Tumor immunoevasion by the conversion of effector NK cells into type 1 innate lymphoid cells.

Avoiding destruction by immune cells is a hallmark of cancer, yet how tumors ultimately evade control by natural killer (NK) cells remains incompletely defined. Using global transcriptomic and flow-cytometry analyses and genetically engineered mouse models, we identified the cytokine-TGF-ß-signaling-dependent conversion of NK cells (CD49a-CD49b+Eomes+) into intermediate type 1 innate lymphoid cell (intILC1) (CD49a+CD49b+Eomes+) populations and ILC1 (CD49a+CD49b-Eomesint) populations in the tumor microenvironment. Strikingly, intILC1s and ILC1s were unable to control local tumor growth and metastasis, whereas NK cells favored tumor immunosurveillance. Experiments with an antibody that neutralizes the cytokine TNF suggested that escape from the innate immune system was partially mediated by TNF-producing ILC1s. Our findings provide new insight into the plasticity of group 1 ILCs in the tumor microenvironment and suggest that the TGF-ß-driven conversion of NK cells into ILC1s is a previously unknown mechanism by which tumors escape surveillance by the innate immune system.

Advancing translational research for colorectal immuno-oncology.

Colorectal cancer (CRC) is a common and deadly disease. Unfortunately, immune checkpoint inhibitors (ICIs) fail to elicit effective anti-tumour responses in the vast majority of CRC patients. Patients that are most likely to respond are those with DNA mismatch repair deficient (dMMR) and microsatellite instability (MSI) disease. However, reliable predictors of ICI response are lacking, even within the dMMR/MSI subtype. This, together with identification of novel mechanisms to increase response rates and prevent resistance, are ongoing and vitally important unmet needs. To address the current challenges with translation of early research findings into effective therapeutic strategies, this review summarises the present state of preclinical testing used to inform the development of immuno-regulatory treatment strategies for CRC. The shortfalls and advantages of commonly utilised mouse models of CRC, including chemically induced, transplant and transgenic approaches are highlighted. Appropriate use of existing models, incorporation of patient-derived data and development of cutting-edge models that recapitulate important features of human disease will be key to accelerating clinically relevant research in this area.

In vivo comparison of local versus systemic delivery of immunostimulating siRNA in HPV-driven tumours.

Small interfering RNAs (siRNAs) to inhibit oncogene expression and also to activate innate immune responses via Toll-like receptor (TLR) recognition have been shown to be beneficial as anti-cancer therapy in certain cancer models. In this study, we investigated the effects of local versus systemic delivery of such immune-stimulating Dicer-substrate siRNAs (IS-DsiRNAs) on a human papillomavirus (HPV)-driven tumour model. Localized siRNA delivery using intratumour injection of siRNA was able to increase siRNA delivery to the tumour compared with intravenous (IV) delivery and potently activated innate immune responses. However, IV injection remained the more effective delivery route for reducing tumour growth. Although IS-DsiRNAs activated innate immune cells and required interferon-α (IFNα) for full effect on tumour growth, we found that potent silencing siRNA acting independently of IFNα were overall more effective at inhibiting TC-1 tumour growth. Other published work utilising IS-siRNAs have been carried out on tumour models with low levels of major histocompatibility complex (MHC)-class 1, a target of natural killer cells that are potently activated by IS-siRNA. As TC-1 cells used in our study express high levels of MHC-class I, the addition of the immunostimulatory motifs may not be as beneficial in this particular tumour model. Our data suggest that selection of siRNA profile and delivery method based on tumour environment is crucial to developing siRNA-based therapies.

Role of the microbiota in response to and recovery from cancer therapy.

