Despite the promising efficacy of adoptive cell therapies (ACT) in melanoma, complete response rates remain relatively low and outcomes in other cancers are less impressive. The immunosuppressive nature of the tumor microenvironment and the expression of immune-inhibitory ligands, such as PD-L1/CD274 by the tumor and stroma are considered key factors limiting efficacy. The addition of checkpoint inhibitors (CPI) to ACT protocols bypasses some mechanisms of immunosuppression, but associated toxicities remain a significant concern. To overcome PD-L1–mediated immunosuppression and reduce CPI-associated toxicities, we used TALEN technology to render tumor-reactive T cells resistant to PD-1 signaling. Here, we demonstrate that inactivation of the PD-1 gene in melanoma-reactive CD8+ T cells and in fibrosarcoma-reactive polyclonal T cells enhanced the persistence of PD-1 gene-modified T cells at the tumor site and increased tumor control. These results illustrate the feasibility and potency of approaches incorporating advanced gene-editing technologies into ACT protocols to silence immune checkpoints as a strategy to overcome locally active immune escape pathways. Cancer Res; 76(8); 2087–93. ©2016 AACR.
Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) or T cells redirected to the tumor by chimeric antigen receptors (CAR) can mediate substantial regression of human cancers (1). Although multiple early-phase studies using ACT have demonstrated durable responses that correlate with persistence of the transferred T cells (2, 3), their in vivo activity and potency can be thwarted by the complex immunosuppressive nature of the tumor microenvironment (4). Combination protocols incorporating checkpoint inhibitors (CPI) such as anti–CTLA-4 or anti–PD-L1/PD-1 are currently being considered (NCT02408861; NCT02210117; NCT02374242; NCT02453620), but the potential exacerbation of the toxicities associated with CPIs may limit their implementation. Advanced genetic engineering of the ACT product offers potential solution to the toxicity issues by specifically targeting tumor-reactive T cells rather than inducing systemic checkpoint blockade. The protocols used for the generation of ACT are compatible with genetic manipulation of cells before transfer, which can be used to increase the proliferative capacity of lymphocytes (5), prolong their in vivo persistence (6), improve tumor infiltration (7), and bypass tumor immune suppression.
Programmed death-1 (PD-1, CD279) is one of the key coinhibitory immune receptors expressed by activated T cells. Initially described on a T-cell hybridoma undergoing cell death (8), PD-1 is a critical regulator of T-cell responses and essential to the maintenance of peripheral self-tolerance (9). In mouse and human cancers, high levels of PD-1 are found on TILs, although its ligand (PD-L1) is upregulated by tumor and stromal cells in response to inflammatory cytokines (e.g., IFNγ; refs. 10, 11). The PDL-1–PD-1 axis plays a major role in tumor growth and immune escape through inhibition of T-cell proliferation, effector function (12), and of T-cell survival by promotion of apoptosis (13). The administration of blocking anti–PD-L1 or PDL-1 antibodies in vivo results in enhanced CD8+ T cell responses and decreases tumor burden in mouse models of cancer (14, 15). In humans, expression of PD-1 by TILs is associated with impaired effector function and/or poor outcome in several tumor types (16, 17). Direct evidence supporting the importance of this pathway in modulating antitumor immunity comes from recent clinical trials in melanoma and non–small cell lung cancer, where anti–PD-1 antibodies have improved both progression-free and overall survival (18, 19). Of relevance, systemic immunotherapy with CPIs can also induce immune-related adverse events (irAE). Specific ablation of negative regulators of T-cell function on tumor-reactive T cells used for ACT offers the potential to exploit the antitumor activity while reducing systemic toxicity.
