Jing Liu

Suppression of Metastases Using a New Lymphocyte Checkpoint Target for Cancer Immunotherapy

UNLABELLED CD96 has recently been shown as a negative regulator of mouse natural killer (NK)-cell activity, with Cd96(-/-)mice displaying hyperresponsive NK cells upon immune challenge. In this study, we have demonstrated that blocking CD96 with a monoclonal antibody inhibited experimental metastases in three different tumor models. The antimetastatic activity of anti-CD96 was dependent on NK cells, CD226 (DNAM-1), and IFNγ, but independent of activating Fc receptors. Anti-CD96 was more effective in combination with anti-CTLA-4, anti-PD-1, or doxorubicin chemotherapy. Blocking CD96 in Tigit(-/-)mice significantly reduced experimental and spontaneous metastases compared with its activity in wild-type mice. Co-blockade of CD96 and PD-1 potently inhibited lung metastases, with the combination increasing local NK-cell IFNγ production and infiltration. Overall, these data demonstrate that blocking CD96 is a new and complementary immunotherapeutic strategy to reduce tumor metastases. SIGNIFICANCE This article illustrates the antimetastatic activity and mechanism of action of an anti-CD96 antibody that inhibits the CD96-CD155 interaction and stimulates NK-cell function. Targeting host CD96 is shown to complement surgery and conventional immune checkpoint blockade.

Improved Efficacy of Neoadjuvant Compared to Adjuvant Immunotherapy to Eradicate Metastatic Disease

Immunotherapy has recently entered a renaissance phase with the approval of multiple agents for the treatment of cancer. Immunotherapy stands ready to join traditional modalities, including surgery, chemotherapy, radiation, and hormone therapy, as a pillar of cancer treatment. Although immunotherapy has begun to have success in advanced cancer treatment, its scheduling and efficacy with surgery to treat earlier stages of cancer and prevent distant metastases has not been systematically examined. Here, we have used two models of spontaneously metastatic breast cancers in mice, to illustrate the significantly greater therapeutic power of neoadjuvant, compared with adjuvant immunotherapies in the context of primary tumor resection. Elevated and sustained peripheral tumor-specific immune responses underpinned the outcome, and blood sampling of tumor-specific CD8+ T cells immediately prior and post surgery may provide a predictor of outcome. These data now provide strong rationale to extensively test and compare neoadjuvant immunotherapy in humans.

Improved mouse models to assess tumour immunity and irAEs after combination cancer immunotherapies

The current excitement surrounding cancer immunotherapy stems particularly from clinical data involving agents mediating immune checkpoint receptor blockade, which have induced unprecedented efficacy against a range of tumours compared with previous immunotherapeutic approaches. However, an important consideration in targeting checkpoint receptors has been the emergence of associated toxicities termed immune‐related adverse events (irAEs). In light of the clinical benefits observed after co‐blockade of checkpoint receptors and data from preclinical mouse models, there is now a strong rationale to combine different checkpoint receptors together, with other immunotherapies or more conventional therapies to assess if clinical benefits to cancer patients can be further improved. However, one may predict the frequency and severity of irAEs will increase with combinations, which may result in premature therapy cessation, thus limiting the realization of such an approach. In addition, there is a limit to how many different combination therapies that can be tested in a timely manner given the legal, regulatory and budgetary issues associated with conducting clinical trials. Thus, there is a need to develop preclinical mouse models that more accurately inform us as to which immunotherapies might combine best to provide the optimal therapeutic index (maximal anti‐tumour efficacy and low level irAEs) in different cancer settings. In this review we will discuss the irAEs observed in patients after checkpoint blockade and discuss which mouse models of cancer can be appropriate to assess the development of tumour immunity and irAEs following combination cancer immunotherapies.

