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. 2020 Apr 7;160(3):280–294. doi: 10.1111/imm.13187

T‐cells expressing a chimeric‐PD1‐Dap10‐CD3zeta receptor reduce tumour burden in multiple murine syngeneic models of solid cancer

Geoffrey Parriott 1, Kelsey Deal 1, Shane Crean 1, Elle Richardson 1, Emily Nylen 1, Amorette Barber 1,✉
PMCID: PMC7341544  PMID: 32144940

Summary

Adoptive transfer of T‐cells is a promising therapy for many cancers. To enhance tumour recognition by T‐cells, chimeric antigen receptors (CARs) consisting of signalling domains fused to receptors that recognize tumour‐associated antigens can be expressed in T‐cells. While CAR T‐cells have shown clinical success for treating haematopoietic malignancies, using CAR T‐cells to treat solid tumours remains a challenge. We developed a chimeric PD1 (chPD1) receptor that recognizes the ligands for the PD1 receptor that are expressed on many types of solid cancer. To determine if this novel CAR could target a wide variety of tumour types, the anti‐tumour efficacy of chPD1 T‐cells against syngeneic murine models of melanoma, renal, pancreatic, liver, colon, breast, prostate and bladder cancer was measured. Of the 14 cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T‐cells. ChPD1 T‐cells lysed the tumour cells and secreted pro‐inflammatory cytokines [interferon (IFN)γ, tumour necrosis factor (TNF)α, interleukin (IL)‐2, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), IL‐17 and IL‐21], but did not secrete the anti‐inflammatory cytokine IL‐10. Furthermore, T‐cells expressing chPD1 receptors reduced an established tumour burden and led to long‐term tumour‐free survival in all types of solid tumours tested. ChPD1 T‐cells did not survive longer than 14 days in vivo; however, treatment with chPD1 T‐cells induced protective host anti‐tumour memory responses in tumour‐bearing mice. Therefore, adoptive transfer of chPD1 T‐cells could be a novel therapeutic strategy to treat multiple types of solid cancer.

Keywords: cancer, CD8 T‐cell, chimeric antigen receptor, Dap10, immunotherapy

A chimeric PD1‐Dap10‐CD3zeta (chPD1) receptor that recognizes the PD1 ligands expressed on many types of solid cancer was developed. T‐cells expressing chPD1 receptors lysed tumour cells, secreted proinflammatory cytokines, reduced an established tumour burden and led to long‐term tumour‐free survival in multiple types of solid tumours. Treatment with chPD1 T‐cells also induced protective host anti‐tumour memory responses in tumour‐bearing mice.

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Abbreviations

CAR

chimeric antigen receptor

ChPD1

chimeric PD1 receptor

GM‐CSF

granulocyte‐macrophage colony‐stimulating factor

ICOS

inducible T‐cell co‐stimulator

IFN

interferon

IL

interleukin

NKG2D

natural killer group 2D

PD1

programmed death receptor 1

PDL

programmed death ligand

scFv

single‐chain variable fragment

TNF

tumour necrosis factor

WtPD1

wild‐type PD1 receptor

Introduction

Expression of chimeric antigen receptors (CARs) in T‐cells has had clinical success in some lymphoid malignancies, including acute lymphoblastic leukaemia and diffuse large B‐cell lymphoma. 1 , 2 CARs are designed to recognize tumour‐associated antigens and allow T‐cell activation independent of the T‐cell receptor. 2 , 4 However, the success of CAR T‐cells in solid tumours has so far been limited. 5 , 6 , 7 There are many factors contributing to the restricted success of CAR T‐cell therapies in solid tumours, which include targeting tumour‐associated antigens that are also expressed on normal tissues, poor T‐cell trafficking and persistence, and the immunosuppressive environment that is present in solid tumours. 5 , 6 , 7 , 8 , 9

During an immune response, negative regulation of T‐cells occurs through many mechanisms. 10 , 11 , 12 Programmed death receptor 1 (PD1, CD279) is an inhibitory receptor that is upregulated during sustained T‐cell activation, and has been shown to inhibit T‐cell function in patients with cancer. 12 , 13 , 14 , 15 The ligands for the PD1 receptor, programmed death ligand 1 (PDL1, B7‐H1, CD274) and programmed death ligand 2 (PDL2, B7‐DC, CD273), are expressed on many types of solid cancers and can also be expressed on tumour‐associated dendritic cells, macrophages and fibroblasts. 12 , 14 , 16 Within the tumour microenvironment, activation of PD1 on T‐cells induces T‐cell exhaustion and causes T‐cells to have reduced cytolytic activity, proliferation and cytokine secretion, and ultimately may cause T‐cell apoptosis. There are a multitude of current trials testing PD1‐ or PDL1‐targeted immune checkpoint blockade therapies. This therapeutic approach aims to reawaken CD8 + T‐cell responses in patients with cancer, and PD1‐targeted therapies have been approved as a first‐ or second‐line therapy for various malignancies, including melanoma, lymphoma, lung cancer, renal cell cancer, bladder cancer and liver cancer. 15 , 17

To enhance targeting of solid tumours, there is a need to develop novel CARs that target tumour‐associated antigens. Due to the expression of PD1 ligands on many different types of solid cancers, we created a CAR that recognizes PD1 ligands. 20 This murine chimeric PD1 receptor (chPD1) consists of the extracellular domain of the PD1 receptor fused to the cytoplasmic domains of the Dap10 co‐stimulatory receptor and CD3ζ. Replacement of the inhibitory domains of PD1 with the activating domains of CD3ζ induces T‐cell activation upon engagement with PD1 ligand‐expressing tumour cells. Previously, it was shown that chPD1‐expressing T‐cells reduced tumour burden and increased survival in an aggressive mouse model of T‐cell lymphoma. 20 However, the therapeutic efficacy of chPD1 T‐cells for solid tumours is unknown.

