Clinical investigation of CAR T cells for solid tumors: Lessons learned and future directions

Journal Pre-proof Clinical investigation of CAR T cells for solid tumors: lessons learned and future directions Stephen J. Bagley, Donald M. O’Rourke

PII:

S0163-7258(19)30171-8

DOI:

https://doi.org/10.1016/j.pharmthera.2019.107419

Reference:

JPT 107419

To appear in: Accepted Date:

3 October 2019

Please cite this article as: Bagley SJ, O’Rourke DM, Clinical investigation of CAR T cells for solid tumors: lessons learned and future directions, Pharmacology and Therapeutics (2019), doi: https://doi.org/10.1016/j.pharmthera.2019.107419

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

P&T #23507 Title: Clinical investigation of CAR T cells for solid tumors: lessons learned and future directions Authors: Stephen J. Bagley, MD, MSCE a,b and Donald M. O’Rourke, MD b,c

ro

of

Author Affiliations: a Division of Hematology/Oncology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104 b Glioblastoma Translational Center of Excellence, Abramson Cancer Center of the University of Pennsylvania, Philadelphia, PA 19104 c Department of Neurosurgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104

-p

Corresponding Author: Stephen J. Bagley, MD, MSCE, Perelman Center for Advanced Medicine 10th Floor South Pavilion 3400 Civic Center Blvd Philadelphia, PA 19104, Email: [email protected], Office: 215-614-1858 Fax: 215-662-2432

re

Abstract

lP

Chimeric antigen receptor (CAR) T cells are a form of autologous immunotherapy that has changed the therapeutic landscape of hematologic malignancies. CAR T cell therapy involves collection of a patient’s T cells by apheresis, ex vivo genetic modification of the T cells to

ur na

encode a synthetic receptor that binds a specific tumor antigen, and infusion of the T cells back into the patient. With the unprecedented success of CAR T cells in leukemia and lymphoma, a growing number of preclinical studies and clinical trials have focused on translating this

Jo

treatment to solid tumors. In non-hematologic malignancies, however, response rates have been much less favorable. Some of the most significant challenges for CAR T cell immunotherapy in solid cancers include a paucity of unique tumor target antigens, limited CAR T cell trafficking to tumor sites, tumor heterogeneity and antigen loss, and the severely immunosuppressive tumor microenvironment. This review article provides an update on completed and ongoing clinical

trials of CAR T cells for solid tumors, as well as an overview of strategies to improve CAR T cell efficacy in non-hematologic malignancies.

Abbreviations AE, Adverse event; ALL, Acute lymphoblastic leukemia; APC, Antigen presenting cell; BCMA,

of

B cell maturation antigen; BiTE, Bispecific T-cell engager; CAIX, Carobxy-anhydrase-IX; CAR, Chimeric antigen receptor; CEA, Carcino-embryonic antigen; CNS, Central nervous system; CR,

ro

Complete remission; CMV, Cytomegalovirus; CRC, Colorectal cancer; CRISPR, Clustered

-p

regularly interspaced short palindromic repeats; CRS, Cytokine release syndrome; CT, Computed tomography; CTLA4, Cytotoxic T lymphocyte associated protein 4; DLBCL, Diffuse

re

large B cell lymphoma; DLT, Dose limiting toxicity; EBV, Epstein-Barr virus; EGFR,

lP

Endothelial growth factor receptor; FDA, Food and Drug Administration; FITC; Fluorescin isothiocyanate; FR, Folate receptor; GBM, Glioblastoma; HER2, Human epidermal growth factor 2; HIF, Hypoxia inducible factor; HLA, Human leukocyte antigen; HLH, hemophagocytic

ur na

lymphohistiocytosis; IDO, Indoleamine 2,3-dioxygenase; MAS, Macrophage activation syndrome; MAV, metabolically active volume; MRI, Magnetic resonance imaging; MPM, Malignant pleural mesothelioma; PD-1, Programmed cell death protein 1; PD-L1, Programmed

Jo

death-ligand 1; PDAC, Pancreatic ductal adenocarcinoma; PET, Positron emission tomography; PSA, Prostate specific antigen; PSMA, Prostate-specific membrane antigen; PSCA, Prostate stem cell antigen; qPCR, Quantitative polymerase chain reaction; ROR1, Receptor tyrosine kinase-like orphan receptor 1; scFV, Single-chain variable fragment; TAA, tumor-associated antigen; TCR, T cell receptor; TGF- (transforming growth factor );

Keywords: CAR T cells; Immunotherapy; Solid Tumors; Adoptive Cell Transfer; Tumor Microenvironment

1. Introduction

of

1.1. CAR T cell structure and function

ro

CARs are genetically engineered, artificial fusion proteins that incorporate an extracellular

antigen-recognition domain, a transmembrane and hinge domain that anchors the receptor on the

-p

cell surface and projects the antigen-targeting moiety out to the extracellular space, and an intracellular T-cell signaling domain that is triggered on antigen engagement.(Sadelain,

re

Brentjens, & Riviere, 2013; Srivastava & Riddell, 2015) After a CAR construct is transfected

lP

into autologous or allogeneic peripheral blood T cells using plasmid transfection, mRNA, or viral vector transduction, the T cells are infused into the patient to target whichever surfaceexposed tumor antigen is specified by the CAR’s antigen-binding domain, usually in the form of

ur na

a single-chain variable fragment (scFv).(Eshhar, Waks, Bendavid, & Schindler, 2001; Willemsen, et al., 2001) Upon CAR engagement of its associated antigen, primary T-cell activation occurs and leads to cytokine release, cytolytic degranulation, and T-cell

Jo

proliferation.(Hombach, et al., 2001) Additional T-cell effector mechanisms and memory responses also occur in a manner dependent on the mechanism of co-stimulation (e.g. 4-1BB or CD28 in the case of “second generation CARs,” or two or more signaling domains for “third generation CARs”).(Jensen & Riddell, 2015; van der Stegen, Hamieh, & Sadelain, 2015) For example, CD28 co-stimulation produces a potent, yet short-lived effector-like phenotype, while 4-1BB yields better expansion, longer persistence in vivo, and an increased capacity to generate

central memory T cells.(Labanieh, Majzner, & Mackall, 2018) Importantly, unlike vaccines and immunomodulatory agents that rely on in vivo priming of endogenous tumor-reactive cells and are therefore human leukocyte-antigen (HLA) restricted, CAR T cells are capable of inducing durable antitumor responses in a universal, HLA-independent manner.(D. Chen & Yang, 2017)

Leukapheresis is the first step in developing CAR T cells for use in an individual patient. Blood

of

is removed, leukocytes are separated, and the remainder of the blood is returned to the

ro

circulation. After a sufficient number of leukocytes have been harvested, the leukapheresis

product is enriched for T cells. The T cells then undergo an activation process during which they

-p

are incubated with the viral vector encoding the CAR, and after several days, the vector is

re

washed out of the culture. Lentiviral vectors are used most frequently, but other methods of gene transfer, such as Sleeping Beauty synthetic DNA or mRNA transposon systems, are being

lP

explored. When the cell expansion process is finished, the cell culture is concentrated to a volume that can be infused into the patient and is cryopreserved in infusible medium. When the

ur na

product is released for treatment, the frozen cells are transported to the treatment site and thawed prior to administration. In patients with hematologic malignancies, and increasingly in solid tumor CAR T cell trials, lymphodepleting chemotherapy is administered prior to the CAR T cells. Lymphodepletion can substantially increase the in vivo expansion of the infused CAR T

Jo

cells by multiple effects, including reduction of the patient’s lymphoid cell pool to make “space” for the CAR T cells, increasing homeostatic cytokines, and ameliorating the tumor inhibitory microenvironment.

1.2. CAR T cell therapy in hematologic malignancies

Genetic engineering of T cells to express CARs directed against specific antigens has opened the door to a new era of personalized cancer therapy. The greatest advances for CAR T cells have occurred in the treatment of hematologic malignancies, with the United States Food and Drug Administration (FDA) having approved two therapies. Tisagenlecleucel, a CD19-targeted CAR T-cell therapy formerly known as CTL019, was first approved for the treatment of patients up to 25 years of age with B-cell precursor acute lymphoblastic leukemia (ALL) that is refractory or in

of

second or later relapse.(Buechner, et al., 2017) This approval followed several key studies that

ro

demonstrated complete remission (CR) rates between 60 and 90% in patients with heavily

pretreated, relapsed/refractory B cell ALL.(Maude, et al., 2014; Park, et al., 2018) Subsequently,

-p

axicabtagene ciloleucel, another anti-CD19 CAR T-cell treatment, was approved for large B-cell

re

lymphoma patients who have failed at least 2 prior therapies.(Neelapu, et al., 2017) This therapy results in CR in approximately half of patients with refractory B cell lymphoma, with some

lP

remissions having been sustained for over three years.(Neelapu, et al., 2017) Most recently, tisagenlecleucel was also approved by the FDA for adult patients with relapsed or refractory

ur na

large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL), high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma.(Schuster, et al., 2019; Schuster, et al., 2017) In addition to ALL and DLBCL, CAR T cells have shown promise in early phase trials in other hematologic malignancies, including

Jo

multiple myeloma.(Raje, et al., 2019)

CAR T cell therapies for hematologic malignancies have unique toxicities that are distinct from those of cytotoxic chemotherapy, small-molecule targeted therapies, and even from other immunotherapies. The most commonly observed toxicity with CAR T cells is cytokine-release

syndrome (CRS), which manifests as high fever, hypotension, hypoxia, and/or multiorgan toxicity.(Neelapu, et al., 2018) Rarely, cases of CRS progress to fulminant hemophagocytic lymphohistiocytosis (HLH) (also known as macrophage-activation syndrome [MAS]), which is characterized by severe immune activation, lymphohistiocytic tissue infiltration, and immunemediated multisystem organ failure. CRS is triggered by the activation of T cells upon engagement of their CARs with cognate antigens expressed by tumor cells. When this occurs,

of

both the activated T cells, as well as bystander immune cells including monocytes and

ro

macrophages, release cytokines and chemokines that lead to a systemic inflammatory state that resembles sepsis physiology. CRS usually manifests with constitutional symptoms, such as

-p

fever, malaise, anorexia, and myalgias, but can ultimately impact any organ in the body,

re

including the heart, lungs, gut, liver, kidneys, bone marrow, or nervous system. CRS is managed in accordance with the grade of this toxicity, which can be determined using established

lP

guidelines.(Neelapu, et al., 2018) Detailed discussion of the management of CRS is beyond the scope of this review, but interventions typically include supportive care such as intravenous fluid

ur na

boluses, anti-IL-6 therapy with tocilizumab or siltuximab, and/or corticosteroids in cases refractory to anti-IL6 therapy.

In contrast to the successes observed in hematologic malignancies, consistent clinical efficacy of

Jo

CAR T cells has not been demonstrated for solid tumors.(Long, et al., 2018) The remainder of this article will review key clinical trials of CAR T cells in solid tumors that have been reported to date (Table), obstacles to therapeutic success that have been identified thus far, and the scientific and translational efforts that are being pursued to make CAR T cell therapy a clinical reality for non-hematologic malignancies.

2. CAR T cell therapy in solid tumors: recent clinical advances 2.1 Glioblastoma Glioblastoma (GBM) is the most common malignant primary brain tumor in adults and is near uniformly fatal.(Ostrom, et al., 2018) In part due to the lack of effective therapies for this tumor,

of

as well as the poor efficacy of other immunotherapies in GBM to date,(Reardon, et al., 2017)

ro

CAR T cells have been studied extensively as a novel therapy for in this disease.(Bagley, Desai, Linette, June, & O'Rourke, 2018) One of the most promising targets for CAR T cell therapy in

-p

GBM is IL13Rα2, which is a membrane-bound protein expressed in over 75% of GBMs that is associated with activation of the phosphatidylinositol-3 kinase/Akt/mammalian target of

re

rapamycin (mTOR) pathway,(Mintz, Gibo, Slagle-Webb, Christensen, & Debinski, 2002; Thaci,

lP

et al., 2014; Tu, et al., 2016) increased tumor invasiveness, and poor prognosis.(Brown, et al., 2013) Due to its specificity for GBM tumor cells and limited expression in normal brain and other tissues,(Debinski, Gibo, Slagle, Powers, & Gillespie, 1999) IL13Rα2 has long been

ur na

recognized as an attractive candidate for CAR T-cell targeting.(Rodriguez, Brown, & Badie, 2017) In a safety and feasibility trial of a first-generation IL13Rα2–specific CAR, termed “IL13 zetakine,” repeat doses of autologous CD8+ T cells engineered to express this CAR were

Jo

administered intracranially to 3 patients with recurrent GBM following gross total resection of the tumor.(Brown, et al., 2015) In addition, one subject was subsequently treated with direct intratumoral CAR T-cell infusions at a distant site of tumor recurrence. This first-in-human study demonstrated that IL13Rα2–directed CAR T cells could be successfully manufactured and delivered to patients with recurrent GBM via an implanted reservoir/catheter system. The CAR T cells were well tolerated, with adverse events such as headaches and transient neurologic deficits

being manageable. In addition, potential signs of anti-glioma activity were demonstrated. Patients experienced a rapid increase in necrotic tumor volume by MRI, significant loss of IL13Rα2 tumor cell expression, and encouraging duration of overall survival. In a follow-up trial utilizing a second-generation 4-1BB co-stimulatory IL13 zetakine CAR, one 50-year-old patient with recurrent multifocal GBM, including leptomeningeal disease, received 6 weekly intracavitary infusions of the CAR T-cell product following surgical resection of 3 of his 5

of

progressing intracranial tumors.(Brown, et al., 2016) Although the locally CAR T-cell treated

ro

site remained stable, other intracranial lesions progressed and new spinal lesions developed. The patient was then treated with 10 additional CAR T-cell infusions delivered intraventricularly

-p

through a catheter device placed in the right lateral ventricle. Remarkably, in addition to

re

tolerating the infusions well, this patient experienced regression of all intracranial and spinal tumors lasting for 7.5 months. Although the patient subsequently progressed at new locations

ur na

cells in GBM.

lP

distinct from his previous tumors, this case report highlights the therapeutic potential for CAR T

Human epidermal growth factor receptor 2 (HER2), a receptor tyrosine kinase overexpressed in many human cancers, is also considered an promising tumor-associated antigen for CAR targeting in GBM.(Andersson, et al., 2004; G. Liu, et al., 2004; C. Zhang, et al., 2016) Most

Jo

recently, 17 patients with progressive HER2+ GBM were treated on a phase I trial with peripheral blood infusions of HER2-specific CAR-modified virus-specific T cells.(Ahmed, et al., 2017) Because safety concerns had been raised by the death of a colorectal cancer patient treated in a previous study with a third-generation HER2-CAR T-cell therapy (composed of a trastuzumab-based antigen-recognition domain and a CD28.4-1BB signaling domain),(Morgan,

et al., 2010) the investigators in the GBM study utilized a second-generation CAR with an FRP5based exodomain and a CD28 signaling endodomain. No dose-limiting toxicity was observed. HER2-CAR T cells were detected by qPCR in all patients after the infusion, peaking in 15 of 17 patients at 3 hours after the infusion and at 1 week and 2 weeks in the other 2 patients, respectively. At 6 weeks after the infusion, HER2-CAR T cells were present in 7 of 15 patients, with blood levels declining further every month thereafter (with 2 samples remaining positive

of

out to 12 months, but none positive at 18 months). This suggested that the HER2-CAR T cells

ro

did not expand after infusion but could persist for up to 1 year at a low frequency. Of 16

evaluable patients, 1 had a partial response lasting for more than 9 months, and 7 had stable

-p

disease ranging between 8 weeks and 29 months (with 3 of these remaining free of progression

re

during 24‒29 months of follow-up). A key aspect of this study was that it relied on the expression of CARs in virus-specific T cells. Using this strategy, virus-specific T cells provide

lP

the expected antitumor activity through their CAR but may also receive appropriate costimulation following native T-cell receptor engagement by latent virus antigens presented by

ur na

professional antigen-presenting cells.(Ahmed, et al., 2017; Pule, et al., 2008) The investigators in this study administered CAR-modified T cells specific for adenovirus, Epstein–Barr virus (EBV), or cytomegalovirus (CMV), the safety of which had been previously demonstrated in hematopoietic stem cell transplant recipients.(Leen, et al., 2013) Among the 17 patients treated,

Jo

the CAR T cells of all patients contained adenovirus- and EBV-specific T cells, and all CAR T cells from CMV seropositive patients contained CMVpp65-specific T cells as determined by interferon gamma Elispot assays. Overall, this phase I trial demonstrated the feasibility and safety of peripherally infused virus-specific CAR T cells in GBM and, despite the lack of expansion of the CAR T cells in the blood, displayed encouraging signs of efficacy.