Assessing Immune-Related Adverse Events of Efficacious Combination Immunotherapies in Preclinical Models of Cancer

New combination immunotherapies are displaying both efficacy and immune-related adverse events (irAE) in humans. However, grade 3/4 irAEs occur in a high proportion, which can lead to discontinuation of treatment and can result in fatalities if not promptly treated. Prolonged T regulatory cell (Treg) depletion in tumor-bearing Foxp3-DTR mice using diphtheria toxin (DT) mirrored the spectrum of antitumor responses and severity of irAEs that can occur in ipilimumab/nivolumab-treated patients. In contrast, transient Treg depletion or anti-CTLA-4/PD-1 therapy had equivalent effects in mice, lowering the immune tolerance threshold and allowing irAEs to be more easily induced following treatment with additional immunomodulatory antibodies. Transient Treg depletion of DT in combination with anti-PD-1 or anti-TIM-3 monoclonal antibodies had a high therapeutic window compared with DT plus anti-CD137. In contrast, DT plus anti-CD137-treated mice developed severe irAEs similar to grade 3/4 clinical symptoms. These irAEs appeared because of an infiltration of activated proliferating effector T cells in the tissues producing IFNγ and TNF; however, TNF blockade decreased irAEs severity without impacting on tumor growth. Cancer Res; 76(18); 5288-301. ©2016 AACR.

CD155 loss enhances tumor suppression via combined host and tumor-intrinsic mechanisms

Critical immune-suppressive pathways beyond programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) require greater attention. Nectins and nectin-like molecules might be promising targets for immunotherapy, since they play critical roles in cell proliferation and migration and exert immunomodulatory functions in pathophysiological conditions. Here, we show CD155 expression in both malignant cells and tumor-infiltrating myeloid cells in humans and mice. Cd155–/– mice displayed reduced tumor growth and metastasis via DNAM-1 upregulation and enhanced effector function of CD8+ T and NK cells, respectively. CD155-deleted tumor cells also displayed slower tumor growth and reduced metastases, demonstrating the importance of a tumor-intrinsic role of CD155. CD155 absence on host and tumor cells exerted an even greater inhibition of tumor growth and metastasis. Blockade of PD-1 or both PD-1 and CTLA4 was more effective in settings in which CD155 was limiting, suggesting the clinical potential of cotargeting PD-L1 and CD155 function.

Deficiency of host CD96 and PD-1 or TIGIT enhances tumor immunity without significantly compromising immune homeostasis

ABSTRACT Multiple non-redundant immunosuppressive pathways co-exist in the tumor microenvironment and their co-targeting can increase clinical responses. Indeed, concurrent blockade of CTLA-4 and PD-1 in patients with advanced melanoma increased clinical responses over monotherapy alone although the frequency and severity of immune related adverse events (irAEs) also increased. Nevertheless, a substantial number of patients still display an innate resistance phenotype and are unresponsive to current approved immunotherapies even when utilized in combination. In this study, we generated Pdcd1−/−CD96−/− and Tigit−/−CD96−/− mice to investigate how loss of CD96 in combination with PD-1 or TIGIT impacts on immune homeostasis and hence the potential of inducing immune related toxicities following co-targeting of these pairs of receptors. The ability of Pdcd1−/−CD96−/− and Tigit−/−CD96−/− mice to suppress primary tumor growth was also assessed using the MC38 colon carcinoma and SM1WT1 BRAF-mutated melanoma tumor models. Both Pdcd1−/−CD96−/− or Tigit−/−CD96−/− mice displayed no overt perturbations in immune homeostasis over what was previously reported with Pdcd1−/− or Tigit−/− mice even when aged for 22 months. Interestingly, increased suppression of subcutaneous tumor growth and complete responses was seen in Pdcd1−/−CD96−/− mice compared to Pdcd1−/− or CD96−/− mice depending upon the tumor model. In contrast, in these models, growth suppression in Tigit−/−CD96−/− were similar to Tigit−/− or CD96−/− . This enhanced anti-tumor efficacy of Pdcd1−/−CD96−/− appeared to be due to favorable changes in the ratio of CD8+ T cells to T regulatory cells or CD11b+GR-1hi myeloid cells in the tumor microenvironment. Co-targeting CD96 and PD-1 may increase anti-tumor immunity over targeting PD-1 alone and potentially not induce serious immune-related toxicities and thus appears a promising strategy for clinical development.