Efficient and rapid inactivation of genes in primary antigen-specific T cells is achievable using the transient expression of transcription activator-like effector nucleases (TALEN) mRNA, which offers several safety advantages for clinical applications (20). Using two different mouse models of cancer, we defined the impact of TALEN-mediated PD-1 gene inactivation in adoptively transferred tumor-reactive CD4+ and CD8+ T cells. Our data support a model in which PD-1 gene inactivation increases persistence of T cells at the tumor site, enhancing tumor control of B16 melanoma by gp-100-reactive CD8+ T cells and mediating complete rejection of established MCA205 fibrosarcoma by endogenous CD4+ and CD8+ T cells. This work demonstrates both the feasibility and the potential potency of strategies incorporating immune checkpoint editing of tumor-reactive T cells for use in the context of adoptive T-cell therapies for cancer.
Materials and Methods
Mice and cell lines
Six- to 8-week-old pmel-1/SJL TCR-transgenic, C57BL/6, and C57BL/6-SJL mice were purchased from Charles River Laboratories. All animal use was in accordance with the Home Office guidelines. The B16.BL6 melanoma cell line was obtained from Prof. James Allison (MD Anderson Cancer Center, Houston, TX), the MCA205 fibrosarcoma cell line was provided by Prof. Guido Kroemer (Institut Gustave Roussy, Villejuif, South Paris, France), EL4 thymoma cells were provided by Prof. Henning Walczak (UCL Cancer Institute, London, United Kingdom), and authentication was performed by the ATCC using short tandem repeat DNA profiles. The cells were grown in RPMI-1640 supplemented with 10% FCS, l-glutamine, penicillin/streptomycin (Sigma-Aldrich).
TALEN construction and validation
Three pairs of TALEN targeting the PD-1 murine gene were produced by Cellectis using the solid phase assembly method (21). Their activity was evaluated in 5 × 106 EL4 cells, cotransfected with 10 μg of in vitro transcribed mRNA (mMESSAGE mMACHINE T7 Kit; Ambion) from each half TALEN (Supplementary Fig. S1). Three days after transfection, locus-specific PCRs were performed on genomic DNA (Supplementary Table S1), mutations were assessed by mismatches digestion (T7) and Miseq analysis (20).
Cell culture and adoptive transfer
Splenocytes and lymph nodes cells from pmel-1/SJL transgenic mice were cultured in complete RPMI with gp100 peptide (Pepceuticals) and rhIL-2 (Preprotech) 100 UI/mL for 24 hours before selection of CD8+ T cells using the Dynabeads FlowComp Mouse CD8 Kit. TRL were selected from 100 to 150 mm3 tumors using Dynabeads FlowComp Mouse Pan T (CD90.2) and maintained in complete RPMI with rhIL-2 (1,000 UI/mL). A total of 5 × 106 Pmel-1 CD8+ T cells and MCA205 TRL/180 μL of BTX medium T were mixed with 20 μg of IVT mRNA, before electroporation using an Agile Pulse BTX system (Harvard Apparatus). C57BL/6 mice were subcutaneously implanted with either 2.5 × 105 B16 cells or 4 ×105 MCA205 cells (day 0). On day 6 for the pmel-1 model and on day 4 for the MCA205 model, total body irradiation (TBI) was performed (5 Gy) and 1 × 106 pmel-1 T cells or 1 × 106 MCA205 TRL were adoptively transferred into tumor-bearing mice (n = 5/group), followed by intraperitoneal (i.p.) injection of rhIL-2 (1.2 × 106 IU) for 3 days. Mice were sacrificed when the tumors exceeded 15 mm in diameter. Mice were treated with the anti-mouse CD8a 2.43–depleting antibody (BioXcell) and the anti-MCH class II M5/114 (BioXcell), intraperitoneally on day 0, 2, 4, 6, and 8 after ACT (10 mg/kg).