As the development of CAR T‐cell therapies continues, much of the research is focused on enhancing T‐cell function and trafficking in vivo. One mechanism to improve T‐cell function and longevity is to include co‐stimulatory domains in CAR constructs. Inclusion of CD28, 4‐1BB or other co‐stimulatory domains can enhance T‐cell functions, including cytokine secretion, memory differentiation, cytotoxicity, proliferation and cell survival. 6 , 8 , 21 , 22 Dap10 is a co‐stimulatory receptor that has been shown to enhance T‐cell effector responses and induce differential activation of signal transduction pathways, including β‐catenin, NFκB and mTOR, leading to enhanced cytokine secretion and T‐cell differentiation into memory precursor cells. 23 , 24 , 25 , 26 , 27 , 28 Thus, inclusion of the Dap10 co‐stimulatory domain in CARs may allow for CAR T‐cells to overcome some of the immunosuppressive barriers that are encountered in the tumour microenvironment of solid tumours. 29 , 30

This study determined the anti‐tumour efficacy of chPD1‐expressing T‐cells in multiple syngeneic murine models of solid cancers, including melanoma, renal, pancreatic, liver, colon, breast, prostate and bladder cancer. chPD1 T‐cells lysed PD1‐ligand‐positive tumour cell lines and secreted proinflammatory cytokines in vitro. Treatment with chPD1 T‐cells reduced an established tumour burden and led to long‐term tumour‐free survival in the various syngeneic solid tumour models. Furthermore, chPD1 T‐cell treatment induced protective host memory responses in tumour‐surviving mice.

Materials and methods

Cell lines

Murine melanoma cell line B16‐F1, pancreatic cancer cell lines Pan02 and TGP49, renal cancer cell lines Renca and RAG, colon cancer cell lines MC38 and CT26, liver cancer cell lines Hepa1‐6 and C37, prostate cancer cell lines PTEN‐P8 and TRAMP‐C1, bladder cancer cell line MB49, breast cancer cell lines E0771 and 4T1, and T‐cell lymphoma cell line RMA were used. All cell lines were purchased from ATCC, except for Pan02 (from Frederick National Laboratory for Cancer Research, Frederick, Maryland), MB49 (from Millipore Sigma, St Louis, MO) and E0771 (from CH3 Biosystems, Amherst, NY). RMA cells were kindly provided by Dr Charles Sentman at Dartmouth Medical School (Lebanon, NH). The MC38‐PD‐L1 knockout (KO) cell line was purchased from genOway (Lyon, France). Cell lines were grown in complete DMEM media supplemented with 10% heat‐inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mm pyruvate, 10 mm Hepes, 0·1 mm non‐essential amino acids, with the exception of Pan02, Renca, MC38, CT26, 4T1 and RMA, which were grown in RMPI‐1640 media with the same added supplements as described above.

Generation of wild‐type (wt)‐ and chPD1 constructs and retroviral supernatants

Wt‐ and chPD1 constructs and retroviral supernatants were prepared as previously described. 20 Briefly, murine cDNA clones of CD3ζ, PD1 and Dap10 were purchased from OriGene. The chPD1‐Dap10 receptor was created by overlapping polymerase chain reaction (PCR) using Phusion high‐fidelity DNA polymerase (New England BioLabs, Ipswich, MA). To create the chPD1‐Dap10 receptor, the extracellular domain of the murine PD1 receptor (AA 1−155) was fused in frame to the transmembrane region of CD28 (AA 141−177), and the cytoplasmic domains of Dap10 (AA 57−79) and CD3ζ (AA 52−164). To create the wtPD1 receptor, the extracellular and transmembrane domain of the PD1 receptor (AA 1−190) was used. All constructs were cloned into the pQCXIN retroviral expression vector using NotI and EcoRI digestion of the plasmid and constructs, and were subsequently ligated into the vector. Ecotropic retroviral supernatants were expressed using the EcoPack 2‐293 cell line according to manufacturer’s instructions (Clontech, Mountain View, CA). Xfect polymer was used to co‐transfect EcoPack 2‐293 cells with the pEco envelope vector and the pQCXIN retroviral expression vector from the RetroX‐Q vector set (Clontech). RetroX Concentrator was used to concentrate the ecotropic retroviral supernatants before transduction of primary murine T‐cells. Retroviral supernatants were stored at −80º before being used to transduce murine T‐cells.

