Tumor Antigen Expression and Heterogeneity

A primary challenge in developing CAR T cell therapy is identifying a tumor antigen that can be targeted safely and effectively [reviewed in Ref. (35)]. Ideally, CAR T cell therapy should target a tumor-restricted antigen to avoid the risk of “on-target/off-tumor” toxicity that may result in an immune reaction against healthy tissues, and at least two criteria should be considered (36). First, the proposed TAA should be differentially expressed on tumor cells compared with essential normal tissue. The CAR response is highly specific and can potentially bind to antigens even at low expression levels in normal tissues. Fine-tuning CAR design to recognize differential expression of antigens on tumor cells continues to evolve, and this represents a dynamic area of research aimed to expand the reach of CAR T cell therapy. Second, the TAA should be broadly expressed on the majority of tumor cells, as the success of CAR T cell therapy is largely dependent on expression of antigens on tumor cells (8, 36). Many of the TAAs identified [e.g., EGFR/EGFRvIII, IL13Rα2, HER2, CD171, mesothelin (MSLN), GD2, and carcinoembryonic antigen (CEA)] are expressed by a wide range of solid tumors, and this affords opportunity for combination therapies using CARs targeting multiple antigens. Table ​Table22 lists antigens that have served as targets for solid-tumor T cell therapeutic studies to date.

Table 2

A summary of solid tumor antigens being targeted using CAR T cell therapy.

Antigen	Type of cancer	Endomains	Gene transfer method	Reference
CD171	Recurrent/refractory neuroblastoma	CD3ζ	Electroporation	(37)
EGFRvIII	Glioma	CD28+CD3ζ, 4-1BB	Gamma-retrovirus	(38)
Epidermal growth factor receptor	Gastric cancer	–	Gamma-retrovirus	(39)
Carbonic anhydrase IX	Metastatic renal cell carcinoma	FcRγ	Gamma-retrovirus	(40)
α-folate receptor	Ovarian	FcRγ	Gamma-retrovirus	(41)
HER2	Sarcoma	CD28-CD3ζ	Gamma-retrovirus	(42)
HER2	Glioblastoma	CD28-CD3ζ	pigyBac	(43)
HER2	Osteosarcoma	CD28-CD3ζ	SFG retroviral	(44)
αHER2/CD3	Gastric cancer	CD28-CD3ζ	Gamma-retrovirus	(45)
Carcinoembryonic antigen	Liver metastases	CD28-CD3ζ	Gamma-retrovirus	(46)
IL13Rα2	Glioblastoma	CD3ζ	Electroporation	(47)
IL13Rα2	Glioblastoma	4-1BB, CD3ζ	Lentivirus	NEJM
HER2	Metastatic colon cancer	4-1BB, CD28, CD3ζ	Gamma-retrovirus	(48)
GD2	Neuroblastoma	CD3ζ	Gamma-retrovirus	(49)
GD2	Neuroblastoma	CD28, CD3ζ, OX40	SFG retroviral	(50)
ErbB2+MUC1	Breast cancer	CD28, CD3ζ	SFG retroviral	(51)
Vascular endothelial growth factor receptor 2+gp100+TRP-1+or TRP-2	Melanoma	–	Gamma-retrovirus	(24)
FAP	Colon and ovarian cancer	CD8α, CD3ζ, 4-1BB	Gamma-retrovirus	(17)
HER2+CD19	Medulloblastoma	CD28+CD3ζ	SFG retroviral	(23)
Mesothelin (MSLN)	Malignant Pleural Mesothelioma	CD3ζ and 4-1BB	Lentiviral	(22)
NKG2D	Breast cancer	CD28+CD3ζ	Gamma-retrovirus	(21)
MSLN	Pancreatic cancer	CD3ζ and 4-1BB	Gamma-retrovirus	(8)
MSLN	Malignant pleural mesothelioma	CD3ζ and 4-1BB	Gamma-retrovirus	(8)