Lastly, EGFRvIII, resulting from an in-frame deletion of exons 2 to 7(Li & Wong, 2008) is present in 25-30% newly diagnosed GBMs and represents another potential target for CAR T cell targeting in this disease.(Felsberg, et al., 2017) The amino acid sequence resulting from the EGFRvIII alteration yields a novel glycine residue at the junction of exons 1 and 8, generating a tumor-specific and immunogenic epitope within the extracellular domain of EGFR. In a first-in-

of

human phase I trial, 10 patients with recurrent GBM were treated with a single dose of

ro

peripherally infused EGFRvIII-directed CAR T cells.(O'Rourke, et al., 2017) Manufacturing and infusion of the CAR T cells was feasible and safe, without evidence of off-tumor toxicity or

-p

CRS. While the study was not designed to evaluate for efficacy, no patients experienced tumor

re

regression (although one patient had residual stable disease lasting >18 months). All infused subjects had detectable engraftment of EGFRvIII CAR T cells in the peripheral blood, although

lP

the degree of engraftment was considerably lower than what has been observed with CD19specific CAR T cells bearing the same 4-1BB co-stimulatory domain, lentiviral backbone, and

ur na

manufacturing process.(Rapoport, et al., 2009) Seven of the 10 subjects in this study had postCAR T-cell surgical intervention, allowing for tissue-specific analysis of CAR T-cell trafficking and other “pharmacodynamic” endpoints. In 2 of these subjects, both of whom had their tumors resected within 2 weeks of CAR T-cell infusion, CART-EGFRvIII cells were found at higher

Jo

concentrations in the brain than in the peripheral blood at the same time point. Because CARTEGFRvIII DNA sequences by qPCR were 3 times and 100 times higher, respectively, in brain specimens than in corresponding blood samples from these patients, there was a suggestion that the CAR T cells had effectively trafficked and expanded in situ within active regions of GBM. In addition to determining EGFRvIII CAR T-cell trafficking to the tumor, acquisition of

posttreatment surgical specimens allowed for measurement of EGFRvIII target antigen expression and characterization of the tumor immune microenvironment following CAR T-cell infusion. Most of the subjects had specific loss or decreased expression of EGFRvIII in resected tumors following CAR T-cell infusion. Although it cannot be ruled out that decreased EGFRvIII expression was unrelated to CAR T-cell therapy, as EGFRvIII expression was previously shown to display both spatial and temporal variation,(Del Vecchio, et al., 2013) a more recent study

of

demonstrated that the vast majority of EGFRvIII+ GBMs maintain EGFRvIII positivity at

ro

recurrence.(Felsberg, et al., 2017) This suggests that antigen loss was more likely related to

successful targeting of EGFRvIII+ tumor cells by CAR T cells. Findings related to CAR T cell-

2.2.1. Colorectal Cancer

lP

2.2 Gastrointestinal malignancies

re

-p

induced changes in the tumor microenvironment are detailed in Section 3.3.3.

In colorectal cancer (CRC), the most common cancer of the GI tract, early experience with

ur na

adoptive cell therapy included a phase I trial of CAR T cells targeted against carcino-embryonic antigen (CEA) and administered to liver metastases directly through the hepatic artery, with or without systemic interleukin 2 (IL2) support.(Katz, et al., 2015) This study used a second generation anti-CEA CAR containing the CD28 costimulatory and CD3 domains.(Emtage, et

Jo

al., 2008) There were no grade 3/4 AEs among the six patients treated, but only one patient had prolonged stable disease (23 months). CT guided biopsies to sample liver metastases pre- and post-CAR T cell infusion showed an abundance of CAR T cells in liver metastases compared to normal liver in 5 of 6 patients, and 3 of these patients had an increase in necrosis within the lesion following CAR T cell delivery. In addition, all three patients who received systemic IL2

support with the CAR T cells had decreased CEA levels compared to baseline. Around the same time, a phase I study of first-generation CEA-specific CAR T cells in metastatic CRC and other CEA+ solid tumors was published.(Thistlethwaite, et al., 2017) Three cohorts of patients received increasing doses of CEA-specific CAR T cells after fludarabine +/-cyclophosphamide pre-conditioning. Systemic IL2 support was also administered following T cell infusion, based on preclinical data demonstrating that IL2 therapy may enhance CAR T cell killing.(Lo, Ma, Liu,

of

& Junghans, 2010) CAR T cell engraftment in this trial was short-lived with a rapid decline of

ro

systemic CAR T cells within 14 days, and no objective responses were observed. However, increased intensity of pre-conditioning (cyclophosphamide plus fludarabine, compared to

-p

fludarabine alone) resulted in enhanced CAR T cell engraftment. Another important observation

re

from this trial was the presence of on-target off-tumor toxicity. Patients in the cyclophosphamide-fludarabine pre-conditioning cohort had transient, acute respiratory toxicity,

lP

potentially related to CEA expression on lung epithelium. The presence of CEA expression on healthy lung tissue was controversial prior to this study, but was ultimately confirmed by the

ur na

study’s investigators in this study, underscoring the importance of considering and evaluating for on-target off-tumor toxicity in any organ in CAR T cell trials.

These studies were followed by another phase I trial of peripherally administered CEA-targeting

Jo

CAR T cells,(C. Zhang, et al., 2017) this time using a second-generation CAR with CD28-CD3 and a different scFv targeting moiety for CEA. In 10 patients with CEA-positive CRC and liver and/or lung metastases, five escalating dose levels (1 × 105 to 1 × 108/CAR+/kg cells) were evaluated following treatment with cyclophosphamide (300mg/m2 or 900mg/m2) for three days and fludarabine 25mg/m2 for two days. Severe AEs related to CAR T cell therapy were not

observed. Two patients remained with stable disease for more than 30 weeks, and two patients showed tumor shrinkage by positron emission tomography (PET)/computed tomography (CT) and MRI analysis, respectively. Decline of serum CEA level occurred in most patients, but CAR T cells persisted in the circulation for only a few days to a few weeks, with all patients having undetectable levels by qPCR 4-6 weeks post CAR-T infusion. Additional trials of CEA-targeted

of

CAR T cells are ongoing for metastatic CRC and other CEA+ tumors (NCT03682744).

ro

2.2.2. Pancreatobiliary cancers

In general, pancreatic ductal adenocarcinoma (PDAC) has demonstrated striking resistance to T

-p

cell immunotherapy,(Beatty, et al., 2018) making CAR T cell therapy particularly appealing for

re

this disease. As with other solid tumors, one of the most significant challenges to CAR T cell therapy in pancreatic cancer is the risk of on-target off-tumor toxicities as a result of target

lP

antigen expression by normal healthy tissues. In a phase I trial of CAR T cells in patients with PDAC,(Beatty, et al., 2018) investigators used autologous T cells genetically modified with

ur na

mRNA via electroporation to transiently express a CAR specific for mesothelin, which is overexpressed by PDAC cells but also found on the lining of the peritoneum, pleura, and pericardium. Six patients with chemotherapy-refractory, metastatic PDAC received CAR T cells intravenously three times weekly for three weeks. There were no dose-limiting toxicities (DLTs)

Jo

and no CRS experienced by any of the patients. The best overall response achieved was stable disease by RECIST v1.1, which was not unexpected given the transient CAR expression associated with mRNA-based genetic modification compared to virally transduced DNA-based CARs. However, FDG PET/CT imaging of tumor lesions revealed that the total metabolically active volume (MAV) remained stable in three patients and decreased by 70% in one patient,

suggesting potential anti-tumor activity of the mRNA CAR T cells. In terms of correlative studies, an important finding was that CAR T cell therapy induced a spreading of antibody responses against multiple proteins beyond mesothelin in multiple patients, including immunoregulatory molecules such as PD-1, PD-L1, and BCMA. This suggests a potential vaccine effect by inducing tumor cell death and the release of tumor-associated antigens. Importantly, the investigators demonstrated that mesothelin expression was confined to the

of

tumor cell cytoplasm in one of the patients, highlighting the importance of confirming cell

ro

surface expression of target antigen in CAR T cell studies. A phase I trial of lentiviral transduced

-p

anti-mesothelin CAR T cells in pancreatic cancer is ongoing (NCT03323944).

re

Other targets being clinically evaluated in pancreatobiliary cancers include HER2, Claudin 18.2, and prostate stem cell antigen (PSCA). In a phase I study of HER2-directed CAR T cells in

lP

advanced biliary tract and pancreatic cancers,(Feng, et al., 2018) 11 patients with advanced HER2+ (>50%) disease received a conditioning regimen of nab-paclitaxel (100-200mg/m2) and

ur na

cyclophosphamide (15-35 mg/kg) followed by 1-2 cycles of CART-HER2 cell infusion. Severe AEs related to the CAR T cells included one grade 3 fever, one grade 3 transaminitis, and one grade 3 GI hemorrhage, potentially attributed to expression of HER2 on the GI mucosa. The infusions were otherwise well tolerated. One patient obtained a partial response and five

Jo

achieved stable disease. Rapid elevation of CAR transgene copy numbers in the peripheral blood was observed in 9 of 11 patients following infusion of CART-HER2 cells, and the copy number had a second robust peak in the two patients who received a second infusion. However, persistence of CART-HER2 cells at therapeutic levels in vivo could not be maintained beyond ~30 days. Unfortunately, no tissue samples were taken in this trial, and intra-tumoral infiltration

of CAR T cells and their effect on the tumor microenvironment could not be assessed. Preliminary results of an ongoing first-in-human phase I trial of Claudin 18.2-specific CAR T cells for advanced gastric and pancreatic adenocarcinoma were also recently presented (NCT03159819).(Zhan, et al., 2019) Among twelve subjects with Claudin 18.2-positive metastatic adenocarcinoma (5 pancreatic and 7 gastric) treated with CAR-Claudin18.2 T cell infusions after lymphodepleting chemotherapy, there were no serious AEs, one patient achieved

of

a complete response (gastric cancer), and three patients achieved partial responses (two gastric,

ro

one pancreatic). Lastly preliminary results of an ongoing dose escalation trial of ligand-

inducible, PSCA-directed CAR T cells in PSCA+ metastatic pancreatic cancer were also recently

-p

reported.(Becerra, et al., 2019) The CAR product used in this trial is engineered to contain a

re

PSCA-CD3 CAR, as well as a MyD88/CD40 costimulatory domain that is inducible by administration of the small molecule rimiducid, a lipid-permeable tacrolimus analogue that

lP

homodimerizes the Fv-containing drug-binding domains of genetically engineered receptors to activate the receptor. Nine patients received cyclophosphamide for lymphodepletion, followed

ur na

by CAR T cell administration on day 0 and a single rimiducid dose on day 7. No DLTs, related serious adverse events (SAEs), or CRS events were reported. Rapid cell engraftment by day 4 was observed in all patients, and two of the patients who received rimiducid had cell expansion 10- to 20-fold within seven days and persistence greater than 3 weeks. Two patients achieved a

Jo

minor response (one with matched CA19-9 decrease), and 4 patients achieved stable disease for at least 8 weeks. The next phase of the study will add fludarabine to cyclophosphamide lymphodepletion and will expand to include gastric and prostate cancers (NCT02744287).