Experimental lung metastases in mice are more effectively inhibited by blockade of IL23R than IL23

IL23 is an inflammatory cytokine with protumoral effects and is present in the serum of patients with some malignancies. In three different mouse tumor models, blocking IL23R was more effective at suppressing metastases than was neutralizing IL23. Tumor-induced immunosuppression is mediated through various mechanisms including engagement of immune checkpoint receptors on effector cells, function of immunoregulatory cells such as regulatory T cells and myeloid-derived suppressor cells, and deployment of immunosuppressive cytokines such as TGFβ and IL10. IL23 is a cytokine that negatively affects antitumor immunity. In this study, we investigated whether IL23-deficient (IL23p19−/−) and IL23R-deficient (IL23R−/−) mice phenocopied each other, with respect to their tumor control. We found that IL23R−/− mice had significantly fewer lung metastases compared with IL23p19−/− mice across three different experimental lung metastasis models (B16F10, LWT1, and RM-1). Similarly, IL23R blocking antibodies were more effective than antibodies neutralizing IL23 in suppressing experimental lung metastases. The antimetastatic activity of anti-IL23R was dependent on NK cells and IFNγ but independent of CD8+ T cells, CD4+ T cells, activating Fc receptors, and IL12. Furthermore, our data suggest this increased antitumor efficacy was due to an increase in the proportion of IFNγ-producing NK cells in the lungs of B16F10 tumor-bearing mice. Anti-IL23R, but not anti-IL23p19, partially suppressed lung metastases in tumor-bearing mice neutralized for IL12p40. Collectively, our data imply that IL23R has tumor-promoting effects that are partially independent of IL23p19. Blocking IL23R may be more effective than neutralizing IL23 in the suppression of tumor metastases. Cancer Immunol Res; 6(8); 978–87. ©2018 AACR.

Abstract PR07: Use of a novel mouse model to investigate immune related adverse events arising from immunotherapies

While immunotherapy is rapidly growing as an excellent treatment for many cancer types, most immunotherapies, particularly in combination can induce immune related adverse events (irAEs) which are generally caused by immune cell activation and expansion. Although most irAEs are low grade (1-2), higher grade events (3-4) can prevent patients from completing therapy and can also be life threatening. Thus, there is a strong need to develop pre-clinical mouse models to identify which immunotherapeutic combinations induce the best anti-tumor responses without inducing severe irAEs. To replicate irAEs associated with immunotherapies in humans, we utilized the FoxP3-DTR mouse model where regulatory T-cell (Treg) depletion can be achieved by injecting diphtheria toxin (DT), releasing Treg associated immune suppression. Transient Treg depletion by a single dose of DT induced mild T-cell activation and increased T-cell immune checkpoint expression, making mice more sensitive to irAEs development from clinically relevant immunotherapies. In three tumor models, we demonstrated that transient Treg depletion with anti-PD-1 or TIM-3 had a good anti-tumor efficacy and mild irAEs. Conversely, combining transient Treg depletion with an antibody targeting the co-stimulatory receptor CD137 had a lower therapeutic window due to increased irAEs. This combination was effective in suppressing established tumors, but induced severe irAEs including elevated serum cytokines and acute liver inflammation. These irAEs were similar to those seen in clinical trials of anti-CD137 antibodies where liver toxicity was a major concern and were driven by a rapid systemic CD8+ T-cell expansion. We also demonstrated that irAEs from anti-CD137 therapy could be mitigated by neutralising TNF without impacting on anti-tumor efficacy. We believe this model can provide valuable data on the efficacy and irAEs associated with novel combination immunotherapies which may aid clinicians and pharmaceutical companies in clinical trial design. We are currently utilizing this model to predict the therapeutic irAEs and efficacy of other novel immunotherapies that are in pre-clinical and early stage clinical trials. This abstract is also being presented as Poster B58 . Citation Format: Stephen J. Blake, Jing Liu, Harjunpaa Heidi, Mark J. Smyth, Michele W.L. Teng. Use of a novel mouse model to investigate immune related adverse events arising from immunotherapies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR07.

