Noriko Kawashima

Fractionated but Not Single-Dose Radiotherapy Induces an Immune-Mediated Abscopal Effect when Combined with Anti–CTLA-4 Antibody

Purpose: This study tested the hypothesis that the type of dose fractionation regimen determines the ability of radiotherapy to synergize with anti–CTLA-4 antibody. Experimental Design: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice at two separate sites, defined as a “primary” site that was irradiated and a “secondary” site outside the radiotherapy field. When both tumors were palpable, mice were randomly assigned to eight groups receiving no radiotherapy or three distinct regimens of radiotherapy (20 Gy × 1, 8 Gy × 3, or 6 Gy × 5 fractions in consecutive days) in combination or not with 9H10 monoclonal antibody against CTLA-4. Mice were followed for tumor growth/regression. Similar experiments were conducted in the MCA38 mouse colon carcinoma model. Results: In either of the two models tested, treatment with 9H10 alone had no detectable effect. Each of the radiotherapy regimens caused comparable growth delay of the primary tumors but had no effect on the secondary tumors outside the radiation field. Conversely, the combination of 9H10 and either fractionated radiotherapy regimens achieved enhanced tumor response at the primary site (P < 0.0001). Moreover, an abscopal effect, defined as a significant growth inhibition of the tumor outside the field, occurred only in mice treated with the combination of 9H10 and fractionated radiotherapy (P < 0.01). The frequency of CD8+ T cells showing tumor-specific IFN-γ production was proportional to the inhibition of the secondary tumor. Conclusions: Fractionated but not single-dose radiotherapy induces an abscopal effect when in combination with anti–CTLA-4 antibody in two preclinical carcinoma models. (Clin Cancer Res 2009;15(17):5379–88)

Radiation-Induced CXCL16 Release by Breast Cancer Cells Attracts Effector T Cells

Recruitment of effector T cells to inflamed peripheral tissues is regulated by chemokines and their receptors, but the factors regulating recruitment to tumors remain largely undefined. Ionizing radiation (IR) therapy is a common treatment modality for breast and other cancers. Used as a cytocidal agent for proliferating cancer cells, IR in combination with immunotherapy has been shown to promote immune-mediated tumor destruction in preclinical studies. In this study we demonstrate that IR markedly enhanced the secretion by mouse and human breast cancer cells of CXCL16, a chemokine that binds to CXCR6 on Th1 and activated CD8 effector T cells, and plays an important role in their recruitment to sites of inflammation. Using a poorly immunogenic mouse model of breast cancer, we found that irradiation increased the migration of CD8+CXCR6+ activated T cells to tumors in vitro and in vivo. CXCR6-deficient mice showed reduced infiltration of tumors by activated CD8 T cells and impaired tumor regression following treatment with local IR to the tumor and Abs blocking the negative regulator of T cell activation, CTLA-4. These results provide the first evidence that IR can induce the secretion by cancer cells of proinflammatory chemotactic factors that recruit antitumor effector T cells. The ability of IR to convert tumors into “inflamed” peripheral tissues could be exploited to overcome obstacles at the effector phase of the antitumor immune response and improve the therapeutic efficacy of immunotherapy.

Suppressing T cell motility induced by anti–CTLA-4 monotherapy improves antitumor effects

A promising strategy for cancer immunotherapy is to disrupt key pathways regulating immune tolerance, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4). However, the determinants of response to anti-CTLA-4 mAb treatment remain incompletely understood. In murine models, anti-CTLA-4 mAbs alone fail to induce effective immune responses to poorly immunogenic tumors but are successful when combined with additional interventions, including local ionizing radiation (IR) therapy. We employed an established model based on control of a mouse carcinoma cell line to study endogenous tumor-infiltrating CD8+ T lymphocytes (TILs) following treatment with the anti-CTLA-4 mAb 9H10. Alone, 9H10 monotherapy reversed the arrest of TILs with carcinoma cells in vivo. In contrast, the combination of 9H10 and IR restored MHC class I-dependent arrest. After implantation, the carcinoma cells had reduced expression of retinoic acid early inducible-1 (RAE-1), a ligand for natural killer cell group 2D (NKG2D) receptor. We found that RAE-1 expression was induced by IR in vivo and that anti-NKG2D mAb blocked the TIL arrest induced by IR/9H10 combination therapy. These results demonstrate that anti-CTLA-4 mAb therapy induces motility of TIL and that NKG2D ligation offsets this effect to enhance TILs arrest and antitumor activity.

