Adoptive immunotherapy involving genetic modification of T cells with antigen-specific, chimeric, single-chain receptors is a promising approach for the treatment of cancer. To determine whether gene-modified T cells could induce antitumor effects without associated autoimmune pathology, we assessed the ability of T cells expressing an anti-Her-2 chimeric receptor to eradicate tumor in Her-2 transgenic mice that express human Her-2 as a self-antigen in brain and mammary tissues. In adoptive transfer studies, we demonstrated significant improvement in the survival of mice bearing Her-2+ 24JK tumor following administration of anti-Her-2 T cells compared with control T cells. The incorporation of a lymphoablative step prior to adoptive transfer of anti-Her-2 T cells and administration of IL-2 were both found to further enhance survival. The reduction in tumor growth was also correlated with localization of transferred T cells at the tumor site. Furthermore, an antigen-specific recall response could be induced in long-term surviving mice following rechallenge with Her-2+ tumor. Importantly, antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. This study highlights the therapeutic potential of using gene-engineered T cells as a safe and effective treatment of cancer. Cancer Res; 70(23); 9591–8. ©2010 AACR.

Strategies, including the use of cytokines, antibodies, and various vaccine regimens, aimed at harnessing the power of the immune system have long been investigated for the treatment of cancer (1–3). However, the generation of effective and long-lasting antitumor responses has remained a challenge. In recent studies, the genetic modification of T cells with scFv chimeric receptors that have the ability to target any characterized cell surface tumor antigen has been investigated as a new avenue of redirecting immune effector function (4–9). Adoptive transfer of scFv receptor gene–modified T cells in immunocompromised mouse models has shown great potential and demonstrated long-term protection against primary and secondary tumor challenge (8, 10).

Although the results achieved in the immunocompromised setting have been encouraging, these studies have been performed in models in which the targeted antigen has been expressed exclusively in tumor, which is rarely the case in a clinical setting. Consequently, this has not allowed investigation of whether host immunoregulatory factors may impact on the activity of transferred T cells or whether these cells may also damage normal tissue expressing the target antigen. The latter point is particularly important, given that adoptive cell therapies utilizing tumor-infiltrating lymphocytes (TILs) or gene-modified T cells have in some cases resulted in on-target toxicity, manifested as “autoimmunity” in patients including thyroiditis, uveitis, and respiratory and liver toxicity (11–14).

The generation of human Her-2 transgenic mice represents an ideal model to investigate this issue. These mice have been reported to constitutively express human Her-2 as a self-antigen both in brain tissue and in the mammary gland during lactation under the control of the whey acidic protein promoter (15). Her-2+ tumor cells grow progressively when injected into these mice. Thus, Her-2 transgenic mice offer a physiologically relevant model that can be utilized for the preclinical evaluation of gene-modified T cells in a setting that more closely mimics the situation in patients. In the current study, we utilized this Her-2 transgenic mouse model to evaluate the antitumor efficacy of Her-2-reactive, gene-modified T cells and assess whether these cells induce autoimmune pathology.

Cell culture and mice

The MHC class I–negative C57BL/6 mouse 24JK sarcoma cell line (kindly provided by Dr. Patrick Hwu, National Institutes of Health, Bethesda, MD), 24JK cells retrovirally transduced with the human Her-2 antigen (24JK-Her-2) and GP+E86 retroviral packaging cells were cultured as described previously. The 24JK tumor cell lines were routinely tested and authenticated in the last 6 months by flow cytometry and found to be MHC class I negative as per the original line acquired. C57BL/6 human Her-2 transgenic mice and congenic Thy1.1+ Her-2 mice were bred at the Peter MacCallum Cancer Centre, Victoria, Australia, and used for experimentation at 6 to 16 weeks of age. All animal experimentation was approved in advance by the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee.

Generation of anti-Her-2–transduced T cells

The GP+E86 packaging line expressing the scFv-anti-Her-2 CD28-ζ receptor that contained the extracellular scFv-anti- Her-2 monoclonal antibody (mAb) region (kindly provided by Dr. Winfried Wels) (16) was generated as previously described (17, 18). For transduction, splenocytes from C57BL/6 Her-2 transgenic mice were gene modified as previously described (19). Following transduction, splenocytes were cultured in RPMI, supplemented with 100 U/mL of IL-2 and 2 ng/mL of IL-7, and utilized for in vitro and in vivo experiments on days 7 and 8.

Flow cytometry

Expression of the chimeric anti-Her-2 receptor on the surface of T cells was determined by direct immunofluorescence with a c-myc tag Alexa Fluor 488–conjugated immunoglobulin (Cell Signaling Technology, Beverly, MA). Background immunofluorescence was assessed using an IgG2a Alexa Fluor 488–conjugated mouse isotype immunoglobulin (Invitrogen). Phenotypic characterization of transduced cells was determined using direct staining with fluorescein isothiocyanate–conjugated anti-CD4 (clone RM4-5; BD Pharmingen, San Jose, CA) and phycoerythrin-conjugated anti-CD8 (clone 53-6.7; BD Pharmingen).

Antigen-specific cytokine secretion, proliferation, and cytotoxicity by gene-modified T cells

The ability of transduced anti-Her-2 T cells to secrete IFN-γ in response to Her-2 antigen stimulation was analyzed by enzyme-linked immunosorbent assay (ELISA) following overnight coculture with 24JK-Her-2 or 24JK parental cells as previously described (20). The proliferative potential and capacity of transduced T cells to mediate antigen-specific tumor cell lysis was assessed by [3H]-thymidine incorporation and 5-hour chromium release assays, respectively, as described previously (21).