Expression of wt‐ and chPD1 receptors in T‐cells

Male and female C57BL/6 (B6), BALB/c and B6.SJL‐Ptprca (Ly5.1 congenic) mice were purchased from Taconic Biosciences (Rensselaer, NY). Mice were between 8 and 12 weeks old at the start of each experiment. All animal work was performed in accordance and with approval from Longwood University’s Institutional Animal Use and Care Committee. T‐cells were activated and genetically engineered with chPD1 and wtPD1 receptors as described previously. 20 Briefly, spleens were removed from mice, dissociated and red blood cells were lysed using an ACK lysis buffer. Splenocytes (2 × 106 c/ml) from mice were activated with conA (1 µg/ml) for 18 hr. T‐cells (0·5 × 106 cells/ml) were transduced with retroviral supernatants by centrifugation at 1000 g for 1 hr in the presence of 8 µg/ml polybrene and 25 U/ml recombinant human IL‐2, and were subsequently cultured for 6 hr before retroviral supernatants were removed and replaced with fresh complete RPMI media supplemented with 10% heat‐inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mm pyruvate, 10 mm Hepes, 0·1 mm non‐essential amino acids and 50 μm 2‐mercaptoethanol. Two days after infection, T‐cells were selected in complete RPMI media containing G418 (0·5 mg/ml) plus 25 U/ml recombinant human IL‐2 for an additional 3 days. T‐cells were maintained at a concentration between 0·5 and 2 × 106 c/ml during culture. Viable cells were isolated using Histopaque‐1083 (Millipore Sigma), and were expanded for an additional 2 days with 25 U/ml recombinant human IL‐2 but without G418 before functional analysis.

Flow cytometry

The expression of PDL1 and PDL2 on tumour cells was tested using flow cytometry. Cells were stained with antigen‐presenting cell (APC)‐labelled anti‐PDL1 (clone 10F.9G2) and phycoerythrin (PE)‐labelled anti‐PDL2 (clone TY25) or isotype controls. The expression of PD1 on T‐cells was determined by staining with PE‐labelled anti‐PD1 (clone RMP1‐30) antibodies or isotype controls. For T‐cell differentiation studies, wt‐ or chPD1 T‐cells (2 × 105 cells/well) were stimulated with B16 cells (2 × 105 cells/well) for 24 hr, and were analysed for cell surface marker expression by flow cytometry. Cells were stained with PE‐conjugated‐ anti‐CD127 (clone A7R34) or anti‐KLRG1 (clone 2F1/KLRG1), and APC‐conjugated anti‐CD62L (clone MEL‐14) or isotype controls. All antibodies were purchased from BioLegend (San Diego, CA). Cell fluorescence was measured using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ).

RT‐PCR

Total RNA was isolated from T‐cells using the SV Total RNA isolation kit according to manufacturer’s instructions (Promega). cDNA was created using RevertAid First Strand cDNA synthesis kit using random hexamer primers (Fermetas). One‐hundred nanograms of cDNA was used as a template for reverse transcriptase (RT)‐PCR to measure gene expression of T‐bet, BLIMP1, Eomes, BCL6 and β‐actin. Maxima SYBRGreen qPCR Master Mix (Thermo Scientific) and gene‐specific primers were used. Gene‐specific primers for T‐cell differentiation genes T‐bet, BLIMP1, Eomes and BCL6 were previously described. 28 Primers were purchased from Integrated DNA Technologies.

Cytokine production and cytotoxicity by chPD1 T‐cells

T‐cells transduced with chPD1 or wtPD1 receptors (105) were cultured with tumour cells (105) or media in a round‐bottom 96‐well plate. After 24 hr, cell‐free supernatants were tested for the presence of interferon (IFN)γ, tumour necrosis factor (TNF)α or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) by ELISA according to manufacturer’s instructions (BioLegend). Cytokine secretion was also measured in cell‐free supernatants using mouse T helper cytokine LEGENDPlex assays (BioLegend) according to manufacturer’s instructions.

To determine the response of chPD1 T‐cells against healthy tissues, T‐cells transduced with wtPD1 or chPD1 receptors (105) were cultured with splenocytes, hepatocytes or lung cells isolated from naïve C57BL/6 mice, B16 tumour cells or media in a round‐bottom 96‐well plate. After 24 hr, cell‐free supernatants were tested for the presence of IFNγ by ELISA according to manufacturer’s instructions (BioLegend). Naïve C57BL/6 mice were administered one dose of wt‐ or chPD1‐modified T‐cells (5× 106) i.v., and serum levels of IFNγ, TNFα, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine were determined at various time points by ELISA according to manufacturer’s instructions. IFNγ and TNFα ELISA kits were purchased from BioLegend, ALT and AST ELISA kits were purchased from CusaBio, and the creatinine ELISA kit was purchased from Crystal Chem. Additionally, B16‐tumour‐bearing C57BL/6 mice were administered one dose of wt‐ or chPD1‐modified T‐cells (5 × 106) i.v. 10 days after tumour cell injection, and serum levels of AST, ALT and creatinine were determined at various time points by ELISA.

To determine lysis of tumour cells, chPD1‐ or wtPD1‐transduced T‐cells (105) were cultured with tumour cells at various effector to target ratios (E:T 25:1, 5:1 and 1:1). Specific lysis was measured after 5 hr using an LDH cytotoxicity assay kit (ThermoFisher Scientific, Waltham, MA) according to manufacturer’s instructions.

Treatment of mice with genetically modified T‐cells

B16‐F1 (105), Pan02 (6 × 105), MC38 or MC38‐PDL1 KO (105), PTEN‐P8 (106), MB49 (2 × 105) cells were injected subcutaneously into C57BL/6 mice, or Renca (2 × 105) cells were injected subcutaneously into Balb/c mice. E0771 (2 × 105) were injected into the mammary fat pad of C57BL/6 mice. An equal mix of male and female mice was used for all tumour types, except for prostate cancer experiments that utilized male mice, and breast cancer experiments that utilized female mice. For tumour burden experiments, mice were administered two doses of wt‐ or chPD1‐modified T‐cells (5 × 106) i.v. 5 days and 8 days after tumour injection, or two doses of T‐cells (5 × 106) i.v. 10 and 13 days after tumour injection. Dimensions of palpable tumours were measured every other day using calipers, and the tumour area was calculated by multiplying width times length of the tumours. For survival studies, the health of the mice was monitored closely, and mice were killed when signs of stress (laboured breathing, dragging legs, hunched back or ruffled fur) were observed or when the tumour size exceeded 100 mm2.