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The epidermal growth factor receptor (EGFR) is a trans-membrane receptor tyrosine kinase (170kDa) that belongs to ErbB oncogene family (52–54). A wide range of normal and tumor cells express EGFR, and deregulation of EGFR is associated with epithelial tumors, such as pancreatic cancer, lung cancer, head and neck squamous cell carcinoma, colorectal cancer, and breast cancer (55, 56). Upregulation of EGFR is associated with poor prognosis in clinical settings (57, 58). Many studies have reported genomic alterations of EGFR in glioblastomas affecting both the extracellular and intracellular domains (59, 60). As the most common oncogenic EGFR mutant, with expression on ~30% of glioma cells (60, 61), EGFRvIII contains a deletion of extracellular amino acids 6–273 (62, 63), resulting in constitutive tyrosine kinase activity that promotes aggressive growth and tumor metastasis (64–66). This mutated extracellular EGFRvIII domain presents a tumor-specific, immunogenic epitope for CAR targeting (67, 68). Researchers have evaluated EGFRvIII-CARs for immunotherapy of glioma (38, 68), with the targeting domain derived from EGFRvIII-specific monoclonal antibodies. EGFRvIII-CAR T cells produced interferon-γ, effector cytokines, and were able to kill EGFRvIII+ tumor cells, demonstrating that EGFRvIII-CAR T cells can eliminate glioma cells (38, 67, 68).

Another promising target for brain malignancy is IL13 receptor α2 (IL13Rα2), a monomeric high affinity IL-13 receptor that is overexpressed in the majority of glioblastoma tumors and not expressed at significant levels on normal brain tissue (69, 70). In addition, IL13Rα2 expression is a prognostic indicator of poor patient survival (70). This disease-associated expression profile supports the development of CAR T cells targeting IL13Rα2 for the treatment of glioblastoma and possibly other solid tumors (71). To target IL13Rα2 both antibody- and ligand-based CARs are being evaluated. Our group and others have developed ligand-based CARs utilizing membrane bound IL13 muteins for preferential recognition of IL13Rα2 over the more ubiquitously expressed IL13Rα1 (71). Ligand-based CARs represent a novel class of CAR design. City of Hope is currently in clinical trial evaluating an IL13-ligand CAR T cell platform, and early findings suggest encouraging evidence for safety and therapeutic bioactivity (47, 72).

HER2, a trans-membrane glycoprotein belonging to the EGFR family, is another attractive target antigen for cancer immunotherapy (73, 74). HER2 is overexpressed in osteosarcoma, medulloblastoma, glioblastoma, and ovarian and breast cancer, among others (75–78). Several studies point to the critical role of HER2 in various cancer pathological processes (79), and HER2 overexpression is associated with poor clinical outcomes (80, 81). Ahmed et al. evaluated HER2-CAR T cell therapy for medulloblastoma (78), demonstrating that HER2-CAR T cells are able to target and kill HER2+ medulloblastoma cells in vitro and in an established medulloblastoma orthotopic xenogeneic SCID mouse model (78). The researchers reported in a study of osteosarcoma that HER2-CAR T cells, proliferated, produced immunostimulatory T helper 1 (Th1) cytokines, and killed HER2+ osteosarcoma cells in vitro, and HER2-CAR T cells caused regression of established osteosarcoma xenografts in locoregional as well as metastatic mouse models (44).

Mesothelin is a tumor differentiation antigen (40kDa) that is normally present on the mesothelial cells of pleura, peritoneum, and pericardium (82, 83) and is highly expressed in many human cancers, including malignant mesothelioma, pancreatic, ovarian, and lung adenocarcinoma (84–87). MSLN overexpression is associated with proliferation of tumor cells, invasion, and poor survival rates of patients (88–90). The limited expression in normal tissues and high expression in many cancers renders MSLN a potential CAR T cell target (86). Riese et al. evaluated MSLN-CAR T cell treatment for thymoma in a mouse model (91) using a novel strategy designed to improve T cell function by eliminating negative regulators. Given that CAR signaling derives from TCR intracellular domains that function to initiate signal transduction, deletion of negative regulators may augment CAR signaling and effector T cell function. The researchers examined CAR activity in T cells that lacked one or both isoforms of diacylglycerol (DAG) kinase (dgk), normally highly expressed in T cells. The enzymes dgkα and dgkζ metabolize the second messenger DAG and limit Ras/ERK activation. The researchers found that, similar to pharmacologic inhibition of dgk enzymes, dgk-deficient CAR T cells were more effective in limiting the growth of implanted tumors, both concurrent with and after establishment of the tumor. These results indicate that modification of CAR T cells (herein, deletion of negative regulators of TCR signaling) could improve the activity and function of CAR T cells in a solid tumor model. This work highlights the importance of CAR T cell modifications that extend beyond the CAR molecule to T cell-specific functional machinery—modifications that may broaden clinical use and improve the efficacy of CAR T cells (91).

