Although adoptive transfer of T cells genetically engineered to express chimeric antigen receptor (CAR) or T-cell receptor (TCR) has been actively developed and applied into clinic recently, further improvement of these modalities is highly demanded, especially in terms of its efficacy. Because we previously revealed the profound enhancement of antitumor effects of CAR T cells by concomitant expression of IL7 and CCL19, this study further explored a potential of IL7/CCL19 production technology to augment antitumor effects of TCR T cells. IL7/CCL19-producing P1A tumor antigen-specific TCR T cells (7 × 19 P1A T cells) demonstrated significantly improved antitumor effects, compared with those without IL7/CCL19 production, and generated long-term memory responses. The antitumor effects of 7×19 P1A T cells were further upregulated by combination with anti–PD-1 antibody, in which blockade of PD-1 signal in both 7×19 P1A T cells and endogenous T cells plays an important role. Taken together, our study demonstrated that concomitant production of IL7 and CCL19 by genetically engineered tumor-reactive T cells could synergize with PD-1 blockade therapy to generate potent and long-lasting antitumor immunity.

Gene-modified immune cell therapy has been gaining much attention as a promising modality for treatment of advanced cancers. In particular, adoptive transfer of gene-engineered T cells to express chimeric antigen receptor (CAR) has demonstrated profound clinical benefits in hematologic cancers including leukemia, lymphoma, and multiple myeloma, and thus obtained an approval by authorized agencies for clinical use in some cancers (1–7). Another major approach is a genetic engineering of T cells to express T-cell receptor (TCR) specific to tumor antigens (8, 9). Although advantage of CAR T cells includes a recognition and elimination of tumor cells independent of HLA restriction and a delivery of both CD3 and costimulatory signals by a single CAR receptor, a potential drawback of CAR is a requirement of target molecules to be selectively expressed on cell surface of tumor cells, but not normal cells. The molecules which completely meets such requirement are very limited. On the other hand, TCR T cells can recognize tumor antigen regardless of its localization, that is, cell surface, cytoplasm, or intranuclear, but require presentation of epitope peptide in an HLA-restricted manner. As CAR T- and TCR T-cell therapies possess distinct functional features, technical improvement of both modalities are highly demanded.

Because the clinical development of gene-modified T-cell therapies progressed, their vulnerabilities also became apparent. In CAR T-cell therapy, although short-term clinical responses in hematologic malignancies are remarkably high, tumor recurrence has been observed within 1 year in 30% to 40% of responding cases (5). As potential mechanisms of recurrence, loss of CAR target molecule on tumor cells and dysfunctionality of CAR T cells have been reported (10, 11). In addition, clinical efficacy of CAR T-cell therapy against solid tumors remains much weaker than that in hematologic malignancies, and needs to be further improved (12). Regarding TCR T-cell therapy, tumor types in which clinical efficacy was confirmed remain limited, such as melanoma and synovial sarcoma (13–15). To overcome these issues and promote an importance in cancer treatment, various strategies to improve gene-modified T-cell therapy have been proposed by various groups. For instance, modification of intracellular signaling domain of CAR resulted in potent antitumor efficacy by preventing dysfunction of CAR T cells (16, 17). To avoid exhaustion of CAR T cells, genome-editing technology to knockdown programmed cell death protein 1 (PD-1, encoded by Pdcd1) or a combination with anti–PD-1 antibody (Ab) treatment has been proposed (18–20). CAR T cells which were modified to concomitantly express immune-stimulatory molecules have been developed as fourth generation of CAR technology (21, 22).

In line with these efforts, our group also developed novel CAR T-cell technology to induce concomitant expression of IL7 and CCL19 in addition to CAR (22). We selected IL7 and CCL19, because this particular combination is known to play a crucial role in the development and maintenance of T-cell zone in secondary lymphoid organs (23), and thus hypothesized to accelerate an accumulation of dendritic cells (DCs) and T cells in the tumor microenvironment. In addition, intratumor administration of IL7-transduced DCs or CCL19 protein induced therapeutic effects in animal tumor models (24, 25), suggesting an important role of these factors in antitumor immune responses. As expected, treatment with IL7/CCL19-producing CAR T cells induced massive infiltration of DCs and T cells in tumor tissues, consisting of both transferred CAR T cells and endogenous host T cells, and demonstrated a potent therapeutic efficacy in various solid tumor models with an induction of long-term, tumor-specific immunologic memory responses (22).

We then hypothesized that genetic engineering of TCR T cells to induce concomitant production of IL7 and CCL19 could also reinforce their antitumor efficacy, similarly to CAR T cells. In this study, we addressed this hypothesis and found that tumor regression and long-term mouse survival can be achieved by the treatment with IL7/CCL19-producing TCR T cells. We also revealed that antitumor effects of IL7/CCL19-producing CAR or TCR T cells were significantly enhanced by combination with anti–PD-1 mAb.

Mice and cell lines

Male and female DBA/2 mice (6 to 8 weeks old) were purchased from SLC. P1A-specific TCR-transgenic mice were originally generated and kindly provided by Dr. Yang Liu (26) and backcrossed with DBA/2 mice at least 10 generations in our animal facility. All mice were maintained under specific pathogen-free conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee of Yamaguchi University. P815 mastocytoma was purchased from ATCC and maintained with complete RPMI1640 medium, consisting of RPMI1640 culture medium, 10% heat-inactivated FBS, 1% penicillin–streptomycin, 25 mmol/L HEPES, and 50 mmol/L 2-mercaptoethanol. P815 expressing human CD20 were established as described previously (22) and maintained with DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Both P815 and P815 expressing human CD20 were used for experiments within 10 passages from the original cell line, and confirmed as Mycoplasma-free using e-Myco Mycoplasma PCR Detection Kit (iNtRON Biotechnology).

Plasmids

The sequence of murine IL7, CCL19, and EGFP genes was connected by intercalating F2A self-cleavable peptide sequence (27, 28), and cloned into retroviral vector MSGV1 (22, 29, 30). As a control, EGFP gene alone was also cloned into MSGV1. The construct to express antihuman CD20 CAR concomitantly with murine IL7 and CCL19 was generated as reported previously (22).

Gene modification of T cells

GP2 retroviral packaging cells were transfected with the plasmids by Lipofectamin 3000 reagent (Thermo Fisher Scientific). Forty-eight hours later, the culture supernatants containing retroviral vectors were harvested and used for gene transduction. Spleen cells harvested from P1A-specific TCR-transgenic mouse were incubated in the presence of P1A peptide (LPYLGWLVF) and recombinant human IL2. After 48 hours of incubation, the cultured cells were harvested and enriched to T cells by negative magnetic cell sorting using mouse Pan T-cell Isolation Kit (Miltenyi Biotec). The isolated T cells were exposed to retroviral vectors in the presence of RetroNectin (Takara Bio) and IL2 for 2 hours, followed by incubation for 6 hours. Then, gene-transduced cells were harvested, washed, and incubated for an additional 2 days, prior to use for experiments. In some experiments, the gene-transduced cells were purified for EGFP-positive cells using FACS cell sorting. To confirm the gene transduction, the culture supernatants were harvested and measured for the concentrations of IL7 and CCL19 by ELISA. Production of T cells expressing antihuman CD20 CAR concomitantly with murine IL7 and CCL19 were conducted as described previously (22).

