to target a cancer cell surface antigen. The remarkable clinical response rates achieved by adoptive transfer of T cells that target CD19 in patients with leukemia and lymphoma have led to a growing number of clinical trials exploring CAR T-cell therapy for solid tumors. Herein, we review the evolution of adoptive T-cell therapy, highlight advances in CAR T-cell therapy for thoracic malignancies, and summarize the targets being investigated in clinical trials for patients with lung cancer, malignant pleural mesothelioma, and esophageal cancer. We further discuss the barriers to successfully translating CAR T-cell therapy for solid tumors and present strategies that have been investigated to overcome these hurdles.

INTRODUCTION

Over the past decade, successful translation of immunotherapeutic agents to clinic with remarkable response rates in solid tumors, including lung cancer, has reinvigorated the interest in further exploiting the discoveries in tumor immunology. Immunotherapy uses and/or activates components of the immune system, such as antibodies, dendritic cells, and T lymphocytes, to treat cancer. The first documented attempt of immune stimulation as a cancer treatment was by Dr. William Coley in 1893. Dr. Coley treated patients diagnosed with sarcoma and carcinoma using cultures of Streptococcus pyogenes.¹ This approach was based on this pioneering New York surgeon’s observation of occasional tumor regression after acute infections. Dr. Coley treated >900 patients with “Coley’s toxins” and achieved a response rate of approximately 10% over a century ago.² Despite the promising results, these findings were not adopted by the scientific community and remained unexplored.

Further advancements in tumor immunology occurred in the 1950s when preclinical studies showed that the immune system can induce tumor regression in mice.³ In 1967, Burnet proposed the theory of immunosurveillance, which states that the immune system can recognize and eliminate initial tumor cells.⁴ This theory was confirmed years later in immunodeficient strains of mice that lacked expression of granzyme B (a protein necessary for the cytotoxic activity of CD8+ T lymphocytes) and in mice without B and T lymphocytes (Rag knockout); these animals have an increased rate of tumor formation compared with wild type animals.^{5, 6} In contrast to the role of the immune system in tumor control and eradication, it was clear that it also allows tumor development by selected clones with increased ability to escape from immune recognition and elimination. This paradoxical process was termed “cancer immunoediting” and has three phases—elimination, equilibrium and escape—that were confirmed in murine and human models.⁷

Adoptive T-cell therapy

The demonstration that the immune system can recognize and kill tumor cells in vivo through T lymphocyte activity has prompted researchers to develop protocols for isolation and expansion of tumor-reactive T cells, and to use these cells as treatment by adoptively infusing them back into patients; this process is termed “adoptive T-cell therapy.” The first such approach was developed in the 1990s and involved the isolation and expansion of tumor infiltrating lymphocytes (TILs) from tumors of patients with advanced melanoma. TILs were shown to recognize and kill tumor cells in vitro and can be expanded using anti-CD3/CD28 antibodies in the presence of interleukin-2 (IL-2). While initial studies involved only the infusion of T cells, which achieved limited responses,⁸ subsequent studies combined T cells with a non-myeloablative pre-conditioning regimen designed to induce lymphodepletion. This combination induced clinical responses in 51% of patients.⁹ This indicated that the pre-conditioning regimen lowered the competition for cytokines by endogenous T cells and favored the proliferation and survival of the infused T cells.¹⁰

However, TIL cultures cannot be generated for all melanomas nor other tumor types where there are not enough TILs or TILs do not recognize the tumor; this limits the applicability of this therapy. Advances in cellular and molecular biology have allowed for the isolation of responding T-cell clones and identification and sequencing of their T-cell receptor (TCR). The TCR is responsible for the recognition of peptides bound to the major histocompatibility complex (MHC) and activation of lymphocytes. Cloning the TCR sequence in viral vectors and transducing T lymphocytes isolated from peripheral blood can generate large numbers of tumor-specific T cells without the cumbersome process of isolation and expansion of TILs. TCRs that recognize tumor antigens have been identified and used in clinical trials to treat tumors such as melanoma and synovial sarcoma.¹¹ High-throughput analysis of TILs, in combination with deep sequencing of the autologous tumor, has led to T-cell therapy that targets tumor neoantigens arising from mutations.¹²