Adoptive transfer experiments

Groups of 5 to 10 Her-2 transgenic mice were intravenously injected with 1 × 106 24JK-Her-2 cells followed by transfer of scFv-anti-Her-2–transduced T cells or control LXSN–transduced T cells (1 × 107 per dose on days 0 and 1) or left untreated, and survival of mice was monitored. In some experiments, recipient mice received transduced donor T cells from congenic Thy1.1+ Her-2 mice. For experiments involving lymphodepletion, Her-2 transgenic mice were irradiated (5 Gy) followed by intravenous injection of 24JK-Her-2 tumor cells (1 × 106) and adoptive transfer of transduced T cells at 1 × 107 per dose at days 0 and 1, days 5 and 6, or days 10 and 11. Mice were also given twice-daily intraperitoneal injections of recombinant human IL-2 (Biological Resource Branch, National Cancer Institute, Frederick, MD) involving 9 doses of 50,000 IU/200 μL given subsequent to T-cell transfer. For all experiments, mice were monitored for survival. Mice displaying overt signs of distress were culled, and lungs were harvested and weighed to record tumor burden. To investigate the trafficking and persistence of adoptively transferred T cells in vivo, 1 × 107 donor T cells from congenic Thy1.1+ Her-2 mice were transduced and transferred into irradiated 24JK-Her-2 tumor bearing Her-2 recipient mice (Thy1.2+) on days 0 and 1 and received IL-2 as described previously. Flow cytometry was carried out to determine the frequency of all T cells and Thy1.1+ T cells in the organs.

Histology and immunohistochemistry

Lung, mammary (nonlactating female), and brain tissues (female and male) from Her-2 transgenic mice were removed, fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned. To examine tumor burden in lungs of mice (days 5 and 10 after 24JK-Her-2 tumor inoculation) and toxicity to mammary and brain tissues expressing Her-2 antigen following therapy, sections were stained with hematoxylin and eosin (H&E) and analyzed by a pathologist. To examine expression of the human Her-2 antigen in mammary and brain tissues from Her-2 transgenic mice and the 24JK-Her-2 and 24JK cell lines, sections were stained with a polyclonal rabbit anti-human Her-2 oncoprotein antibody (A 0485; DAKO Corp., Real Carpinteria, CA) following antigen retrieval in 10 mmol/L of citrate buffer (pH 6.0). Images were visualized with an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan), acquired using RT SE Diagnostics Instruments SPOT camera (Diagnostic Instruments, Sterling Heights, MI) in conjunction with SPOT Advanced Version 4.6 (Diagnostic Instruments), and compiled with Adobe Photoshop and Illustrator CS4 (Adobe systems, San Jose, CA).

Statistical analysis

Differences between mice survival distributions were analyzed using log-rank (Mantel–Cox) test to determine statistical significance. P2 < 0.05 was considered significant.

Expression of the anti-Her-2 chimeric receptor in Her-2 transgenic mouse T cells

Using flow cytometry, we observed reproducibly high-level receptor expression on transduced T cells (Fig. 1A) (46 ± 3.2% SEM, n = 16). There was no detectable anti-tag staining on the surface of T cells transduced with an empty LXSN retroviral vector (Fig. 1B). These transduced T cells were predominantly CD8+ (79.5 ± 4.4% SEM, n = 8), with only a small percentage of CD4+ T cells present in the culture (7.3 ± 2.7% SEM, n = 8) (Fig. 1C and D).

Figure 1.

Expression of the scFv-anti-Her-2 chimeric receptor in transduced Her-2 transgenic mouse T cells. Splenic T cells derived from Her-2 transgenic mice were retrovirally transduced with the scFv-anti-Her-2-CD28-ζ receptor (A) or empty LXSN vector (B). scFv chimeric receptor expression was detected in T cells by flow cytometry following staining with an Alexa Fluor 488–conjugated anti-tag mAb or Alexa Fluor 488–conjugated mouse isotype control antibody. T cells were also analyzed for expression of CD4 and CD8 (C, D).

Figure 1.

Expression of the scFv-anti-Her-2 chimeric receptor in transduced Her-2 transgenic mouse T cells. Splenic T cells derived from Her-2 transgenic mice were retrovirally transduced with the scFv-anti-Her-2-CD28-ζ receptor (A) or empty LXSN vector (B). scFv chimeric receptor expression was detected in T cells by flow cytometry following staining with an Alexa Fluor 488–conjugated anti-tag mAb or Alexa Fluor 488–conjugated mouse isotype control antibody. T cells were also analyzed for expression of CD4 and CD8 (C, D).

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Antigen-specific cytokine secretion, tumor lysis, and proliferation by transduced Her-2 transgenic mouse T cells

The functional capacity of transduced transgenic T cells was tested in several in vitro assays. Following stimulation by overnight coculture with the C57BL/6 sarcoma cell line 24JK expressing human Her-2 antigen (24JK-Her-2), transgenic anti-Her-2 T cells secreted high levels of IFN-γ as determined by ELISA (Fig. 2A), whereas coculture with the Her-2 24JK parental tumor cell line demonstrated no significant release of IFN-γ. Anti-Her-2 T cells could also significantly lyse 24JK-Her-2 target cells compared with Her-2 24JK tumor cells (Fig. 2B). The proliferative capacity of transduced transgenic T cells was measured by a [3H]-thymidine incorporation assay following stimulation with plate-bound anti-c-myc antibody. Anti-Her-2 T cells could specifically proliferate following stimulation with anti-c-myc antibody but not with isotype control antibody (Fig. 2C). Taken together, these results showed that transgenic T cells could be effectively transduced with the scFv-anti-Her-2 receptor and mediate antigen- specific cytokine secretion, killing, and proliferation.

Figure 2.