2.3. Genitourinary malignancies

2.3.1 Renal cell carcinoma One of the first efforts to develop CAR T cells for solid tumors was in renal cell carcinoma (RCC). In a phase I trial of carboxy-anhydrase-IX (CAIX)-targeted CAR T cells, twelve patients with metastatic disease were treated with a maximum of 10 daily infusions of 2 x 107 to 2 x 109 CAR T cells.(Lamers, Klaver, Gratama, Sleijfer, & Debets, 2016; Lamers, et al., 2013) Although

of

no efficacy was demonstrated, importance lessons were learned from this trial. First, patients developed anti-CAR T cell antibodies, thus informing future decisions regarding the format and

ro

immunogenicity of CARs in development. Second, the CAR T cells induced severe liver enzyme disturbances, which were seen on liver biopsy to result from on-target off-tumor binding of CAR

-p

T cells to CAIX expressed on bile duct epithelium. Other trials of CAR T cells for renal cell

re

carcinoma are ongoing, including CCT301-38 (anti-AXL CAR T cells; NCT03393936) and

2.3.2. Prostate Cancer

lP

CCT301-59 (anti-ROR2 CAR T cells; NCT03960060).

ur na

In prostate cancer, several groups have generated early phase clinical data. In one phase I trial, five patients with metastatic, castrate-resistant prostate cancer received chemotherapy conditioning with cyclophosphamide 60mg/kg/day for 2 days and fludarabine 25mg/m2/day for 5 days followed by first-generation CAR T cells targeted against prostate specific membrane

Jo

antigen (PSMA).(Junghans, et al., 2016) In addition, patients received low dose IL2 by continuous intravenous infusion at 75,000 IU/Kg/d for 4 weeks. Engraftment was confirmed in all subjects with 2.5-22% of circulating T cells being PSMA-CART cells after reconstitution at 2 weeks. The peak absolute number of CAR T cells was at day 14, with a leveling off at lower total levels that were stable by day 21 through the end of the study period on day 28. No anti-

PSMA toxicities were noted. Two patients achieved prostate-specific antigen (PSA) responses, with 50% and 70% declines at their nadirs, respectively. However, these patients did not have measurable tumor radiographically (other than a bone scan). The PSA responses were correlated directly with plasma IL2 and inversely with engraftment, suggesting that high engraftment in certain patients may have depleted IL2 to the point of limiting CAR T cell anti-tumor activity.

of

This hypothesis warrants further exploration in additional studies.

ro

Another phase I trial in prostate cancer, which is currently ongoing, utilizes PSMA-directed, transforming growth factor  (TFG-)-insensitive CAR T cells for the first time in human

-p

subjects. Because prostate cancer secretes TFG- as an immunosuppressive factor, the

re

investigators hypothesize that using a PSMA CAR with co-expression of a dominant-negative TFG- receptor will enhance antitumor immunity.(Kloss, et al., 2018) Early results

lP

(NCT03089203) demonstrated no DLT in the first six subjects treated (1-3 x 107 and 1-3 x 108/m2 CAR T cells) without lymphodepleting chemotherapy.(Narayan, et al., 2019) Cohorts 1

ur na

and 2 have been completed without observed DLT. A case of reversible CRS was observed that was responsive to anti-IL6 therapy with tocilizumab. Additional cohorts will evaluate the same CAR T cells following a lymphodepleting regimen of cyclophosphamide and fludarabine.

Jo

2.3.3. Ovarian Cancer

The only published ovarian cancer study of CAR T cells was a phase I trial of peripheral administration of a first-generation anti-ovarian cancer-associated antigen -folate receptor (FR) CAR administered to patients with FR+ ovarian cancer that was refractory to platinum/paclitaxel-based chemotherapy.(Kershaw, et al., 2006) A total of 14 patients were

treated; eight received FR-specific CAR T cells plus high-dose IL2 (720,000 IU/kg), and six received dual-specific T cells (reactive to both FR and allogenic antigen) followed by subcutaneous immunization with allogenic peripheral blood mononuclear cells from the same donor that was used to stimulate T cells during manufacturing. The only serious toxicities encountered were those related to high dose IL2 therapy. No reduction in tumor burden was seen in any patient, which was explained by several observations. First, imaging of radiolabeled

of

CAR T cells revealed lack of specific localization to tumor expect in one patient. Second, PCR

ro

analysis showed that CAR T cells were present in the circulation in large numbers for only 2 days after transfer, rapidly declining thereafter and barely detectable by 1 month. Lastly, an

-p

inhibitory factor against the CAR T cells developed in the serum of three of the patients. Other

re

trials of CAR T cells in ovarian cancer are ongoing, some of which are utilizing direct peritoneal

2.4. Breast Cancer

lP

administration to improve CAR T cell tumor trafficking (NCT03585764; NCT02498912).

ur na

The cell-surface molecule c-Met is expressed in ~50% of breast tumors regardless of hormone receptor/HER2 expression status and was thus identified as a promising target for CAR T cell therapy in this disease.(Ghoussoub, et al., 1998; Tchou, et al., 2017) However, c-Met is also present, albeit at low levels, on healthy tissues. To limit on-target off-tumor toxicity, a phase 0

Jo

clinical trial was conducted using intratumorally delivered, mRNA-transfected c-MET-CAR T cells. In this trial, six patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 x 107 (n=3) or 3 x 108 (n=3) cells. Treatment was well-tolerated, with no CAR T cell-related SAEs. No measurable clinical responses were observed. CAR T mRNA was detectable in peripheral blood and in the injected

tumor tissues after intratumoral injection in two and four patients, respectively. Following treatment, tumors were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis, cellular debris, loss of c-Met immunoreactivity, and extensive macrophage infiltration at the leading edges and within necrotic zones. Taken together, these data suggest that intratumoral mRNA c-Met-CAR T cells are well tolerated and are capable of evoking an inflammatory response within breast cancers. A study evaluating intravenous delivery of RNA

of

CART-cMET cells is currently enrolling (NCT03060356). Other ongoing trials of CAR T cell

ro

therapy in breast cancer include mesothelin-specific CAR T cells for patients with HER2-

re

with CNS disease (NCT02442297; NCT03696030).

-p

negative disease (NCT02792114) and HER2-specific CAR T cells for HER2 positive tumors

2.5. Lung cancer

lP

CAR T cell trials for lung cancers have focused thus far on malignant pleural mesothelioma (MPM). In a phase I dose escalation first-in-human trial, CD28-costimulated mesothelin CAR T

ur na

cells with the I-caspase-9 safety gene were administered intrapleurally to 18 patients with mesothelin-expressing MPM (NCT02414269).(Adusumilli, et al., 2019) Patients were treated in dose escalating cohorts (3 x 105 to 1 x 107 cells/kg) following IV cyclophosphamide lymphodepletion. No CAR T cell-related toxicities higher than grade 1 or any evidence of on-

Jo

target off-tumor toxicity were observed. Fourteen patients subsequently received anti-PD1 therapy off protocol; two of these patients achieved complete metabolic response on PET imaging, and five achieved a partial response by investigator assessment. Ongoing trials of other CAR T cells in primary lung cancers include receptor tyrosine kinase-like orphan receptor 1 (ROR1)-specific CAR T cells for stage IV non-small cell lung cancer (NSCLC)

(NCT02706392), and mesothelin-specific CAR T cells for recurrent lung adenocarcinoma (NCT03054298).

3. Challenges to CAR T cell therapy in solid tumors 3.1. Paucity of tumor-specific targets

of

One of the most significant challenges to CAR T cell therapy for solid tumors is difficulty in

ro

identifying an ideal target antigen. Unlike hematologic malignancies, most solid tumors do not express one tumor specific antigen. Instead, it is common to find tumor-associated antigens that

-p

are enriched on tumors but also expressed at low levels on normal tissues.(Martinez & Moon, 2019) As described in the above sections, this is the case for many of the antigens previously

re

studied in human clinical trials of CAR T cells for solid tumors, including CEA, ERBB2, EGFR,

lP

mesothelin, and PSMA.(Ahmed, et al., 2017; Beatty, et al., 2018; Lamers, et al., 2016; Narayan, et al., 2019; O'Rourke, et al., 2017) When CAR T cells are designed to target an antigen that is not specific to tumor cells, patients are subjected to the potential risk of on-target off-tumor

ur na

toxicity. One way to mitigate this risk is to genetically engineer the T cells so that recognition of one antigen expressed only on tumor cells (i.e, a tumor-specific antigen) can induce expression of a CAR directed towards a second antigen that may be expressed on both tumor cells and non-

Jo

tumor cells (i.e, a tumor-associated antigen), thereby initiating full CAR T cell activity only in the tumor site and not systemically on healthy tissues. This can be accomplished using a T cell “circuit” in which a synthetic Notch receptor (“SynNotch” receptor), when bound by its cognate antigen, translocates to the nucleus and induces expression of a CAR for a second antigen.(Roybal, et al., 2016) Other methods of attempting to limit CAR T cell activation to tumor sites include (1) modifying the affinity of antigen binding of the CAR, (2) using two

separate CARs on the same T cell (one to provide antigen-specific zeta-signaling and one to provide antigen-specific co-stimulation, thus restricting full T cell activation to tumors that express both antigens), (3) using “switchable” CAR T cells that are only activated in the presence of an exogenously administered “switch”, which can be a small molecule(Becerra, et al., 2019) that induces the CAR T cell’s costimulatory domain or an antibody that bridges the CAR to the antigen of interest,(Raj, et al., 2019) or (4) designing CARs to include an inhibitory

of

peptide that reversibly blocks the scFv and keeps the T cells in an off state until they reach the

ro

tumor microenvironment, where local conditions (locally secreted proteases, or hypoxic

re

activation of the T cells.(Labanieh, et al., 2018)

-p

conditions, e.g.) result in cleavage of the inhibitory peptide, unmasking of the CAR, and

Another problem related to antigen selection in solid tumors is that traditional CAR T cells

lP

require a target antigen that is expressed on the cell surface. Unfortunately, only about 1% of total cellular proteins are actually expressed on the cell surface,(Walseng, et al., 2017) meaning

ur na

that a large number of potential tumor target antigens are not available to CAR T cells. TCR gene therapy is one way to address this issue,(Garber, 2018) but this approach is beyond the scope of this CAR T cell-focused review. One CAR T cell-based strategy that is being explored to tackle the lack of appropriate antigens in solid tumors is the use of nanobody-based CAR T

Jo

cells.(Xie, et al., 2019) Nanobodies, composed of the variable regions of heavy-chain-only antibodies, are small, stable, camelid-derived single-domain antibody fragments with affinities comparable to traditional scFvs.(Revets, De Baetselier, & Muyldermans, 2005) Unlike scFvs, which are composed of a heavy-chain variable fragment connected to a light-chain variable fragment by a flexible linker, nanobodies do not require additional folding and assembly steps

that come with variable region pairing, and they can access epitopes different from those seen by scFvs. In addition, nanobodies allow surface display on CAR T cells without the requirement for extensive linker optimization or other types of reformatting typically needed to develop a new conventional antibody into an scFv for use in CAR T cell therapy. Thus, different nanobody recognition domains can be switched out easily to allow for more nimble development of CAR T cells for novel targets. In elegant work by Xie and colleagues, this approach was used to target

of

markers in the tumor microenvironment, such as PD-L1, rather than targeting antigens on the

ro

surface of tumor cells.(Xie, et al., 2019) The investigators also targeted the tumor stroma and vasculature through the EIIIB+ fibronectin splice variant, which is expressed by multiple tumor

-p

types and on neovasculature. In B16 melanoma-bearing mice, as well as mice bearing tumors

re

from a colon adenocarcinoma line (MC38), PD-L1- and EIIIB-targeted CAR T cells led to a significant delay in tumor growth and improved survival. Since solid tumors rarely display

lP

unique antigenic markers on their surface, this work may ultimately improve CAR T cell therapy in solid tumors by exploiting a variety of microenvironment markers that are shared across many

ur na

malignancies.

3.2. Trafficking to tumor

Following peripheral infusion, CAR T cells targeting a given antigen must localize to the tumor

Jo

bed. However, T cells often do not express the cognate chemokine receptors for the chemokines produced by tumors, and tumors produce only very small amounts of the chemokines needed for successful CAR T cell trafficking.(Long, et al., 2018) In addition, there are environmental barriers to CAR T cell extravasation and tumor infiltration, including endothelial cell dysfunction, downregulation of key adhesion molecules,(Dirkx, et al., 2003; Gust, et al., 2017)

and extracellular matrix barriers.(Overstreet, et al., 2013) One logical way to improve CAR T cell recruitment to tumor cells is to target the tumor bed itself with direct injection of CAR T cells. As described in the above sections, this has been performed in previous clinical trials with hepatic artery infusions of CEA-targeted CAR T cells for CEA+ liver metastases,(Katz, et al., 2015) intraventricular delivery of IL-13 Rα2-targeted CAR T cells for glioblastoma, and intrapleural delivery of mesothelin-targeted CAR T cells for MPM.(Adusumilli, et al., 2019)

of

Peritoneal administration for ovarian cancers is also being assessed (NCT03585764;

ro

NCT02498912). In addition, emerging preclinical data has suggested the possibility of using

implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors,

-p

thereby exposing them to high concentrations of immune cells for a substantial period of

re

time.(Smith, et al., 2017)

lP

To optimize intravenous delivery of CAR T cell therapy for non-hematologic malignancies, proper trafficking of CAR T cells into tumors must occur. One potential way to improve CAR T

ur na

cell trafficking is to augment chemokine communication, and various preclinical approaches are being taken in this vein. Recognizing that the chemokine CCL2 is highly secreted by malignant pleural mesothelioma (MPM), but that the corresponding chemokine receptor (CCR2) is minimally activated mesothelin-targeted CAR T cells, Moon and colleagues transduced the

Jo

chemokine receptor CCR2b into mesothelin-targeted CAR T cells using a lentiviral vector.(E. K. Moon, et al., 2011) This led to increased T cell transmigration and augmentation of in vitro Tcell killing ability, with a single intravenous dose of 20 million mesothlin-CCR2b CAR T cells resulting in a 12.5-fold increase in T-cell tumor infiltration and increased antitumor activity compared to mesothelin-CAR T cells without CCR2b transduction. Other methods of increasing

T cell tumor trafficking that have been explored include normalization of the vasculature by lowdose angiogenesis inhibitors,(Shrimali, et al., 2010) or engineering CAR T cells to express the enzyme heparanase, which degrades sulfate proteoglycans in the extracellular matrix and can promote tumor T cell infiltration into stroma-rich solid tumors.(Caruana, et al., 2015)

of

3.3. CAR T cell proliferation and function in the immunosuppressive microenvironment 3.3.1. Role for lymphodepletion

ro

Assuming that CAR T cells have successfully trafficked to the tumor site and encountered their

-p

cognate antigen, they must then undergo rapid expansion. Importantly, the extent of in vivo expansion has been closely corelated with CAR T cell efficacy across multiple previous

re

trials.(Kalos, et al., 2011; Maude, et al., 2014; Porter, et al., 2015) In patients with hematologic malignancies, lymphodepleting chemotherapy is typically administered prior to the CAR T cells.

lP

Lymphodepletion can substantially increase the in vivo expansion of the infused CAR T cells by multiple effects, including reducing the patient’s lymphoid cell pool to make “space” for the

ur na

CAR T cells, increasing homeostatic cytokines, depleting immunosuppressive regulatory T cells (Tregs), and ameliorating the tumor inhibitory microenvironment.(Anthony, et al., 2016; Gattinoni, et al., 2005; Heczey, et al., 2017; Muranski, et al., 2006) In solid tumors, the value of lymphodepletion and the optimal chemotherapy regimens to achieve it are not well elucidated.

Jo

For certain solid tumors, chemotherapy used for standard treatment of the tumor can also be used to provide lymphodepletion prior to CAR T cell therapy. One example of this is the use of temozolomide for glioblastoma.(Suryadevara, et al., 2018) However, for most solid tumors there is no obvious lymphodepleting chemotherapy used for standard treatment, and typical lymphodepleting regimens, such as cyclophosphamide plus fludarabine, can be exceedingly

toxic. One approach under evaluation to circumvent the need for lymphodepletion is the design of CAR T cells to eliminate their secretion of IL2, which potentiates the immunosuppressive activity of Tregs. This was accomplished in one study by introducing amino acid substitutions in the CAR transgene to prevent lymphocyte-specific tyrosine kinase (Lck) ligation to the CD28 cytosolic tail, which controls net IL2 secretion.(Suryadevara, et al., 2019) Additional studies are needed to better delineate the necessity of lymphodepleting chemotherapy in solid tumor CAR T

ro

of

cell trials, as well as the optimal regimens to be used.