Despite facilitating the generation of tumor-specific T cells, TCR immunotherapy is limited by MHC restriction where each TCR recognizes a specific MHC haplotype. This means that expansion of TCR immunotherapy would involve the identification and cloning of a large number of TCRs to cover the most represented haplotypes in different patient populations; this would, in turn, increase the complexity of the therapy. Moreover, tumors generally downregulate MHC class I as an escape mechanism and become resistant to this type of therapy.¹³ These limitations can be overcome with use of chimeric antigen receptors (CARs). CARs are synthetic membrane receptors that activate T lymphocytes upon interaction with the target antigen (Table 1). These receptors were first developed by Eshhar et al.¹⁴ and have a structure comprised of an antigen-binding domain that is commonly a single-chain variable fragment (scFv) derived from an antibody, a transmembrane domain, and an intracellular signaling domain. The scFv confers high specificity and affinity, and allows for binding to the antigen that is expressed on the membrane of the target cell in a MHC-independent fashion (Fig. 1). Over the past two decades, CAR T cells have evolved from the first generation that contained only a signaling domain, such as the TCR-derived CD3ζ chain, to the second and third generations that have incorporated costimulatory domains such as CD28 and 4-1BB. These costimulatory domains recruit downstream effector molecules that result in the activation of multiple signaling pathways and lead to increased proliferation and improved functional persistence (Fig. 2).

Table 1.

Differences between TCR and CAR immunotherapy

	TCR complex	CAR
Typical affinity (range)	10−4–10−6 M	10−6–10−9 M
Target antigens	Surface and intracellular	Surfacea
Type of target antigen	Proteins	Proteins, carbohydrates, lipids
MHC restriction	Yes	No
Antigen processing required	Yes	No
MHC expression by target cell	Yes	No
Include costimulatory signals by design	No	Yes (second and third generation)

^aRecent research has shown that CAR T cells can also target intracellular antigens by using a scFv that recognizes a peptide epitope in context of the MHC complex on the cell surface.⁴⁸

CAR, chimeric antigen receptor; MHC, major histocompatibility complex; TCR, T-cell receptor

Fig. 1.

Scanning electron micrograph of CAR T cells pseudo colored (green) attacking a cultured MSLN-overexpressing malignant mesothelioma cell (blue). The T cells were genetically engineered to express a scFv that specifically targets MSLN.

CAR, chimeric antigen receptor; MSLN, mesothelin

Fig. 2.

Structure of CAR T cells. The extracellular domain is typically an antibody-derived scFv fused to a transmembrane and an intracellular signaling domain. While first generation CAR T cells contain only one intracellular signaling domain, such as the TCR-derived CD3ζ chain, second and third generation CAR T cells combine the signaling domain with one (second generation) or two or more (third generation) costimulatory domains such as CD28 or 4-1BB. The addition of costimulatory domains leads to increased proliferation and resistance to exhaustion.

CAR, chimeric antigen receptor; scFv, single-chain variable fragment; TCR, T-cell receptor

CAR T-cell therapy clinical successes

The first clinical trials that evaluated CAR T cells targeting CD19-positive hematologic malignancies, such as acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, showed remarkable response rates; 68–100% of ALL patients achieved complete remission.¹⁵ More importantly, these studies included relapsed, chemotherapy refractory patients and made use of second generation anti-CD19 CARs. Promising results have also been achieved with anti-CD22 CAR T cells in pediatric patients with CD22-expressing ALL that relapsed due to CD19 antigen loss in tumor escape variants after previously receiving anti-CD19 CAR T cells.¹⁶ CAR T-cell therapy for hematologic malignancies employs cytotoxic, preconditioning lymphodepletion, without which, the clinical benefits would be limited. The dramatic responses observed were not without toxicities such as cytokine release syndrome (CRS) and neurotoxicity. While CRS that results from T-cell activation is managed effectively by administering interleukin receptor-6 blockade agents or corticosteroids, the mechanism of neurotoxicity remains elusive.¹⁷ Novel CAR designs that incorporate molecular safety switches, which are suicide genes that can be activated to kill infused CAR T cells, are already in use in clinical trials to manage unexpected severe adverse events.