Transduced mouse T cells expressing the anti-Her-2 receptor mediate antigen-specific cytokine secretion, proliferation, and tumor cell lysis. To assess cytokine secretion, T cells were cocultured with media alone, 24JK-Her-2, or 24JK parental cells or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs. Supernatants were harvested and level of IFN-γ secreted by transduced T cells was measured by ELISA (A). Results are expressed as mean ± SEM values of IFN-γ secreted from duplicate samples of 3 representative experiments. Antigen-specific cytolytic activity of transduced T cells was examined in a 5-hour 51Cr release assay (B). Results are expressed as percentage specific 51Cr release ± SEM of triplicate samples representative of 3 experiments. The proliferative ability of transduced T cells was evaluated in a 72-hour [3H]-thymidine incorporation assay (C). Chimeric receptor or empty LXSN vector–transduced T cells were incubated with media alone or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs, isotype control mAb, or anti-c-myc mAb. Results are expressed as mean ± SEM values of [3H]-thymidine [counts per minute (cpm)] of triplicate samples and are representative of 3 experiments.

Figure 2.

Transduced mouse T cells expressing the anti-Her-2 receptor mediate antigen-specific cytokine secretion, proliferation, and tumor cell lysis. To assess cytokine secretion, T cells were cocultured with media alone, 24JK-Her-2, or 24JK parental cells or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs. Supernatants were harvested and level of IFN-γ secreted by transduced T cells was measured by ELISA (A). Results are expressed as mean ± SEM values of IFN-γ secreted from duplicate samples of 3 representative experiments. Antigen-specific cytolytic activity of transduced T cells was examined in a 5-hour 51Cr release assay (B). Results are expressed as percentage specific 51Cr release ± SEM of triplicate samples representative of 3 experiments. The proliferative ability of transduced T cells was evaluated in a 72-hour [3H]-thymidine incorporation assay (C). Chimeric receptor or empty LXSN vector–transduced T cells were incubated with media alone or stimulated with plate-bound anti-CD3 and anti-CD28 mAbs, isotype control mAb, or anti-c-myc mAb. Results are expressed as mean ± SEM values of [3H]-thymidine [counts per minute (cpm)] of triplicate samples and are representative of 3 experiments.

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Adoptive transfer of anti-Her-2 T cells can mediate enhanced survival of tumor-bearing mice and localize to the tumor site

We have previously shown that adoptive transfer of T cells gene-modified with the scFv-anti-Her-2 receptor could specifically eradicate 5-day established lung metastases in immunocompromised SCID mice (10). Thus, we wanted to assess whether transduced T cells could impact on Her-2+ tumor in an immunocompetent setting in a similar way. In this experiment, we compared the survival of Her-2 transgenic mice bearing 24JK-Her-2 lung metastases treated with anti-Her-2 or control LXSN–transduced T cells administered intravenously on days 0 and 1. A significant increase in survival of mice (∼20% long-term, tumor-free survivors) was observed following transfer of anti-Her-2 T cells compared with mice that received control T cells or no T cells, which all succumbed to lung disease (P2 < 0.05; Fig. 3A). It has been reported both in mouse studies and in patients that the use of lymphodepletion as a preconditioning treatment prior to adoptive T-cell transfer can enhance their therapeutic efficacy (22–25). To test whether this was the case in our model using gene-engineered T cells, Her-2 transgenic mice were irradiated prior to tumor challenge and transfer of transduced T cells and administered IL-2 between days 0 and 4. In this experiment, a striking increase in survival was observed in recipient mice treated with anti-Her-2 T cells in combination with irradiation and IL-2 (∼70% long-term, tumor-free survivors; Fig. 3B) compared with mice given anti-Her-2 T cells with no preconditioning (only 20% survivors; Fig. 3A). Interestingly, the combination of both irradiation and IL-2 along with anti-Her-2 T cells was critical for this therapeutic effect, given the decreased survival that was observed in groups of mice that received anti-Her-2 T cells with either IL-2 or irradiation. There was no survival of mice that received control T cells with the combined regimen (Fig. 3B). Importantly, treatment of mice with irradiation or IL-2 alone did not significantly alter the growth kinetics of the 24JK-Her-2 tumor in recipient mice and no overt toxicity was observed in mice following therapy (Fig. 3B). To determine whether transduced T cells could be detected following transfer, mice were challenged with 24JK-Her-2+ tumor and irradiated before receiving transduced donor T cells from congenic Thy1.1+ Her-2 mice and IL-2. In this experiment, we observed a significant increase in percentage of Thy1.1+ donor T cells in lungs of anti-Her-2 T-cell–treated mice compared with the mice that received control T cells at day 18 following T-cell transfer (Fig. 4A). There was no significant difference in Thy1.1+ T cells observed in spleens of mice treated with anti-Her-2 or control T cells. We could also detect Thy1.1+ donor T cells in lungs and spleen of mice at day 53 in mice treated with anti-Her-2 T cells, indicating persistence of these cells (Fig. 4B). Collectively, these set of experiments showed that adoptively transferred anti-Her-2 T cells could be detected at the tumor site and significantly impact on Her-2+ tumor growth in immunocompetent mice and that combined treatment with irradiation and IL-2 could further increase the therapeutic effect similar to that observed in a patient setting receiving TIL and lymphodepletion with IL-2.

Figure 3.

Adoptive transfer of anti-Her-2 T cells combined with lymphodepletion and IL-2 mediates enhanced survival of mice. A, Her-2 transgenic mice were intravenously injected with 24JK-Her-2 cells (1 × 106) at day 0 prior to intravenous injection of transduced T cells 6 hours later and on day 1 (1 × 107/dose). *, P2 = 0.0001. B, to examine the effect of lymphodepletion and IL-2 on adoptively transferred, gene-engineered T cells, mice were irradiated (5 Gy) at day 0 prior to intravenous injection of 24JK-Her-2 tumor cells. Mice were then treated with transduced T cells (1 × 107 cells on days 0 and 1) in combination with IL-2 (9 doses of 50,000 IU between days 0 and 4). *, P2 < 0.05. Arrows represent days of T-cell transfer and data are representative of 2 experiments.

Figure 3.