For analysis of in vivo T‐cell survival, tumour‐bearing mice were treated 10 days after tumour cell injection with congenic Ly5.1 + chPD1 T‐cells (5 × 106) i.v., and mice were killed 1, 3, 7, 10, 14 or 18 days after T‐cell injection. The lymphoid tissues were mechanically teased, and red blood cells were lysed with ACK lysis buffer (0·15 m NH4Cl, 1 mm KHCO3, 0·1 mm). Spleen and lymph node cells were incubated with FcR block and mouse gamma globulin (Jackson ImmunoResearch, West Grove, PA) to prevent non‐specific binding, and stained with PE‐conjugated anti‐CD3 and APC‐conjugated anti‐CD45.1 (clone A20), and analysed by flow cytometry. The total number of tumour cells was determined by multiplying the percentage of Ly5.1 + cells by the total number of cells.

Seventy days after tumour cell injection, splenocytes were isolated from naïve mice (n = 4) or tumour‐surviving mice (n = 4) that were previously treated i.v. with two doses of chPD1 T‐cells (5 × 106) 10 and 13 days after tumour cell injection. Splenocytes (2.5 × 106) were cultured with tumour cells (2.5 × 105) pretreated with mitomycin‐C (30 min, 50 µg/ml) or media for 72 hr. After 72 hr, secretion of IFNγ was measured in cell‐free supernatants by ELISA according to manufacturer’s instructions.

For the tumour rechallenge experiments, tumour cells were injected subcutaneously into tumour‐surviving mice that were previously treated i.v. with two doses of chPD1 T‐cells (5 × 106) 10 and 13 days after tumour cell injection or naïve mice. RMA cells were also injected subcutaneously (105) into groups of tumour‐surviving and naïve mice as a control. Tumour area and survival was determined.

Statistical analysis

Statistical analysis was conducted using an unpaired, two‐tailed Student’s t‐test or anova with a post hoc Tukey test when comparing multiple groups. The data were determined to be normally distributed using the Shapiro−Wilk test. The program R was used for statistical analysis of the data. All experiments were run in triplicate on at least three independent sets of T‐cells, and P‐values < 0·05 were considered significant. For survival studies, Kaplan−Meier survival curves were plotted and analysed using the Log rank test and Prism software (GraphPad Software, San Diego, CA).

Results

ChPD1 T‐cells secrete proinflammatory cytokines and lyse multiple types of PDL‐expressing tumour cells

Previously, a chPD1 receptor consisting of the extracellular region of the PD1 receptor fused with the intracellular regions of the Dap10 co‐stimulatory receptor and CD3ζ was created, and T‐cells expressing the chPD1‐Dap10‐CD3ζ receptor decreased tumour burden and increased survival in a murine model of T‐cell lymphoma. 20 The chPD1 and control wtPD1 receptors were successfully expressed in activated murine T‐cells, and chPD1‐ and wtPD1‐expressing T‐cells consisted of a mix of activated CD4+ (~10%) and CD8+ (~90%) T‐cells. 20 Furthermore, chPD1‐expressing T‐cells expressed markers associated with a central memory phenotype including having elevated expression of transcription factors Eomes and BCL‐6, and expression of cell surface CD127hi, CD62Lhi and KLRG1lo markers (Fig. S1). 20

However, the ability of chPD1 T‐cells to target solid tumours is still unknown. Therefore, the anti‐tumour efficacy of chPD1‐expressing T‐cells against various types of solid cancers was determined. ChPD1 T‐cell responses against murine tumour cell lines of several cancer types including melanoma (B16), pancreatic cancer (Pan02 and TGP49), renal cancer (Renca and RAG), colon cancer (MC38 and CT26), liver cancer (Hepa1‐6 and C37), prostate cancer (PTEN‐C8 and TRAMP‐C1), bladder cancer (MB49) and breast cancer (E0771 and 4T1) were evaluated. All cancer cell lines tested expressed cell surface PDL1 and PDL2 as determined by flow cytometry and RT‐PCR, indicating that these cell lines are potential targets of chPD1 T‐cells (Fig. 1a and data not shown). When cultured with tumour cells, chPD1 T‐cells lysed the tumour cells significantly more than T‐cells expressing a control wtPD1 receptor consisting only of the extracellular and transmembrane domains of the PD1 receptor (Fig. 1b). ChPD1 T‐cell lysis of MC38‐PDL1 KO cells (MC38‐PDL1 KO), which do not express PDL1 and have low expression of PDL2, was reduced compared with lysis of parental MC38 cells (Fig. 1b). Blocking the chPD1 receptor with anti‐PD1 blocking antibodies also prevented the lysis of the tumour cells, indicating that the lysis was dependent on recognition by the chPD1 receptor (data not shown).

Figure 1.

Figure 1

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Chimeric PD1 (chPD1) T‐cells lyse programmed death ligand (PDL)‐expressing tumour cells. (a) Murine tumour cells were stained with anti‐PDL1 (black) or ‐PDL2 (grey) antibodies, and were analysed using flow cytometry. (b) Wild‐type (wt)PD1 (circles) or chPD1 (squares) T‐cells were used as effector cells with tumour cell targets at the indicated E:T ratios (1:1, 5:1, 25:1), and cell lysis was measured using an LDH assay. ChPD1 T‐cells had significantly higher specific lysis at all E:T ratios compared with wtPD1 T‐cells (*P < 0·001). Data are presented as mean + SD, and are representative of at least three experiments.