The manufacturing of autologous CAR T cells represents a multifaceted process that involves the collection of peripheral blood mononuclear cells from patients via apheresis, enrichment and activation of T cells, gene transfer and ex vivo expansion of CAR T cells, end-of-process formulation, and release testing.¹⁸ The chimeric receptor itself can be introduced into the T-cell either via stable insertion into the genome using viral (retroviral or lentiviral) and nonviral (transposon) based gene expression vectors or using messenger RNA for transient expression of the transgene.¹⁸ The next frontiers in CAR T-cell therapy includes third generation CARs, newer antigen targets, and effective management of CAR T-cell functional persistence.¹⁹

Despite differences in CAR T-cell antigen binding and costimulatory domains, gene transfer methods, and manufacturing processes across different clinical centers, there was a very high success rate in the eradication of hematologic malignancies in the majority of Phase I clinical trials.¹⁵ These successes have paved the way for translating CAR T-cell therapy to solid tumors including thoracic malignancies.

Rationale for promoting TILs in thoracic malignancies

Publications from our laboratory and others have demonstrated the survival benefits associated with higher ratio of effector to suppressive TILs in lung adenocarcinoma (ADC),²⁰ lung squamous cell carcinoma (LSCC),²¹ and malignant pleural mesothelioma (MPM).²² These observations, combined with the identification of tertiary lymphoid structures within non-small cell lung cancer (NSCLC) as specific chemoattractant signatures associated with TILs and prognosis,²³ and recent successes and development of strategies to increase functional TILs in NSCLC underscores the need to promote antigen-specific T-cell responses in thoracic tumors.²⁴ Unlike in hematologic malignancies where CD19 has been an ideal target for CAR T-cell therapy, identification of an ideal target for thoracic malignancies is challenging.

Targets for CAR T-cell therapy in clinical trials for thoracic malignancies

The ideal target for CAR T-cell therapy is either exclusively expressed or overexpressed on all or the majority of tumor cells with no or very limited expression on normal tissue. Targets for CAR T-cell therapy of thoracic malignancies that are currently being evaluated include overexpressed or aberrantly expressed antigens (epidermal growth factor receptor [EGFR], human epidermal growth factor receptor 2 [HER2], mesothelin [MSLN], glypican-3 [GPC3], epithelial cell adhesion molecule [EpCAM], prostate stem cell antigen [PSCA]), aberrantly glycosylated proteins (mucin 1 [MUC1]), oncofetal antigens (carcinoembryonic antigen [CEA], receptor tyrosine kinase-like orphan receptor 1 [ROR1]), tumor-associated stroma proteins (fibroblast activating protein [FAP]), and immunomodulatory antigens (programmed death-ligand 1 [PD-L1], CD80/CD86) (Fig. 3). Clinical studies investigating CAR T cells for thoracic malignancies are listed in Table 2.

Fig. 3.

Antigen targets in CAR T-cell therapy clinical trials for thoracic malignancies. The targets shown are compiled from clinical studies listed on ClinicalTrials.gov (last assessed on 7/20/17) that specifically target thoracic malignancies with CAR T cells. There are no targets currently employed for thymic cancer; however, MSLN is a potential therapeutic target for thymic carcinoma.³³

CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; FAP, fibroblast activating protein; GPC3, glypican-3; HER2, human epidermal growth factor receptor 2; MSLN, mesothelin; MUC1, mucin 1; PD-L1, programmed death-ligand 1; PSCA, prostate stem cell antigen; ROR1, receptor tyrosine kinase-like orphan receptor 1

Table 2. CAR T-cell clinical trials for thoracic malignancies.

These are clinical studies listed on ClinicalTrials.gov (last assessed on 7/20/17) that specifically defined thoracic tumors in the inclusion criteria or study outline.