Adoptive transfer of anti-Her-2 T cells combined with lymphodepletion and IL-2 mediates enhanced survival of mice. A, Her-2 transgenic mice were intravenously injected with 24JK-Her-2 cells (1 × 106) at day 0 prior to intravenous injection of transduced T cells 6 hours later and on day 1 (1 × 107/dose). *, P2 = 0.0001. B, to examine the effect of lymphodepletion and IL-2 on adoptively transferred, gene-engineered T cells, mice were irradiated (5 Gy) at day 0 prior to intravenous injection of 24JK-Her-2 tumor cells. Mice were then treated with transduced T cells (1 × 107 cells on days 0 and 1) in combination with IL-2 (9 doses of 50,000 IU between days 0 and 4). *, P2 < 0.05. Arrows represent days of T-cell transfer and data are representative of 2 experiments.

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Figure 4.

Adoptively transferred, transduced T cells can be detected in the lungs and spleens of recipient mice. The frequency of all T cells (black bars) and adoptively transferred T cells (striped bars) in the spleens and lungs of irradiated 24JK-Her-2 tumor-bearing mice that were treated with transduced congenic Thy1.1+ T cells and IL-2 was investigated by flow cytometry and expressed as a percentage of total viable cells. A, a significant increase in the percentage of anti-Her-2 T cells could be detected in the lungs of mice compared with mice treated with control LXSN T cells at day 18 following transfer (*, P2 < 0.05). There was no significant difference (ns) in the percentage of transferred T cells in the spleens of mice treated with control or anti-Her-2 T cells or in the total numbers of T cells analyzed from both organs. B, adoptively transferred anti-Her-2 T cells could also be detected in the lungs and spleens of mice at day 53, indicating persistence of these cells. Data shown are average ± SEM from 5 mice analyzed.

Figure 4.

Adoptively transferred, transduced T cells can be detected in the lungs and spleens of recipient mice. The frequency of all T cells (black bars) and adoptively transferred T cells (striped bars) in the spleens and lungs of irradiated 24JK-Her-2 tumor-bearing mice that were treated with transduced congenic Thy1.1+ T cells and IL-2 was investigated by flow cytometry and expressed as a percentage of total viable cells. A, a significant increase in the percentage of anti-Her-2 T cells could be detected in the lungs of mice compared with mice treated with control LXSN T cells at day 18 following transfer (*, P2 < 0.05). There was no significant difference (ns) in the percentage of transferred T cells in the spleens of mice treated with control or anti-Her-2 T cells or in the total numbers of T cells analyzed from both organs. B, adoptively transferred anti-Her-2 T cells could also be detected in the lungs and spleens of mice at day 53, indicating persistence of these cells. Data shown are average ± SEM from 5 mice analyzed.

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Adoptive transfer of anti-Her-2 T cells can mediate regression of established tumor

Given that the majority of patients will present with established disease, we next assessed whether our therapy could impact on advanced lung metastases in mice. In 2 separate experiments, the antitumor ability of anti-Her-2 T cells in combination with irradiation and IL-2 was examined against day 5 and day 10 established disease. Immunohistochemical staining using H&E demonstrated increased 24JK-Her-2 metastases in lung sections of mice at days 5 and 10 compared with untreated mice (Fig. 5A). In the first experiment, mice were irradiated and challenged with 24JK-Her-2 tumor on day 0 followed by transfer of transduced T cells on days 5 and 6 and administered IL-2 between days 5 and 9. The majority of mice that received anti-Her-2 T cells combined with irradiation and IL-2 survived (∼80% long-term, tumor-free survivors; Fig. 5B). This effect was dependent on T cells modified with the scFv anti-Her-2 receptor, as mice treated with control LXSN–transduced T cells succumbed to lung metastases similar to mice challenged with 24JK-Her-2 tumor alone (Fig. 5B). In the next experiment, mice were irradiated on day 10 and challenged with 24JK-Her-2 tumor followed by transfer of transduced T cells on days 10 and 11 and administered IL-2 between days 10 and 14. Strikingly, a significant increase in survival of mice was observed in mice with day 10 established disease that received anti-Her-2 T cells in combination with irradiation and IL-2 compared with control treated mice (Fig. 5C). Again, no overt toxicity was observed in mice following therapy. These experiments showed that gene-modified T cells could significantly impact on established disease in immunocompetent Her-2 transgenic mice, which has important implications for development of this approach for the treatment of patients with advanced cancer.

Figure 5.

Adoptive transfer of anti-Her-2 T cells can mediate regression of established tumor. A, Her-2 transgenic mice were injected intravenously with 1 × 106 24JK-Her-2 tumor cells. Lungs from mice were harvested at days 5 and 10 and stained with H&E and compared with lungs from untreated mice to determine the level of tumor burden. Representative images of 3 mice analyzed for each time point are shown and the presence of tumor is indicated by arrows (magnification × 200). B, Her-2 transgenic mice were irradiated (5 Gy) followed by intravenous injection of 24JK-Her-2 cells (1 × 106) on day 0 and then administered transduced T cells on days 5 and 6 (1 × 107 per dose) and IL-2 (9 doses of 50,000 IU between days 5 and 9). C, Her-2 transgenic mice were intravenously injected with 24JK-Her-2 cells (1 × 106) at day 0 and irradiated at day 10 just prior to the administration of transduced T cells at days 10 and 11 and IL-2 (9 doses of 50,000 IU between days 10 and 14). *, P2 < 0.05. Arrows represent days of T-cell transfer, and data are representative of 2 experiments.

Figure 5.

Adoptive transfer of anti-Her-2 T cells can mediate regression of established tumor. A, Her-2 transgenic mice were injected intravenously with 1 × 106 24JK-Her-2 tumor cells. Lungs from mice were harvested at days 5 and 10 and stained with H&E and compared with lungs from untreated mice to determine the level of tumor burden. Representative images of 3 mice analyzed for each time point are shown and the presence of tumor is indicated by arrows (magnification × 200). B, Her-2 transgenic mice were irradiated (5 Gy) followed by intravenous injection of 24JK-Her-2 cells (1 × 106) on day 0 and then administered transduced T cells on days 5 and 6 (1 × 107 per dose) and IL-2 (9 doses of 50,000 IU between days 5 and 9). C, Her-2 transgenic mice were intravenously injected with 24JK-Her-2 cells (1 × 106) at day 0 and irradiated at day 10 just prior to the administration of transduced T cells at days 10 and 11 and IL-2 (9 doses of 50,000 IU between days 10 and 14). *, P2 < 0.05. Arrows represent days of T-cell transfer, and data are representative of 2 experiments.