Many preclinical studies of CAR T cells that target stroma and/or TAAs in solid tumor models have evaluated such targets as carbonic anhydrase IX (CAIX), GD2, vascular endothelial growth factor receptor 2 (VEGFR2), folate receptor alpha (FR-α), FAP, and CEA. Several platforms have advanced to the clinic, and Table ​Table33 lists clinical trials corresponding to solid-tumor targets, many of which used first-generation CARs. For example, Lamers et al. assessed first-generation CAIX–CAR T cell therapy in renal carcinoma patients and observed “on-target/off-tumor” side effects and a low persistence of CAR T cells, possibly due to host immune response against CARs (92, 93). Other studies have reported low persistence of first-generation CD171–CAR T cells in neuroblastoma patients and FR-α-CAR T cells in ovarian cancer patients (37, 41). Although results from first-generation CAR T cell therapy trials were disappointing (37, 92, 93), the studies provided data and insights on CAR optimization, leading to the generation of second- and third-generation CARs that may overcome some of the challenges in solid tumor therapy.

A large number of surface antigens are variably expressed by tumor cells, and among barriers associated with solid-tumor CAR T cell therapy, cell surface antigen heterogeneity features prominently in failures to achieve durable responses (36). O’Rourke et al. found heterogeneity of EGFRvIII expression to be a major barrier in targeting it as a single antigen (67). In a clinical study of EGFRvIII–CAR T cell therapy for glioblastoma, they noted wide regional variation of EGFRvIII expression in tumor samples after EGFRvIII–CAR T cell infusion. Most subjects had loss or decreased expression of EGFRvIII in tumors despite no change in the degree of EGFR amplification or other tumor mutations. The study poses the question whether targeting EGFRvIII alone can provide durable clinical benefits or whether antigen escape will negate the clinical impact. CAR T cell targeting of the tumor antigen IL13Rα2 in patients with glioblastoma has encountered similar hurdles with antigen heterogeneity. Brown et al. reported that treatment with IL13Rα2–CAR T cells mediated a CR in one patient, but the disease eventually recurred 228days after the first CAR T cell treatment at sites distinct and nonadjacent to the original tumors. Preliminary results suggest the cause of recurrence is decreased expression of IL13Rα2 (72). These and many other studies point to the importance of target antigen overexpression and distribution on most, if not all, tumor cells. As a criterion for patient enrollment on CAR T cell therapy trials, prescreening for the intensity and percentage of target antigen expression on tumor cells by immunohistochemistry and/or immunofluorescent techniques may be predictive of response (17, 23, 24, 51).

Targeting of multiple tumor antigens simultaneously or in a combinatorial strategy could lead to better “killing coverage” and potentially block the emergence of target antigen-null tumor cells (17, 23, 24, 51). Preclinical experiments with trivalent CAR T cells co-targeting HER2, IL13Rα2, and EphA2 showed promise in overcoming glioblastoma variability (94). Analysis of primary glioblastoma patient samples demonstrated the trivalent CARs captured nearly 100% of tumor cells in most tumors and exhibited improved cytotoxicity and cytokine release over monospecific and bispecific CAR T cells. Treatment with the multi-specific CAR T cells in vivo controlled established autologous glioblastoma patient-derived xenografts and improved survival of treated animals (94). Another study of dual-targeted CAR T cells specific for MUC1 and ErbB2 demonstrated their effectiveness against solid tumors, particularly breast cancer (51). Proliferation of the dual MUC1/ErbB2 CAR T cells required coexpression of MUC1 and ErbB2 on target tumor cells, and the CAR T cells were effective in killing ErbB2(+) tumor cells. These findings suggest that multivalent CARs may be an effective strategy to box-in heterogeneous tumors and thereby block resistance through tumor escape (51). However, tumor antigen expression loss in glioblastoma patients following CAR T cell therapy specific to one antigen implies that selection of clonal variants resistant to treatment occurs. With the integration of multivalent targets, there may be potential for further selection and the development of treatment resistance over time.