For gene knockdown of Pdcd1, spleen cells of P1A-specific TCR-transgenic mouse were stimulated with P1A peptide and enriched to T cells by magnetic cell sorting as described previously, and then suspended with Buffer R (Thermo Fisher Scientific) in the presence of Cas9-RNP complex consisting of TrueCut Cas9 Protein v2 (Thermo Fisher Scientific) and gene-specific guide RNA. The Cas9–RNP complex were electroporated into the T cells using Neon Transfection System (Invitrogen). Immediately after electroporation, T cells were exposed to retroviral gene transduction as described above. For targeting mouse Pdcd1, specific guide RNA was designed as previously reported (31). As a control, guide RNA targeting mouse ROSA26 (Thermo Fisher Scientific) was used. The guide RNA sequences targeting mouse Pdcd1 and mouse ROSA26 genes were 5′-UCUGGGCAUGUGGGUCCGGC-3′ and 5′-CUCCAGUCUUUCUAGAAGAU-3′, respectively.

Flow cytometry

Reagents and Abs used for flow cytometric analysis were as follows: Zombie Yellow Fixable Viability Kit (BioLegend), phycoerythrin (PE)-, allophycocyanin (APC)-Cy7-, or Brilliant Violet (BV) 421-conjugated anti-CD3ϵ Ab (BioLegend or BD Biosciences), BV421- or APC-conjugated anti-CD8a Ab (BioLegend), APC-Cy7-conjugated anti-CD4 Ab (BD Biosciences), PerCP-Cy5.5-conjugated anti-CD11b Ab (BioLegend), BV421- or APC-conjugated anti-CD11c Ab (BD Biosciences or eBioscience), PE-conjugated anti–Gr-1 Ab (eBioscience), PE-Cy7- or biotin-conjugated anti-CD117 (c-kit) Ab (BioLegend), PerCP-Cy5.5-conjugated anti-CD44 Ab (BioLegend), APC-conjugated anti-CD62 L Ab (eBioscience), PE-conjugated anti–PD-1 Ab (eBioscience), PE-or biotin-conjugated anti-Vα8.3 Ab (eBioscience), PE-conjugated anti-IFNγ Ab (eBioscience), PerCP-Cy5.5-conjugated anti-IL2 Ab (BioLegend), BV421-conjugated anti-TNFα Ab (BioLegend), and BV421-conjugated anti-human CD20 Ab (BD Biosciences). Anti-CD16/CD32 Ab was purified from the culture supernatant from the hybridoma (clone 2.4G2) in our laboratory. We took advantage of anti-CD16/CD32 Ab for blocking nonspecific binding of Abs via Fcγ receptors. For intracellular cytokine staining, cells were fixed with Fixation Buffer (BioLegend), followed by washing and permeabilization with Intracellular Staining Permeabilization Wash Buffer (BioLegend). Flow cytometric data were acquired by EC800 (Sony), BD LSRFortessa X-20 cell analyzer (BD Biosciences) or CytoFLEX (Beckman Coulter), and analyzed using FlowJo software (FlowJo, LLC). Flow cytometric cell sorting of EGFP-positive cells was conducted by SH800 (Sony).

In vivo models to assess antitumor effects of gene-modified T cells

In the model using T cells from P1A-specific TCR-transgenic mouse (hereafter referred to as P1A T cells), DBA/2 mice were inoculated subcutaneously (s.c.) in the right flank with 5×105 P815 tumor cells on day 0, and then exposed to sublethal irradiation (3 or 5 Gy) on day 6. On day 7, the mice were injected intravenously (i.v.) with 1×106 gene-modified P1A T cells. Tumor size was measured twice a week by digital caliper. The tumor volume was calculated as the following: (major axis of tumor) × (minor axis of tumor)2/2. The survivals of the mice were also assessed. In some experiments, the mice inoculated with P815 tumor cells were treated with gene-modified P1A T cells followed by intraperitoneal (i.p.) injection of anti–PD-1 mAb at 150 μg/mouse once a week for a total of six times starting on day 10. In other experiments, the mice inoculated with P815 tumor cells were injected with gene-modified P1A T cells, which were pretreated with either Pdcd1 gene knockdown or ROSA26 gene knockdown, with or without i.p. injections of anti–PD-1 mAb.

To analyze tumor-infiltrating immune cells, DBA/2 mice were inoculated s.c. in the right flank with 5 × 105 P815 tumor cells on day 0, and then injected i.v. with 1.5 × 106 gene-modified P1A T cells, which had been enriched to EGFP-positive cells by flow cytometric cell sorting on day 7. On day 12, tumor tissues were resected, digested, and homogenized as described previously (32). Single-cell suspensions of tumor tissue were then counted and analyzed by flow cytometry.

In the model using CAR T cells, DBA/2 mice were inoculated s.c. in the right flank with 5×105 human CD20-expessing P815 tumor cells on day 0, and then injected i.p. with 100 mg/kg cyclophosphamide (CPA) on day 11. On day 14, the mice were injected i.v. with 2.5×105 T cells, which were genetically modified to express antihuman CD20 CAR concomitantly with murine IL7 and CCL19, followed by i.p. injections with anti–PD-1 mAb or control hamster IgG at 100 μg/mouse every 4 or 5 days for a total of five times starting on day 17.

Persistence and functions of memory T cells

The mice that achieved complete tumor regression by the treatment with gene-modified P1A T cells were euthanized on day 119. The spleen cells were harvested from the euthanized mice, counted, and analyzed by flow cytometry to determine the existing EGFP-positive gene-modified P1A T cells. To assess antitumor responses, T cells were isolated from the spleen cells by magnetic cell sorting and incubated with mitomycin C-treated P815 tumor cells. After 3 and 5 days, culture supernatants were harvested and analyzed to measure IFNγ concentration by ELISA. In addition, the number of gene-modified P1A T cells expressing EGFP and CD8 were analyzed by flow cytometry.

In some experiments, spleen cells from the mice that achieved complete tumor regression by the treatment with 7×19 P1A T cells alone or in combination with anti–PD-1 mAb were harvested on day 67 and analyzed by flow cytometry to determine the population and memory phenotype of 7×19 P1A T cells and endogenous T cells. In addition, these T cells were isolated from the spleen cells by magnetic cell sorting and incubated with mitomycin C-treated P815 tumor cells. For intracellular staining of IFNγ, Brefeldin A (BioLegend) was added 18 hours after the initiation of culture, and the cells were harvested after an additional 6 hours of incubation. For analysis of memory T-cell expansion, these cells were incubated for 3 days, and analyzed by flow cytometry to measure the number of 7×19 P1A T cells and endogenous T cells.

Statistical analysis

Statistical analyses of in vitro experiments were examined by two-sided Student t tests. Log-rank test was applied for the analyses of mouse survival. P < 0.05 was considered as statistically significant.