The combination of ionizing radiation and peripheral vaccination produces long-term survival of mice bearing established invasive GL261 gliomas.

Purpose: High-grade glioma treatment includes ionizing radiation therapy. The high invasiveness of glioma cells precludes their eradication and is responsible for the dismal prognosis. Recently, we reported the down-regulation of MHC class I (MHC-I) products in invading tumor cells in human and mouse GL261 gliomas. Here, we tested the hypothesis that whole-brain radiotherapy (WBRT) up-regulates MHC-I expression on GL261 tumors and enhances the effectiveness of immunotherapy. Experimental Design: MHC-I molecule expression on GL261 cells was analyzed in vitro and in vivo by flow cytometry and immunohistochemistry, respectively. To test the response of established GL261 gliomas to treatment, mice with measurable (at CT imaging) brain tumors were randomly assigned to four groups receiving (a) no treatment, (b) WBRT in two fractions of 4 Gy, (c) vaccination with irradiated GL261 cells secreting granulocyte-macrophage colony-stimulating factor, or (d) WBRT and vaccination. Endpoints were tumor response and survival. Results: An ionizing radiation dose of 4 Gy maximally up-regulated MHC-I molecules on GL261 cells in vitro. In vivo, WBRT induced the expression of the β2-microglobulin light chain subunit of the MHC class I complex on glioma cells invading normal brain and increased CD4+ and CD8+ T cell infiltration. However, the survival advantage obtained with WBRT or vaccination alone was minimal. In contrast, WBRT in combination with vaccination increased long-term survival to 40% to 80%, compared with 0% to 10% in the other groups (P < 0.002). Surviving animals showed antitumor immunity by rejecting challenge tumors. Conclusion: Ionizing radiation can be successfully combined with peripheral vaccination for the treatment of established high-grade gliomas.

Synergy of topical Toll-Like Receptor 7 agonist with radiation and low dose cyclophosphamide in a mouse model of cutaneous breast cancer

Purpose: This study tested the hypothesis that topical Toll-like receptor (TLR) 7 agonist imiquimod promotes antitumor immunity and synergizes with other treatments in a model of skin-involving breast cancer. Experimental Design: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice. Imiquimod 5% or placebo cream was applied topically on the shaved skin overlying tumors three times/wk. In some experiments, local ionizing radiation therapy (RT) was delivered to the tumor in three fractions of 8 Gy, given on consecutive days. Cyclophosphamide was given intraperitoneally (i.p.) in one dose of 2 mg/mouse. Mice were followed for tumor growth and survival. Results: Treatment with imiquimod significantly inhibited tumor growth, an effect that was associated with increased tumor infiltration by CD11c+, CD4+, and CD8+ cells, and abolished by depletion of CD8+ cells. Administration of imiquimod in combination with RT enhanced significantly tumor response compared with either treatment alone (P < 0.005), and 11% to 66% of irradiated tumors completely regressed. Importantly, the addition of topical imiquimod also resulted in growth inhibition of a secondary tumor outside of the radiation field. Low-dose cyclophosphamide given before start of treatment with imiquimod and RT further improved tumor inhibition and reduced tumor recurrence. Mice that remained tumor-free rejected a tumorigenic inoculum of TSA cells, showing long-term immunologic memory. Conclusions: Topical imiquimod inhibits tumor growth and synergizes with RT. Addition of cyclophosphamide further increases the therapeutic effect and induces protective immunologic memory, suggesting that this combination is a promising strategy for cutaneous breast cancer metastases. Clin Cancer Res; 18(24); 6668–78. ©2012 AACR.