Target(s)	Thoracic malignancy	Sponsor	Clinical trial ID
CD80/CD86, MUC1, PD-L1, PSCA	Advanced lung cancer	Second Affiliated Hospital of Guangzhou Medical University	NCT03198052
CD80/CD86, PD-L1	NSCLC	Yu Fenglei, Second Xiangya Hospital of Central South University	NCT03060343
CEA	Metastatic or unresectable lung cancer	Cancer Research UK	NCT01212887a
CEA	Lung ADC	Roger Williams Medical Center	NCT01723306b
CEA	Relapsed or refractory lung cancer	Southwest Hospital, China	NCT02349724
EGFR	Relapsed and refractory NSCLC	Chinese PLA General Hospital	NCT01869166
EGFR	Advanced lung cancer	Shanghai International Medical Center	NCT02862028
EpCAM	Relapsed or refractory esophageal cancer	First Affiliated Hospital of Chengdu Medical College	NCT03013712
FAP	MPM with pleural effusion	University of Zurich	NCT01722149
GPC3	Refractory, recurrent, metastatic, advanced LSCC	Carsgen Therapeutics, Ltd	NCT02876978
GPC3	LSCC	Second Affiliated Hospital of Guangzhou Medical University	NCT03198546
HER2	≥Stage IIIb esophageal cancer, stage IV lung cancer	Baylor College of Medicine	NCT00889954c
HER2	Chemotherapy refractory lung cancer	Chinese PLA General Hospital	NCT01935843
HER2	Relapsed or refractory lung cancer	Zhi Yang, Southwest Hospital, China	NCT02713984
MSLN	Epithelial or biphasic mesothelioma	University of Pennsylvania	NCT01355965c
MSLN	Metastatic or unresectable lung cancer, mesothelioma	National Cancer Institute	NCT01583686
MSLN	Epithelial mesothelioma	University of Pennsylvania	NCT02159716d
MSLN	NSCLC metastatic to the pleura, mesothelioma treated with at least one prior treatment regimen	Memorial Sloan Kettering Cancer Center	NCT02414269
MSLN	Chemotherapy refractory or relapsed mesothelioma	Chinese PLA General Hospital	NCT02580747
MSLN	Recurrent or metastatic mesothelioma	China Meitan General Hospital	NCT02930993
MSLN	Metastatic or recurrent lung ADC, Epithelial mesothelioma	University of Pennsylvania	NCT03054298
MUC1	Refractory or recurrent NSCLC	PersonGen BioTherapeutics (Suzhou) Co., Ltd.	NCT02587689
ROR1	Stage IV NSCLC treated with at least one prior treatment regimen	Fred Hutchinson Cancer Research Center	NCT02706392

^aThe study has been terminated due to lack of efficacy and safety concerns.

^bThe study is suspended due to funding.

^cThe study is active but not recruiting.

^dThe study has been completed.

ADC, adenocarcinoma; CEA, carcinoembryonic antigen; EGFR, epidermal growth factor receptor; EpCAM, epithelial cell adhesion molecule; FAP, fibroblast activating protein; GPC3, glypican-3; HER2, human epidermal growth factor receptor 2; LSCC, lung squamous cell carcinoma; MSLN, mesothelin; MPM, malignant pleural mesothelioma; MUC1, mucin 1; NSCLC, non-small cell lung cancer; PD-L1, programmed death-ligand 1; PSCA, prostate stem cell antigen; ROR1, receptor tyrosine kinase-like orphan receptor 1

In contrast to CD19, which is a B-cell lineage-specific target and consistently expressed in leukemia, most solid tumor antigen targets are also expressed at low levels on healthy tissue and may lead to on-target/off-tumor effects. A fatal adverse event of pulmonary toxicity was reported for a patient with colon cancer that metastasized to the lungs and liver immediately after administration of 1×10¹⁰ HER2-targeted CAR T cells that was ascribed to HER2 expression in the lung epithelium.²⁵ A subsequent dose-escalation Phase I/II study established feasibility and safety of administering HER2-targeted CAR T cells (10⁴/m²–10⁸/m²) to patients with recurrent or refractory HER2-positive tumors; however, the clinical benefit was limited as only 4 of 17 evaluable patients had stable disease.²⁶ Baylor College of Medicine, Southwest Hospital in China, and the Chinese PLA General Hospital are currently conducting further clinical studies using HER2-targeted CAR T cells to test their safety and efficacy for HER2-positive solid tumors including lung and esophageal cancer.