Generation of IL7/CCL19-producing P1A-specific T cells

We recently reported that CAR T cells, which simultaneously produce IL7 and CCL19 demonstrate potent therapeutic effects against solid tumors (22). To study whether similar gene modification can also enhance the functions of tumor-specific TCR T cells, we employed a murine model of P815 mastocytoma expressing P1A tumor antigen (33) in the context of H-2Ld, and P1A T cells derived from P1A-specific TCR-transgenic mice (26). We constructed a retroviral vector encoding IL7, CCL19, and EGFP genes, which were intercalated with F2A self-cleavable peptide sequence (Fig. 1A, right; ref. 22). We also generated a retroviral vector encoding EGFP alone as a control (Fig. 1A, left). After transduction of these retroviral vectors, activated P1A T cells expressed IL7, CCL19, and EGFP (hereafter referred to as 7×19 P1A T cells) or EGFP alone (hereafter referred as Conv. P1A T cells), where expression levels of EGFP in these cells were approximately 70% to 80% (Fig. 1B). Expression intensity of EGFP on 7×19 P1A T cells was lower than that on Conv. P1A T cells, probably due to a longer size of coding region and location of EGFP after IL7/CCL19 in the 7×19 vector plasmid. We confirmed significant production of IL7 and CCL19 secretion from 7×19 P1A T cells, but not Conv. P1A T cells, by ELISA (Fig. 1C).

Figure 1.

Gene modification of P1A T cells. A, Representations of control vector encoding EGFP gene (for Conv. P1A T cells) and 7×19 vector encoding IL7, CCL19, and EGFP genes connected by F2A peptide sequence (for 7×19 P1A T cells) are shown. B, CD8 and EGFP expressions of the Conv. P1A T cells and 7×19 P1A T cells were analyzed by flow cytometry. C, Concentrations of IL7 and CCL19 in the culture supernatants 2 days after gene transduction were analyzed by ELISA. Data are shown as mean and SD of triplicate wells. Similar results were obtained by five independent experiments. N.D.; not detected (***, P < 0.001).

Figure 1.

Gene modification of P1A T cells. A, Representations of control vector encoding EGFP gene (for Conv. P1A T cells) and 7×19 vector encoding IL7, CCL19, and EGFP genes connected by F2A peptide sequence (for 7×19 P1A T cells) are shown. B, CD8 and EGFP expressions of the Conv. P1A T cells and 7×19 P1A T cells were analyzed by flow cytometry. C, Concentrations of IL7 and CCL19 in the culture supernatants 2 days after gene transduction were analyzed by ELISA. Data are shown as mean and SD of triplicate wells. Similar results were obtained by five independent experiments. N.D.; not detected (***, P < 0.001).

Close modal

Enhanced antitumor effect of 7×19 P1A T cells with tumor regression and prolonged mouse survival

To evaluate the effects of IL7 and CCL19 production in antitumor potential of P1A T cells in vivo, DBA/2 mice inoculated s.c. with P815 tumor cells were treated with preconditioning by sublethal irradiation followed by i.v. transfer of either Conv. P1A T cells or 7×19 P1A T cells, or left untreated. Compared with the mice treated with preconditioning alone, those treated with preconditioning plus Conv. P1A T-cell injection showed no significant survival improvement (Fig. 2A) and hardly any inhibition regarding tumor growth (Fig. 2B, left top and middle). In sharp contrast, the mice treated with preconditioning with 7×19 P1A T-cell injection demonstrated significant prolongation of survival (Fig. 2A) along with remarkable inhibition of tumor growth. This resulted in a complete tumor regression in 50% of mice (Fig. 2B, left bottom). These findings clearly indicated that genetic modification of tumor Ag-specific P1A T cells to simultaneously produce IL7 and CCL19 significantly enhanced an antitumor therapeutic activity, even under the experimental condition in which Conv. P1A T cells induced almost no antitumor effects.

Figure 2.

Antitumor activity of 7×19 P1A T cells in combination with or without anti–PD-1 mAb. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0, and then exposed to sublethal irradiation on day 6 as preconditioning. On day 7, the mice were injected i.v. with either Conv. P1A T cells or 7×19 P1A T cells, or left untreated. In some groups, anti–PD-1 mAb was injected i.p. once a week for a total of six times starting on day 10. Survival (A) and tumor volume (B) of the mice were assessed. Data are pooled from five independent experiments. The number of mice that achieved complete tumor regression on day 100 out of the total mouse number is indicated in B. Symbols indicate the following: preconditioning only (open square), preconditioning plus Conv. P1A T cells (open triangle), preconditioning plus 7×19 P1A T cells (open circle), preconditioning plus anti–PD-1 mAb (solid square), preconditioning plus Conv. P1A T cells in combination with anti–PD-1 mAb (solid triangle), preconditioning plus 7×19 P1A T cells in combination with anti–PD-1 mAb (solid circle). P values of log-rank tests are as follows: open square vs. solid square; P = 0.8213, open square vs. open triangle; P = 0.1538, open triangle vs. solid triangle; P = 0.7284, open triangle vs. open circle; P = 0.0005, solid triangle vs. solid circle; P = 0.0002, open circle vs. solid circle; P = 0.0253.

Figure 2.

Antitumor activity of 7×19 P1A T cells in combination with or without anti–PD-1 mAb. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0, and then exposed to sublethal irradiation on day 6 as preconditioning. On day 7, the mice were injected i.v. with either Conv. P1A T cells or 7×19 P1A T cells, or left untreated. In some groups, anti–PD-1 mAb was injected i.p. once a week for a total of six times starting on day 10. Survival (A) and tumor volume (B) of the mice were assessed. Data are pooled from five independent experiments. The number of mice that achieved complete tumor regression on day 100 out of the total mouse number is indicated in B. Symbols indicate the following: preconditioning only (open square), preconditioning plus Conv. P1A T cells (open triangle), preconditioning plus 7×19 P1A T cells (open circle), preconditioning plus anti–PD-1 mAb (solid square), preconditioning plus Conv. P1A T cells in combination with anti–PD-1 mAb (solid triangle), preconditioning plus 7×19 P1A T cells in combination with anti–PD-1 mAb (solid circle). P values of log-rank tests are as follows: open square vs. solid square; P = 0.8213, open square vs. open triangle; P = 0.1538, open triangle vs. solid triangle; P = 0.7284, open triangle vs. open circle; P = 0.0005, solid triangle vs. solid circle; P = 0.0002, open circle vs. solid circle; P = 0.0253.