3.3.2. Addressing intrinsic T cell deficits

-p

In addition to creating “space” for the CAR T cells to expand via lymphodepleting

re

chemotherapy, there are also intrinsic T cell deficits in patients with solid tumors that impair CAR T cell proliferation and persistence.(Leick & Maus, 2019) In a study of 195 children with

lP

both hematologic and solid tumors, Das and colleagues performed an extensive analysis of T-cell fitness and phenotyping in relation to receipt of progressive cycles of cytotoxic

ur na

chemotherapy.(Das, Vernau, Grupp, & Barrett, 2019) Using single-cell techniques and a functional expansion assay that is similar to the culture process used for manufacturing CAR T cells, the investigators found that patients with the percentage of T cells that “pass” a previously defined threshold for successful CAR T cell manufacturing declined significantly with

Jo

subsequent cycles of chemotherapy. Thus, certain patients with heavily pre-treated solid tumors may be not be optimal candidates or CAR T cells therapy. Adult patients with glioblastoma serve as an example of a patient population that often carries severe intrinsic T cell deficits. In patients with glioblastoma, severe lymphopenia is often present even prior to treatment, which was recently demonstrated to be due to T cell sequestration in the bone marrow.(Chongsathidkiet, et

al., 2018) In addition, the standard of care for this disease is radiation in combination with temozolomide chemotherapy, which is extremely lymphodepleting and leads to CD4 counts less than 200/mm3 in over one-third of patients.(Grossman, et al., 2011) Collection of an adequate number of viable T cells from such patients can prove challenging. Ongoing efforts are under way to improve intrinsic T-cell fitness in patients with solid tumors such as glioblastoma. One option is to use allogeneic CAR T cells from healthy donors, although this approach has been

of

limited by concerns regarding rejection in both the host and graft directions.(Leick & Maus,

ro

2019) Another strategy involves simply shifting CAR T cell therapy (or at least apheresis for cell collection) to the first line setting, prior to receipt of cytotoxic chemotherapy. Clinical trials

re

-p

employing this approach are ongoing (NCT03792633; NCT03726515).

A separate but related issue is that preclinical studies have indicated that some T cell subtypes

lP

show superior antitumor activity in vivo compared to others. In ALL and non-Hodgkin lymphoma, for example, the presence of early-lineage T cells (naïve and early memory) prior to

ur na

genetic engineering has been found to correlate with better expansion.(Singh, Perazzelli, Grupp, & Barrett, 2016) Similarly, in preclinical high-grade glioma models CAR CD4+ T cells have superior antitumor activity compared to CAR CD8+ T cells, and this advantage is lost upon mixing the CAR CD4+ cells with CAR CD8+ T cells.(D. Wang, et al., 2018) These results

Jo

suggest that the differentiation status of CAR T cells is an important consideration for optimizing their expansion and persistence. To date, most clinical trials using CAR T cells have used cell products prepared from unselected “bulk” T cells.(S. Guedan, et al., 2018) Moving forward, additional focus will be placed on the starting lymphocyte material used to generate CAR T cells. One strategy being explored to influence T cells subsets is the use of a PI3K inhibitor during

manufacturing, which has been shown to preserve a less differentiated T cell state.(Zheng, et al., 2018) In addition, it has been shown that epigenetic reprogramming may be used to alter CAR T cell differentiation and result in a central memory phenotype.(Fraietta, et al., 2018)

3.3.3. Immune checkpoints and other immunosuppressive microenvironment factors

of

Even if CAR T cells traffic to the tumor appropriately and initially expand upon antigen recognition, most solid tumors present additional challenges related to a hostile,

ro

immunosuppressive tumor microenvironment. One issue is that CAR T cells require

immunostimulatory cytokines for optimal killing activity, but these are often downregulated in

-p

the solid tumor microenvironment.(Labanieh, et al., 2018) Since systemic delivery of cytokine

re

therapy is carries excessive toxicity, several transgenic-based strategies have been developed to deliver higher local levels of key cytokines, including IL-12,(L. Zhang, et al., 2015) IL-

lP

15,(Krenciute, et al., 2017) IL-18,(Hu, et al., 2017) and IL-21.(Spolski & Leonard, 2014) However, because these cytokines carry multiple context-dependent roles that can sometimes be

ur na

immunosuppressive, it remains controversial as to whether they would help or hinder the CAR T cells.(Labanieh, et al., 2018)

While key cytokines are often limited in the solid tumor microenvironment, immune checkpoints

Jo

and soluble immune inhibitory factors are highly upregulated(Ngwa, Edwards, Philip, & Chen, 2019) and can severely impact T cell proliferation and function. With regard to impairing CAR T cells specifically, the critical role of immune checkpoints and other immunosuppressive molecules in the tumor microenvironment was highlighted in a human study of EGFRvIIItargeted CAR T cells for glioblastoma.(O'Rourke, et al., 2017) Immunohistochemical stains in

post-CAR T cell surgical specimens compared to matched pre-treatment samples displayed consistent and significant upregulation of immune checkpoints and other soluble immunosuppressive molecules, including indoleamine 2,3-dioxygenase (IDO) 1, programmed death (PD) ligand 1 (PD-L1), transforming growth factor (TGF)–β, and IL-10. These observations suggest that CAR T-cell targeting of EGFRvIII+ tumor cells induced a compensatory immunosuppressive response in the tumor microenvironment, and that pairing

of

CAR T cells with immune checkpoint inhibitors and/or small molecule inhibitors (IDO, TGF-β,

ro

etc) is a logical next step for solid tumors. A clinical trial of CART-EGFRvIII cells in

combination with the PD-1 inhibitor pembrolizumab for newly diagnosed glioblastoma is

-p

currently ongoing (NCT03726515), as well a trial of IL13R2-targeted CAR T cells with or

re

without nivolumab and ipilimumab in recurrent glioblastoma (NCT04003649).

lP

Beyond administering additional systemic agents such as immune checkpoint inhibitors, CAR T cell persistence and function in the solid tumor microenvironment can also be augmented by

ur na

additional genetic engineering of the T cells. One option is to transduce T cells with both a CAR specific for a tumor antigen, as well as a chimeric switch receptor containing the extracellular domain of an immune inhibitory signaling molecule fused to the transmembrane and cytoplasmic domains of a co-stimulatory molecule. This was described by Liu and colleagues using a PD1

Jo

switch receptor.(X. Liu, et al., 2016) When the PD1 portion of the switch-receptor engages the PDL1 ligand, it transmits an activating signal via the CD28 cytoplasmic domain instead of the inhibitory signal normally transduced by the PD1 cytoplasmic domain. This approach augmented CAR T cell control of large, established solid tumors in multiple models of aggressive human solid tumors expressing relevant tumor antigens.(X. Liu, et al., 2016) Another

T cell engineering strategy to overcome the immunosuppressive solid tumor microenvironment is to selectively knock-out or knock-in key endogenous genes through targeted nucleases, e.g. via CRISPR-Cas9 gene editing.(J. Liu, Zhou, Zhang, & Zhao, 2019) Several strategies based on CRISPR are being applied to develop novel CAR T cells by multiplexed genome editing, including (a) knockout of endogenous genes (TCRs, MHC, or self-antigens) to build allogenic universal CAR T cells,(Ren, et al., 2017) (b) disruption of inhibitory receptors such as PD-1 or

of

CTLA-4,(Rupp, et al., 2017) and (c) generation of T cells with a CAR knocked into the T cell

ro

receptor  constant (TRAC) locus, which places the CAR expression under control of the TCR promoter and results in enhanced potency and uniform CAR expression.(Eyquem, et al., 2017)

-p

A third way to engineer CAR T cells to overcome immune checkpoints and other inhibitory

re

molecules is to include dominant-negative receptors for key immunosuppressive ligands. This has been performed by engineering CAR T cells to overexpress a truncated receptor for PD-1

lP

that lacks the usual PD-1 transmembrane and intracellular signaling domains,(N. Chen, Morello, Tano, & Adusumilli, 2017) and a similar approach has been taken for the TGF- receptor.(Kloss,

ur na

et al., 2018) Thus, when the immunosuppressive ligands (PD-1, TGF-, or other ligand of choice) bind the dominant negative receptors on the CAR T cells in the tumor microenvironment, no immunosuppressive signal is transduced.

Jo

Lastly, in addition to immune checkpoints, competition for key nutrients including glucose, amino acids, and fatty acids, as well as the hypoxic tumor microenvironment, have also emerged as critical factors that restrict the antitumor activity of CAR T cells.(Schurich, Magalhaes, & Mattsson, 2019) This is a relatively underexplored, but rapidly growing area of research. One approach already being developed is to generate CAR T cells that are only effective in the

presence of hypoxia. Juillerat and colleagues fused an oxygen sensitive subdomain of HIF1 to a CAR scaffold, resulting in a CAR that is sub-optimally presented at the surface of the T cell under normal oxygen concentration, but increased in expression along with improved cytolytic properties under hypoxic conditions.(Juillerat, et al., 2017)

of

3.3.4. Combination treatments with vaccines or viral therapies CAR T cell therapies are also being explored in combination with vaccines, oncolytic viruses,

ro

and other immunotherapeutic classes of treatment in an effort to augment their efficacy. Both native and genetically engineered viruses are being developed for treatment across a variety of

-p

solid tumors, including an FDA-approved herpes simplex virus (HSV-1) for

re

melanoma(Andtbacka, et al., 2015) and numerous viruses in clinical trials for glioblastoma.(Cloughesy, et al., 2018; Desjardins, et al., 2018) Because oncolytic viral therapies

lP

can lead to increased immune cell infiltration and pro-inflammatory cytokines and have the potential to lyse tumor cells and release additional tumor-associated antigens, there is strong

ur na

rational for combining these treatments with CAR T cells.(Sonia Guedan & Alemany, 2018) In addition, it is feasible that viral therapies can be armed with therapeutic transgenes that could further enhance the effector functions of T cells. A number of preclinical studies have started to explore the combination of viral therapies with CAR T cells,(Edmund K. Moon, et al., 2018;

Jo

Nishio, et al., 2014; Rosewell Shaw, et al., 2017; Tanoue, et al., 2017; Watanabe, et al., 2018) and clinical trials are likely to follow soon.

Due to the limited efficacy of CAR T cells in solid tumors to date, coupled with the known ability of therapeutic vaccination to enhance endogenous T cell responses against cancer,(van der

Burg, Arens, Ossendorp, van Hall, & Melief, 2016) CAR T cells are also being studied in combination with vaccination. Multiple studies have demonstrated that introducing a CAR together with a second antigen receptor specific for a target peptide (or using virus-specific endogenous T cells to prepare the CAR T cells) and then vaccinating recipients against the secondary or viral antigen can improve CAR T cell efficacy.(Slaney, et al., 2017; Tanaka, et al., 2017; Wang, et al., 2015) A more recent study took this concept a step further by designing a

of

vaccine strategy to re-stimulate CAR T cells directly within the native lymph node

ro

microenvironment.(Ma, et al., 2019) This was accomplished by using a membrane-integrating phospholipid polymer that can be linked to small molecules or peptides, resulting in expression

-p

of a desired target antigen on the cell surface after immunization with this molecule. By binding

re

to albumin after injection, these amphiphilic polymers are directed to lymph nodes, where they are preferentially displayed on resident antigen-presenting cells (APCs). Mice in this study were

lP

treated with CAR T cells targeted against the antigen fluorescin isothiocyanate (FITC), followed by immunization with the amphiphilic polymers linked to FITC. In vivo T cell proliferation was

ur na

significantly improved by this approach. The experiment was then repeated using the tumor antigen EGFRvIII in glioma-bearing mice and demonstrated improved CAR T cell proliferation and survival, as well as improved infiltration of activated CAR T cells into tumor sites.(Ma, et

Jo

al., 2019)

3.4. Heterogeneity, Tumor Antigen Escape Clinical trials of CD19 CAR T cells in ALL have demonstrated cases of outgrowth of CD19negative leukemia, resulting in refractoriness to CAR T cell therapy.(Maude, et al., 2014) Thus, even in the setting of a uniformly expressed TAA, there is the possibility of antigen loss or

antigen escape. This problem is even more pressing in solid tumors, which tend to display significant antigen heterogeneity.(Martinez & Moon, 2019) Key examples of tumor heterogeneity and its detrimental impact on CAR T cell therapy in solid tumors can be found from glioblastoma trials. With both EGFRvIII and IL13R2 CAR T cells in glioblastoma, patients had progressive disease with antigen-negative tumors despite successful initial antigen

of

targeting by CAR T cells.(Brown, et al., 2016; O'Rourke, et al., 2017) Although it is possible that CAR T cell therapy targeted against a heterogeneously expressed antigen could result in a

ro

secondary immune response against other tumor-specific neoantigens or epitopes, a phenomenon termed epitope spreading,(Gulley, et al., 2017) this has not yet been proven to occur in humans.

re

heterogeneity in CAR T cell solid tumor trials.

-p

In the meantime, additional efforts should be undertaken to address the problem of tumor

lP

One strategy to begin to address tumor heterogeneity is to design bi-specific or multivalent CAR T cells. While the same challenges associated with choosing a single target antigen remain or are

ur na

even more significant when choosing multiple antigens, targeting more than one antigen with CAR T cells may ultimately reduce or delay antigen escape. There are several ways to engineer a T cell product for multispecificity.(Majzner & Mackall, 2018) The most straightforward approach is to simultaneously or sequentially administer T cell products that are separately

Jo

transduced for different CARs. It is also possible to combine vectors for two different CARs during cell production to achieve a mixed product. A different approach is to engineer a CAR molecule to recognize multiple antigens. This can be accomplished by linking two different antigen binders on a single molecule (i.e., a single “tandem” CAR),(Hegde, et al., 2016) or by using a single vector to encode two or three separate CARs that can be expressed on a single T

cell.(Bielamowicz, et al., 2018) A unique strategy altogether has been proposed by Choi and colleagues, who developed a bicistronic construct to drive dual expression of an EGFRvIIIspecific CAR and a bispecific T-cell engager (BiTE) against wild-type EGFR.(Choi, et al., 2019) The engineered cells secreted EGFR-specific BiTEs in the local tumor microenvironment that redirected CAR T cells and recruited untransduced bystander T cells against wild-type EGFR. As a result, these cells eliminated mouse tumors that heterogeneously expressed EGFRvIII.

of

Importantly, toxicity against human skin grafts, a potential concern with targeting wild-type

ro

EGFR, was not observed in vivo. Multiple clinical trials are underway testing different variations

re

NCT0448393, NCT03019055, NCT0330691).