The Suppressive Solid Tumor Microenvironment

Clinical and preclinical studies have shown that reversing immune inhibitory pathways triggered in many cancers may require CAR T cell modifications beyond the inclusion of co-stimulatory signaling. In contrast to certain blood cancers that have responded well to CAR T cell therapy, solid tumors not only lack conventional co-stimulatory molecules, which are expressed on malignant and normal B lymphocyte targets in hematological malignancies, but also have evolved mechanisms to actively suppress the immune system (95, 96). A number of immunosuppressive pathways can limit the full potential of adoptive CAR T cell therapy. Inhibitory immune receptors are often expressed on T cells following persistent tumor antigen encounter, and these include T-cell membrane protein-3 (TIM-3), lymphocyte-activation protein-3 (LAG-3), T cell Ig and ITIM domain (TIGIT), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and programmed death-1 (PD-1). The upregulation of these receptors limit the persistence and activity of the antitumor response of CAR T cells (36).

Tumors employ multiple tactics to evade or misdirect tumor-specific immune response. Many soluble factors that suppress antitumor immune responses have been identified in tissue extracts, serum, and ascites fluid of cancer patients. Tumor cells and macrophages express prostaglandin E2 (PGE2), a soluble factor derived from arachidonic acid and produced by inducible cyclo-oxygenase 2 enzyme (8, 36) that exerts its immunosuppressive effect through subversion of CD8 differentiation, suppression of T cell proliferation, and inhibition of CD4 T cell helper functions (97). The PGE2/EP2/protein kinase A (PKA) signaling pathway mediates immunosuppression through PGE2 (98), which in combination with adenosine activates PKA and blocks TCR activation. A small peptide called the “regulatory subunit I anchoring disruptor” (RIAD) dampens the negative effects of PKA on TCR activation—a function that researchers leveraged to improve T cell function. Through generation of CAR T cells expressing RIAD, Albelda and colleagues showed that inhibition of upstream immunosuppressive mediators of PKA activation such as PGE2 and adenosine could lead to increased TCR signaling, more cytokine release, and increased CAR T cell infiltration, leading to enhanced tumor cells killing (19). Increased inflammatory activity is a hallmark of the tumor microenvironment and creates an abundance of reactive oxygen species (ROS) that substantially impair antitumor activity. Ligtenberg and colleagues hypothesized that CAR T cells coexpressing catalase (CAT) would perform better than regular CAR T cells. They showed that CAT–CAR T cells produced more intracellular catalase, leading to a reduced oxidative state with less ROS accumulation in both the basal and activated states. The CAT–CAR T cells maintained antitumor activity despite an inhospitable environment of high H₂O₂ (99).

Many tumors produce transforming growth factor beta (TGFβ), which inhibits T cell activation, proliferation, and cytotoxicity (Figure ​(Figure1).1). Chou et al. demonstrated that cell-intrinsic abrogation of TGFβ signaling can enhance T cell persistence and function in a murine model of autochthonous prostate cancer. Moreover, it has been shown that T cells rendered insensitive to TGFβ by transduction with TGFβ dominant negative receptor II were highly effective in eliminating established melanoma-bearing mice (100). This idea has been translated to clinical studies of tumor-infiltrating lymphocytes engineered to express a TGFβ1-dominant negative transgene (NCT01955460). This approach offers an alternative to therapeutic anti-TGFβ monoclonal antibodies (fresolimumab/GC1008).

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Figure 1

A schematic representation of the immunosuppressive tumor microenvironment.

The recent development of checkpoint inhibitors such as ipilimumab targeting CTLA-4 and nivolumab targeting PD-1 provide further opportunities to enhance antitumor immune response with the potential to produce durable clinical responses though opportunistic autoimmunity (36). John et al. demonstrated that combining CAR T cells and PD-1 blocking antibodies could potently enhance CAR T cell therapy. They found that such combination can significantly decrease the percentage of Gr1+ CD11b+ myeloid-derived suppressor cells, and this was strongly correlated with therapeutic responses in established disease (9). Alternatively, CAR T cells can be further modified intrinsically to enhance their function particularly in the context of the targeted tumor microenvironment. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely engineer specific genomic sequences. The CRISPR/Cas9 system and the simpler Cas9/sgRNA system enable the efficient construction of knockout alleles through the induction of frameshift mutations. With this gene-editing tool, it is possible to generate knock out PD-1 and/or CTLA-4 CAR T cells. This strategy not only prevents potential toxicity of anti-PD-1 or CTLA-4 administration (e.g., opportunistic autoimmunity) but it also would not interfere with normal homeostatic functions of these molecules within the body. Table ​Table11 illustrates various challenges faced by CAR T cell therapy in solid tumors.