The secretion of cytokines from CAR T‐cells also plays an important role in reducing tumour burden and modulating immune responses to cancer. 21 , 30 For all tumour cell lines tested, chPD1 T‐cells secreted significant levels of proinflammatory cytokines IFNγ, TNFα, GM‐CSF, IL‐2, IL‐17 and IL‐21, but did not secrete the anti‐inflammatory cytokine IL‐10 (Fig. 2). However, chPD1 T‐cells did not have significant cytokine secretion when cultured with MC38‐PDL1 KO cells. These in vitro data demonstrate that chPD1 T‐cells secrete proinflammatory cytokines and lyse multiple types of PD1‐ligand‐expressing tumour cells, and therefore may potentially be used to treat solid tumours.

Figure 2.

Figure 2

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Culture of tumour cells with chimeric PD1 (chPD1) T‐cells results in secretion of proinflammatory cytokines. Tumour cells were cultured with media (white), wild‐type (wt)PD1‐ (grey) or chPD1‐ (black) expressing T‐cells. After 24 hr, secretion of cytokines was measured in cell‐free supernatants by (a) ELISA or (b) LEGENDPlex analysis. chPD1 T‐cells produced higher levels of proinflammatory cytokines compared with wtPD1 T‐cells when cultured with tumour cells (* P < 0·0001). Data are presented as mean + SD, and are representative of at least three experiments.

Treatment with chPD1 T‐cells leads to a reduction in tumour burden and an increase in survival of tumour‐bearing mice

Previous studies demonstrated that treatment with chPD1 T‐cells significantly reduced an established tumour burden and increased survival in an aggressive mouse model of T‐cell lymphoma. 20 However, in this mouse model of lymphoma the tumour cells traffic to the spleen and lymph nodes, and this may not be representative of the immunosuppressive tumour microenvironment that occurs in solid tumours. 8 , 29 Therefore, the in vivo therapeutic efficacy of chPD1 T‐cells in solid tumours including melanoma, pancreatic, renal, colon, prostate, bladder and breast cancer was investigated. Tumour‐bearing mice were treated with two doses of chPD1 T‐cells 5 and 8 days after tumour cell injection, and tumour burden and survival was measured (Fig. 3a). Compared with mice treated with wtPD1 T‐cells, tumour burden was significantly decreased in mice treated with chPD1 T‐cells in all tumour models tested. Furthermore, 100% of mice treated with chPD1 T‐cells demonstrated long‐term tumour‐free survival. However, chPD1 T‐cells did not induce significant anti‐tumour responses in mice bearing MC38‐PDL1 KO tumours, suggesting the chPD1 T‐cells likely require interaction with PDL1 on the surface of the tumour cells to have strong anti‐tumour efficacy. To test the in vivo therapeutic efficacy of chPD1 T‐cells against a more established tumour burden, mice were treated with wt‐ or chPD1 T‐cells 10 and 13 days after tumour cell injection when a visible tumour was detected. Treatment with chPD1 T‐cells significantly reduced these established tumours and led to long‐term tumour‐free survival in all models of solid cancers tested (Fig. 3b). These data demonstrate that treatment with chPD1 T‐cells reduced an established tumour burden and increased survival in multiple types of cancer.

Figure 3.

Figure 3

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Treatment with chimeric PD1 (chPD1) T‐cells leads to a reduction in tumour burden and an increase in survival of tumour‐bearing mice. Tumour cells were injected s.c. into mice on day 0. Mice were treated i.v. with two doses of wild‐type (wt)PD1 (circles) or chPD1 (squares) T‐cells (5 × 106) after (a) 5 and 8 days, or (b) 10 and 13 days (n = 6 for all groups), and tumour burden and survival were measured. chPD1 T‐cells significantly reduced tumour burden and increased survival compared with wtPD1 T‐cells (*P < 0·01). Data are presented as mean + SD, and are representative of three independent experiments.

The interaction of chPD1 T‐cells with PD1 ligands expressed on healthy tissues is a potential safety concern for this type of therapy. Therefore, the response of chPD1 T‐cells to healthy tissues was evaluated. ChPD1 T‐cells did not secrete a significant amount of IFNγ when cultured with splenocytes, liver cells or lung cells isolated from a naïve mouse (Fig. 4a). 20 In addition, when chPD1 T‐cells were injected intravenously into naïve mice, no adverse symptoms were observed, and serum levels of IFNγ, TNFα, AST, ALT and creatinine did not increase significantly (Fig. 4b). 20 Furthermore, in B16 tumour‐bearing mice treated intravenously with chPD1 T‐cells, AST, ALT and creatinine levels were also not elevated compared with untreated mice (Fig. 4c). These preliminary data suggest that intravenous injection of chPD1 T‐cells does not respond to healthy tissues and may not cause significant damage to healthy tissues. However, a more thorough pathology analysis of tissue samples should be conducted in the future to further determine the possibility of on‐target, off‐tumour tissue damage induced by chPD1 T‐cells.

Figure 4.