EGFR, along with HER2, belongs to the ErbB family of receptor tyrosine kinases and is an established target for antibody-based therapies and receptor tyrosine kinase inhibitors (RTKIs). The clinical response of EGFR-directed CAR T cells was investigated in a Phase I study (NCT01869166) in relapsed/refractory NSCLC patients with EGFR expression of ≥50% (as determined by immunohistochemistry). There were 2 of 11 patients with a partial response and 5 patients with stable disease after infusion of a median dose of 0.97×10⁷ CAR T cells/kg. This study also showed that patients who received no benefit from RTKI therapy can benefit from CAR T-cell therapy.²⁷

Mesothelin is a cell surface glycoprotein that is expressed at very low levels on normal mesothelial cells of the pleura, peritoneum, and pericardium. Our laboratory and others²⁸ have shown that MSLN is overexpressed on cancer cells in 80–90% of MPM,²⁹ 40–60% of lung ADC,³⁰ 36% of triple-negative breast cancer,³¹ 40% of esophageal cancer,³² and 79% of thymic cancer.³³ Overexpression of MSLN is correlated with tumor aggressiveness, KRAS mutation positivity, and decreased survival in lung ADC.³⁰ Additionally, we have shown that MSLN expression correlates with invasion in MPM²⁹ and malignant transformation in Barrett’s esophagus.³² This justifies our rationale for targeting MSLN in thoracic tumors. A case from a clinical trial (NCT01355965) conducted by the University of Pennsylvania that tested the safety and feasibility of repetitive intravenous or intratumoral administration of mRNA engineered T cells that transiently expressed an anti-MSLN CAR did not reveal evidence of off-tumor toxicities; however, epitope spreading was observed as a result of the antitumor efficacy. An anaphylactic event caused by immunogenicity against the scFv of murine origin occurred after repeated infusions.³⁴ Our laboratory has developed a CAR construct with a fully human scFv that targets MSLN and exerts long-lasting tumor immunity following regional administration of MSLN-targeted, second generation CAR T cells in an orthotopic mouse model of human pleural disease.³⁵ Based on our preclinical data of enhanced antitumor efficacy following regional, rather than systemic, administration of CAR T cells, we are conducting a Phase I clinical trial to evaluate the safety of regionally administered MSLN-targeted CAR T cells in patients with primary or secondary pleural malignancies (NCT02414269).

GPC3, EpCAM, and PSCA are additional tumor-associated antigens that are overexpressed in tumors cells and are currently being investigated as CAR T-cell targets for thoracic malignancies. GPC3 was present in 66% LSCC and 3.3% of lung ADC samples, but not in normal lung tissue,³⁶ and it has been targeted in two clinical studies (NCT02876978, NCT03198546). EpCAM overexpression is associated with poor survival in patients with esophageal squamous cell carcinoma³⁷ and explored as a target for CAR T-cell therapy of esophageal cancers (NCT03013712). PSCA is being studied in one clinical trial (NCT03198052) as a candidate target and a recent study in patient-derived xenograft mouse models suggests that dual targeting of PSCA and MUC1 can synergistically enhance antitumor efficacy in NSCLC.³⁸ Tumor-selective antigens that arise from deregulated post-translational modifications, such as MUC1, provide another class of targets for immunotherapy. MUC1 is found aberrantly glycosylated in multiple ADCs and is associated with poor overall survival in patients with NSCLC.³⁹ Currently, two ongoing clinical trials are using MUC1-targeted CAR T cells to treat lung cancer patients (NCT02587689, NCT03198052).

Oncofetal antigens are comprised of a class of targets that are typically only present during fetal development but are found on some cancer variants in adults. A Phase I clinical trial (NCT01212887) of first generation CEA-specific CAR T cells with preconditioning chemotherapy and intravenous IL-2 in patients with CEA-positive solid tumors was terminated. The reasons for termination included safety concerns and lack of efficacy, which was attributed to poor persistence of the CAR T cells and pre-conditioning-dependent respiratory toxicity.⁴⁰ Another ongoing clinical trial targeting CEA in lung cancer and other solid tumors (NCT02349724) is using a second generation CAR T-cell and has shown encouraging results in patients with CEA-positive metastatic colorectal cancer.⁴¹ The receptor tyrosine kinase ROR1 is an oncofetal antigen overexpressed in lung ADC and is an independent prognostic biomarker for overall survival.⁴² Fred Hutchinson Cancer Research Center is currently conducting a Phase I clinical trial of CAR T cells in patients with stage IV ROR-positive NSCLC (NCT02706392).