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Long-term surviving mice can respond to tumor rechallenge in an antigen-specific manner

Given that tumor relapse can often occur in patients following treatment, the ability for cancer immunotherapies to induce a strong recall response is an important issue. To test whether this was the case for our therapy, long-term surviving mice (120 days after primary 24JK-Her-2 tumor inoculation) that were irradiated and treated with anti-Her-2 T cells and IL-2 were rechallenged with either 24JK-Her-2 or parental 24JK tumor cells. A significant increase in survival of mice was observed following rechallenge with 24JK-Her-2+ compared with 24JK-Her-2 tumor cells (Fig. 6). These experiments demonstrated for the first time in an immunocompetent setting that adoptively transferred, gene-engineered T cells could induce a strong recall response in an antigen-specific manner.

Figure 6.

Long-term surviving mice can respond to tumor rechallenge in an antigen-specific manner. Long-term surviving mice (120 days after primary 24JK-Her-2 tumor inoculation) that were treated with transduced anti-Her-2 T cells were rechallenged with intravenous injection of 24JK-Her-2 or 24JK parental cells (1 × 106). *, P2 < 0.05.

Figure 6.

Long-term surviving mice can respond to tumor rechallenge in an antigen-specific manner. Long-term surviving mice (120 days after primary 24JK-Her-2 tumor inoculation) that were treated with transduced anti-Her-2 T cells were rechallenged with intravenous injection of 24JK-Her-2 or 24JK parental cells (1 × 106). *, P2 < 0.05.

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Adoptive transfer of transduced T cells does not cause autoimmunity in mice

An important issue with regard to adoptive immunotherapy using gene-engineered T cells is whether these cells may cause severe autoimmune effects against normal tissue expressing the target antigen. In Her-2 transgenic mice, the human Her-2 antigen has been shown to be expressed as a self-antigen in parts of the brain and mammary tissue under lactating conditions (15). We confirmed these results and showed with a specific antibody recognizing Her-2 positive staining for this antigen in the cerebellum of brain sections from transgenic mice but not in nontransgenic littermates (Fig. 7A). Interestingly, we could also detect expression of Her-2 antigen in the mammary gland of nonlactating Her-2 transgenic mice but not in nontransgenic littermates for which staining was confined predominantly in adipocytes surrounding the ducts (Fig. 7A). As a positive control for antibody recognition of Her-2 antigen, we showed intense staining of 24JK-Her-2 tumor cells but not 24JK parental cells (Fig. 7A). The expression of Her-2 antigen in transgenic mice approximates the situation in humans in whom Her-2 can be expressed on a range of normal tissues including epithelial cells of the breast, neuronal cells, and gastrointestinal tract (26, 27). To determine whether genetically redirected T cells caused autoimmunity against normal tissue, we performed H&E staining of brain and mammary sections from Her-2 transgenic mice that were irradiated and challenged with 24JK-Her-2 tumor and received anti-Her-2 engineered T cells and IL-2 as previously. Brain and mammary tissues were then assessed for any indication of T-cell–mediated damage. Interestingly, all mice treated with T cells expressing the scFv-anti-Her-2 receptor demonstrated no tissue damage, as shown by H&E staining of mammary or brain sections from representative mice at days 18 and 50 following T-cell transfer (Fig. 7B and C). The morphologic appearance of sections from these tissues was comparable with sections either from mice treated with control T cells or from untreated mice (Fig. 7B). Given that the presence of Her-2+ tumor may act as a sink for transferred T cells, we also treated Her-2 transgenic mice with transduced T cells in the absence of tumor and found no evidence of autoimmune damage to brain and mammary tissues in these mice (data not shown). Thus, the transfer of genetically redirected T cells did not seem to induce any significant autoimmunity against normal host tissue expressing Her-2 target antigen at the time points analyzed.

Figure 7.

Adoptive transfer of anti-Her-2 T cells does not induce toxicity to normal tissue expressing Her-2 antigen. A, localization of the Her-2 antigen in 24JK-Her-2 cells, mammary gland from nonlactating female, and brain tissue from Her-2 transgenic mice were analyzed using immunohistochemical diaminobenzidine staining. The 24JK-Her-2 cell line displayed intense staining for human Her-2 antigen. Positive staining for Her-2 was detected on adipocytes in the mammary tissue (closed arrow) and confined to the molecular layer of the cerebellum along the Bergman glial fibers (open arrow) and membranes of the Purkinje cells (closed arrow). No staining was observed in 24JK parental cells or in mammary and brain tissues from nontransgenic mouse littermates. Sections are representative of 5 mice analyzed. B and C, Her-2 transgenic mice were irradiated (5 Gy) and treated with 24JK-Her-2 tumor cells (1 × 106) at day 0, followed by adoptively transferred, transduced T cells (1 × 107 per dose) at days 0 and 1 and IL-2 (9 doses of 50,000 IU between days 0 and 4). Mammary and brain tissues were harvested and stained with H&E at days 18 and 50. Analysis of tissue sections revealed no structural damage or increased immune cell infiltration for mice treated with chimeric receptor–transduced T cells in mammary or brain tissues at day 18 (B) or at the later time point day 50 (C) and were morphologically similar to sections from mice treated with control T cells or nontreated mice. Mammary and brain sections are representative of 5 mice analyzed for each time point.

Figure 7.