Figure 4

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Treatment with chimeric PD1 (chPD1) T‐cells does not induce significant responses against healthy tissues. (a) T‐cells transduced with wild‐type (wt)PD1 or chPD1 receptors were cultured with splenocytes, hepatocytes or lung cells isolated from naïve C57BL/6 mice, B16 tumour cells, or media. After 24 hr, cell‐free supernatants were tested for the presence of interferon (IFN)γ by ELISA. (b) Naïve C57BL/6 mice were administered one dose of wt‐ or chPD1‐modified T‐cells (5 × 106) i.v., and serum levels of IFNγ, tumour necrosis factor (TNF)α, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine were determined at various time points by ELISA. (c) B16‐tumour‐bearing C57BL/6 mice were administered one dose of wt‐ or chPD1‐modified T‐cells (5 × 106) i.v. 10 days after tumour cell injection, and serum levels of AST, ALT and creatinine were determined at various time points by ELISA (n = 6 for all groups). Treatment with chPD1 T‐cells did not significantly increase the release of markers of kidney or liver damage, and also did not increase serum levels of inflammatory cytokines (* P < 0·01). Data are presented as mean + SD, and are representative of three independent experiments.

Treatment with chPD1 T‐cells induces protective host anti‐tumour memory responses in multiple tumour models

In vivo persistence of CAR T‐cells is often correlated with anti‐tumour efficacy. 35 , 36 , 37 To determine in vivo persistence of chPD1 T‐cells, the presence of Ly5.1 + chPD1 T‐cells was determined in the spleens and lymph nodes of tumour‐bearing mice that received one dose of T‐cells 10 days after tumour cell injection (Fig. 5a). Ly5.1+ chPD1‐Dap10 T‐cells were detectable by flow cytometry in the spleen and lymph nodes up to 14 days after T‐cell injection; however, they were not detected after day 18. This indicated that even though mice had long‐term tumour‐free survival, the chPD1 T‐cells were not maintained long term in vivo.

Figure 5.

Figure 5

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Treatment with chimeric PD1 (chPD1) T‐cells induces host anti‐tumour memory responses in multiple tumour models. (a) Tumour‐bearing mice were treated with 5 × 106 Ly5.1+ chPD1 T‐cells i.v. 10 days after tumour cell injection. Spleen and lymph node cells were isolated at various time points after T‐cell injection, and the percentage of Ly5.1 + CD3+ cells was calculated (n = 4). (b) Seventy days after tumour cell injection, splenocytes were isolated from tumour‐surviving mice that were treated i.v. with two doses of chPD1 T‐cells (5 × 106) 10 and 13 days after tumour cell injection or from naïve mice. Splenocytes were then cultured with tumour cells or media (n = 4). After 72 hr, secretion of interferon (IFN)γ was measured in cell‐free supernatants by ELISA. Splenocytes from surviving chPD1 T‐cell‐treated mice produced higher levels of IFNγ when cultured with tumour cells the mice originally rejected compared with naïve mice (*P < 0·0001). Data are presented as mean + SD, and are representative of three experiments.

Some CAR T‐cell therapies cause a ‘vaccine‐like’ response and can induce host anti‐tumour memory responses. 31 , 38 , 39 To determine if tumour‐surviving mice treated with chPD1 T‐cells developed host immune responses to tumour antigens, splenocytes from tumour‐surviving mice (70 days after tumour cell injection) or from naïve mice were cultured with tumour cells or with media alone. Spleen cells from surviving mice secreted IFNγ when cultured with tumour cells they had previously rejected, but not with other tumour types or with media (Fig. 5b). Spleen cells from naïve mice did not secrete IFNγ when cultured with any of the tumour cells. These data suggest that chPD1‐treated mice develop long‐lived host memory responses that are specific to tumour antigens.

To test whether these host memory responses were protective against a tumour rechallenge, naïve and tumour‐surviving mice (Fig. 3b) were challenged with the tumour type they had originally rejected. Compared with naïve mice that had significant tumour growth, 100% of tumour‐surviving mice rejected the tumour rechallenge and had long‐term tumour‐free survival (Fig. 6a). The rejection of a tumour rechallenge was antigen specific because tumour‐surviving mice rechallenged with a tumour they had not previously been exposed to (RMA lymphoma cell line) all had significant tumour growth that was similar to tumour growth in naïve mice (Fig. 6b). Taken together, these data show that treatment with chPD1 T‐cells led to tumour‐free survival and the induction of protective host memory responses in multiple models of solid tumours.

Figure 6.

Figure 6

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Tumour‐surviving mice previously treated with chimeric PD1 (chPD1) T‐cells successfully reject a tumour rechallenge in multiple tumour models. (a) Seventy days after tumour cell injection, tumour‐surviving mice that were treated i.v. with two doses of chPD1 T‐cells (5 × 106) 10 and 13 days after tumour cell injection were rechallenged with (a) the tumour cell type that was originally rejected or (b) RMA tumour cells, which is a tumour cell type the mice had not been previously exposed to. Tumour cells were also injected into naïve mice as a control (n = 6 for all groups). Tumour burden and survival were measured. Splenocytes from surviving chPD1 T‐cell‐treated mice had improved tumour rejection compared with naïve mice (*P < 0·0001). Data are presented as mean + SD, and are representative of three experiments.

Discussion

Therapy with CAR‐expressing T‐cells has shown remarkable efficacy for treatment of B‐cell malignancies. 1 , 2 , 3 , 4 However, success of CAR T‐cell therapy in solid tumours is diminished in part due to increased immunosuppressive factors, problems with T‐cell trafficking and persistence, and a limited number of tumour‐associated antigens that can be targeted. 7 , 9 , 29 , 30 This study demonstrated that murine T‐cells expressing the chPD1‐Dap10‐CD3ζ receptor can target multiple types of solid cancers. Our results suggest that chPD1‐expressing T‐cells are able to target a wide variety of PD1‐ligand‐expressing tumours. ChPD1 T‐cells secreted proinflammatory cytokines and lysed PDL‐expressing tumour cells, and also induced long‐term tumour‐free survival in mice with established tumours. In addition, chPD1 T‐cell‐treated mice induced long‐lived protective host anti‐tumour immune responses against tumour antigens.