-p

of multispecific CAR T cells to address tumor heterogeneity (NCT03241940, NCT03233854,

Lastly, another proposed method for tackling tumor heterogeneity is to target cancer stem cells,

lP

which are largely responsible for heterogeneity of tumor cells. (Badrinath & Yoo, 2019) CD133, a transmembrane glycoprotein that is a well-recognized marker of cancer stem cells, is

ur na

overexpressed in many solid tumors and has emerged as an attractive target for CAR T cell therapy.(Ferrandina, Petrillo, Bonanno, & Scambia, 2009) Initial results were recently reported for a phase I trial of CD133-directed CAR T cells in advanced GI malignancies, including hepatocellular carcinoma, pancreatic carcinoma, and colorectal carcinoma.(Y. Wang, et al.,

Jo

2018) A total of 23 patients were treated in a cell dose escalation scheme. Three partial responses were observed, and post-treatment tissue demonstrated that CD133+ cells were eliminated after CAR T cell infusions. Despite this, however, relapses occurred with CD133-negative tumors, suggesting that heterogeneity remains a problem with CAR T cell therapy even when specifically targeting cancer stem cell markers.

4. Conclusions CAR T cell therapy has revolutionized the care of patients with hematologic malignancies. Although efficacy in solid tumors has lagged significantly behind, an unprecedented number of solid tumor CAR T cells trials are currently ongoing, and additional clinical data is being

of

generated rapidly. Challenges related to CAR T cell trafficking to the tumor, expansion and persistence in a severely immunosuppressive tumor microenvironment, and antigen

ro

heterogeneity are among the main barriers to success identified in solid tumor studies thus far. It

-p

is imperative that strong cross-institutional and academia-industry collaborations are forged as this complex field moves forward, and that we continue to learn from each and every clinical

lP

Conflict of Interest

re

trial of CAR T cell therapy that is performed.

S.J.B. and D.M.O. are co-inventors of intellectual property licensed by the University of

Jo

ur na

Pennsylvania to Novartis.

References Adusumilli, P. S., Zauderer, M. G., Rusch, V. W., O'Cearbhaill, R., Zhu, A., Ngai, D., McGee, E., Chintala, N., Messinger, J., Cheema, W., Halton, E., Diamonte, C., Pineda, J., Vincent, A., Modi, S., Solomon, S. B., Jones, D. R., Brentjens, R. J., Riviere, I., & Sadelain, M. (2019). Regional delivery of mesothelin-targeted CAR T cells for pleural

of

cancers: Safety and preliminary efficacy in combination with anti-PD-1 agent. Journal of

ro

Clinical Oncology, 37, 2511-2511.

Ahmed, N., Brawley, V., Hegde, M., Bielamowicz, K., Kalra, M., Landi, D., Robertson, C.,

-p

Gray, T. L., Diouf, O., Wakefield, A., Ghazi, A., Gerken, C., Yi, Z., Ashoori, A., Wu, M.

re

F., Liu, H., Rooney, C., Dotti, G., Gee, A., Su, J., Kew, Y., Baskin, D., Zhang, Y. J., New, P., Grilley, B., Stojakovic, M., Hicks, J., Powell, S. Z., Brenner, M. K., Heslop, H.

lP

E., Grossman, R., Wels, W. S., & Gottschalk, S. (2017). HER2-Specific Chimeric Antigen Receptor-Modified Virus-Specific T Cells for Progressive Glioblastoma: A

ur na

Phase 1 Dose-Escalation Trial. JAMA Oncol, 3, 1094-1101. Andersson, U., Guo, D., Malmer, B., Bergenheim, A. T., Brannstrom, T., Hedman, H., & Henriksson, R. (2004). Epidermal growth factor receptor family (EGFR, ErbB2-4) in gliomas and meningiomas. Acta Neuropathol, 108, 135-142.

Jo

Andtbacka, R. H., Kaufman, H. L., Collichio, F., Amatruda, T., Senzer, N., Chesney, J., Delman, K. A., Spitler, L. E., Puzanov, I., Agarwala, S. S., Milhem, M., Cranmer, L., Curti, B., Lewis, K., Ross, M., Guthrie, T., Linette, G. P., Daniels, G. A., Harrington, K., Middleton, M. R., Miller, W. H., Jr., Zager, J. S., Ye, Y., Yao, B., Li, A., Doleman, S., VanderWalde, A., Gansert, J., & Coffin, R. S. (2015). Talimogene Laherparepvec

Improves Durable Response Rate in Patients With Advanced Melanoma. J Clin Oncol, 33, 2780-2788. Anthony, S. M., Rivas, S. C., Colpitts, S. L., Howard, M. E., Stonier, S. W., & Schluns, K. S. (2016). Inflammatory Signals Regulate IL-15 in Response to Lymphodepletion. J Immunol, 196, 4544-4552. Badrinath, N., & Yoo, S. Y. (2019). Recent Advances in Cancer Stem Cell-Targeted

of

Immunotherapy. Cancers, 11, 310.

ro

Bagley, S. J., Desai, A. S., Linette, G. P., June, C. H., & O'Rourke, D. M. (2018). CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-

-p

Oncology, 20, 1429-1438.

re

Beatty, G. L., O'Hara, M. H., Lacey, S. F., Torigian, D. A., Nazimuddin, F., Chen, F., Kulikovskaya, I. M., Soulen, M. C., McGarvey, M., Nelson, A. M., Gladney, W. L.,

lP

Levine, B. L., Melenhorst, J. J., Plesa, G., & June, C. H. (2018). Activity of MesothelinSpecific Chimeric Antigen Receptor T Cells Against Pancreatic Carcinoma Metastases in

ur na

a Phase 1 Trial. Gastroenterology, 155, 29-32. Becerra, C. R., Hoof, P., Paulson, A. S., Manji, G. A., Gardner, O., Malankar, A., Shaw, J., Blass, D., Ballard, B., Yi, X., Anumula, M., Foster, A. E., Senesac, J., & Woodard, P. (2019). Ligand-inducible, prostate stem cell antigen (PSCA)-directed GoCAR-T cells in

Jo

advanced solid tumors: Preliminary results from a dose escalation. Journal of Clinical Oncology, 37, 283-283.

Bielamowicz, K., Fousek, K., Byrd, T. T., Samaha, H., Mukherjee, M., Aware, N., Wu, M. F., Orange, J. S., Sumazin, P., Man, T. K., Joseph, S. K., Hegde, M., & Ahmed, N. (2018).

Trivalent CAR T cells overcome interpatient antigenic variability in glioblastoma. NeuroOncology, 20, 506-518. Brown, C. E., Alizadeh, D., Starr, R., Weng, L., Wagner, J. R., Naranjo, A., Ostberg, J. R., Blanchard, M. S., Kilpatrick, J., Simpson, J., Kurien, A., Priceman, S. J., Wang, X., Harshbarger, T. L., D'Apuzzo, M., Ressler, J. A., Jensen, M. C., Barish, M. E., Chen, M., Portnow, J., Forman, S. J., & Badie, B. (2016). Regression of Glioblastoma after

of

Chimeric Antigen Receptor T-Cell Therapy. N Engl J Med, 375, 2561-2569.

ro

Brown, C. E., Badie, B., Barish, M. E., Weng, L., Ostberg, J. R., Chang, W. C., Naranjo, A., Starr, R., Wagner, J., Wright, C., Zhai, Y., Bading, J. R., Ressler, J. A., Portnow, J.,

-p

D'Apuzzo, M., Forman, S. J., & Jensen, M. C. (2015). Bioactivity and Safety of

re

IL13Ralpha2-Redirected Chimeric Antigen Receptor CD8+ T Cells in Patients with Recurrent Glioblastoma. Clin Cancer Res, 21, 4062-4072.

lP

Brown, C. E., Warden, C. D., Starr, R., Deng, X., Badie, B., Yuan, Y. C., Forman, S. J., & Barish, M. E. (2013). Glioma IL13Ralpha2 is associated with mesenchymal signature

ur na

gene expression and poor patient prognosis. PLoS One, 8, e77769. Buechner, J., Grupp, S. A., Maude, S. L., Boyer, M., Bittencourt, H., Laetsch, T. W., Bader, P., Verneris, M. R., Stefanski, H., Myers, G. D., Qayed, M., Pulsipher, M. A., De Moerloose, B., Hiramatsu, H., Schlis, K., Davis, K., Martin, P. L., Nemecek, E., Peters,

Jo

C., Wood, P., Taran, T., Mueller, K. T., Zhang, Y., & Rives, S. (2017). Global Registration Trial of Efficacy and Safety of CTL019 in Pediatric and Young Adult Patients with Relapsed/Refractory (R/R) Acute Lymphoblastic Leukemia (ALL): Update to the Interim Analysis. Clinical Lymphoma, Myeloma and Leukemia, 17, S263-S264.

Caruana, I., Savoldo, B., Hoyos, V., Weber, G., Liu, H., Kim, E. S., Ittmann, M. M., Marchetti, D., & Dotti, G. (2015). Heparanase promotes tumor infiltration and antitumor activity of CAR-redirected T lymphocytes. Nat Med, 21, 524-529. Chen, D., & Yang, J. (2017). Development of novel antigen receptors for CAR T-cell therapy directed toward solid malignancies. Transl Res, 187, 11-21. Chen, N., Morello, A., Tano, Z., & Adusumilli, P. S. (2017). CAR T-cell intrinsic PD-1

of

checkpoint blockade: A two-in-one approach for solid tumor immunotherapy.

ro

Oncoimmunology, 6, e1273302.

Choi, B. D., Yu, X., Castano, A. P., Bouffard, A. A., Schmidts, A., Larson, R. C., Bailey, S. R.,

-p

Boroughs, A. C., Frigault, M. J., Leick, M. B., Scarfo, I., Cetrulo, C. L., Demehri, S.,

re

Nahed, B. V., Cahill, D. P., Wakimoto, H., Curry, W. T., Carter, B. S., & Maus, M. V. (2019). CAR-T cells secreting BiTEs circumvent antigen escape without detectable

lP

toxicity. Nat Biotechnol.

Chongsathidkiet, P., Jackson, C., Koyama, S., Loebel, F., Cui, X., Farber, S. H., Woroniecka, K.,

ur na

Elsamadicy, A. A., Dechant, C. A., Kemeny, H. R., Sanchez-Perez, L., Cheema, T. A., Souders, N. C., Herndon, J. E., Coumans, J. V., Everitt, J. I., Nahed, B. V., Sampson, J. H., Gunn, M. D., Martuza, R. L., Dranoff, G., Curry, W. T., & Fecci, P. E. (2018). Sequestration of T cells in bone marrow in the setting of glioblastoma and other

Jo

intracranial tumors. Nat Med, 24, 1459-1468.

Cloughesy, T. F., Landolfi, J., Vogelbaum, M. A., Ostertag, D., Elder, J. B., Bloomfield, S., Carter, B., Chen, C. C., Kalkanis, S. N., Kesari, S., Lai, A., Lee, I. Y., Liau, L. M., Mikkelsen, T., Nghiemphu, P., Piccioni, D., Accomando, W., Diago, O. R., Hogan, D. J., Gammon, D., Kasahara, N., Kheoh, T., Jolly, D. J., Gruber, H. E., Das, A., & Walbert, T.

(2018). Durable complete responses in some recurrent high-grade glioma patients treated with Toca 511 + Toca FC. Neuro-Oncology, 20, 1383-1392. Das, R. K., Vernau, L., Grupp, S. A., & Barrett, D. M. (2019). Naive T-cell Deficits at Diagnosis and after Chemotherapy Impair Cell Therapy Potential in Pediatric Cancers. Cancer Discov, 9, 492-499. Debinski, W., Gibo, D. M., Slagle, B., Powers, S. K., & Gillespie, G. Y. (1999). Receptor for

of

interleukin 13 is abundantly and specifically over-expressed in patients with glioblastoma

ro

multiforme. Int J Oncol, 15, 481-486.

Del Vecchio, C. A., Giacomini, C. P., Vogel, H., Jensen, K. C., Florio, T., Merlo, A., Pollack, J.

-p

R., & Wong, A. J. (2013). EGFRvIII gene rearrangement is an early event in

re

glioblastoma tumorigenesis and expression defines a hierarchy modulated by epigenetic mechanisms. Oncogene, 32, 2670-2681.

lP

Desjardins, A., Gromeier, M., Herndon, J. E., 2nd, Beaubier, N., Bolognesi, D. P., Friedman, A. H., Friedman, H. S., McSherry, F., Muscat, A. M., Nair, S., Peters, K. B., Randazzo, D.,

ur na

Sampson, J. H., Vlahovic, G., Harrison, W. T., McLendon, R. E., Ashley, D., & Bigner, D. D. (2018). Recurrent Glioblastoma Treated with Recombinant Poliovirus. N Engl J Med, 379, 150-161.

Dirkx, A. E., Oude Egbrink, M. G., Kuijpers, M. J., van der Niet, S. T., Heijnen, V. V., Bouma-

Jo

ter Steege, J. C., Wagstaff, J., & Griffioen, A. W. (2003). Tumor angiogenesis modulates leukocyte-vessel wall interactions in vivo by reducing endothelial adhesion molecule expression. Cancer Res, 63, 2322-2329.

Emtage, P. C., Lo, A. S., Gomes, E. M., Liu, D. L., Gonzalo-Daganzo, R. M., & Junghans, R. P. (2008). Second-generation anti-carcinoembryonic antigen designer T cells resist

activation-induced cell death, proliferate on tumor contact, secrete cytokines, and exhibit superior antitumor activity in vivo: a preclinical evaluation. Clin Cancer Res, 14, 81128122. Eshhar, Z., Waks, T., Bendavid, A., & Schindler, D. G. (2001). Functional expression of chimeric receptor genes in human T cells. J Immunol Methods, 248, 67-76. Eyquem, J., Mansilla-Soto, J., Giavridis, T., van der Stegen, S. J., Hamieh, M., Cunanan, K. M.,

ro

CRISPR/Cas9 enhances tumour rejection. Nature, 543, 113-117.

of

Odak, A., Gonen, M., & Sadelain, M. (2017). Targeting a CAR to the TRAC locus with

Felsberg, J., Hentschel, B., Kaulich, K., Gramatzki, D., Zacher, A., Malzkorn, B., Kamp, M.,

-p

Sabel, M., Simon, M., Westphal, M., Schackert, G., Tonn, J. C., Pietsch, T., von

re

Deimling, A., Loeffler, M., Reifenberger, G., & Weller, M. (2017). Epidermal Growth Factor Receptor Variant III (EGFRvIII) Positivity in EGFR-Amplified Glioblastomas:

Res, 23, 6846-6855.

lP

Prognostic Role and Comparison between Primary and Recurrent Tumors. Clin Cancer

ur na

Feng, K., Liu, Y., Guo, Y., Qiu, J., Wu, Z., Dai, H., Yang, Q., Wang, Y., & Han, W. (2018). Phase I study of chimeric antigen receptor modified T cells in treating HER2-positive advanced biliary tract cancers and pancreatic cancers. Protein Cell, 9, 838-847. Ferrandina, G., Petrillo, M., Bonanno, G., & Scambia, G. (2009). Targeting CD133 antigen in

Jo

cancer. Expert Opin Ther Targets, 13, 823-837.