Adoptive transfer of anti-Her-2 T cells does not induce toxicity to normal tissue expressing Her-2 antigen. A, localization of the Her-2 antigen in 24JK-Her-2 cells, mammary gland from nonlactating female, and brain tissue from Her-2 transgenic mice were analyzed using immunohistochemical diaminobenzidine staining. The 24JK-Her-2 cell line displayed intense staining for human Her-2 antigen. Positive staining for Her-2 was detected on adipocytes in the mammary tissue (closed arrow) and confined to the molecular layer of the cerebellum along the Bergman glial fibers (open arrow) and membranes of the Purkinje cells (closed arrow). No staining was observed in 24JK parental cells or in mammary and brain tissues from nontransgenic mouse littermates. Sections are representative of 5 mice analyzed. B and C, Her-2 transgenic mice were irradiated (5 Gy) and treated with 24JK-Her-2 tumor cells (1 × 106) at day 0, followed by adoptively transferred, transduced T cells (1 × 107 per dose) at days 0 and 1 and IL-2 (9 doses of 50,000 IU between days 0 and 4). Mammary and brain tissues were harvested and stained with H&E at days 18 and 50. Analysis of tissue sections revealed no structural damage or increased immune cell infiltration for mice treated with chimeric receptor–transduced T cells in mammary or brain tissues at day 18 (B) or at the later time point day 50 (C) and were morphologically similar to sections from mice treated with control T cells or nontreated mice. Mammary and brain sections are representative of 5 mice analyzed for each time point.

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The above-described model closely approximates the clinical setting in which the Her-2 antigen is overexpressed in ∼30% of breast cancer patients as well as several other cancers including ovarian, lung, and head and neck cancer (28–30) while also present on normal tissue including those of epithelial, mesenchymal, and neuronal origin (26, 27). In this study, we showed that anti-Her-2 T cells could significantly impact on 24JK-Her-2 lung metastases in Her-2 transgenic mice compared with control T cells.

An important consideration for the therapeutic use of adoptively transferred, gene-engineered T cells is whether they may induce extensive autoimmune damage to normal tissues expressing the target antigen (31). In this study, we investigated whether the transfer of gene-modified T cells in combination with lymphoablation and IL-2 into tumor-bearing Her-2 transgenic mice caused any autoimmune toxicity to brain and/or mammary tissues expressing the Her-2 target antigen. In these experiments, no autoimmune damage to these tissues was observed by engineered T cells at early and late time points analyzed. The fact that we did not observe any autoimmunity may be in part due to differences in Her-2 antigen levels between tumor and normal brain and mammary tissues. Our immunohistochemical analysis indicated comparably stronger Her-2 antigen staining on 24JK-Her-2 tumor cells relative to brain and mammary tissues, which is generally the case seen in the patient setting. Our results correlate well with a recent report showing no overt toxicity in mice following adoptive transfer of scFv-transduced T cells targeting mouse CD19 antigen, although in this study, scFv-transduced T cells did not persist long term in mice. This may have been due to lack of costimulation with the use of a chimeric receptor that contained only the CD3-ζ signaling domain (19). In the present study, we used an scFv receptor containing both the CD28 costimulatory and CD3-ζ domains. Our results are also consistent with another study that showed that nonmyeloablative preconditioning and adoptive transfer of carcinoembryonic antigen–specific, MHC-restricted, T cells mediated antitumor activity without associated autoimmunity (32). However, autoimmunity was observed in this model following increased lymphoablation at a myeloablative dose (9.5 Gy) or the addition of anti-IL-10 receptor blocking antibody, indicating that the type of immunomodulation employed must be treated with caution.

Despite these reports, several studies have reported mild to severe autoimmune toxicity following transfer of tumor reactive T cells (33–35) including liver toxicity in patients involving transfer of T cells gene-modified with an anti-CAIX scFv receptor (13). Toxicity, manifested by increased levels of transaminase in serum, was thought to be due to CAIX expression on bile duct epithelium. Resolution of toxicity was achieved by cessation of therapy and administration of corticosteroids.

A recent trial involving adoptive transfer of gene-modified T cells targeting the Her-2 antigen resulted in the death of a patient 5 days after treatment; this might have been due to a cytokine storm resulting from recognition of Her-2 on normal tissue (14). Pulmonary edema occurred within 1 hour of cell infusion requiring intubation. Hypotension, bradycardia, and gastrointestinal bleeding led to cardiac arrest and death. The herceptin-based scFv receptor utilized in this trial incorporated the CD28, 4-1BB, and CD3-ζ domains, which may have led to increased secretion of cytokines from T cells following antigen recognition and subsequent toxicity (14). Interestingly, in another study, no toxicity was reported in patients following transfer of autologous anti-Her-2 CTL clones targeting the same antigen (36).

In another phase I clinical study, a serious adverse event involving death of a patient was observed following adoptive transfer of T cells gene-modified to respond against CD19 (37). The patient, with chronic lymphocytic leukemia, received (1.2–3.0) × 107 T cells/kg following cyclophosphamide conditioning. Hypotension and acute renal failure led to respiratory arrest. However, a definitive role for the infused T cells was not indicated in this case and death was thought to be as a result of sepsis due to infection leading to hypotension.

Collectively, studies both in preclinical mouse models and in patients have indicated that the location and levels of antigen expressed on normal tissue, the number of T cells transferred, the type of signaling domains incorporated into chimeric receptors, and the level of immune preconditioning used have to be carefully considered for adoptively transferred T-cell therapy. Given the potential severity of autoimmunity that has been reported in some studies, it would be prudent in the future to use scFv chimeric receptors targeting carefully selected antigens that are either tumor-specific or expressed at relatively low levels on normal tissue compared with tumor. In support of this idea, we have found that T cells engineered with an scFv-anti-LewisY chimeric receptor could strongly react with tumor cells expressing high LewisY antigen but did not react against endogenous neutrophils expressing low levels of LewisY (8). The possibility of redirecting T cells to multiple tumor-associated antigens expressed on tumor cells may further enhance their specificity for tumor while minimizing their activity against healthy tissue. Another alternative may be the inclusion of a regulated “suicide” gene function such as hsv-tk or the cytoplasmic domain of Fas or an inducible caspase incorporated into genetically engineered T cells to abort any aberrant T-cell responses (38–40).