Blockade of the PD1 pathway is one approach that can be used to reawaken anti‐tumour CD8 + T‐cell responses, and the use of PD1 checkpoint blockade antibodies has shown clinical success in many types of cancer. 17 , 18 Monoclonal antibody blockade of PD1 or PDL1 can restore the function of autologous tumour‐reactive T‐cells, and may also improve the efficacy of adoptively transferred T‐cells. However, even if the PD1 pathway is inhibited, the success of this therapy in part depends on the existence, activation and efficacy of host anti‐tumour T‐cells. In addition, the ligands for T‐cell co‐stimulatory receptors are often absent in the tumour microenvironment, thus effector T‐cell functions, persistence and differentiation into long‐lived memory cells is likely limited. 40 The combination of antigen‐specific adoptively transferred T‐cells, such as CAR T‐cells, and PD1/PDL blockade may therefore be a logical approach to combine the positive effects of PD1 blockade with the tumour antigen specificity and enhanced functions of modified T‐cells.

The chPD1‐Dap10‐CD3ζ receptor was designed to take advantage of the tumour‐associated expression of PD1 ligands to allow for the development of a CAR that can target multiple tumour types. In addition, this receptor converts the normally inhibitory PD1 signal into an activating signal for T‐cells. Other approaches have also combined PD1 targeting with genetically modified T‐cells. For example, expression of PD1‐CD28 and PD1‐CD28‐41BB switch receptors that replace the cytoplasmic domain of PD1 with the cytoplasmic domain of CD28 and/or 4‐1BB can prevent T‐cell inhibition. 41 , 42 , 43 , 44 , 45 , 46 In addition, CAR T‐cells engineered to secrete anti‐PD‐L1 or anti‐PD1 antibodies or anti‐PD1 single‐chain antibody fragments (scFvs) demonstrate enhanced anti‐tumour efficacy. 29 , 47 However, the chPD1 receptor is different than the PD1‐based switch receptors or the combination of CAR T‐cells with PD1 checkpoint blockade antibodies because our chPD1 receptor provides both the activation (CD3ζ) and co‐stimulatory signal (Dap10) all within the same receptor. This eliminates the need for the co‐expression of two or more genes in T‐cells. However, unlike the PD1‐based switch‐receptors or PD1 checkpoint blockade, the chPD1‐Dap10‐CD3ζ receptor removes the need for tumour‐antigen recognition. This will likely greatly increase the chance that chPD1 T‐cells will react with many types of healthy tissues that express PD1 ligands and cause on‐target, off‐tumour side‐effects. Our preliminary work investigating the safety of chPD1 T‐cells injected intravenously in mice showed that there was no increase in inflammatory cytokines IFNγ or TNFα, or markers of liver and kidney damage, AST, ALT or creatinine. Future studies should continue to investigate the effect of chPD1 T‐cells on healthy cells. In addition, a suicide gene could be added to the chPD1 construct to allow for the selective depletion of chPD1 T‐cells if adverse side‐effects were observed. 51 Another approach could be to modulate the functions of chPD1 T‐cells by controlling the expression of the chPD1 receptor with an on‐ or off‐ switch. 51 , 52

One critical barrier of using CAR T‐cells for the treatment of solid cancers is the immunosuppressive tumour microenvironment. For optimal anti‐tumour efficacy, CAR T‐cells must be able to overcome the harsh tumour environment that has multiple factors that suppress the infiltration, persistence, activation and effector activity of T‐cells. Specifically, inhibitory cell types, including tumour‐associated macrophages, myeloid‐derived suppressor cells and regulatory T‐cells, immunosuppressive cytokines and expression of inhibitory immune checkpoint molecules can suppress the anti‐tumour activities of tumour‐infiltrating CAR T‐cells. 8 , 29 In addition to tumour cells, many cell types in the tumour microenvironment express PDL‐1 and PDL‐2, including myeloid‐derived suppressor cells, tumour‐associated macrophages, regulatory T‐cells, tumour‐associated fibroblasts and angiogenic vascular endothelial cells. 12 , 17 , 18 The data in this study demonstrate that chPD1 T‐cells target various tumour types that express PDL‐1 or PDL‐2, and that lysis of the tumour cells is dependent on recognition by the chPD1 receptor. Therefore, it is possible that chPD1 T‐cells may also be able to target and lyse other PD1‐ligand‐expressing cells in the tumour microenvironment, including immunosuppressive myeloid cells, regulatory T‐cells, fibroblasts and angiogenic blood vessels. This could result in the concurrent lysis of tumour cells and reduction of immunosuppressive cells within the tumour mass. Furthermore, chPD1 T‐cells secrete IFNγ during co‐culture with PD1‐ligand‐positive cells. IFNγ has been shown to increase expression of PD‐1 ligands on myeloid cells, endothelial cells and tumour cells, therefore activation of chPD1 T‐cells in the tumour may lead to increased expression of PD1 ligands and lead to sustained targeting of tumours and other cells in solid tumours by chPD1 T‐cells. 12 , 53 , 54 It will be interesting to investigate the targeting of tumour‐associated cells and manipulation of the tumour microenvironment by chPD1 T‐cells in future studies.