Tumors were weighed, digested in Liberase D (Roche) and mechanically disaggregated through 70-μm filters. Lymph nodes cells and lymphocytes from tumor samples, enriched on a Ficoll gradient were incubated with murine antibodies: anti–CD4-V500 (BD Horizon), anti–CD8-BV650, anti–PD-1-PE-Cy7 (Biolegend), anti–CD45.1-APC-Cy7, anti–FoxP3-AF700, anti–IFNγ-FITC (restimulation with gp100-pulsed dendritic cells for pmel cells or PMA/ionomycin for TRL), anti–KI67-PerCP-eFluor 710, Fixable Viability Dye eFluor 450 (eBioscience), anti-FLICA (Vybrant FAM Poly Caspases Assay Kit; ThermoFisher), Annexin-V–PE Apoptosis Detection Kit II (BD Pharmingen), Cell Sorting Set-up Beads (Life Technologies), and anti-human Granzyme B-APC (Invitrogen) in a blocking solution: 5% mouse serum, 5% rat serum (Thermo Scientific), 2% FCS, and 2% anti-FcR 2.4G2 (BioXcell). Samples were permeabilized using a Fixation/Permeabilization kit (eBioscience), acquired on a LSRII Fortessa and analyzed with FlowJo software (Tree Star).
Western blot analysis
Proteins were separated and subjected to the following antibodies: anti-TALEN (1 mg/mL), anti-GAPDH (D16H11; Cell Signaling Technology; 1:2,000), HRP-coupled secondary antibody anti-Rabbit IgG1 HRP-linked (Cell Signaling Technology).
One-way ANOVA, two-way ANOVA, or t tests with P < 0.05 were performed using Prism 6.0 software (GraphPad). Multiple comparisons were corrected with the Bonferroni coefficient and Kaplan–Meier survival curves were compared with the log-rank test.
For additional information, see Supplementary Data Section.
Results and Discussion
TALEN-mediated PD-1 gene inactivation in melanoma reactive CD8+ pmel-1 T cells increases persistence of CD8PD-1Ex2 pmel-1 cells at the tumor site
Three pairs of TALENs targeting the murine PD-1 gene were tested in EL4 cells using an optimized mRNA–electroporation protocol. Only the pair targeting the exon 2 sequence (Fig. 1A) caused detectable mismatch-identified mutagenesis (up to 27%) in the PD-1 gene, correlating with loss of PD-1 expression in up to 15% of electroporated EL4 cells (Supplementary Fig. S1). To investigate whether PD-1 inactivation of primary tumor-reactive T cells provided superior antitumor activity against the poorly immunogenic mouse melanoma B16 model, we used CD8+ T-cell receptor transgenic (TCR Tg) cells specific to the melanoma differentiation antigen gp-100 (pmel-1, Supplementary Fig. S2A; ref. 22). In vitro activated pmel-1 T cells were electroporated with control GFP mRNA (CD8wt) or PD-1–targeting TALEN mRNA (CD8PD-1Ex2), delivering a high transfection efficiency of >85% as confirmed by GFP expression (Supplementary Fig. S2B). Western blot analysis confirmed transient TALEN expression one day (d1) after transfection (Fig. 1B) and Miseq analysis of the targeted sequence showed a high frequency of mutations (53% non-homologous end joining, NHEJ; Fig. 1C). Three days after transfection, PD-1–negative cells were enriched using magnetic beads (Supplementary Fig. S2C) before ACT of 1 × 106 CD8wt or CD8PD-1Ex2 pmel-1 cells and rhIL-2 into B16 tumor-bearing mice. Significantly, enhanced tumor control was consistently observed in mice receiving CD8PD-1Ex2 cells compared with those treated with CD8wt cells (Fig. 1D). Six days after transfer in vivo, a significant enrichment of tumor-infiltrating PD-1–negative pmel-1 cells was observed in mice treated with CD8PD-1Ex2 T cells (48.2%; Fig. 1E, top), which contributed to a 2-fold increase in the total number of tumor-infiltrating pmel-1 cells (Fig. 1E, bottom). In contrast, the absolute number of