Close modal

Synergistic antitumor effects induced by 7×19 P1A T cells and anti–PD-1 mAb treatment

Although 7×19 P1A T cells significantly prolonged the survival of tumor-inoculated mice, half of the mice experienced tumor progression and eventual death until 100 days. To further improve antitumor efficacy of 7×19 P1A T cells, we investigated combining with anti–PD-1 mAb treatment to unleash an inhibitory signal by PD-1. The combination of anti–PD-1 mAb with preconditioning alone or preconditioning plus Conv. P1A T cells showed no significant improvement of mouse survival compared with those without anti–PD-1 mAb (Fig. 2A and B). On the other hand, the mice treated with anti–PD-1 mAb combined with preconditioning plus 7×19 P1A T cells showed significantly improved survival rates compared with 7×19 P1A T cells alone and induced complete tumor regression in more than 80% of mice (Fig. 2A and B). These results revealed that resistance of preestablished P815 tumor to endogenous T cells as well as Conv. P1A T cells was not overcome by PD-1 signal blockade alone, whereas production of IL7 and CCL19 by P1A T cells could visualize the effects of anti–PD-1 mAb. It was also found that IL7/CCL19-producing T cells without specificity to P1A induced neither prolongation of mouse survival nor inhibition of tumor growth even when combined with a concurrent injection of anti–PD-1 mAb (Supplementary Fig. S1), suggesting that a specificity of T cells against a tumor is an essential factor to generate antitumor efficacy of 7×19 TCR T cells plus anti–PD-1 mAb. On the basis of our previous findings that treatment with CAR T cells expressing IL7 and CCL19 induce massive infiltration of endogenous T cells in the tumor microenvironment (22), a potential mechanism of the synergistic antitumor effects by anti–PD-1 mAb could be explained by a dual blockade of PD-1 signal in 7×19 P1A T cells and infiltrating endogenous T cells.

Inhibitory effects of PD-1 signal in 7×19 P1A T cells and endogenous T cells

To explore whether 7×19 P1A T cells or endogenous tumor-infiltrating T cells from recipient DBA/2 mice play a role in the combination with anti–PD-1 mAb, Pdcd1 gene knockdown 7×19 P1A T cells were prepared to assess their antitumor effects in the presence or absence of anti–PD-1 mAb. Knockdown of the Pdcd1 gene was conducted by CRISPR/Cas9 genome editing, which successfully decreased PD-1 expression to 2.8% (Supplementary Fig. S2). First, without anti–PD-1 mAb treatment, mice injected with Pdcd1 gene knockdown 7×19 P1A T cells showed a significantly prolonged survival and a persistent tumor growth inhibition, compared with those injected with control ROSA26 gene knockdown 7×19 P1A T cells (Fig. 3A and B). This result indicated that PD-1 signal in 7×19 P1A T cells could deliver inhibitory effects in the antitumor responses. Although antitumor effects of control ROSA26 gene knockdown 7×19 P1A T cells were less than those of 7×19 P1A T cells in Fig. 2, this was probably due to cellular damages caused by electroporation. Second, mice treated with Pdcd1 gene knockdown 7×19 P1A T cells together with anti–PD-1 mAb demonstrated further prolonged survival and increased rate of complete tumor regression, compared with those with Pdcd1 gene knockdown 7×19 P1A T cells without anti–PD-1 mAb (Fig. 3A and B). This finding indicated that tumor-infiltrating endogenous T cells are also potential targets for anti–PD-1 mAb. Together, our findings suggested that both 7×19 P1A T cells and endogenous T cells are responsible for the enhanced antitumor efficacy induced by combination with anti–PD-1 mAb.

Figure 3.

Antitumor activity of Pdcd1 gene knockdown 7×19 P1A T cells together with anti–PD-1 mAb. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0, and then exposed to sublethal irradiation on day 6 as preconditioning. On day 7, the mice were injected i.v. with either ROSA26 gene knockdown 7×19 P1A T cells or Pdcd1 gene knockdown 7×19 P1A T cells, or left untreated. In some groups, anti–PD-1 mAb was injected i.p. once a week for a total of six times starting on day 10. Survival (A) and tumor volume (B) of the mice are shown. The number of mice that achieved complete tumor regression on day 65 out of the total mouse number is indicated in B. Symbols indicate the following: preconditioning only (cross), preconditioning plus ROSA26 gene knockdown 7×19 P1A T cells (open square), preconditioning plus Pdcd1 gene knockdown 7×19 P1A T cells (open diamond), preconditioning plus Pdcd1 gene knockdown 7×19 P1A T cells in combination with anti–PD-1 mAb (solid diamond). P values of log-rank tests indicate the following: cross vs. open square; P = 0.001, open square vs. open diamond; P = 0.0431, open diamond vs. solid diamond; P = 0.0402.

Figure 3.

Antitumor activity of Pdcd1 gene knockdown 7×19 P1A T cells together with anti–PD-1 mAb. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0, and then exposed to sublethal irradiation on day 6 as preconditioning. On day 7, the mice were injected i.v. with either ROSA26 gene knockdown 7×19 P1A T cells or Pdcd1 gene knockdown 7×19 P1A T cells, or left untreated. In some groups, anti–PD-1 mAb was injected i.p. once a week for a total of six times starting on day 10. Survival (A) and tumor volume (B) of the mice are shown. The number of mice that achieved complete tumor regression on day 65 out of the total mouse number is indicated in B. Symbols indicate the following: preconditioning only (cross), preconditioning plus ROSA26 gene knockdown 7×19 P1A T cells (open square), preconditioning plus Pdcd1 gene knockdown 7×19 P1A T cells (open diamond), preconditioning plus Pdcd1 gene knockdown 7×19 P1A T cells in combination with anti–PD-1 mAb (solid diamond). P values of log-rank tests indicate the following: cross vs. open square; P = 0.001, open square vs. open diamond; P = 0.0431, open diamond vs. solid diamond; P = 0.0402.

Close modal

To further confirm the importance of endogenous T cells in antitumor effects of 7×19 P1A T cells, phenotypes of tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. P815-bearing mice were injected with either Conv. or 7×19 P1A T cells, which had been enriched to more than 95% purity of EGFP-positive cells by FACS cell sorting before the injection. Tumor tissues were harvested to examine the cell number and the presence of DCs, endogenous T cells, and transferred P1A T cells. To separate nontumor immune cells from P815 tumor cells, we utilized c-kit staining, which is known to be high positive in P815 mastocytoma (34) but not on mature T cells and only weakly expressed on subsets of DCs (35, 36). First, we found that the percentage of the c-kit–negative subset in the mice injected with 7×19 P1A T cells was higher than those with Conv. P1A T cells (Fig. 4A). Among the c-kit–negative subset, the percentages of CD3-negative/CD11c-positive DCs, CD3-positive/EGFP-negative endogenous T cells, and CD3-positive/EGFP-positive transferred P1A T cells were increased in the mice injected with 7×19 P1A T cells compared with those with Conv. P1A T cells. From further analyses, it was found that the number of tumor-infiltrating DCs, especially CD8-positive DCs, CD4-positive and CD8-positive endogenous T cells, and CD8-positive P1A T cells were significantly increased in the mice treated with 7×19 P1A T cells, compared with those with Conv. P1A T cells (Fig. 4A–C). The numbers of P815 tumor cells identified as a c-kit-positive subset were decreased in the mice treated with 7×19 P1A T cells, although it did not reach statistical significance (Fig. 4D). These results underscored an important role of endogenous T cells and DCs in the treatment by 7×19 P1A T cells.

Figure 4.