Another appealing strategy for CAR T-cell therapy is to simultaneously target tumor and associated stroma cells. A potential target is FAP and it is expressed in all of the major MPM histologic subtypes (epithelioid, sarcomatoid, and biphasic) as well as the tumor stroma. Based on this observation, Schubert et al. developed an intraperitoneal xenograft model for testing FAP-specific CAR T cells and showed that treated mice bearing FAP-positive tumors had a significant survival benefit.⁴³ This provided the rationale for a clinical trial that tests the safety of a single dose of 1×10⁶ FAP-specific T cells administered directly into the pleural effusion (NCT01722149). Recent research has shown that FAP-targeting CAR T cells disrupt stromagenesis, diminish tumor angiogenesis, and may increase immune cell infiltration in highly desmoplastic tumors.⁴⁴ Conversely, a preclinical study raised concerns of possible bone toxicity due to the strong expression of FAP on bone marrow stromal cells.⁴⁵

There are currently two clinical trials (NCT03198052, NCT03060343) that target PD-L1 and CD80/CD86 in lung cancer. Although PD-L1 blocking antibodies (checkpoint inhibitors) are successfully used in the clinic to treat lung cancer,⁴⁶ no preclinical data exists for CAR T cells that target PD-L1. CD80 and CD86 that are expressed on the surface of antigen-presenting cells either bind to CD28 on T cells and provide the costimulatory signal necessary for T-cell activation or, depending on the context, lead to downregulation of T-cell activity by binding to cytotoxic T-lymphocyte antigen 4 (CTLA-4).⁴⁷ Little is known about CD80/CD86 expression on lung tumor cells and, due to the expression of CD80/CD86 and PD-L1 on normal immune cells, possible on-target/off-tumor toxicities need to be carefully elucidated.

It is noteworthy to mention that CAR T cells can also be engineered to target intracellular antigens. This is exemplified by the CAR T cells that bind to a peptide epitope from the zinc finger transcription factor WT1 that is presented in the context of MHC on the cell surface.⁴⁸ This work provided proof-of-concept that the scope of possible CAR targets can be expanded beyond surface antigens.

Hurdles in successful translation of CAR T-cell therapy for solid tumors

Despite the encouraging clinical results in hematologic malignancies, successful use of CAR T cells for the treatment of solid tumors has proven to be challenging. While several T-cell parameters can still be optimized, it is clear that tumor-intrinsic mechanisms and the associated tumor microenvironment have an important role in the inhibition of antitumor response. Tumor cell heterogeneity that results from genomic instability induces the generation of clonal populations that harbor different mutations and antigen expressions, and can facilitate the generation of escape variants upon CAR T-cell therapy. Variability of antigen expression on the surface of the tumor cell constitutes one of the major hurdles in CAR T-cell therapy, possibly rendering tumor cells with low antigen expression less susceptible to CAR T-cell therapy (Fig. 4A). The use of T cells that target two antigens simultaneously can help prevent tumor evasion.⁴⁹ Despite the fact that this strategy may be hampered by the limited number of described antigens and potential on-target/off-tumor toxicity, ongoing studies are investigating the utility in the context of thoracic malignancies.

Fig. 4.

Multiplex immunofluorescence staining of patient tumors demonstrate key obstacles to CAR T-cell therapy for thoracic malignancies. (A) Tumor heterogeneity: Shown are MSLN-positive tumor cells in yellow. The arrowhead points to high antigen-expressing tumor cells and the arrows point to low antigen-expressing tumor cells. (B) Stroma as a barrier: Shown are tumor cells in yellow, TILs in green, and stromal components in red. (C) Immunosuppressive cells, such as tumor-associated macrophages, are shown in red interacting with TILs (green). (D) Immune checkpoint suppression via PD-1/PD-L1 axis. The arrowheads point to PD-L1-positive cells in magenta and the arrows point to PD-1-positive TILs.