Fraietta, J. A., Nobles, C. L., Sammons, M. A., Lundh, S., Carty, S. A., Reich, T. J., Cogdill, A. P., Morrissette, J. J. D., DeNizio, J. E., Reddy, S., Hwang, Y., Gohil, M., Kulikovskaya, I., Nazimuddin, F., Gupta, M., Chen, F., Everett, J. K., Alexander, K. A., Lin-Shiao, E., Gee, M. H., Liu, X., Young, R. M., Ambrose, D., Wang, Y., Xu, J., Jordan, M. S.,

Marcucci, K. T., Levine, B. L., Garcia, K. C., Zhao, Y., Kalos, M., Porter, D. L., Kohli, R. M., Lacey, S. F., Berger, S. L., Bushman, F. D., June, C. H., & Melenhorst, J. J. (2018). Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature, 558, 307-312. Garber, K. (2018). Driving T-cell immunotherapy to solid tumors. Nat Biotechnol, 36, 215-219. Gattinoni, L., Finkelstein, S. E., Klebanoff, C. A., Antony, P. A., Palmer, D. C., Spiess, P. J.,

of

Hwang, L. N., Yu, Z., Wrzesinski, C., Heimann, D. M., Surh, C. D., Rosenberg, S. A., &

ro

Restifo, N. P. (2005). Removal of homeostatic cytokine sinks by lymphodepletion

enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med,

-p

202, 907-912.

re

Ghoussoub, R. A., Dillon, D. A., D'Aquila, T., Rimm, E. B., Fearon, E. R., & Rimm, D. L. (1998). Expression of c-met is a strong independent prognostic factor in breast

lP

carcinoma. Cancer, 82, 1513-1520.

Goff, S. L., Morgan, R. A., Yang, J. C., Sherry, R. M., Robbins, P. F., Restifo, N. P., Feldman, S.

ur na

A., Lu, Y. C., Lu, L., Zheng, Z., Xi, L., Epstein, M., McIntyre, L. S., Malekzadeh, P., Raffeld, M., Fine, H. A., & Rosenberg, S. A. (2019). Pilot Trial of Adoptive Transfer of Chimeric Antigen Receptor-transduced T Cells Targeting EGFRvIII in Patients With Glioblastoma. J Immunother, 42, 126-135.

Jo

Grossman, S. A., Ye, X., Lesser, G., Sloan, A., Carraway, H., Desideri, S., & Piantadosi, S. (2011). Immunosuppression in patients with high-grade gliomas treated with radiation and temozolomide. Clin Cancer Res, 17, 5473-5480.

Guedan, S., & Alemany, R. (2018). CAR-T Cells and Oncolytic Viruses: Joining Forces to Overcome the Solid Tumor Challenge. Frontiers in immunology, 9, 2460-2460.

Guedan, S., Posey, A. D., Jr., Shaw, C., Wing, A., Da, T., Patel, P. R., McGettigan, S. E., Casado-Medrano, V., Kawalekar, O. U., Uribe-Herranz, M., Song, D., Melenhorst, J. J., Lacey, S. F., Scholler, J., Keith, B., Young, R. M., & June, C. H. (2018). Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight, 3. Gulley, J. L., Madan, R. A., Pachynski, R., Mulders, P., Sheikh, N. A., Trager, J., & Drake, C. G. (2017). Role of Antigen Spread and Distinctive Characteristics of Immunotherapy in

of

Cancer Treatment. J Natl Cancer Inst, 109.

ro

Gust, J., Hay, K. A., Hanafi, L. A., Li, D., Myerson, D., Gonzalez-Cuyar, L. F., Yeung, C., Liles, W. C., Wurfel, M., Lopez, J. A., Chen, J., Chung, D., Harju-Baker, S., Ozpolat, T., Fink,

-p

K. R., Riddell, S. R., Maloney, D. G., & Turtle, C. J. (2017). Endothelial Activation and

re

Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells. Cancer Discov, 7, 1404-1419.

lP

Heczey, A., Louis, C. U., Savoldo, B., Dakhova, O., Durett, A., Grilley, B., Liu, H., Wu, M. F., Mei, Z., Gee, A., Mehta, B., Zhang, H., Mahmood, N., Tashiro, H., Heslop, H. E., Dotti,

ur na

G., Rooney, C. M., & Brenner, M. K. (2017). CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma. Mol Ther, 25, 2214-2224.

Hegde, M., Mukherjee, M., Grada, Z., Pignata, A., Landi, D., Navai, S. A., Wakefield, A.,

Jo

Fousek, K., Bielamowicz, K., Chow, K. K., Brawley, V. S., Byrd, T. T., Krebs, S., Gottschalk, S., Wels, W. S., Baker, M. L., Dotti, G., Mamonkin, M., Brenner, M. K., Orange, J. S., & Ahmed, N. (2016). Tandem CAR T cells targeting HER2 and IL13Ralpha2 mitigate tumor antigen escape. J Clin Invest, 126, 3036-3052.

Hombach, A., Wieczarkowiecz, A., Marquardt, T., Heuser, C., Usai, L., Pohl, C., Seliger, B., & Abken, H. (2001). Tumor-specific T cell activation by recombinant immunoreceptors: CD3 zeta signaling and CD28 costimulation are simultaneously required for efficient IL2 secretion and can be integrated into one combined CD28/CD3 zeta signaling receptor molecule. J Immunol, 167, 6123-6131. Hu, B., Ren, J., Luo, Y., Keith, B., Young, R. M., Scholler, J., Zhao, Y., & June, C. H. (2017).

of

Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-

ro

18. Cell Rep, 20, 3025-3033.

Jensen, M. C., & Riddell, S. R. (2015). Designing chimeric antigen receptors to effectively and

-p

safely target tumors. Curr Opin Immunol, 33, 9-15.

re

Juillerat, A., Marechal, A., Filhol, J. M., Valogne, Y., Valton, J., Duclert, A., Duchateau, P., & Poirot, L. (2017). An oxygen sensitive self-decision making engineered CAR T-cell.

lP

Scientific reports, 7, 39833-39833.

Junghans, R. P., Ma, Q., Rathore, R., Gomes, E. M., Bais, A. J., Lo, A. S., Abedi, M., Davies, R.

ur na

A., Cabral, H. J., Al-Homsi, A. S., & Cohen, S. I. (2016). Phase I Trial of Anti-PSMA Designer CAR-T Cells in Prostate Cancer: Possible Role for Interacting Interleukin 2-T Cell Pharmacodynamics as a Determinant of Clinical Response. Prostate, 76, 1257-1270. Kalos, M., Levine, B. L., Porter, D. L., Katz, S., Grupp, S. A., Bagg, A., & June, C. H. (2011). T

Jo

cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci Transl Med, 3, 95ra73.

Katz, S. C., Burga, R. A., McCormack, E., Wang, L. J., Mooring, W., Point, G. R., Khare, P. D., Thorn, M., Ma, Q., Stainken, B. F., Assanah, E. O., Davies, R., Espat, N. J., & Junghans, R. P. (2015). Phase I Hepatic Immunotherapy for Metastases Study of Intra-Arterial

Chimeric Antigen Receptor-Modified T-cell Therapy for CEA+ Liver Metastases. Clin Cancer Res, 21, 3149-3159. Kershaw, M. H., Westwood, J. A., Parker, L. L., Wang, G., Eshhar, Z., Mavroukakis, S. A., White, D. E., Wunderlich, J. R., Canevari, S., Rogers-Freezer, L., Chen, C. C., Yang, J. C., Rosenberg, S. A., & Hwu, P. (2006). A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res, 12, 6106-6115.

of

Kloss, C. C., Lee, J., Zhang, A., Chen, F., Melenhorst, J. J., Lacey, S. F., Maus, M. V., Fraietta,

ro

J. A., Zhao, Y., & June, C. H. (2018). Dominant-Negative TGF-beta Receptor Enhances

Eradication. Mol Ther, 26, 1855-1866.

-p

PSMA-Targeted Human CAR T Cell Proliferation And Augments Prostate Cancer

re

Krenciute, G., Prinzing, B. L., Yi, Z., Wu, M. F., Liu, H., Dotti, G., Balyasnikova, I. V., & Gottschalk, S. (2017). Transgenic Expression of IL15 Improves Antiglioma Activity of

5, 571-581.

lP

IL13Ralpha2-CAR T Cells but Results in Antigen Loss Variants. Cancer Immunol Res,

ur na

Labanieh, L., Majzner, R. G., & Mackall, C. L. (2018). Programming CAR-T cells to kill cancer. Nat Biomed Eng, 2, 377-391.

Lamers, C. H., Klaver, Y., Gratama, J. W., Sleijfer, S., & Debets, R. (2016). Treatment of metastatic renal cell carcinoma (mRCC) with CAIX CAR-engineered T-cells-a

Jo

completed study overview. Biochem Soc Trans, 44, 951-959.

Lamers, C. H., Sleijfer, S., van Steenbergen, S., van Elzakker, P., van Krimpen, B., Groot, C., Vulto, A., den Bakker, M., Oosterwijk, E., Debets, R., & Gratama, J. W. (2013). Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol Ther, 21, 904-912.

Leen, A. M., Bollard, C. M., Mendizabal, A. M., Shpall, E. J., Szabolcs, P., Antin, J. H., Kapoor, N., Pai, S. Y., Rowley, S. D., Kebriaei, P., Dey, B. R., Grilley, B. J., Gee, A. P., Brenner, M. K., Rooney, C. M., & Heslop, H. E. (2013). Multicenter study of banked third-party virus-specific T cells to treat severe viral infections after hematopoietic stem cell transplantation. Blood, 121, 5113-5123.

cell Deficits in Patients with Cancer. Cancer Discov, 9, 466-468.

of

Leick, M., & Maus, M. V. (2019). Wishing on a CAR: Understanding the Scope of Intrinsic T-

immunotherapy. Expert Rev Vaccines, 7, 977-985.

ro

Li, G., & Wong, A. J. (2008). EGF receptor variant III as a target antigen for tumor

-p

Liu, G., Ying, H., Zeng, G., Wheeler, C. J., Black, K. L., & Yu, J. S. (2004). HER-2, gp100, and

Cancer Res, 64, 4980-4986.

re

MAGE-1 are expressed in human glioblastoma and recognized by cytotoxic T cells.

lP

Liu, J., Zhou, G., Zhang, L., & Zhao, Q. (2019). Building Potent Chimeric Antigen Receptor T Cells With CRISPR Genome Editing. Frontiers in immunology, 10, 456-456.

ur na

Liu, X., Ranganathan, R., Jiang, S., Fang, C., Sun, J., Kim, S., Newick, K., Lo, A., June, C. H., Zhao, Y., & Moon, E. K. (2016). A Chimeric Switch-Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors. Cancer Res, 76, 1578-1590.

Jo

Lo, A. S., Ma, Q., Liu, D. L., & Junghans, R. P. (2010). Anti-GD3 chimeric sFv-CD28/T-cell receptor zeta designer T cells for treatment of metastatic melanoma and other neuroectodermal tumors. Clin Cancer Res, 16, 2769-2780.

Long, K. B., Young, R. M., Boesteanu, A. C., Davis, M. M., Melenhorst, J. J., Lacey, S. F., DeGaramo, D. A., Levine, B. L., & Fraietta, J. A. (2018). CAR T Cell Therapy of Non-

hematopoietic Malignancies: Detours on the Road to Clinical Success. Front Immunol, 9, 2740. Ma, L., Dichwalkar, T., Chang, J. Y. H., Cossette, B., Garafola, D., Zhang, A. Q., Fichter, M., Wang, C., Liang, S., Silva, M., Kumari, S., Mehta, N. K., Abraham, W., Thai, N., Li, N., Wittrup, K. D., & Irvine, D. J. (2019). Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor. Science, 365, 162-168.

of

Majzner, R. G., & Mackall, C. L. (2018). Tumor Antigen Escape from CAR T-cell Therapy.

ro

Cancer Discov, 8, 1219-1226.

Martinez, M., & Moon, E. K. (2019). CAR T Cells for Solid Tumors: New Strategies for

-p

Finding, Infiltrating, and Surviving in the Tumor Microenvironment. Front Immunol, 10,

re

128.

Maude, S. L., Frey, N., Shaw, P. A., Aplenc, R., Barrett, D. M., Bunin, N. J., Chew, A.,

lP

Gonzalez, V. E., Zheng, Z., Lacey, S. F., Mahnke, Y. D., Melenhorst, J. J., Rheingold, S. R., Shen, A., Teachey, D. T., Levine, B. L., June, C. H., Porter, D. L., & Grupp, S. A.

ur na

(2014). Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med, 371, 1507-1517.

Mintz, A., Gibo, D. M., Slagle-Webb, B., Christensen, N. D., & Debinski, W. (2002). IL13Ralpha2 is a glioma-restricted receptor for interleukin-13. Neoplasia, 4, 388-399.

Jo

Moon, E. K., Carpenito, C., Sun, J., Wang, L. C., Kapoor, V., Predina, J., Powell, D. J., Jr., Riley, J. L., June, C. H., & Albelda, S. M. (2011). 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, 17, 47194730.