Infiltration of DCs, endogenous T cells, and transferred P1A T cells in tumor tissues. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0 and injected i.v. with Conv. P1A T cells or 7×19 P1A T cells on day 7, which were purified as over 95% EGFP-positive cells prior to injection. Tumor tissues were harvested, prepared into single cell suspension, and examined to count cell number and by flow cytometric analysis on the expression of c-kit, EGFP, CD11c, CD3, CD4, and CD8 on day 12. A, Representative dot plots are shown. Percentages of c-kit–negative cells identified as nontumor subset are indicated (top left). Among c-kit–negative subsets, the percentages of CD3-negative/CD11c-positive DCs (top right), CD3-positive/EGFP-negative endogenous T cells, and CD3-positive/EGFP-positive transferred P1A T cells (middle left) are indicated. Percentages of CD8-positive cells in the subsets of DCs (middle right), CD4-positive or CD8-positive cells in the subsets of endogenous T cells (bottom left) and transferred P1A T cells (bottom right) are indicated. B and C, Cell numbers of each TIL subset (B) and CD8-positive DCs (C) per 1 × 105 P815 tumor cells are shown. D, Total cell number of c-kit positive P815 tumor cells in each tumor tissue is shown. Cell numbers of tumor cells and each immune cell subsets are shown as mean and SD (n = 5). Open bars and solid bars indicate TIL subsets in the tumor tissues of Conv. P1A T-cell–treated mice and 7×19 P1A T-cell–treated mice, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

Infiltration of DCs, endogenous T cells, and transferred P1A T cells in tumor tissues. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0 and injected i.v. with Conv. P1A T cells or 7×19 P1A T cells on day 7, which were purified as over 95% EGFP-positive cells prior to injection. Tumor tissues were harvested, prepared into single cell suspension, and examined to count cell number and by flow cytometric analysis on the expression of c-kit, EGFP, CD11c, CD3, CD4, and CD8 on day 12. A, Representative dot plots are shown. Percentages of c-kit–negative cells identified as nontumor subset are indicated (top left). Among c-kit–negative subsets, the percentages of CD3-negative/CD11c-positive DCs (top right), CD3-positive/EGFP-negative endogenous T cells, and CD3-positive/EGFP-positive transferred P1A T cells (middle left) are indicated. Percentages of CD8-positive cells in the subsets of DCs (middle right), CD4-positive or CD8-positive cells in the subsets of endogenous T cells (bottom left) and transferred P1A T cells (bottom right) are indicated. B and C, Cell numbers of each TIL subset (B) and CD8-positive DCs (C) per 1 × 105 P815 tumor cells are shown. D, Total cell number of c-kit positive P815 tumor cells in each tumor tissue is shown. Cell numbers of tumor cells and each immune cell subsets are shown as mean and SD (n = 5). Open bars and solid bars indicate TIL subsets in the tumor tissues of Conv. P1A T-cell–treated mice and 7×19 P1A T-cell–treated mice, respectively (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

We next explored the effects of anti–PD-1 mAb combination with P1A T-cell treatment on the phenotypes and functions of TILs. Administration of anti–PD-1 mAb indicated a tendency to increase the numbers of both CD3-positive/EGFP-positive transferred P1A T cells and CD3-positive/EGFP-negative endogenous T cells when combined with 7×19 P1A T cells, but not Conv. P1A T cells (Supplementary Fig. S3A). This observation was the same as the results described in Fig. 2, where the addition of anti–PD-1 mAb further improved the antitumor effects of 7×19 P1A T cells, but not Conv. P1A T cells. The numbers of DCs and tumor-associated macrophage (TAM) in tumor tissues also showed a tendency to increase by the addition of anti–PD-1 mAb when combined with 7×19 P1A T cells, but not Conv. P1A T cells, whereas mechanisms underlying this finding need further investigation. Interestingly, there were no significant differences in the intracellular expression of IFNγ, IL2, and TNFα among the treatment with Conv. P1A T cells or 7×19 P1A T cells with or without anti–PD-1 mAb, whereas the percentages of cytokine-producing cells were generally higher in P1A T cells compared with endogenous T cells (Supplementary Fig. S3B). These results suggested that 7×19 P1A T cells combined with anti–PD-1 mAb treatment could increase the quantity of tumor-reactive T cells in the tumor tissue rather than their effector functions, such as cytokine production and cytotoxic activity.

Long-term persistence of 7×19 P1A T cells with antitumor memory responses

One of the important features in cancer immunotherapy is a generation of long-term memory responses against tumors that could establish protective immunity to prevent tumor recurrence. Therefore, we next examined whether the treatment with 7×19 P1A T cells efficiently induces antitumor memory responses in our model. The spleen cells were extracted from mice that survived more than 100 days after tumor inoculation by the treatment with either Conv. P1A T cells or 7×19 P1A T cells and examined the persistence of EGFP-positive P1A T cells by flow cytometry. The presence of EGFP-positive cells including both CD8-positive and -negative subsets was clearly detected in the spleen cells from the mice treated with 7×19 P1A T cells, but not Conv. P1A T cells (Fig. 5A and B). When these spleen cells were co-cultured with P815 tumor cells, apparent expansion of EGFP-positive/CD8-positive cells (Fig. 5C) and massive production of IFNγ (Fig. 5D) was detected in the spleen cells from 7×19 P1A T-cell–treated mice. These findings revealed that 7×19 P1A T cells persisted long term in the tumor-rejected mice and fulfilled memory functions in response to the P815 tumor.

Figure 5.

Generation of long-term memory responses by 7×19 P1A T-cell therapy. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0, exposed to sublethal irradiation as preconditioning on day 6, and then injected i.v. with Conv. or 7×19 P1A T cells on day 7. The mice that survived for 119 days with a complete tumor regression were euthanized to harvest spleen cells for analyses on the persistence and function of the P1A T cells. A and B, Expression of CD8 and EGFP on the spleen cells were determined by flow cytometry. Representative dot plots are shown in A. Percentages of EGFP-positive/CD8-negative and EGFP-positive/CD8-positive subsets are assessed, and their averages are shown in B. C and D, T cells were isolated from the spleen cells by magnetic cell sorting and co-cultured with mitomycin C-treated P815 tumor cells. The numbers of EGFP-positive/CD8-positive cells assessed by flow cytometry on day 0, 3, and 5 are shown in C. The concentrations of IFNγ in the culture supernatants assessed by ELISA on day 3 and 5 are shown in D. Open bars and solid bars indicate T cells from Conv. P1A T-cell–treated mice (n = 2) and 7×19 P1A T-cell–treated mice (n = 3), respectively. Data are shown as mean of samples with indication of individual value as an open circle.

Figure 5.

Generation of long-term memory responses by 7×19 P1A T-cell therapy. DBA/2 mice were inoculated s.c. with P815 tumor cells on day 0, exposed to sublethal irradiation as preconditioning on day 6, and then injected i.v. with Conv. or 7×19 P1A T cells on day 7. The mice that survived for 119 days with a complete tumor regression were euthanized to harvest spleen cells for analyses on the persistence and function of the P1A T cells. A and B, Expression of CD8 and EGFP on the spleen cells were determined by flow cytometry. Representative dot plots are shown in A. Percentages of EGFP-positive/CD8-negative and EGFP-positive/CD8-positive subsets are assessed, and their averages are shown in B. C and D, T cells were isolated from the spleen cells by magnetic cell sorting and co-cultured with mitomycin C-treated P815 tumor cells. The numbers of EGFP-positive/CD8-positive cells assessed by flow cytometry on day 0, 3, and 5 are shown in C. The concentrations of IFNγ in the culture supernatants assessed by ELISA on day 3 and 5 are shown in D. Open bars and solid bars indicate T cells from Conv. P1A T-cell–treated mice (n = 2) and 7×19 P1A T-cell–treated mice (n = 3), respectively. Data are shown as mean of samples with indication of individual value as an open circle.