CAR, chimeric antigen receptor; MSLN, mesothelin; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; TILs: tumor infiltrating lymphocytes

The successful trafficking of CAR T cells to the tumor site is dependent on the expression of adhesion molecules on the T-cell and tumor endothelium, as well as chemokines secreted by the tumor that match the chemokine receptor expressed on the T-cell.⁵⁰ The fact that MPM secretes high levels of the chemokine CCL2 has provided the necessary evidence to generate CAR T cells that overexpress CCR2 (the receptor for CCL2), which have shown increased migration to the tumor and improved antitumor activity in a mesothelioma model.⁵¹

Regional delivery of CAR T cells is another promising approach to bypass the pulmonary sequestration of lymphocytes. Our published observations that intrapleural administration of anti-MSLN CAR T cells yields long-term antitumor activity, even at a 30-fold lower doses to achieve complete response in a preclinical model,³⁵ provides a clinically significant opportunity to treat pleural malignancies; the current annual incidence in the U.S. alone is 150 000 patients.⁵² Administration of CAR T cells into the pleural space is currently being tested in clinical trials for MSLN- and FAP-targeted CAR T cells (NCT02414269, NCT01722149).

Once trafficked to the tumor site, CAR T cells are exposed to anatomical and metabolic barriers. Cancer-associated stroma represents an anatomical barrier that CAR T cells need to overcome to successfully infiltrate the tumor (Fig. 4B). Targeting stroma cells using FAP-specific CAR T cells have been shown to increase CD8+ T-cell infiltration and augment antitumor immunity by disrupting extracellular matrix components.⁴⁴ The limited supply of oxygen and nutrients found in the tumor microenvironment also affects the function of T lymphocytes. A glucose-poor microenvironment limits the capacity of effector T cells to engage in glycolysis and results in inhibition of effector functions and lower levels of the glycolytic metabolite phosphoenolpyruvate that sustains the Ca2+/NFAT signaling essential for T-cell function.⁵³ Byproducts of metabolism, such as lactate, can also inhibit the activity of CD8+ T cells.⁵⁴ Limited availability of amino acids, such as arginine, used by lymphocytes as an alternative source of energy for protein synthesis during the expansion phase of activated T cells also impairs T-cell function and antitumor activity.⁵⁵

Immunosuppressive cells found in the microenvironment of solid tumors also play an important role in T-cell therapy resistance. Tumor-associated macrophages (M1 and M2 phenotype) are a large fraction of the infiltrating myeloid cells in MPM and lung cancer (Fig. 4C).⁵⁶ While M1 macrophages are associated with Th1 responses and antitumor activity, M2 macrophages are involved in angiogenesis and extracellular matrix remodelling. M2 macrophages inhibit differentiation to M1 macrophages and secrete several inhibitory cytokines that results in inhibition of T-cell function when their frequency increases.^{57, 58} Myeloid-derived suppressor cells are a heterogeneous population of immature myeloid cells that are also involved in inhibiting T-cell response by changing the amino acid composition in the tumor microenvironment and recruiting regulatory T cells (Tregs).⁵⁹ Tregs can also suppress T-cell function via direct cell-to-cell interaction, checkpoint regulation, or secretion of inhibitory cytokines.⁶⁰

Several soluble factors and cytokines secreted by the tumor and immunosuppressive cells have inhibitory effects on T cells. One of the best studied factors is transforming growth factor β (TGF-β) and it has described roles in NSCLC,⁶¹ mesothelioma,⁶² and thymoma⁶³ progression. TGF-β induces the polarization of macrophages into a M2-like phenotype and, in the case of T lymphocytes, promotes Treg differentiation⁶⁴ and inhibits CD8+ T-cell function.⁶⁵ Cytotoxic lymphocytes that overexpress a dominant negative TGF-β receptor were resistant to these inhibitory functions,⁶⁶ thereby providing a promising strategy to enhance CAR T-cell activity. Other factors, such as prostaglandin E2 and adenosine, exist within the tumor microenvironment and also contribute to T-cell immunosuppression. Both molecules bind to G protein-coupled receptors expressed in T cells and inhibit their function mainly through cyclic AMP/PKA signaling.⁶⁷ A recent study has shown that anti-MSLN CAR T cells that express a small peptide that inhibits PKA signaling have increased antitumor activity in vitro and in vivo.⁶⁸