Invariant Natural Killer T Cells Regulate Breast Cancer Response to Radiation and CTLA-4 Blockade

Purpose: Immunoregulatory and suppressive mechanisms represent major obstacles to the success of immunotherapy in cancer patients. We have shown that the combination of radiotherapy to the primary tumor and CTL-associated protein 4 (CTLA-4) blockade induces antitumor immunity, inhibiting metastases and extending the survival of mice bearing the poorly immunogenic and highly metastatic 4T1 mammary carcinoma. Similarly to patients with metastatic cancer, however, mice were seldom cured. Here we tested the hypothesis that invariant natural killer T (iNKT) cells, a subset with unique regulatory functions, can regulate the response to radiotherapy and CTLA-4 blockade. Experimental Design: The growth of 4T1 primary tumors and lung metastases was compared in wild-type and iNKT cell–deficient (iNKT-/-) mice. Treatment was started on day 13 when the primary tumors were palpable. Mice received radiotherapy to the primary tumor in two doses of 12 Gy in combination or not with 9H10 monoclonal antibody against CTLA-4. Response to treatment was assessed by measuring primary tumor growth delay/regression, survival, and number of lung metastases. Results: The response to radiotherapy plus 9H10 was markedly enhanced in the absence of iNKT cells, with 50% of iNKT-/- versus 0% of wild-type mice showing complete tumor regression, long-term survival, and resistance to a challenge with 4T1 cells. Administration of the iNKT cell activator α-galactosylceramide did not enhance the response of wild-type mice to radiotherapy plus 9H10. Tumor-infiltrating iNKT cells were markedly reduced in wild-type mice treated with radiotherapy plus 9H10. Conclusions: iNKT cells play a major role in regulating the response to treatment with local radiotherapy and CTLA-4 blockade.

Radiotherapy Enhances Antitumor Effect of Anti-CD137 Therapy in a Mouse Glioma Model

Abstract Previously, we reported that peripheral vaccination of mice with modified autologous tumor cells secreting granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with ionizing radiation to the whole brain cured 50% of mice using a syngeneic, intracranial model of murine high-grade glioma. Here, we tested the combination of radiotherapy (4 Gy × 2) with an immunotherapeutic approach using an anti-CD137 antibody directed to the co-stimulatory molecule CD137. The CD137 antibody has shown promise in generating effective antitumor responses in several animal models and has demonstrated a favorable toxicity profile in the clinic. The combination of radiation and anti-CD137 therapy resulted in complete tumor eradication and prolonged survival in six of nine (67%) mice with established brain tumors (P  =  0.0009). Five of six (83%) long-term survivors in the combination group demonstrated antitumor immunity by rejecting challenge tumors. Antitumor immunity was associated with an increased number of tumor-infiltrating lymphocytes (TILs) in brain tumors and increased tumor-specific production of γIFN. In view of the finding that radiation enhanced the antitumor effect of anti-CD137 therapy, this approach should be studied further for clinical translation.