T cells in draining lymph nodes (DLN) was similar between the two groups, suggesting that accumulation of CD8PD-1Ex2 T cells at the tumor site was not due to differences in engraftment nor infiltration from the DLNs (Supplementary Fig. S2D). Whereas use of RNA and electroporation to deliver TALENs is compatible with future clinical translation, it does not allow incorporation of a reporter gene to distinguish PD-1–edited cells. Nevertheless, the rate of mutagenesis, investigated ex vivo 7 days after ACT demonstrates an effective PD-1 gene editing (70%) in the PD-1–negative fraction from the CD8PD-1Ex2 group (Supplementary Fig. S2E). To investigate the mechanisms underpinning enhanced tumor control by pmel-1 cells in the CD8PD-1Ex2 group, we characterized the proliferation and effector function of CD8wt and CD8PD-1Ex2 T cells in both PD-1+ and PD-1− tumor-infiltrating pmel-1 populations. Contrary to previous work suggesting that PD-1 signaling reduces effector activity at the tumor site (23), no differences were observed in KI67, granzyme B (GZB), or IFNγ expression between PD-1+ and PD-1− cells. When we compared the adjusted percentages (percentage of cells in PD-1+ and PD-1− subpopulations) of tumor-infiltrating KI67+, GZB+, and IFNγ+ cells, we observed that the TALEN-induced PD-1 gene inactivation did not impact the proliferative or functional characteristics of pmel-1 CD8+T cells in vivo (Supplementary Fig. S2F–S2H). Instead, we consistently observed the appearance of a PD-1− pmel-1 population in the CD8PD-1Ex2 group, which contributed to an increase in the total number of KI67-, GZB-, and IFNγ-expressing pmel-1 cells in tumors from mice receiving CD8PD-1Ex2 T cells (Fig. 1F and G). Together, these data strongly suggest that, in the context of ACT, PD-1 expression primarily controls number of tumor-reactive T cells at the tumor site rather than their proliferative or functional status.
PD-1 gene inactivation of polyclonal tumor-reactive lymphocytes promotes persistence of activated CD4+ and CD8+ at the tumor site
To define the therapeutic potential and functional impact of PD-1 gene inactivation in a more physiologically relevant T cell population, we performed PD-1 gene editing in polyclonal tumor-reactive lymphocytes (TRL) from C57BL/6SJL mice challenged with MCA205 cells (24). CD4+ and CD8+ T cells were isolated from the tumor (TILs) and tumor-draining lymph node (TDLN) via positive selection. After electroporation of the donor TRL, we observed a transfection efficiency >65% and we identified TALEN-induced PD-1 gene mutations by Miseq analysis in 26% of CD4+ T cells and almost 40% of CD8+ T cells (Supplementary Fig. S3A and S3B).
In parallel to the generation of the donor mock-transfected (TRLwt) and PD-1–edited TRL cells (TRLPD-1Ex2), we challenged a separate group of C57BL/6 mice with MCA205 tumors. Four days later, tumor-bearing mice were treated with TBI followed by ACT with TRLwt or TRLPD-1Ex2 cells and rhIL-2. Six days after transfer, analysis of PD-1 expression on TILs from mice receiving TRLwt or TRLPD-1Ex2 revealed a 15% to 20% average increase in PD-1− CD4+ and PD-1− CD8+ T cells (Fig. 2A and B). Consistent with our findings in the TCR Tg model, a significant accumulation of PD-1− cells was observed only at the tumor site (Fig. 2C; Supplementary Fig. S3C). Although PD-1 gene inactivation did not alter TRL proliferation (KI67) or function (GZB and IFNγ; Supplementary Fig. S3D–S3F), the absolute number of PD1− TRL–expressing KI67, GZB, and IFNγ was significantly higher in mice receiving TRLPD-1Ex2 (Fig. 2D–F).