In summary, this study has demonstrated that transfer of gene-engineered T cells combined with lymphoablation and IL-2 could mediate significant antitumor effects in a self-antigen setting without causing damaging autoimmune effects. These results suggest that redirected T-cell therapy has great potential as a safe and viable option for the treatment of cancer patients in the future.

No potential conflicts of interest were disclosed.

The authors thank the Peter MacCallum Cancer Centre Experimental Animal Facility technicians for animal care. They also thank pathologist Fenella Long for analysis of H&E sections of brain and mammary tissues following therapy.

Grant Support This work was funded by Project Grants from the National Health and Medical Research Council (NHMRC), Cancer Council of Victoria, Bob Parker Memorial Fund, and the Susan Komen Breast Cancer Foundation. M.H. Kershaw and P.K. Darcy were supported by an NHMRC Senior Research Fellowship and Career Development Award, respectively. M.J. Smyth and J.A. Trapani were supported by an NHMRC Australia Research Fellowship and Senior Principal Research Fellowship, respectively.

1.
Rosenberg
SA
,
Yang
JC
,
Restifo
NP
. 
Cancer immunotherapy: moving beyond current vaccines
.
Nat Med
2004
;
10
:
909
15
.
2.
Malmberg
KJ
. 
Effective immunotherapy against cancer: a question of overcoming immune suppression and immune escape?
Cancer Immunol Immunother
2004
;
53
:
879
92
.
3.
Blattman
JN
,
Greenberg
PD
. 
Cancer immunotherapy: a treatment for the masses
.
Science
2004
;
305
:
200
5
.
4.
Kershaw
MH
,
Jackson
JT
,
Haynes
NM
, et al
Gene-engineered T cells as a superior adjuvant therapy for metastatic cancer
.
J Immunol
2004
;
173
:
2143
50
.
5.
Kershaw
MH
,
Westwood
JA
,
Hwu
P
. 
Dual-specific T cells combine proliferation and antitumor activity
.
Nat Biotechnol
2002
;
20
:
1221
7
.
6.
Cheadle
EJ
,
Gilham
DE
,
Thistlethwaite
FC
,
Radford
JA
,
Hawkins
RE
. 
Killing of non-Hodgkin lymphoma cells by autologous CD19 engineered T cells
.
Br J Haematol
2005
;
129
:
322
32
.
7.
Maher
J
,
Brentjens
RJ
,
Gunset
G
,
Riviere
I
,
Sadelain
M
. 
Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta/CD28 receptor
.
Nat Biotechnol
2002
;
20
:
70
5
.
8.
Westwood
JA
,
Smyth
MJ
,
Teng
MW
,
Moeller
M
,
Trapani
JA
,
Scott
AM
, et al
Adoptive transfer of T cells modified with a humanized chimeric receptor gene inhibits growth of Lewis-Y-expressing tumors in mice
.
Proc Natl Acad Sci U S A
2005
;
102
:
19051
6
.
9.
Hombach
A
,
Schlimper
C
,
Sievers
E
,
Frank
S
,
Schild
HH
,
Sauerbruch
T
, et al
A recombinant anti-carcinoembryonic antigen immunoreceptor with combined CD3zeta-CD28 signalling targets T cells from colorectal cancer patients against their tumour cells
.
Gut
2006
;
55
:
1156
64
.
10.
Moeller
M
,
Haynes
NM
,
Kershaw
MH
,
Jackson
JT
,
Teng
MW
,
Street
SE
, et al
Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection
.
Blood
2005
;
106
:
2995
3003
.
11.
Ridolfi
L
,
Ridolfi
R
,
Riccobon
A
,
De Paola
F
,
Petrini
M
,
Stefanelli
M
, et al
Adjuvant immunotherapy with tumor infiltrating lymphocytes and interleukin-2 in patients with resected stage III and IV melanoma
.
J Immunother
2003
;
26
:
156
62
.
12.
Johnson
LA
,
Morgan
RA
,
Dudley
ME
,
Cassard
L
,
Yang
JC
,
Hughes
MS
, et al
Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen
.
Blood
2009
;
114
:
535
46
.
13.
Lamers
CH
,
Sleijfer
S
,
Vulto
AG
,
Kruit
WH
,
Kliffen
M
,
Debets
R
, et al
Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience
.
J Clin Oncol
2006
;
24
:
e20
2
.
14.
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
.
15.
Piechocki
MP
,
Ho
YS
,
Pilon
S
,
Wei
WZ
. 
Human ErbB-2 (Her-2) transgenic mice: a model system for testing Her-2 based vaccines
.
J Immunol
2003
;
171
:
5787
94
.
16.
Wels
W
,
Harwerth
IM
,
Mueller
M
,
Groner
B
,
Hynes
NE
. 
Selective inhibition of tumor cell growth by a recombinant single-chain antibody toxin specific for the erbB-2 receptor
.
Cancer Res
1992
;
52
:
6310
7
.
17.
Darcy
PK
,
Haynes
NM
,
Snook
MB
,
Trapani
JA
,
Cerruti
L
,
Jane
SM
, et al
Redirected perforin-dependent lysis of colon carcinoma by ex vivo genetically engineered CTL
.
J Immunol
2000
;
164
:
3705
12
.
18.
Haynes
NM
,
Snook
MB
,
Trapani
JA
,
Cerruti
L
,
Jane
SM
,
Smyth
MJ
, et al