The inclusion of co‐stimulatory domains in CARs enhances T‐cell functions, including cytokine secretion, differentiation and persistence. A great majority of CAR T‐cell constructs currently being developed are using second‐generation or third‐generation CARs consisting of CD3ζ and CD28, 4‐1BB, or other co‐stimulatory domains. 3 , 6 , 8 , 21 , 22 , 32 Each co‐stimulatory domain induces a unique set of effector functions and differentiation. For example, CARs that include 4‐1BB differentiate into a central memory phenotype and have enhanced persistence and anti‐tumour activity in vivo, whereas T‐cells with a CD28‐ containing CAR do not persist as long in vivo. 55 A previous study demonstrated that a Dap10‐containing chPD1 CAR had enhanced persistence, development of a central memory phenotype and superior anti‐tumour immunity in vivo compared with the same chPD1‐CAR containing a CD28 co‐stimulation domain. 20 While natural killer group 2D (NKG2D)/Dap10 and CD28 signalling are similar in many aspects, stimulation of NKG2D/Dap10 induces differential activation of mTOR in effector CD8 T‐cells and supports the development of a central memory phenotype. 28 Therefore, it would be interesting to directly compare the inclusion of alternative co‐stimulatory domains, including CD28, 4‐1BB or inducible T‐cell co‐stimulator (ICOS), in chPD1 receptors to determine which co‐stimulatory receptors induce superior anti‐tumour responses for solid tumours. Interestingly, a similar anti‐PD‐L1 CAR composed of a scFv against PD‐L1, CD3ζ, and a combination of CD28 and 4‐1BB co‐stimulatory domains has shown in vitro cytotoxicity against lung adenocarcinoma cells, although it has not yet been determined if this CAR construct is effective in vivo. 55

In addition, CAR T‐cell persistence may be enhanced by expressing CARs containing different co‐stimulatory domains in CD4 and CD8 T‐cells. 37 , 56 A recent study in a lung cancer model demonstrated that CAR T‐cells had enhanced in vivo persistence when CD4‐ICOS‐ and CD8‐4‐1BB‐CAR T‐cells were mixed. 56 These results highlight the different co‐stimulation requirements of CD4 and CD8 T‐cells. ChPD1‐Dap10 T‐cells are a mix of ~10% CD4 T‐cells and ~90% CD8 T‐cells (data not shown), and it would be interesting to investigate the differential signalling induced in chPD1‐Dap10‐expressing CD4 and CD8 T‐cells.

Many of the studies investigating anti‐tumour efficacy of PD1‐based switch receptors and combination therapy of anti‐PD1 checkpoint blockade with CAR T‐cells utilize immunodeficient mouse models. 41 , 42 , 46 However, immunodeficient mouse models do not accurately represent the immunosuppressive tumour microenvironment observed in solid tumours or the role of host immunity in tumour reduction. Because the induction of host immune responses is often required for full anti‐tumour efficacy of CAR T‐cells, the use of immunodeficient mouse models may not be ideal for analysing the potential success of this type of therapy. 21 , 31 , 38 , 57 Therefore, the evaluation of a murine chPD1‐Dap10‐CD3ζ receptor in syngeneic immunocompetent mouse models allows for the analysis of chPD1 T‐cell efficacy, and better represents the suppressive tumour microenvironment in solid tumours. Although chPD1 T‐cells did not persist long term in the tumour‐surviving mice, this treatment leads to the induction of long‐lived host memory responses to tumour antigens. This tumour‐antigen‐specific host memory response was protective against a tumour rechallenge in various models of solid tumours and mice that were re‐injected with tumour cells 70 days after the original tumour cell injection rejected the tumour rechallenge. This suggests that chPD1 T‐cell treatment activated strong host immune responses to tumour antigens, likely through tumour cell lysis, release of tumour antigens and danger‐associated molecules, and secretion of cytokines. The host immune response was specific to tumour antigens because chPD1 T‐cell‐treated surviving mice were able to reject tumour cells that they had originally rejected, but not a tumour they had not previously been exposed to (RMA lymphoma cells). The RMA lymphoma cell line expresses PDL1, demonstrating that the host immune responses are specific for tumour antigens and not specific for PD‐1 ligands. 20 The development of long‐lived host memory responses to tumour antigens is likely important for durable responses in CAR T‐cell therapies, as immune responses to additional tumour antigens would be beneficial due to heterogeneous expression of PD1 ligands and other tumour‐associated antigens within a tumour.

In summary, chPD1 receptor‐expressing T‐cells may be able to target multiple types of solid tumours due to the expression of PD1 ligands in many cancer types. Furthermore, the secretion of proinflammatory cytokines and induction of protective host memory responses against tumour antigens may help to overcome some of the challenges that are currently encountered in CAR T‐cell therapy of solid tumours.

Disclosures

The authors declare no conflict of interest.

Supporting information

Figure S1. ChPD1 T‐cells express memory‐associated differentiation markers.

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Acknowledgements

Geoffrey Parriott, Kelsey Deal, Shane Crean, Elle Richardson, Emily Nylen and Amorette Barber performed the experiments and designed the study, and Amorette Barber wrote the paper. This work was supported in part by Longwood University’s Faculty Research Grants, Department of Biological and Environmental Sciences, and Virginia Academy of Science’s Mary Louise Olds Andrews Cancer Grant.

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Associated Data

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Supplementary Materials

Figure S1. ChPD1 T‐cells express memory‐associated differentiation markers.

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