Our understanding of how the microbiota affects the balance between response to and failure of cancer treatment by modulating the tumour microenvironment and systemic immune system has advanced rapidly in recent years. Microbiota-targeting interventions in patients with cancer are an area of intensive investigation. Promisingly, phase I-II clinical trials have shown that interventions such as faecal microbiota transplantation can overcome resistance to immune checkpoint blockade in patients with melanoma, improve therapeutic outcomes in treatment-naive patients and reduce therapy-induced immunotoxicities. Here, we synthesize the evidence showing that the microbiota is an important determinant of both cancer treatment efficacy and treatment-induced acute and long-term toxicity, and we discuss the complex and inter-related mechanisms involved. We also assess the potential of microbiota-targeting interventions, including bacterial engineering and phage therapy, to optimize the response to and recovery from cancer therapy.

A systems immunology study comparing innate and adaptive immune responses in adults to COVID-19 mRNA and adenovirus vectored vaccines.

Identifying the molecular mechanisms that promote optimal immune responses to coronavirus disease 2019 (COVID-19) vaccination is critical for future rational vaccine design. Here, we longitudinally profile innate and adaptive immune responses in 102 adults after the first, second, and third doses of mRNA or adenovirus-vectored COVID-19 vaccines. Using a multi-omics approach, we identify key differences in the immune responses induced by ChAdOx1-S and BNT162b2 that correlate with antigen-specific antibody and T cell responses or vaccine reactogenicity. Unexpectedly, we observe that vaccination with ChAdOx1-S, but not BNT162b2, induces an adenoviral vector-specific memory response after the first dose, which correlates with the expression of proteins with roles in thrombosis with potential implications for thrombosis with thrombocytopenia syndrome (TTS), a rare but serious adverse event linked to adenovirus-vectored vaccines. The COVID-19 Vaccine Immune Responses Study thus represents a major resource that can be used to understand the immunogenicity and reactogenicity of these COVID-19 vaccines.

siRNA-induced immunostimulation through TLR7 promotes antitumoral activity against HPV-driven tumors in vivo.

Oncogene-specific downregulation mediated by RNA interference (RNAi) is a promising avenue for cancer therapy. In addition to specific gene silencing, in vivo RNAi treatment with short interfering RNAs (siRNAs) can initiate immune activation through innate immune receptors including Toll-like receptors, (TLRs) 7 and 8. Two recent studies have shown that activation of innate immunity by addition of tri-phosphate motifs to oncogene-specific siRNAs, or by co-treatment with CpG oligos, can potentiate siRNA antitumor effects. To date, there are no reports on applying such approach against human papillomavirus (HPV)-driven cancers. Here, we characterized the antitumor effects of non-modified siRNAs that can target a specific oncogene and/or recruit the innate immune system against HPV-driven tumors. Following the characterization of silencing efficacy and TLR7 immunostimulatory potential of 15 siRNAs targeting the HPV type 16 E6/E7 oncogenes, we identified a bifunctional siRNA sequence that displayed both potent gene silencing and active immunostimulation effect. In vivo systemic administration of this siRNA resulted in reduced growth of established TC-1 tumors in C57BL/6 mice. Ablation of TLR7 recruitment via 2'O-methyl modification of the oligo backbone reduced these antitumor effects. Further, a highly immunostimulatory, but non-HPV targeting siRNA was also able to exert antitumoral effects although for less prolonged time compared with the bifunctional siRNA. Collectively, our work demonstrates for the first time that siRNA-induced immunostimulation can have antitumoral effects against HPV-driven tumors in vivo, even independent of gene silencing efficacy.

OX40-targeted immune agonist antibodies induce potent antitumor immune responses without inducing liver damage in mice.

Despite promising preclinical and clinical data demonstrating that immune agonist antibody immunotherapies (IAAs) such as αOX40 induce strong antitumor immune responses, clinical translation has been significantly hampered by the propensity of some IAAs to induce dose-limiting and sometimes life-threatening immunotoxicities such as cytokine release syndrome and hepatotoxicity. For example, in a recent study αOX40 was shown to induce significant liver damage in mice by inducing the pyroptosis of liver natural killer T cells (NKT) cells. Surprisingly; however, given these previous reports, αOX40 treatment in our hands did not induce NKT cell pyroptosis or liver damage. We investigated numerous potential confounding factors including age, sex, tumor burden, dosing strategy, and the gut microbiota, which could have explained this discrepancy with the previous study. In none of these experiments did we find that αOX40 induced any more than very mild inflammation in the liver. Our study therefore suggests that, preclinically, αOX40 is a safe and effective immunotherapy and further studies into the clinical benefit of αOX40 are warranted.