Close modal

Next, to explore the effects of the anti–PD-1 mAb combination on the induction of antitumor memory responses, we analyzed the T-cell phenotypes and functions in the spleen cells extracted from the mice that survived 67 days after tumor inoculation by the injection of 7×19 P1A T cells with or without anti–PD-1 mAb. Although the addition of anti–PD-1 mAb induced no significant differences in percentages and memory phenotypes of residual 7×19 P1A T cells (Supplementary Fig. S4A and S4B, left), percentages of central and effector memory subsets in endogenous T cells were significantly increased in the mice treated with 7×19 P1A T cells in combination anti–PD-1 mAb, compared with those treated with 7×19 P1A T cells only (Supplementary Fig. S4B, right). In addition, when these cells were re-stimulated by tumor cells, endogenous T cells, but not residual 7×19 P1A T cells, demonstrated an increased proliferation (Supplementary Fig. S4C) and IFNγ production (Supplementary Fig. S4D) by the combination with anti–PD-1 mAb. These results suggested that the combined treatment of anti–PD-1 mAb with 7×19 P1A T cells could enhance memory generation predominantly in endogenous tumor-reactive T cells.

To further evaluate memory functions in antitumor immune responses in our models, the mice that survived more than 80 days by the treatment with 7×19 P1A T cells or 7×19 P1A T cells in combination with anti–PD-1 mAb were rechallenged with s.c. inoculation of P815 tumor. All mice in both groups achieved complete tumor rejection, whereas control naïve DBA/2 mice showed a massive tumor growth after the same tumor inoculation (Supplementary Fig. S5). This result indicated that the tumor-rejected mice by the treatment with 7×19 P1A T cells can develop long-term antitumor protective immunity regardless of the combination with anti–PD-1 mAb treatment, probably due to generation of memory functions in 7×19 P1A T cells.

Synergistic antitumor effects induced by IL7/CCL19-producing CAR T cells and anti–PD-1 mAb treatment

As the combination of 7×19 P1A T cells with anti–PD-1 mAb showed a synergy in antitumor effects, we further investigated whether this combination is also effective when IL7/CCL19-producing CAR T cells (hereafter referred to as 7×19 CAR T cells) are used. To this end, 7×19 CAR T cells specific to human CD20 (hCD20) as a target molecule were generated as reported previously (22) and injected i.v. into the mice that were previously inoculated s.c. with P815 tumor cells genetically modified to express hCD20 (P815-hCD20) at high level (Supplementary Fig. S6). The mice were treated with CPA as preconditioning prior to CAR T-cell injection, and also treated with anti–PD-1 mAb or control IgG after CAR T-cell injection. Compared with the mice treated with CPA alone, those treated with CPA plus anti–PD-1 mAb showed neither survival benefit nor tumor growth inhibition (Fig. 6A and B). Treatment with CPA and 7×19 CAR T cells plus control IgG induced only a weak survival benefit and tumor growth inhibition, leading to eventual death of 90% mice by day 70. This result was ascribed to a relatively low number of transferred 7×19 CAR T cells, compared with our previous report (22). On the other hand, the mice treated with CPA and 7×19 CAR T cells plus anti–PD-1 mAb showed profound tumor growth inhibition with a complete regression and survival in 90% of mice on day 70. These results indicate combination of anti–PD-1 mAb can improve the efficacy of 7×19 CAR T cells as well as 7×19 TCR T cells.

Figure 6.

Combined immunotherapy of 7×19 CAR T cells with anti–PD-1 mAb. DBA/2 mice were inoculated s.c. with P815-hCD20 tumor cells on day 0, and then injected i.p. with low-dose CPA on day 11 as preconditioning. On day 14, the mice were injected i.v. with 7×19 anti-hCD20 CAR T cells, followed by i.p. injection of anti–PD-1 mAb or control IgG every 4 to 5 days for a total of five times starting on day 17. Survival (A) and tumor volume (B) of the mice were assessed. Data are pooled from two independent experiments. The number of mice that achieved complete tumor regression on day 70 out of the total mouse number is indicated in B. Symbols indicate the following: CPA only (cross), CPA plus anti–PD-1 mAb (open square), CPA and anti-hCD20 7×19 CAR T cells plus control IgG (open circle), CPA and anti-hCD20 7×19 CAR T cells plus anti–PD-1 mAb (solid circle). P values of log-rank tests indicate the following: cross vs. open square; P = 0.1995, cross vs. open circle; P = 0.062, open square vs. solid circle; P < 0.0001, open circle vs. solid circle; P < 0.0001.

Figure 6.

Combined immunotherapy of 7×19 CAR T cells with anti–PD-1 mAb. DBA/2 mice were inoculated s.c. with P815-hCD20 tumor cells on day 0, and then injected i.p. with low-dose CPA on day 11 as preconditioning. On day 14, the mice were injected i.v. with 7×19 anti-hCD20 CAR T cells, followed by i.p. injection of anti–PD-1 mAb or control IgG every 4 to 5 days for a total of five times starting on day 17. Survival (A) and tumor volume (B) of the mice were assessed. Data are pooled from two independent experiments. The number of mice that achieved complete tumor regression on day 70 out of the total mouse number is indicated in B. Symbols indicate the following: CPA only (cross), CPA plus anti–PD-1 mAb (open square), CPA and anti-hCD20 7×19 CAR T cells plus control IgG (open circle), CPA and anti-hCD20 7×19 CAR T cells plus anti–PD-1 mAb (solid circle). P values of log-rank tests indicate the following: cross vs. open square; P = 0.1995, cross vs. open circle; P = 0.062, open square vs. solid circle; P < 0.0001, open circle vs. solid circle; P < 0.0001.

Close modal

In the present study, we developed genetically engineered P1A T cells, which produce IL-7 and CCL19, and revealed their potent therapeutic effects against pre-established P815 tumor by increasing infiltration of DCs and T cells consisting of endogenous T cells as well as transferred P1A T cells in tumor tissues. In addition, the combination of anti–PD-1 mAb with 7×19 P1A T or 7×19 CAR T-cell therapies showed synergistic effects to achieve complete regression of pre-established tumors in majority of mice, leading to superior immune memory generation by endogenous T cells, and long-term protection from tumor recurrence. Thus, this study highlighted a general importance and applicability of IL-7/CCL19 production technologies in gene-modified T-cell therapies.