Moon, E. K., Wang, L.-C. S., Bekdache, K., Lynn, R. C., Lo, A., Thorne, S. H., & Albelda, S. M. (2018). Intra-tumoral delivery of CXCL11 via a vaccinia virus, but not by modified T cells, enhances the efficacy of adoptive T cell therapy and vaccines. Oncoimmunology, 7, e1395997. Morgan, R. A., Yang, J. C., Kitano, M., Dudley, M. E., Laurencot, C. M., & Rosenberg, S. A. (2010). Case report of a serious adverse event following the administration of T cells

of

transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther, 18, 843-851.

ro

Muranski, P., Boni, A., Wrzesinski, C., Citrin, D. E., Rosenberg, S. A., Childs, R., & Restifo, N. P. (2006). Increased intensity lymphodepletion and adoptive immunotherapy--how far

-p

can we go? Nature clinical practice. Oncology, 3, 668-681.

re

Narayan, V., Gladney, W., Plesa, G., Vapiwala, N., Carpenter, E., Maude, S. L., Lal, P., Lacey, S. F., Melenhorst, J. J., Sebro, R., Farwell, M., Hwang, W.-T., Moniak, M., Gilmore, J.,

lP

Lledo, L., Dengel, K., Marshall, A., Coughlin, C. M., June, C. H., & Haas, N. B. (2019). A phase I clinical trial of PSMA-directed/TGFβ-insensitive CAR-T cells in metastatic

ur na

castration-resistant prostate cancer. Journal of Clinical Oncology, 37, TPS347-TPS347. Neelapu, S. S., Locke, F. L., Bartlett, N. L., Lekakis, L. J., Miklos, D. B., Jacobson, C. A., Braunschweig, I., Oluwole, O. O., Siddiqi, T., Lin, Y., Timmerman, J. M., Stiff, P. J., Friedberg, J. W., Flinn, I. W., Goy, A., Hill, B. T., Smith, M. R., Deol, A., Farooq, U.,

Jo

McSweeney, P., Munoz, J., Avivi, I., Castro, J. E., Westin, J. R., Chavez, J. C., Ghobadi, A., Komanduri, K. V., Levy, R., Jacobsen, E. D., Witzig, T. E., Reagan, P., Bot, A., Rossi, J., Navale, L., Jiang, Y., Aycock, J., Elias, M., Chang, D., Wiezorek, J., & Go, W. Y. (2017). Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med, 377, 2531-2544.

Neelapu, S. S., Tummala, S., Kebriaei, P., Wierda, W., Gutierrez, C., Locke, F. L., Komanduri, K. V., Lin, Y., Jain, N., Daver, N., Westin, J., Gulbis, A. M., Loghin, M. E., de Groot, J. F., Adkins, S., Davis, S. E., Rezvani, K., Hwu, P., & Shpall, E. J. (2018). Chimeric antigen receptor T-cell therapy - assessment and management of toxicities. Nat Rev Clin Oncol, 15, 47-62. Ngwa, V. M., Edwards, D. N., Philip, M., & Chen, J. (2019). Microenvironmental Metabolism

of

Regulates Antitumor Immunity. Cancer Res.

ro

Nishio, N., Diaconu, I., Liu, H., Cerullo, V., Caruana, I., Hoyos, V., Bouchier-Hayes, L.,

Savoldo, B., & Dotti, G. (2014). Armed oncolytic virus enhances immune functions of

-p

chimeric antigen receptor-modified T cells in solid tumors. Cancer Res, 74, 5195-5205.

re

O'Rourke, D. M., Nasrallah, M. P., Desai, A., Melenhorst, J. J., Mansfield, K., Morrissette, J. J. D., Martinez-Lage, M., Brem, S., Maloney, E., Shen, A., Isaacs, R., Mohan, S., Plesa, G.,

lP

Lacey, S. F., Navenot, J. M., Zheng, Z., Levine, B. L., Okada, H., June, C. H., Brogdon, J. L., & Maus, M. V. (2017). A single dose of peripherally infused EGFRvIII-directed

ur na

CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med, 9. Ostrom, Q. T., Gittleman, H., Truitt, G., Boscia, A., Kruchko, C., & Barnholtz-Sloan, J. S. (2018). CBTRUS Statistical Report: Primary Brain and Other Central Nervous System

Jo

Tumors Diagnosed in the United States in 2011–2015. Neuro-Oncology, 20, iv1-iv86.

Overstreet, M. G., Gaylo, A., Angermann, B. R., Hughson, A., Hyun, Y. M., Lambert, K., Acharya, M., Billroth-Maclurg, A. C., Rosenberg, A. F., Topham, D. J., Yagita, H., Kim, M., Lacy-Hulbert, A., Meier-Schellersheim, M., & Fowell, D. J. (2013). Inflammation-

induced interstitial migration of effector CD4(+) T cells is dependent on integrin alphaV. Nat Immunol, 14, 949-958. Park, J. H., Riviere, I., Gonen, M., Wang, X., Senechal, B., Curran, K. J., Sauter, C., Wang, Y., Santomasso, B., Mead, E., Roshal, M., Maslak, P., Davila, M., Brentjens, R. J., & Sadelain, M. (2018). Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. N Engl J Med, 378, 449-459.

of

Porter, D. L., Hwang, W. T., Frey, N. V., Lacey, S. F., Shaw, P. A., Loren, A. W., Bagg, A.,

ro

Marcucci, K. T., Shen, A., Gonzalez, V., Ambrose, D., Grupp, S. A., Chew, A., Zheng, Z., Milone, M. C., Levine, B. L., Melenhorst, J. J., & June, C. H. (2015). Chimeric

-p

antigen receptor T cells persist and induce sustained remissions in relapsed refractory

re

chronic lymphocytic leukemia. Sci Transl Med, 7, 303ra139.

Pule, M. A., Savoldo, B., Myers, G. D., Rossig, C., Russell, H. V., Dotti, G., Huls, M. H., Liu,

lP

E., Gee, A. P., Mei, Z., Yvon, E., Weiss, H. L., Liu, H., Rooney, C. M., Heslop, H. E., & Brenner, M. K. (2008). Virus-specific T cells engineered to coexpress tumor-specific

ur na

receptors: persistence and antitumor activity in individuals with neuroblastoma. Nat Med, 14, 1264-1270.

Raj, D., Yang, M. H., Rodgers, D., Hampton, E. N., Begum, J., Mustafa, A., Lorizio, D., Garces, I., Propper, D., Kench, J. G., Kocher, H. M., Young, T. S., Aicher, A., & Heeschen, C.

Jo

(2019). Switchable CAR-T cells mediate remission in metastatic pancreatic ductal adenocarcinoma. Gut, 68, 1052-1064.

Raje, N., Berdeja, J., Lin, Y., Siegel, D., Jagannath, S., Madduri, D., Liedtke, M., Rosenblatt, J., Maus, M. V., Turka, A., Lam, L. P., Morgan, R. A., Friedman, K., Massaro, M., Wang, J., Russotti, G., Yang, Z., Campbell, T., Hege, K., Petrocca, F., Quigley, M. T., Munshi,

N., & Kochenderfer, J. N. (2019). Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma. N Engl J Med, 380, 1726-1737. Rapoport, A. P., Stadtmauer, E. A., Aqui, N., Vogl, D., Chew, A., Fang, H. B., Janofsky, S., Yager, K., Veloso, E., Zheng, Z., Milliron, T., Westphal, S., Cotte, J., Huynh, H., Cannon, A., Yanovich, S., Akpek, G., Tan, M., Virts, K., Ruehle, K., Harris, C., Philip, S., Vonderheide, R. H., Levine, B. L., & June, C. H. (2009). Rapid immune recovery and

of

graft-versus-host disease-like engraftment syndrome following adoptive transfer of

ro

Costimulated autologous T cells. Clin Cancer Res, 15, 4499-4507.

Reardon, D. A., Omuro, A., Brandes, A. A., Rieger, J., Wick, A., Sepulveda, J., Phuphanich, S.,

-p

de Souza, P., Ahluwalia, M. S., Lim, M., Vlahovic, G., & Sampson, J. (2017). OS10.3

re

Randomized Phase 3 Study Evaluating the Efficacy and Safety of Nivolumab vs Bevacizumab in Patients With Recurrent Glioblastoma: CheckMate 143. Neuro-

lP

Oncology, 19, iii21-iii21.

Ren, J., Liu, X., Fang, C., Jiang, S., June, C. H., & Zhao, Y. (2017). Multiplex Genome Editing

ur na

to Generate Universal CAR T Cells Resistant to PD1 Inhibition. Clin Cancer Res, 23, 2255-2266.

Revets, H., De Baetselier, P., & Muyldermans, S. (2005). Nanobodies as novel agents for cancer therapy. Expert Opin Biol Ther, 5, 111-124.

Jo

Rodriguez, A., Brown, C., & Badie, B. (2017). Chimeric antigen receptor T-cell therapy for glioblastoma. Transl Res, 187, 93-102.

Rosewell Shaw, A., Porter, C. E., Watanabe, N., Tanoue, K., Sikora, A., Gottschalk, S., Brenner, M. K., & Suzuki, M. (2017). Adenovirotherapy Delivering Cytokine and Checkpoint

Inhibitor Augments CAR T Cells against Metastatic Head and Neck Cancer. Mol Ther, 25, 2440-2451. Roybal, K. T., Rupp, L. J., Morsut, L., Walker, W. J., McNally, K. A., Park, J. S., & Lim, W. A. (2016). Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits. Cell, 164, 770-779. Rupp, L. J., Schumann, K., Roybal, K. T., Gate, R. E., Ye, C. J., Lim, W. A., & Marson, A.

of

(2017). CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human

ro

chimeric antigen receptor T cells. Sci Rep, 7, 737.

receptor design. Cancer Discov, 3, 388-398.

-p

Sadelain, M., Brentjens, R., & Riviere, I. (2013). The basic principles of chimeric antigen

re

Schurich, A., Magalhaes, I., & Mattsson, J. (2019). Metabolic regulation of CAR T cell function by the hypoxic microenvironment in solid tumors. Immunotherapy, 11, 335-345.

lP

Schuster, S. J., Bishop, M. R., Tam, C. S., Waller, E. K., Borchmann, P., McGuirk, J. P., Jager, U., Jaglowski, S., Andreadis, C., Westin, J. R., Fleury, I., Bachanova, V., Foley, S. R.,

ur na

Ho, P. J., Mielke, S., Magenau, J. M., Holte, H., Pantano, S., Pacaud, L. B., Awasthi, R., Chu, J., Anak, O., Salles, G., & Maziarz, R. T. (2019). Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell Lymphoma. N Engl J Med, 380, 45-56. Schuster, S. J., Svoboda, J., Chong, E. A., Nasta, S. D., Mato, A. R., Anak, O., Brogdon, J. L.,

Jo

Pruteanu-Malinici, I., Bhoj, V., Landsburg, D., Wasik, M., Levine, B. L., Lacey, S. F., Melenhorst, J. J., Porter, D. L., & June, C. H. (2017). Chimeric Antigen Receptor T Cells in Refractory B-Cell Lymphomas. N Engl J Med, 377, 2545-2554.

Shrimali, R. K., Yu, Z., Theoret, M. R., Chinnasamy, D., Restifo, N. P., & Rosenberg, S. A. (2010). Antiangiogenic agents can increase lymphocyte infiltration into tumor and

enhance the effectiveness of adoptive immunotherapy of cancer. Cancer Res, 70, 61716180. Singh, N., Perazzelli, J., Grupp, S. A., & Barrett, D. M. (2016). Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies. Sci Transl Med, 8, 320ra323. Slaney, C. Y., von Scheidt, B., Davenport, A. J., Beavis, P. A., Westwood, J. A., Mardiana, S., Tscharke, D. C., Ellis, S., Prince, H. M., Trapani, J. A., Johnstone, R. W., Smyth, M. J.,

of

Teng, M. W., Ali, A., Yu, Z., Rosenberg, S. A., Restifo, N. P., Neeson, P., Darcy, P. K.,

ro

& Kershaw, M. H. (2017). Dual-specific Chimeric Antigen Receptor T Cells and an

Indirect Vaccine Eradicate a Variety of Large Solid Tumors in an Immunocompetent,

-p

Self-antigen Setting. Clin Cancer Res, 23, 2478-2490.

re

Smith, T. T., Moffett, H. F., Stephan, S. B., Opel, C. F., Dumigan, A. G., Jiang, X., Pillarisetty, V. G., Pillai, S. P. S., Wittrup, K. D., & Stephan, M. T. (2017). Biopolymers codelivering

Invest, 127, 2176-2191.

lP

engineered T cells and STING agonists can eliminate heterogeneous tumors. J Clin

ur na

Spolski, R., & Leonard, W. J. (2014). Interleukin-21: a double-edged sword with therapeutic potential. Nat Rev Drug Discov, 13, 379-395. Srivastava, S., & Riddell, S. R. (2015). Engineering CAR-T cells: Design concepts. Trends Immunol, 36, 494-502.

Jo

Suryadevara, C. M., Desai, R., Abel, M. L., Riccione, K. A., Batich, K. A., Shen, S. H., Chongsathidkiet, P., Gedeon, P. C., Elsamadicy, A. A., Snyder, D. J., Herndon, J. E., 2nd, Healy, P., Archer, G. E., Choi, B. D., Fecci, P. E., Sampson, J. H., & Sanchez-Perez, L. (2018). Temozolomide lymphodepletion enhances CAR abundance and correlates with antitumor efficacy against established glioblastoma. Oncoimmunology, 7, e1434464.

Suryadevara, C. M., Desai, R., Farber, S. H., Choi, B. D., Swartz, A. M., Shen, S. H., Gedeon, P. C., Snyder, D. J., Herndon, J. E., 2nd, Healy, P., Reap, E. A., Archer, G. E., Fecci, P. E., Sampson, J. H., & Sanchez-Perez, L. (2019). Preventing Lck Activation in CAR T Cells Confers Treg Resistance but Requires 4-1BB Signaling for Them to Persist and Treat Solid Tumors in Nonlymphodepleted Hosts. Clin Cancer Res, 25, 358-368. Tanaka, M., Tashiro, H., Omer, B., Lapteva, N., Ando, J., Ngo, M., Mehta, B., Dotti, G.,

of

Kinchington, P. R., Leen, A. M., Rossig, C., & Rooney, C. M. (2017). Vaccination

ro

Targeting Native Receptors to Enhance the Function and Proliferation of Chimeric Antigen Receptor (CAR)-Modified T Cells. Clin Cancer Res, 23, 3499-3509.

-p

Tanoue, K., Rosewell Shaw, A., Watanabe, N., Porter, C., Rana, B., Gottschalk, S., Brenner, M.,

re

& Suzuki, M. (2017). Armed Oncolytic Adenovirus-Expressing PD-L1 Mini-Body Enhances Antitumor Effects of Chimeric Antigen Receptor T Cells in Solid Tumors.

lP

Cancer Res, 77, 2040-2051.

Tchou, J., Zhao, Y., Levine, B. L., Zhang, P. J., Davis, M. M., Melenhorst, J. J., Kulikovskaya,

ur na

I., Brennan, A. L., Liu, X., Lacey, S. F., Posey, A. D., Jr., Williams, A. D., So, A., Conejo-Garcia, J. R., Plesa, G., Young, R. M., McGettigan, S., Campbell, J., Pierce, R. H., Matro, J. M., DeMichele, A. M., Clark, A. S., Cooper, L. J., Schuchter, L. M., Vonderheide, R. H., & June, C. H. (2017). Safety and Efficacy of Intratumoral Injections

Jo

of Chimeric Antigen Receptor (CAR) T Cells in Metastatic Breast Cancer. Cancer Immunol Res, 5, 1152-1161.

Thaci, B., Brown, C. E., Binello, E., Werbaneth, K., Sampath, P., & Sengupta, S. (2014). Significance of interleukin-13 receptor alpha 2-targeted glioblastoma therapy. NeuroOncology, 16, 1304-1312.