Given that the increase in the number of PD-1− cells in mice receiving TRLPD-1Ex2 cells was not associated with increased proliferation, we evaluated whether this could be explained by a reduced rate of PD-1–induced cell death. Using Annexin-V and a fluorescent caspase substrate kit (FLICA), we observed an overall reduction in the frequency of intratumoral apoptotic cells in PD-1− compared with PD-1+ CD4+ and CD8+ T cells (Fig. 3A–D). Whereas we observed no difference in the frequency of apoptotic cells between mice treated with TRLwt and TRLPD-1Ex2, a significant increase in the absolute number of Annexin-V− and FLICA− TRLPD-1Ex2cells per mg of tumor was reported (Fig. 3E and F). The data suggest that instead of increasing T-cell reactivity against tumors, TRLPD-1Ex2 cells are rendered resistant to PD-1–mediated cell death at the tumor site.
PD-1 gene inactivation drives CD4- and CD8-dependent rejection of established tumors and long-term memory
PD-1 gene inactivation significantly increased the activity of our ACT protocol (P = 0.0052), and antitumor activity was dependent on both CD8+ and CD4+ T cells as administration of depleting anti-CD8 or MHC-II–blocking antibodies ablated antitumor responses (Fig. 4A and B). No evidence of tumor recurrence was observed after MCA205 rechallenge on day 50 in mice that had previously rejected tumors (data not shown). The development of long-term memory, as demonstrated by protection against a subsequent tumor challenge, is an important feature of successful T-cell–adoptive immunotherapy (25). Our data show high levels of KI67, consistent with high proliferative capacity for both TRL and TRLPD-1Ex2 cells, which also displayed a T-bethi CD62LlowCD44hi phenotype consistent with effector memory T cells (Supplementary Fig. S4A and S4B; ref. 26). Finally, ex vivo stimulation of circulating T cells from surviving mice (40 days after tumor rejection) demonstrated high levels of IFNγ production in both TRL and TRLPD-1Ex2 groups arguing against terminal differentiation (Supplementary Fig. S4C; ref. 27).
In summary, our data demonstrate the feasibility and potential clinical relevance of gene-editing approaches, conferring superior in vivo activity in the context of adoptive cell therapy protocols. Of relevance, we observed no evidence of toxicity (weight and physiologic alterations) after ACT. To the best of our knowledge, this is the first proof-of-concept study illustrating enhancement and persistence of antitumor responses using targeted genome editing of primary tumor reactive T cells. Our data indicate that the primary mechanism by which PD-1 gene inactivation affects tumor-reactive T cells is by regulating their ability to survive rather than increasing their activity on a per cell basis, which aligns with the original description of the role of PD-1 signaling in T-cell apoptosis (8). Although we investigated PD-1 as the initial proof-of-concept target, the technology and approach described potentially allow the permanent disruption of other inhibitory checkpoints (one or more), considerably advancing the design of the next generation of cancer immunotherapies.
Disclosure of Potential Conflicts of Interest
M. Pule is a CSO at Autolus, reports receiving a commercial research grant from Cellectis, has received speakers bureau honoraria from Amgen and Roche, and has ownership interest (including patents) in Autolus. No potential conflicts of interest were disclosed by the other authors.
Conception and design: L. Menger, L. Poirot, M. Pule, K.S. Peggs, S.A. Quezada
Development of methodology: L. Menger, A. Sledzinska, F. Arce Vargas, L. Poirot, M. Pule
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Menger, A. Sledzinska, K. Bergerhoff, K.S. Peggs
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Menger, J. Herrero, K.S. Peggs
Writing, review, and/or revision of the manuscript: L. Menger, A. Sledzinska, L. Poirot, M. Pule, J. Herrero, K.S. Peggs, S.A. Quezada
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Menger, F. Arce Vargas, J. Smith
Study supervision: K.S. Peggs, S.A. Quezada
S.A. Quezada is funded by a CRUK Career Development Fellowship and a Cancer Research Institute Investigator Award. K.S. Peggs receives funding from CRUK, Leukemia and Lymphoma Research. This work was funded in part by an EU FP7 grant and undertaken at UCL Hospitals/UCL with support from the Department of Health and CRUK funding schemes for National Institute for Health Research Biomedical Research Centers and Experimental Cancer Medicine Centers. The research leading to these results has received funding from the European Union Seventh Framework Programme under grant amendment no 602239 ATECT.