The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life.

Studies investigating whether there is a causative link between the gut microbiota and lifespan have largely been restricted to invertebrates or to mice with a reduced lifespan because of a genetic deficiency. We investigate the effect of early-life antibiotic exposure on otherwise healthy, normal chow-fed, wild-type mice, monitoring these mice for more than 700 days in comparison with untreated control mice. We demonstrate the emergence of two different low-diversity community types, post-antibiotic microbiota (PAM) I and PAM II, following antibiotic exposure. PAM II but not PAM I mice have impaired immunity, increased insulin resistance, and evidence of increased inflammaging in later life as well as a reduced lifespan. Our data suggest that differences in the composition of the gut microbiota following antibiotic exposure differentially affect host health and longevity in later life.

The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota.

Immune agonist antibodies (IAAs) are promising immunotherapies that target co-stimulatory receptors to induce potent anti-tumor immune responses, particularly when combined with checkpoint inhibitors. Unfortunately, their clinical translation is hampered by serious dose-limiting, immune-mediated toxicities, including high-grade and sometimes fatal liver damage, cytokine release syndrome (CRS), and colitis. We show that the immunotoxicity, induced by the IAAs anti-CD40 and anti-CD137, is dependent on the gut microbiota. Germ-free or antibiotic-treated mice have significantly reduced colitis, CRS, and liver damage following IAA treatment compared with conventional mice or germ-free mice recolonized via fecal microbiota transplant. MyD88 signaling is required for IAA-induced CRS and for anti-CD137-induced, but not anti-CD40-induced, liver damage. Importantly, antibiotic treatment does not impair IAA anti-tumor efficacy, alone or in combination with anti-PD1. Our results suggest that microbiota-targeted therapies could overcome the toxicity induced by IAAs without impairing their anti-tumor activity.

Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy.

BACKGROUND: Concomitant tumor necrosis factor (TNF) neutralization in combination with immune checkpoint inhibitors (ICIs) reduces clinical immune-related adverse events (irAEs) and appears to improve antitumor efficacy in preclinical tumor models. Agonistic antibodies targeting costimulatory receptors such as CD40 represent an additional strategy to boost antitumor immune response and potentiate the activity of ICIs. However, the dose-limiting toxicities observed in anti-CD40-treated cancer patients have hindered its clinical development. METHODS: We previously described a mouse model to assess both antitumor activity and irAEs induced by various effective combination immunotherapies. Using the BALB/c and C57BL/6 strains of FoxP3-GFP-DTR (FoxP3DTR) mice, transient depletion of T regulatory cells (Tregs) prior to immunotherapy with additional immunomodulatory antibodies, lowered immune self-tolerance, resulting in the development of a spectrum of physical and biochemical irAEs similar to that reported clinically. In MC38 and 4T1.2 tumor models, following transient Treg depletion, we evaluated the impact of anti-CD40 on antitumor efficacy and the development of irAEs and the impact of concomitant or delayed TNF blockade on both these parameters. Physical irAEs were scored and biochemical irAEs were measured in the serum (ALT and cytokine levels). Histopathological liver and colon tissue analysis were performed to assess immune cell infiltration and tissue damage. RESULTS: Similar to early clinical trials of CD40 agonists, in our tumor models we observed liver toxicities and rapid release of proinflammatory cytokines (TNF, interleukin 6, interferon-γ). In the BALB/c strain, anti-CD40 induced severe physical and biochemical irAEs. Concomitant anti-TNF treatment abrogated weight loss, liver damage and colitis, which consequently resulted in an improved clinical score. However, concomitant anti-TNF impaired antitumor response in a proportion of anti-CD40-treated C57BL/6 FoxP3DTR mice. Delaying TNF blockade in these mice reduced biochemical but not physical irAEs while preserving antitumor efficacy. CONCLUSIONS: Our results suggest concomitant rather than delayed anti-TNF is most effective in reducing biochemical and physical irAEs induced by anti-CD40, although it had the potential to negatively impact antitumor efficacy. Furthermore, our findings highlight the utility of our mouse model to assess the severity of irAEs induced by novel immunotherapeutic agents and evaluate whether their toxicity and antitumor efficacy can be uncoupled.