Redirecting mouse CTL against colon carcinoma: superior signaling efficacy of single-chain variable domain chimeras containing TCR-zeta vs Fc epsilon RI-gamma
.
J Immunol
2001
;
166
:
182
7
.
19.
Cheadle
EJ
,
Hawkins
RE
,
Batha
H
,
O'Neill
AL
,
Dovedi
SJ
,
Gilham
DE
. 
Natural expression of the CD19 antigen impacts the long-term engraftment but not antitumor activity of CD19-specific engineered T cells
.
J Immunol
2010
;
184
:
1885
96
.
20.
Haynes
NM
,
Trapani
JA
,
Teng
MW
,
Jackson
JT
,
Cerruti
L
,
Jane
SM
, et al
Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors
.
Blood
2002
;
100
:
3155
63
.
21.
Moeller
M
,
Haynes
NM
,
Trapani
JA
,
Teng
MW
,
Jackson
JT
,
Tanner
JE
, et al
A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells
.
Cancer Gene Ther
2004
;
11
:
371
9
.
22.
Dudley
ME
,
Wunderlich
JR
,
Yang
JC
,
Sherry
RM
,
Topalian
SL
,
Restifo
NP
, et al
Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma
.
J Clin Oncol
2005
;
23
:
2346
57
.
23.
Chang
AE
,
Shu
SY
,
Chou
T
,
Lafreniere
R
,
Rosenberg
SA
. 
Differences in the effects of host suppression on the adoptive immunotherapy of subcutaneous and visceral tumors
.
Cancer Res
1986
;
46
:
3426
30
.
24.
Hu
HM
,
Poehlein
CH
,
Urba
WJ
,
Fox
BA
. 
Development of antitumor immune responses in reconstituted lymphopenic hosts
.
Cancer Res
2002
;
62
:
3914
9
.
25.
Lenz
DC
,
Kurz
SK
,
Lemmens
E
,
Schoenberger
SP
,
Sprent
J
,
Oldstone
MB
, et al
IL-7 regulates basal homeostatic proliferation of antiviral CD4+ T cell memory
.
Proc Natl Acad Sci U S A
2004
;
101
:
9357
62
.
26.
Press
MF
,
Cordon-Cardo
C
,
Slamon
DJ
. 
Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues
.
Oncogene
1990
;
5
:
953
62
.
27.
Olayioye
MA
. 
Update on HER-2 as a target for cancer therapy: intracellular signaling pathways of ErbB2/HER-2 and family members
.
Breast Cancer Res
2001
;
3
:
385
9
.
28.
Slamon
DJ
,
Godolphin
W
,
Jones
LA
,
Holt
JA
,
Wong
SG
,
Keith
DE
, et al
Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer
.
Science
1989
;
244
:
707
12
.
29.
Yu
D
,
Hung
MC
. 
Overexpression of ErbB2 in cancer and ErbB2-targeting strategies
.
Oncogene
2000
;
19
:
6115
21
.
30.
Tzahar
E
,
Yarden
Y
. 
The ErbB-2/HER2 oncogenic receptor of adenocarcinomas: from orphanhood to multiple stromal ligands
.
Biochim Biophys Acta
1998
;
1377
:
M25
37
.
31.
de Witte
MA
,
Coccoris
M
,
Wolkers
MC
,
van den Boom
MD
,
Mesman
EM
,
Song
JY
, et al
Targeting self-antigens through allogeneic TCR gene transfer
.
Blood
2006
;
108
:
870
7
.
32.
Bos
R
,
van Duikeren
S
,
Morreau
H
,
Van den boom
MD
,
Mesman
EM
,
Song
JY
, et al
Balancing between antitumor efficacy and autoimmune pathology in T-cell-mediated targeting of carcinoembryonic antigen
.
Cancer Res
2008
;
68
:
8446
55
.
33.
Ugel
S
,
Scarselli
E
,
Iezzi
M
,
Mennuni
C
,
Pannellini
T
,
Calvaruso
F
, et al
Autoimmune B-cell lymphopenia after successful adoptive therapy with telomerase-specific T lymphocytes
.
Blood
2010
;
115
:
1374
84
.
34.
Yee
C
,
Thompson
JA
,
Roche
P
,
Byrd
DR
,
Lee
PP
,
Piepkorn
M
, et al
Melanocyte destruction after antigen-specific immunotherapy of melanoma: direct evidence of t cell-mediated vitiligo
.
J Exp Med
2000
;
192
:
1637
44
.
35.
Palmer
DC
,
Chan
CC
,
Gattinoni
L
,
Wrzesinski
C
,
Paulos
CM
,
Hinrichs
CS
, et al
Effective tumor treatment targeting a melanoma/melanocyte-associated antigen triggers severe ocular autoimmunity
.
Proc Natl Acad Sci U S A
2008
;
105
:
8061
6
.
36.
Bernhard
H
,
Neudorfer
J
,
Gebhard
K
,
Conrad
H
,
Hermann
C
,
Nahrig
J
, et al
Adoptive transfer of autologous, HER2-specific, cytotoxic T lymphocytes for the treatment of HER2-overexpressing breast cancer
.
Cancer Immunol Immunother
2008
;
57
:
271
80
.
37.
Brentjens
R
,
Yeh
R
,
Bernal
Y
,
Riviere
I
,
Sadelain
M
. 
Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial
.
Mol Ther
2010
;
18
:
666
8
.
38.
Cohen
JL
,
Boyer
O
,
Thomas-Vaslin
V
,
Klatzmann
D
. 
Suicide gene-mediated modulation of graft-versus-host disease
.
Leuk Lymphoma
1999
;
34
:
473
80
.
39.
Thomis
DC
,
Marktel
S
,
Bonini
C
,
Traversari
C
,
Gilman
M
,
Bordignon
C
, et al
A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease
.
Blood
2001
;
97
:
1249
57
.
40.
Tey
SK
,
Dotti
G
,
Rooney
CM
,
Heslop
HE
,
Brenner
MK
. 
Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation
.
Biol Blood Marrow Transplant
2007
;
13
:
913
24
.