Thistlethwaite, F. C., Gilham, D. E., Guest, R. D., Rothwell, D. G., Pillai, M., Burt, D. J., Byatte, A. J., Kirillova, N., Valle, J. W., Sharma, S. K., Chester, K. A., Westwood, N. B., Halford, S. E. R., Nabarro, S., Wan, S., Austin, E., & Hawkins, R. E. (2017). The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity. Cancer Immunol Immunother, 66, 1425-1436.

of

Tu, M., Wange, W., Cai, L., Zhu, P., Gao, Z., & Zheng, W. (2016). IL-13 receptor alpha2

signaling pathway. Tumour Biol, 37, 14701-14709.

ro

stimulates human glioma cell growth and metastasis through the Src/PI3K/Akt/mTOR

-p

van der Burg, S. H., Arens, R., Ossendorp, F., van Hall, T., & Melief, C. J. (2016). Vaccines for

re

established cancer: overcoming the challenges posed by immune evasion. Nat Rev Cancer, 16, 219-233.

lP

van der Stegen, S. J., Hamieh, M., & Sadelain, M. (2015). The pharmacology of secondgeneration chimeric antigen receptors. Nat Rev Drug Discov, 14, 499-509.

ur na

Walseng, E., Koksal, H., Sektioglu, I. M., Fane, A., Skorstad, G., Kvalheim, G., Gaudernack, G., Inderberg, E. M., & Walchli, S. (2017). A TCR-based Chimeric Antigen Receptor. Sci Rep, 7, 10713.

Wang, D., Aguilar, B., Starr, R., Alizadeh, D., Brito, A., Sarkissian, A., Ostberg, J. R., Forman,

Jo

S. J., & Brown, C. E. (2018). Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JCI Insight, 3.

Wang, X., Wong, C. W., Urak, R., Mardiros, A., Budde, L. E., Chang, W. C., Thomas, S. H., Brown, C. E., La Rosa, C., Diamond, D. J., Jensen, M. C., Nakamura, R., Zaia, J. A., &

Forman, S. J. (2015). CMVpp65 Vaccine Enhances the Antitumor Efficacy of Adoptively Transferred CD19-Redirected CMV-Specific T Cells. Clin Cancer Res, 21, 2993-3002. Wang, Y., Chen, M., Wu, Z., Tong, C., Dai, H., Guo, Y., Liu, Y., Huang, J., Lv, H., Luo, C., Feng, K.-C., Yang, Q.-M., Li, X.-L., & Han, W. (2018). CD133-directed CAR T cells for advanced metastasis malignancies: A phase I trial. Oncoimmunology, 7, e1440169e1440169.

of

Watanabe, K., Luo, Y., Da, T., Guedan, S., Ruella, M., Scholler, J., Keith, B., Young, R. M.,

ro

Engels, B., Sorsa, S., Siurala, M., Havunen, R., Tähtinen, S., Hemminki, A., & June, C. H. (2018). Pancreatic cancer therapy with combined mesothelin-redirected chimeric

-p

antigen receptor T cells and cytokine-armed oncolytic adenoviruses. JCI Insight, 3.

re

Willemsen, R. A., Debets, R., Hart, E., Hoogenboom, H. R., Bolhuis, R. L., & Chames, P. (2001). A phage display selected fab fragment with MHC class I-restricted specificity for

1608.

lP

MAGE-A1 allows for retargeting of primary human T lymphocytes. Gene Ther, 8, 1601-

ur na

Xie, Y. J., Dougan, M., Jailkhani, N., Ingram, J., Fang, T., Kummer, L., Momin, N., Pishesha, N., Rickelt, S., Hynes, R. O., & Ploegh, H. (2019). Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice. Proc Natl Acad Sci U S A, 116, 7624-7631.

Jo

Zhan, X., Wang, B., Li, Z., Li, J., Wang, H., Chen, L., Jiang, H., Wu, M., Xiao, J., Peng, X., Ma, H., Feng, D., Wang, D., Fu, Q., Wang, M., Shen, F., Hao, Q., Zhang, L., Xu, J., & Zhang, Y. (2019). Phase I trial of Claudin 18.2-specific chimeric antigen receptor T cells for advanced gastric and pancreatic adenocarcinoma. Journal of Clinical Oncology, 37, 2509-2509.

Zhang, C., Burger, M. C., Jennewein, L., Genssler, S., Schonfeld, K., Zeiner, P., Hattingen, E., Harter, P. N., Mittelbronn, M., Tonn, T., Steinbach, J. P., & Wels, W. S. (2016). ErbB2/HER2-Specific NK Cells for Targeted Therapy of Glioblastoma. J Natl Cancer Inst, 108. Zhang, C., Wang, Z., Yang, Z., Wang, M., Li, S., Li, Y., Zhang, R., Xiong, Z., Wei, Z., Shen, J., Luo, Y., Zhang, Q., Liu, L., Qin, H., Liu, W., Wu, F., Chen, W., Pan, F., Zhang, X., Bie,

of

P., Liang, H., Pecher, G., & Qian, C. (2017). Phase I Escalating-Dose Trial of CAR-T

ro

Therapy Targeting CEA(+) Metastatic Colorectal Cancers. Mol Ther, 25, 1248-1258. Zhang, L., Morgan, R. A., Beane, J. D., Zheng, Z., Dudley, M. E., Kassim, S. H., Nahvi, A. V.,

-p

Ngo, L. T., Sherry, R. M., Phan, G. Q., Hughes, M. S., Kammula, U. S., Feldman, S. A.,

re

Toomey, M. A., Kerkar, S. P., Restifo, N. P., Yang, J. C., & Rosenberg, S. A. (2015). Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding

2278-2288.

lP

interleukin-12 for the immunotherapy of metastatic melanoma. Clin Cancer Res, 21,

ur na

Zheng, W., O'Hear, C. E., Alli, R., Basham, J. H., Abdelsamed, H. A., Palmer, L. E., Jones, L. L., Youngblood, B., & Geiger, T. L. (2018). PI3K orchestration of the in vivo persistence

Jo

of chimeric antigen receptor-modified T cells. Leukemia, 32, 1157-1167.

Glioblastoma (GBM)

Target

Trial

Study Summary

Antigen

Status

(key results included if available)

IL13R2

Ongoing

Three patients treated with intracranial delivery. One

ro

Cancer Type

of

Table. Key completed and ongoing phase I clinical trials of CAR T cells in adult solid tumors* NCT Number

References

NCT02208362

(Brown, et al., 2015)

infusions. Completed

Peripheral delivery for recurrent GBM. One patient with

re

EGFRvIII

-p

exceptional responder treated with intraventricular

(Brown, et al., 2016)

NCT02209376

(O'Rourke, et al., 2017)

NCT03726515

N/A

durable stable disease. Post-treatment tissue revealed

lP

trafficking of CAR T cells to tumor, reduction in target antigen, and increased PD-L1 expression and regulatory

ur na

T cell infiltration.

EGFRvIII

Ongoing

Peripheral delivery in combination with pembrolizumab for newly diagnosed GBM

Ongoing

Intracerebral CAR T cells for recurrent GBM

NCT03283631

N/A

EGFRvIII

Ongoing

Peripheral delivery in combination with dose-intensified

NCT02664363

N/A

NCT01454596

(Goff, et al., 2019)

Jo

EGFRvIII

EGFRvIII

temozolomide for newly diagnosed GBM Completed

Peripheral delivery for recurrent GBM following lymphodepleting chemotherapy. Two patients

of

experienced severe hypoxia. One patient with survival of 59 months.

Completed

Virus-specific T cells delivered peripherally for

ro

HER2

NCT01109095

(Ahmed, et al., 2017)

NCT01373047

(Katz, et al., 2015)

NCT01212887

(Thistlethwaite, et al.,

recurrent GBM. Poor expansion of CAR T cells, but one

Gastrointestinal Cancers

CEA

Completed

-p

partial response observed.

Direct delivery to colorectal cancer liver metastases via

re

hepatic artery. Well tolerated; one patient with durable stable disease. Completed

Following lymphodepleting chemotherapy, CAR T cells

lP

CEA

delivered peripherally in combination with systemic IL-

2017)

ur na

2. No efficacy, but increased intensity of

Jo

CEA

Ongoing

lymphodepletion correlated with increased CAR T cell engraftment. On-target off-tumor toxicity on lung epithelium. Following lymphodepleting chemotherapy, CAR T cells delivered peripherally. Poor CAR T cell persistence, but two patients experienced tumor shrinkage.

NCT02349724

(C. Zhang, et al., 2017)

Ongoing

Intraperitoneal infusions for CEA-expressing

NCT03682744

N/A

NCT01897415

(Beatty, et al., 2018)

NCT03323944

N/A

NCT01935843

(Feng, et al., 2018)

NCT03159819

(Zhan, et al., 2019)

of

CEA

adenocarcinoma peritoneal metastases or malignant

Mesothelin

Completed

ro

ascites

mRNA CAR T cells delivered peripherally in

-p

chemotherapy-refractory pancreatic ductal

adenocarcinoma. No dose-limiting toxicity. FDG

re

PET/CT imaging revealed significant decrease in total metabolically active volume in one patient. Ongoing

Lentiviral transduced anti-mesothelin CAR T cells in

lP

Mesothelin

pancreatic ductal adenocarcinoma, with or without cyclophosphamide lymphodepletion.

Completed

Jo

ur na

HER2

Claudin 18.2

Ongoing

Peripherally delivered CAR T cells following 1-2 conditioning with nab-paclitaxel and cyclophosphamide. Several severe AEs potentially attributed to HER2 expression on GI mucosa. One partial response achieved. Twelve subjects (5 pancreatic cancer; 7 gastric cancer) treated with peripheral CAR T cells following lymphodepleting chemotherapy. No serious AEs; one complete response and three partial responses.

Ongoing

CAR T cells contain a costimulatory domain that is

NCT02744287

(Becerra, et al., 2019)

NCT02905188

N/A

N/A

(Lamers, et al., 2016)

NCT03393936

N/A

N/A

(Junghans, et al., 2016)

of

PSCA

inducible by administration of small molecule

ro

rimiducid. Peripheral CAR T cell delivery following cyclophosphamide lymphodepletion in patients with

-p

metastatic/refractory pancreatic or gastric cancer. No dose-limiting toxicity; CAR T cells engrafted rapidly;

Glypican 3

Ongoing

re

two minor response achieved.

Peripheral delivery of CAR T cells following

lP

fludarabine/cyclophosphamide lymphodepletion for patients with recurrent hepatocellular carcinoma.

Genitourinary cancers

Carboxy-

ur na

anhydrase IX

Completed

(CAIX)

Jo

AXL

PSMA

Ongoing

Ongoing

Up to 10 daily peripheral infusions of CAR T cells. No efficacy demonstrated. Patients developed anti-CAR T antibodies, and severe liver enzyme disturbances due to on-target off-tumor toxicity on bile duct epithelium. Peripheral infusion of CAR T cells for patients with relapsed/refractory AXL+ renal cell carcinoma Peripheral infusions of CAR T cells for patients with metastatic castrate-resistant prostate cancer in combination with low dose IL2 by continuous infusion.

of

Two patients achieved prostate-specific antigen (PSA)

PSMA

Ongoing

ro

responses.

PSMA-directed, transforming growth factor beta (TGF-

NCT03089203

(Narayan, et al., 2019)

NCT03873805

N/A

N/A

(Kershaw, et al., 2006)

-p

beta)-insensitive CAR T cells (cells are equipped with a dominant-negative TGF-beta receptor to enhance

re

antitumor immunity) in patients with metastatic castrateresistant prostate cancer . No dose limiting toxicity in

lP

first 6 patients; one reversible case of cytokine release syndrome.

Ongoing

ur na

PSCA

Folate

Jo

receptor-alpha

Completed

Peripherally delivered CAR T cells following fludarabine/cyclophosphamide lymphodepletion for patients with PSCA+ metastatic castration resistance prostate cancer. First-generation CAR T cells administered peripherally to patients with platinum-refractory ovarian cancer in combination with high-dose IL2; a subset of patients received dual-specific T cells that were also reactive to allogenic antigen. No efficacy demonstrated; CAR T cells did not appear to traffic to tumor appropriately and

of

did not persist at meaningful levels in circulation

Ongoing

receptor-alpha

Intraperitoneal delivery of CAR T cells with or without

-p

Folate

ro

beyond 2 days.

NCT03585764

N/A

NCT02498912

N/A

NCT01837602

(Tchou, et al., 2017)

NCT03060356

N/A

NCT02792114

N/A

cyclophosphamide/fludarabine lymphodepletion in

Ongoing

Intravenous and intraperitoneal infusion of CAR T cells

lP

MUC16

re

patients with recurrent high grade serious ovarian cancer

engineered to secrete IL-12 and to target MUC16 in patients with recurrent MUC16+ ovarian, peritoneal, or

Breast Cancer

ur na

fallopian tube cancers

c-Met

Jo

c-Met

Mesothelin

Completed

Ongoing

Intratumoral delivery of mRNA CAR T cells followed by tumor excision. Histopathology revealed extensive tumor necrosis and loss of c-Met immunoreactivity. Peripheral delivery of mRNA CAR T cells for advanced c-Met+ breast cancer

Ongoing

Single intravenous infusion of CAR T cells

Ongoing

Intracranial injection of CAR T cells for patients with HER2+ brain metastases

Ongoing

Intraventricular delivery of CAR T cells for patients

ro

HER2

of

HER2

NCT02442297

N/A

NCT03696030

N/A

NCT04020575

N/A

NCT02414269

(Adusumilli, et al., 2019)

NCT03054298

N/A

NCT02706392

N/A

with HER2+ brain metastases Ongoing

Peripheral delivery of CAR T cells for patients with

-p

MUC1

metastatic MUC1+ breast cancer Mesothelin

Ongoing

Mesothelin CAR T cells with I-caspase-9 safety gene

re

Lung Cancer

administered intrapleurally following cyclophosphamide

lP

lymphodepletion to patients with mesothelioma or with pleural metastases from non-small cell lung cancer. No

ur na

evidence of on-target off-tumor toxicity observed. Five

Jo

Mesothelin

ROR1

Ongoing

Ongoing

patients treated with CAR T cells in combination with anti-PD-1 therapy showed partial responses. Intravenous or intrapleural delivery of lentiviraltransduced anti-mesothelin CAR T cells to patients with metastatic/recurrent lung adenocarcinoma Peripheral delivery of CAR T cells following fludarabine/cyclophosphamide lymphodepletion in patients with recurrent ROR1+ non-small cell lung cancer and various other solid tumors

of

* This list is not exhaustive and includes only the phase I trials felt by the authors to provide the most salient clinical data for CAR T

Jo

ur na

lP

re

-p

ro

cell therapy in adult solid tumors