Immune Stimulatory CAR T Cell Modifications

T cell costimulation mediated by CAR second-generation intracellular domains, such as CD28, CD137, CD27, or OX40, fail to overcome many inhibitory effects, especially once T cell anergy/exhaustion have taken effect. Several proposed countermeasures to immunosuppression may enhance antitumor CAR T cell activity in the solid tumor microenvironment, and these include increasing intrinsic CAR T cell activity via intracellular modifications, selecting immature or memory T cell subsets for T cell product manufacturing, and targeting cellular and molecular components of the tumor microenvironment. One promising approach entails the use of immunostimulatory cytokines that may revert or block tumor-associated inhibition and activate adoptively transferred T cells. Strategies that provide high levels of immunostimulatory cytokines locally at the antigen site have demonstrated preclinical and clinical efficacy. Animal models using poorly immunogenic tumors revealed that T cells genetically engineered to produce cytokines like IL-7, Il-15, and Il-12 were effective in eradicating tumors (101, 102). Another approach combines blocking immune suppression simultaneous with enhancing stimulatory cytokine production. To protect CAR T cells from the immunosuppressive cytokine IL-4, Mohammed et al. generated a hybrid cytokine receptor in which an IL-4-receptor exodomain was fused to an IL-7-receptor endodomain (103). Transgenic expression of this molecule in CAR T cells reverted the inhibitory effects of tumor-derived IL-4 and promoted T cell proliferation, resulting in enhanced antitumor activity.

Research has shown that locally produced IL-15 improved CAR T cell expansion and prolonged persistence in vivo by increasing the expression of antiapoptotic molecules, such as Bcl-2, through activation of the phosphoinositide 3-kinase signaling pathway (104). Local production of other cytokines, such as IL-7 and IL-12, have shown promising results in preclinical studies and clinical studies. Preclinical models showed that IL-7 (and IL-15) induced expansion of CAR T memory stem cells (CD8+ CD45RA+ CCR7+) with greater antitumor activity, which is mediated by increased resistance to cell death following repetitive exposures to the antigen, and maintenance of their migration ability to secondary lymphoid organs. Some studies also coupled CAR T cells with the constitutive or inducible release of IL-12. CAR T cells expressing IL-12 promote a Th1 immune response, reverse anergy in tumor-infiltrating cells, and inhibit Treg-mediated suppression of antitumor effector functions of T cells. CAR T cell expression of IL-12 also dampened production of immunosuppressive cytokines such as IL-10 and TGF-β by tumor-associated myeloid cells. Koneru et al. showed that IL-12 secreting tumor-targeted chimeric antigen receptor T cells (also known as armored-CAR T cells) could eradicate human ovarian xenografts. The authors showed that IL-12 secreting CAR T cells exhibit enhanced antitumor efficacy as determined by increased survival, prolonged persistence of T cells, and higher systemic IFN-γ (14). However, it should be noted that the authors measured the secretion of human IL-12 (p70) in the serum of CAR T cell-treated SCID-Beige mice [with impaired lymphoid development and reduced NK cell activity but normal macrophage and dendritic cells (DCs)] with established ovarian tumors. Because SCID-Beige mice have normal macrophages and DC populations and these cells are endogenous sources of mouse IL-12 production upon tumor challenge, these mice could in fact produce IL-12 after tumor challenge. Therefore, the total concentration of serum IL-12 may have consisted of both endogenous and exogenous IL-12, and may not have reflected IL-12 solely produced by armored-CAR T cells (105). Pegram and colleagues demonstrated that IL-12 producing CD19-CAR T cells eradicate systemic tumors without the need for prior conditioning. Moreover, they showed that such engineered T cells acquire intrinsic resistance to Treg cell-mediated inhibition (106). Chinnasamy et al. demonstrated that adoptive transfer of syngeneic CAR T cells co-transduced with VEGFR2 and constitutively expressing single-chain IL-12 resulted in the regression of established tumors of different histologies without the need for IL-2 administration. Indeed, the VEGFR2–CAR T cells changed the immunosuppressive tumor environment by altering/reducing both the systemic and the intratumoral CD11b+ Gr1+ myeloid suppressor cell subsets that expressed VEGFR2 (107). Alternatively, CAR T cells can be engineered to express cytokine receptors such as IL-7Ra that drive proliferation in response to endogenous IL-7. Perna et al. showed that IL-7 supports the proliferation and antitumor activity of IL-7Rα expressing CAR-GD2+ EBV-CTLs both in vitro and in vivo even in the presence of fully functional Tregs (108).