Adenosine regulates radiation therapy-induced anti-tumor immunity

Radiation therapy (RT) induces immunogenic cell death and dose-dependent release of ATP in the tumor microenvironment (TME), triggering maturation and activation of tumor-resident dendritic cells (DCs). However, extracellular ATP is rapidly catabolized to adenosine by ectonucleotidases CD39 and CD73, which are expressed by tumor cells and immune cells in the TME. Adenosine has pleiotropic immunosuppressive effects and inhibits activation of DC and effector T cells, while promoting regulatory T cells (Tregs). Here, we tested the hypothesis that conversion of ATP to adenosine hinders generation of effective anti-tumor immunity by high dose RT, reducing its synergy with anti-CTLA-4 antibody. BALB/c mice were inoculated s.c. with 1 x 105 TSA carcinoma cells on day 0 and assigned to treatment with: (1) control mAb; (2) anti-CD73 (TY/23); (3) TY/23+anti-CTLA-4 (9H10); (4) RT; (5) RT+TY/23; (6) RT+9H10; (6) RT+TY/23+9H10. TY/23 (200 µg) was administered i.p. every 4 days starting on day 11. RT was given locally as single 20 Gy dose on day 12. 9H10 (200 µg) was given i.p. on days 11, 14 and 17. On day 18, some tumors were harvested for flow cytometry analysis of DC and T cells. Mice were monitored for tumor growth/regression. In irradiated tumors, CD73-blockade reduced the percentage of Tregs within the tumor-infiltrating CD4+ T cell population (7.9±2.5% in RT+TY/23 vs 20±0.8% in RT, p < 0.01) while increasing CD8+T cells (38.3±0.1% in RT+TY/23 vs 17.3±4% in RT, p < 0.05). Among intratumoral DCs (CD11c+MHCII+), the CD8a+ DC subpopulation was increased after CD73-blockade (37.9±15.7% in TY/23+RT vs 11.3±4.9% in RT, p < 0.01). Importantly, in irradiated mice, TY/23-administration enhanced activation of DCs and effector T cells, shown by increased CD40 expression on CD8a+ DCs (MFI: 218±1 in RT+TY/23 vs 54±41 in RT, p < 0.05) and increased CD69 expression on CD8+ T cells (MFI: 513±126 in RT+TY/23 vs 148±59 in RT, p < 0.01). TY/23 and 9H10 given alone or in combination had no effect on tumor growth. However, each antibody potentiated tumor inhibition obtained with RT (p=0.08 for RT+TY/23 and p < 0.05 for RT+9H10 vs RT). Moreover, blockade of both CD73 and CTLA-4 in combination with RT further improved tumor control resulting in complete tumor regression in 2/5 mice (p < 0.01 for RT+TY/23+9H10 vs RT). Our findings indicate that adenosine regulates the ability of RT to induce anti-tumor immunity, affecting both DC maturation and T cell activation. Data suggest that CD73-blockade is a promising strategy to improve synergy of RT and immunotherapy.

Abstract A21: Adenosine is a rheostat for radiation therapy-induced immune activation

Background: Administration of anti-CTLA-4 antibodies has shown clinical benefit in a small percentage of cancer patients. However, most patients are unresponsive to anti-CTLA-4 antibody treatment, due to immunosuppressive mechanisms and/or the absence of sufficient spontaneous anti-tumor T cells that can be activated by CTLA-4 blockade. We have previously shown that radiotherapy (RT) induces immunogenic tumor cell death and priming of anti-tumor T cells making tumors susceptible to anti-CTLA-4 treatment. RT also triggers the release of ATP in the tumor microenvironment, which has proinflammatory effects. However, ATP is rapidly converted into adenosine, a pleiotropic immunosuppressive mediator, by ectonucleotidases CD39 and CD73 expressed on cancer cells and tumor stromal cells. We hypothesized that adenosine generation in the tumor microenvironment acts as a rheostat modulating RT ability to prime effective anti-tumor immunity. To test this hypothesis we used a mouse tumor model where we have previously characterized two RT regimens that were efficient (8Gy x 3) or inefficient (20 Gy) in inducing anti-tumor immunity in combination with anti-CTLA-4 antibodies. Method: BALB/c mice were inoculated s.c. with 1 x 105 TSA cells, a poorly immunogenic breast cancer cell line on day 0. Anti-CD73 mAb TY/23 (200 µg) was administered i.p. every 4 days starting on day 11. RT was given locally to the tumor as a single 20 Gy dose on day 12 or in three doses of 8 Gy each given on day 12, 13 and 14. At day 19, tumors were harvested and infiltrating immune cells were isolated for analysis of DC and T cell phenotype by flow cytometry. In another experiment, mice were randomly assigned (n=5/group) to be treated with: (1) Isotype control mAb (2) TY/23 (3) TY/23 + anti-CTLA-4 mAb 9H10 (4) RT 20 Gy (5) RT 20 Gy + TY/23 (6) RT 20 Gy + 9H10 (6) RT 20 Gy + TY/23+ 9H10. RT was given on day 12 and mAbs were administered i.p. on day 11, 14, 17 and 20 after tumor inoculation. Mice were monitored for tumor growth. Results: In tumors that had received 8GyX3 RT there was a four-fold increase in CD70 expression (MFI 436 ± 72 in 8GyX3 vs. 110 ± 30 in control, p Conclusions: Data indicate that the ability of RT to promote immune activation is dependent on the radiation regimen employed and is regulated by adenosine generation by the ectonucleotidase CD73. Importantly, data suggest that blocking CD73 can improve the ability of RT to synergize with immunotherapy. Citation Format: Erik Wennerberg, Noriko Kawashima, Sandra Demaria. Adenosine is a rheostat for radiation therapy-induced immune activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A21.