It was previously reported that P1A T cells failed to reject pre-established P815 tumors in vivo, although they exhibited a direct cytotoxicity against P1A-expressing tumor cell lines including P815 in vitro (37). In this study, such inability of P1A T cells was also confirmed as shown in Fig. 2. These results suggest that P815 tumor has at least to some degree of refractory trait against endogenous immune surveillance in DBA/2 mouse and a transfer of P1A T cells. We also found that the addition of PD-1 blockade yielded no survival benefits in P815-bearing mice even in the presence or absence of Conv. P1A T-cell transfer. Thus, our models resemble anti–PD-1 Ab-resistant tumors in the clinical conditions. Although various biomarkers correlating with the responses to anti–PD-1 Ab therapy has been reported, one of the well-recognized factors is the degree of T-cell infiltration in the tumor microenvironment (38). That is, when tumor tissues contain adequate T-cell infiltration, they are referred to as “hot tumors,” which correlates with better clinical responses to anti–PD-1 Ab. In contrast, when there is a lack or scarcity of T-cell infiltration, the term is “cold tumors,” which are resistant to anti–PD-1 Ab. Thus, it is crucial to convert cold tumors into hot tumors to enhance clinical efficacy of anti–PD-1 Ab. For this purpose, various therapeutic approaches have been proposed, including bispecific Ab, oncolytic viruses, and adoptive transfer of tumor-specific T cells (38). Our results in this study indicated that P815 tumor is categorized into cold tumors, and adoptive transfer of Conv. P1A T cells could not convert the tumor microenvironment from cold to hot. On the other hand, significant survival improvement of P815-bearing mice by the combination of anti–PD-1 mAb with 7×19 P1A T cells or 7×19 CAR T cells strongly suggested that production of IL7 and CCL19 by tumor-reactive T cells in the tumor microenvironment successfully convert cold tumors into hot tumors. In fact, our results support this concept, as TIL analysis in the mice treated with 7×19 P1A T cells revealed a significant increase of DCs and T cells derived from endogenous T cells as well as transferred 7×19 P1A T cells. In addition, our results in the experiments using PD-1-knockdown 7×19 P1A T cells in combination with anti–PD-1 mAb further indicated that endogenous T cells not only accumulated to tumor tissues but also played an important role in antitumor responses against P815 tumor. These results are consistent with our previous findings in the models using 7×19 CAR T cells (22). Although the precise mechanisms how IL7 and CCL19 enhance antitumor effects remain unexplored, it is likely that these factors act on P1A T cells and endogenous T cells by an autocrine and paracrine mechanism, respectively, in the tumor microenvironment. Endogenous T cells are expected to augment antitumor effects via direct cytotoxicity against tumor cells and/or indirect help including cytokine production.

In conclusion, we presented two important findings in this study. First, genetic modification of TCR T cells to introduce concomitant production of IL7 and CCL19 can augment antitumor immune responses and induce regression of tumors that are otherwise resistant to immunotherapies, including anti–PD-1 Ab, conventional adoptive T-cell therapy, and even their combination. Second, the antitumor responses of IL7/CCL19-producing TCR T and CAR T cells are further strengthened by combination with anti–PD-1 Ab therapy. These findings will provide more treatment options for patients who are refractory to the current tumor immunotherapies.

K. Tamada reports grants from Japan Agency for Medical Research and Development and grants and personal fees from Noile-Immune Biotech Inc. during the conduct of the study. No disclosures were reported by the other authors.

Y. Tokunaga: Investigation, writing–original draft. T. Sasaki: Investigation. S. Goto: Investigation. K. Adachi: Supervision. Y. Sakoda: Supervision. K. Tamada: Conceptualization, supervision, funding acquisition, writing–review and editing.