Both infiltrating myeloid cells and tumor cells express several inhibitory ligands in their membrane that can interact with T lymphocytes and inhibit their activation (Fig. 4D). One of the most studied is PD-L1, which is upregulated in response to interferon gamma signaling and interacts with the programmed cell death protein 1 (PD-1) inhibitory receptor on T cells.⁴⁷ Chronic PD-1 signaling in T lymphocytes induces an exhaustion phenotype that is characterized by decreased effector function and proliferation. Our group has shown that, upon repeated antigen stimulation in vitro and in vivo, anti-MSLN CAR T cells become exhausted and upregulate PD-1.⁶⁹ Moreover, tumor cells upregulate PD-L1 and PD-L2 in vivo after treatment with CAR T cells; this is directly related to the cytokines produced by the activated lymphocytes. The administration of PD-1 blocking antibodies can rescue infused T-cell activity; however, multiple doses are required. The use of a PD-1 dominant negative receptor co-expressed with a CAR enhanced T-cell function and increased tumor-free survival, while simultaneously avoiding repeated doses of blocking antibodies.⁶⁹ A similar strategy was used by Liu et al. who co-expressed a CAR with a switch receptor that harbored the extracellular domain of PD-1 and the transmembrane and intracellular domain of the costimulatory molecule CD28. The inhibitory PD-L1/PD-1 signaling was blocked while providing CD28 costimulation and led to augmented antitumor efficacy.⁷⁰

Future perspective and conclusion

With recent unanimous FDA approval of CD19-targeted CAR T cells for the treatment of relapsed or refractory ALL patients (80–90% overall response rates), CAR T-cell therapy for hematologic malignancies has become mainstream. Although treatment will be conducted at accredited treatment centers in the U.S. with staff who are appropriately trained on a comprehensive risk-mitigation strategy, the advances in cell manufacturing and production (failures reduced to 2% and turnaround time 3 weeks), the efficient management of CRS, and the infrastructure for clinical implementation of CAR T-cell therapy are already well established.⁷¹

The constant interplay between T cells, tumor cells, and cells present in the tumor microenvironment induce immunosuppressive networks that are reinforced, which ultimately inhibits the antitumor response. The aforementioned strategies were developed from understanding the solid tumor immune microenvironment, can help overcome the suppression of CAR T cells, and are being translated to the clinic.⁷² Combination CAR T-cell therapy and checkpoint blockade has already been implemented by our group in the clinic and agents that target the PD-1/PD-L1 axis only mark the beginning of many possible future combination regimens.⁴⁷ A more complete understanding of the dominant immunosuppressive pathways in thoracic malignancies will help us define the optimal CAR design in terms of antigen targeting, signaling domains, and additional genetic modifications to improve and sustain clinical responses in future studies. The study of additional inhibitory receptors, such as lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin domain, and mucin domain containing molecule-3 (TIM-3),⁷³ may improve our understanding of immune activation and response, and also provide new strategies in combination therapy. The integration of advances in T-cell biology, tumor biology, and the tumor microenvironment will provide us with the necessary tools to enhance CAR T-cell therapy function and efficacy, and ultimately improve clinical responses of patients with thoracic malignancies.

Abbreviations

ADC

adenocarcinoma

ALL

acute lymphocytic leukemia

CAR

chimeric antigen receptor

CEA

carcinoembryonic antigen

CRS

cytokine release syndrome

CTLA-4

cytotoxic T-lymphocyte antigen4

EGFR

epidermal growth factor receptor

EpCAM

epithelial cell adhesion molecule

FAP

fibroblast activating protein

FDA

Food and Drug Administration

GPC3

glypican-3

HER2

human epidermal growth factor receptor 2

IL-2

interleukin-2

LAG-3

lymphocyte-activation gene 3

LSCC

lung squamous cell carcinoma

MHC

major histocompatibility complex

MPM

malignant pleural mesothelioma

MSLN

mesothelin

MUC1

mucin 1

NSCLC

non-small cell lung cancer

PD-1

programmed cell death protein 1

PD-L1

programmed death-ligand 1

PSCA

prostate stem cell antigen

ROR1

receptor tyrosine kinase-like orphan receptor 1

RTKIs

receptor tyrosine kinase inhibitors

scFv

single chain variable fragment

TCR

T-cell receptor

TGF-β

transforming growth factor β

TILs

tumor infiltrating lymphocytes

TIM-3

T-cell immunoglobulin domain and mucin domain containing molecule-3

Tregs

regulatory T cells