Local Radiotherapy Rescues Responsiveness to Anti-CD137 Immunotherapy in a Mouse Model of Advanced Breast Cancer.

BACKGROUND: The benefits of immunotherapy are often hampered by immunosuppressive networks in the tumor microenvironment. Accumulating evidence indicates that local ionizing radiation (IR) can synergize with immunotherapy and improve tumor response.CD137/4-1BB is a co-stimulatory molecule expressed on activated T-cells that provides a survival signal to promote the expansion and differentiation of antigen-specific T-cells. Pre-clinical studies have shown the ability of agonistic anti-CD137 antibodies to enhance anti-tumor immunity. 4T1 is a poorly immunogenic experimental mouse model of metastatic breast cancer. 4T1 cells injected s.c. form a tumor and rapidly spread systemically giving rise to lung metastases that kill the animals. We have previously shown that IR as single modality can delay growth of the irradiated 4T1 s.c. tumors but not extend mice survival.METHODS: BALB/c mice were inoculated s.c. with 4T1 cells on day 0. Treatment with anti-CD137 mAb (200 mg, BMS-469492 from Bristol Myers Squibb) was started on day 7 or 15 and repeated every 3 days for two or three times. For radiotherapy mice were randomized into 4 groups receiving (1) no treatment, (2) IR alone, (3) anti-CD137 mAb and (4) IR+ anti-CD137 mAb. IR was delivered exclusively to the tumor in 2 fractions of 12 Gy at 24 hours interval starting on day 13. Mice were followed for tumor growth and survival.RESULTS: Administration of anti-CD137 mAb to 4T1 tumor-bearing mice starting at day 7 resulted in significant tumor growth delay which correlated with an 8-fold increase in the ratio of intratumural effector CD8 T cells (Teff) to regulatory T cells (Treg) at day 15. In contrast, when administration of anti-CD137 mAb was delayed until day 15 no anti-tumor effect was observed.The median survival of mice (39 days) was not increased by treatment with anti-CD137 mAb or IR used as single modalities (39 days). On the other hand, the combination of IR and anti-CD137 mAb increased survival to 49 days, although no complete tumor remission was observed.CONCLUSIONS:Overall, our studies indicate that with tumor progression mice became resistant to immunotherapy with single modality anti-CD137 mAb. However, a therapeutic effect was seen when anti-CD137 mAb was combined with local radiotherapy. We are currently investigating the mechanisms of resistance to anti-CD137 mAb treatment that develop with 4T1 tumor progression. Accumulation of Treg and myeloid-derived suppressor cells (MDSC) within the tumors likely contribute to resistance to immunotherapy by creating a highly immunosuppressive microenvironment. Identification of these mechanisms will provide new therapeutic targets to improve the response to treatment. Importantly, data support the combination of local radiotherapy with immunotherapy in advanced breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4134.