Abstract B115: Improved efficacy of neoadjuvant compared to adjuvant immunotherapy to eradicate metastatic disease

Immunotherapy has recently entered a renaissance phase with the approval of multiple agents for the treatment of cancer. Immunotherapy stands ready to join traditional modalities, including surgery, chemotherapy, radiation, and hormone therapy, as a pillar of cancer treatment. Although immunotherapy has begun to have success in advanced cancer treatment, its scheduling and efficacy with surgery to treat earlier stages of cancer and prevent distant metastases have not been systematically examined. Here, we have used two models of spontaneously metastatic breast cancers in mice to illustrate the significantly greater therapeutic power of neoadjuvant, compared with adjuvant, immunotherapies in the context of primary tumor resection. Elevated and sustained peripheral tumor-specific immune responses underpinned the outcome, and blood sampling of tumor-specific CD8+ T cells immediately prior to and post surgery may provide a predictor of outcome. These data now provide a strong rationale to extensively test and compare neoadjuvant immunotherapy in humans. SIGNIFICANCE We demonstrate the significantly greater therapeutic efficacy of neoadjuvant, compared with adjuvant, immunotherapies to eradicate distant metastases following primary tumor resection. Elevated and sustained peripheral tumor-specific immune responses underpinned the outcome, and blood sampling of tumor-specific CD8+ T cells immediately prior to and post surgery may provide a predictor of outcome. Cancer Discov; 6(12); 1382-99. ©2016 AACR.See related commentary by Melero et al., p. 1312This article is highlighted in the In This Issue feature, p. 1293.

Abstract B122: A preclinical mouse model to assess antitumor efficacy and development of immune related adverse events (irAEs) following combination immunotherapies

Given that new immunotherapies in combination are displaying both efficacy and immune related adverse events (irAEs) in humans, there is a need to promptly develop a pre-clinical mouse model that can evaluate the therapeutic index (efficacy versus safety) of current and novel immunotherapies. We demonstrated that transient or prolonged T regulatory cell (Treg) depletion in tumor bearing Foxp3-DTR mice using diphtheria toxin (DT) mirrored the spectrum of anti-tumor responses and severity of irAEs that can occur in ipilimumab/nivolumab treated patients. We showed that transient Treg depletion with DT in combination with anti-PD-1 or anti-TIM-3 monoclonal antibodies (mAbs) had a high therapeutic index compared to DT plus anti-CD137. While all three mAbs suppressed tumor growth, tumor rejection was more frequently observed in the DT plus anti-PD-1 or DT plus anti-TIM-3 combinations, depending on the tumor type, and these mice developed only mild irAEs. In contrast, mice treated with DT plus anti-CD137 developed severe irAEs characterized by infiltration of activated T cells into peripheral organs, liver toxicities and elevated levels of inflammatory cytokines, similar to grade 3/4 clinical symptoms. These irAEs appeared due to an infiltration of activated proliferating effector T cells in the tissues producing IFNγ and TNF, however, TNF blockade decreased the severity of irAEs in these mice without impacting on tumor growth. This model may now be used to pre-clinically assess the therapeutic indices of novel immunotherapy combinations to understand their mechanism of action and safety. Citation Format: Jing Liu, Stephten J. Blake, Kirsten A. Fairfax, Michelle C.R. Yong, Stacey Allen, Mark J. Smyth, Michele W.L. Teng. A preclinical mouse model to assess antitumor efficacy and development of immune related adverse events (irAEs) following combination immunotherapies. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B122.