The authors thank Mr. Satoshi Tatekabe and Ms. Makiko Miyamoto, Nana Okada, Mihoko Ida, Hiromi Kurosawa, Nanami Nakamura, Reiko Ohashi, and Aki Kawai for excellent technical assistance. This study was supported by research funds from Practical Research for Innovative Cancer Control, and Project for Cancer Research and Therapeutic Evolution (P-CREATE) 16770206 (to K. Tamada), by Japan Agency for Medical Research and Development (AMED), and Noile-Immune Biotech Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Maude
SL
,
Laetsch
TW
,
Buechner
J
,
Rives
S
,
Boyer
M
,
Bittencourt
H
, et al
Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia
.
N Engl J Med
2018
;
378
:
439
48
.
2.
Park
JH
,
Rivière
I
,
Gonen
M
,
Wang
X
,
Sénéchal
B
,
Curran
KJ
, et al
Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia
.
N Engl J Med
2018
;
378
:
449
59
.
3.
Fry
TJ
,
Shah
NN
,
Orentas
RJ
,
Stetler-Stevenson
M
,
Yuan
CM
,
Ramakrishna
S
, et al
CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy
.
Nat Med
2018
;
24
:
20
8
.
4.
Neelapu
SS
,
Locke
FL
,
Bartlett
NL
,
Lekakis
LJ
,
Miklos
DB
,
Jacobson
CA
, et al
Axicabtagene Ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma
.
N Engl J Med
2017
;
377
:
2531
44
.
5.
Schuster
SJ
,
Bishop
MR
,
Tam
CS
,
Waller
EK
,
Borchmann
P
,
McGuirk
JP
, et al
Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma
.
N Engl J Med
2019
;
380
:
45
56
.
6.
Raje
N
,
Berdeja
J
,
Lin
Y
,
Siegel
D
,
Jagannath
S
,
Madduri
D
, et al
Anti-BCMA CAR T-cell therapy bb2121 in relapsed or refractory multiple myeloma
.
N Engl J Med
2019
;
380
:
1726
37
.
7.
Xu
J
,
Chen
LJ
,
Yang
SS
,
Sun
Y
,
Wu
W
,
Liu
YF
, et al
Exploratory trial of a biepitopic CAR T-targeting B cell maturation antigen in relapsed/refractory multiple myeloma
.
Proc Natl Acad Sci U S A
2019
;
116
:
9543
51
.
8.
Zhang
J
,
Wang
L
. 
The emerging world of TCR-T cell trials against cancer: a systematic review
.
Technol Cancer Res Treat
2019
;
18
:
1533033819831068
.
9.
Chandran
SS
,
Klebanoff
CA
. 
T cell receptor-based cancer immunotherapy: emerging efficacy and pathways of resistance
.
Immunol Rev
2019
;
290
:
127
47
.
10.
Song
MK
,
Park
BB
,
Uhm
JE
. 
Resistance mechanisms to CAR T-cell therapy and overcoming strategy in B-cell hematologic malignancies
.
Int J Mol Sci
2019
;
20
:
5010
.
11.
Cheng
J
,
Zhao
L
,
Zhang
Y
,
Qin
Y
,
Guan
Y
,
Zhang
T
, et al
Understanding the mechanisms of resistance to CAR T-cell therapy in malignancies
.
Front Oncol
2019
;
9
:
1237
.
12.
Yeku
O
,
Li
X
,
Brentjens
RJ
. 
Adoptive T-cell therapy for solid tumors
.
Am Soc Clin Oncol Educ Book
2017
;
37
:
193
204
.
13.
Morgan
RA
,
Dudley
ME
,
Wunderlich
JR
,
Hughes
MS
,
Yang
JC
,
Sherry
RM
, et al
Cancer regression in patients after transfer of genetically engineered lymphocytes
.
Science
2006
;
314
:
126
9
.
14.
Robbins
PF
,
Morgan
RA
,
Feldman
SA
,
Yang
JC
,
Sherry
RM
,
Dudley
ME
, et al
Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1
.
J Clin Oncol
2011
;
29
:
917
24
.
15.
Robbins
PF
,
Kassim
SH
,
Tran
TL
,
Crystal
JS
,
Morgan
RA
,
Feldman
SA
, et al
A pilot trial using lymphocytes genetically engineered with an NY-ESO-1-reactive T-cell receptor: long-term follow-up and correlates with response
.
Clin Cancer Res
2015
;
21
:
1019
27
.
16.
Guedan
S
,
Posey
AD
 Jr
,
Shaw
C
,
Wing
A
,
Da
T
,
Patel
PR
, et al
Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation
.
JCI Insight
2018
;
3
:
e96976
.
17.
Quintarelli
C
,
Orlando
D
,
Boffa
I
,
Guercio
M
,
Polito
VA
,
Petretto
A
, et al
Choice of costimulatory domains and of cytokines determines CAR T-cell activity in neuroblastoma
.
Oncoimmunology
2018
;
7
:
e1433518
.
18.
Rupp
LJ
,
Schumann
K
,
Roybal
KT
,
Gate
RE
,
Ye
CJ
,
Lim
WA
, et al
CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells
.
Sci Rep
2017
;
7
:
737
.
19.
John
LB
,
Devaud
C
,
Duong
CP
,
Yong
CS
,
Beavis
PA
,
Haynes
NM
, et al
Anti-PD-1 antibody therapy potently enhances the eradication of established tumors by gene-modified T cells
.
Clin Cancer Res
2013
;
19
:
5636
46
.
20.
Cherkassky
L
,
Morello
A
,
Villena-Vargas
J
,
Feng
Y
,
Dimitrov
DS
,
Jones
DR
, et al
Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition
.
J Clin Invest
2016
;
126
:
3130
44
.
21.
Koneru
M
,
Purdon
TJ
,
Spriggs
D
,
Koneru
S
,
Brentjens
RJ
. 
IL-12 secreting tumor-targeted chimeric antigen receptor T cells eradicate ovarian tumors in vivo
.
Oncoimmunology
2015
;
4
:
e994446
.
22.
Adachi
K
,
Kano
Y
,
Nagai
T
,
Okuyama
N
,
Sakoda
Y
,
Tamada
K
. 
IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor
.
Nat Biotechnol
2018
;
36
:
346
51
.
23.
Luther
SA
,
Bidgol
A
,
Hargreaves
DC
,
Schmidt
A
,
Xu
Y
,
Paniyadi
J
, et al
Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis
.
J Immunol
2002
;
169
:
424
33
.
24.
Miller
PW
,
Sharma
S
,
Stolina
M
,
Butterfield
LH
,
Luo
J
,
Lin
Y
, et al
Intratumoral administration of adenoviral interleukin 7 gene-modified dendritic cells augments specific antitumor immunity and achieves tumor eradication
.
Hum Gene Ther
2000
;
11
:
53
65
.
25.
Hillinger
S
,
Yang
SC
,
Zhu
L
,
Huang
M
,
Duckett
R
,
Atianzar
K
, et al
EBV-induced molecule 1 ligand chemokine (ELC/CCL19) promotes IFN-gamma-dependent antitumor responses in a lung cancer model
.
J Immunol
2003
;
171
:
6457
65
.
26.
Sarma
S
,
Guo
Y
,
Guilloux
Y
,
Lee
C
,
Bai
XF
,
Liu
Y
. 
Cytotoxic T lymphocytes to an unmutated tumor rejection antigen P1A: normal development but restrained effector function in vivo
.
J Exp Med
1999
;
189
:
811
20
.
27.
Kim
JH
,
Lee
SR
,
Li
LH
,
Park
HJ
,
Park
JH
,
Lee
KY
, et al
High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice
.
PLoS One
2011
;
6
:
e18556
.
28.
Deng
W
,
Yang
D
,
Zhao
B
,
Ouyang
Z
,
Song
J
,
Fan
N
, et al
Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer
.
PLoS One
2011
;
6
:
e19986
.
29.
Morgan
RA
,
Yang
JC
,
Kitano
M
,
Dudley
ME
,
Laurencot
CM
,
Rosenberg
SA
. 
Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2
.
Mol Ther
2010
;
18
:
843
51
.
30.
Tamada
K
,
Geng
D
,
Sakoda
Y
,
Bansal
N
,
Srivastava
R
,
Li
Z
, et al
Redirecting gene-modified T cells toward various cancer types using tagged antibodies
.
Clin Cancer Res
2012
;
18
:
6436
45
.
31.
Okada
M
,
Chikuma
S
,
Kondo
T
,
Hibino
S
,
Machiyama
H
,
Yokosuka
T
, et al
Blockage of core fucosylation reduces cell-surface expression of PD-1 and promotes anti-tumor immune responses of T cells
.
Cell Rep
2017
;
20
:
1017
28
.
32.
Umezu
D
,
Okada
N
,
Sakoda
Y
,
Adachi
K
,
Ojima
T
,
Yamaue
H
, et al
Inhibitory functions of PD-L1 and PD-L2 in the regulation of anti-tumor immunity in murine tumor microenvironment
.
Cancer Immunol Immunother
2019
;
68
:
201
11
.
33.
Van den Eynde
B
,
Lethe
B
,
Van Pel
A
,
De Plaen
E
,
Boon
T
. 
The gene coding for a major tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2 mice
.
J Exp Med
1991
;
173
:
1373
84
.
34.
Tsujimura
T
,
Furitsu
T
,
Morimoto
M
,
Isozaki
K
,
Nomura
S
,
Matsuzawa
Y
, et al
Ligand-independent activation of c-kit receptor tyrosine kinase in a murine mastocytoma cell line P-815 generated by a point mutation
.
Blood
1994
;
83
:
2619
26
.
35.
Okada
S
,
Nakauchi
H
,
Nagayoshi
K
,
Nishikawa
S
,
Nishikawa
S
,
Miura
Y
, et al
Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule
.
Blood
1991
;
78
:
1706
12
.
36.
Tan
JK
,
Periasamy
P
,
O'Neill
HC
. 
Delineation of precursors in murine spleen that develop in contact with splenic endothelium to give novel dendritic-like cells
.
Blood
2010
;
115
:
3678
85
.
37.
Bai
XF
,
Liu
JQ
,
Joshi
PS
,
Wang
L
,
Yin
L
,
Labanowska
J
, et al
Different lineages of P1A-expressing cancer cells use divergent modes of immune evasion for T-cell adoptive therapy
.
Cancer Res
2006
;
66
:
8241
9
.
38.
Bonaventura
P
,
Shekarian
T
,
Alcazer
V
,
Valladeau-Guilemond
J
,
Valsesia-Wittmann
S
,
Amigorena
S
, et al
Cold tumors: a therapeutic challenge for immunotherapy
.
Front Immunol
2019
;
10
:
168
.