CD96 Is an Immune Checkpoint That Regulates CD8+ T-cell Antitumor Function.

CD96 is a novel target for cancer immunotherapy shown to regulate NK cell effector function and metastasis. Here, we demonstrated that blocking CD96 suppressed primary tumor growth in a number of experimental mouse tumor models in a CD8+ T cell-dependent manner. DNAM-1/CD226, Batf3, IL12p35, and IFNγ were also critical, and CD96-deficient CD8+ T cells promoted greater tumor control than CD96-sufficient CD8+ T cells. The antitumor activity of anti-CD96 therapy was independent of Fc-mediated effector function and was more effective in dual combination with blockade of a number of immune checkpoints, including PD-1, PD-L1, TIGIT, and CTLA-4. We consistently observed coexpression of PD-1 with CD96 on CD8+ T lymphocytes in tumor-infiltrating leukocytes both in mouse and human cancers using mRNA analysis, flow cytometry, and multiplex IHF. The combination of anti-CD96 with anti-PD-1 increased the percentage of IFNγ-expressing CD8+ T lymphocytes. Addition of anti-CD96 to anti-PD-1 and anti-TIGIT resulted in superior antitumor responses, regardless of the ability of the anti-TIGIT isotype to engage FcR. The optimal triple combination was also dependent upon CD8+ T cells and IFNγ. Overall, these data demonstrate that CD96 is an immune checkpoint on CD8+ T cells and that blocking CD96 in combination with other immune-checkpoint inhibitors is a strategy to enhance T-cell activity and suppress tumor growth.

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

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.

Molecular Pathways: Targeting CD96 and TIGIT for Cancer Immunotherapy.

The receptors CD96 and TIGIT are expressed on the surface of T and natural killer (NK) cells, and recent studies suggest both play important inhibitory roles in immune function. CD96 has been shown to modulate immune cell activity in mice, with Cd96-/- mice displaying hypersensitive NK-cell responses to immune challenge and significant tumor resistance. TIGIT overexpression has been shown to reduce NK-cell-mediated cytotoxicity. TIGIT is also upregulated on T cells during cancer and chronic viral infection, with expression associated with effector T-cell exhaustion and increased regulatory T-cell suppression. The counterbalance between the putative inhibitory CD96 and TIGIT receptors and the activating receptor, CD226, offers unique strategies for immuno-oncology drug development. Blocking CD96 or TIGIT with mAbs has been shown to improve tumor control in mice, in particular when used in combination with PD-1/PD-L1 blockade. These results have highlighted these pathways as promising new targets for immune modulation. This review will examine the rationale behind targeting CD96 and TIGIT, and discuss the potential approaches in translating these preclinical findings into novel clinical agents. Clin Cancer Res; 22(21); 5183-8. ©2016 AACR.

Anti-CD137 enhances anti-CD20 therapy of systemic B-cell lymphoma with altered immune homeostasis but negligible toxicity.

Studies of sequential anti-CD137/anti-CD20 therapy have previously shown that the efficacy of anti-CD20 was heavily reliant upon anti-CD137; however, the exact mechanism of the anti-B-cell lymphoma efficacy, and whether this correlates with enhanced adverse effects or toxicity, had not been elucidated. Here, we observed that sequential anti-CD137 administration with anti-CD20 resulted in a synergistic therapy, largely dependent upon Fc receptors (FcR), to prolong survival in an experimental B-cell lymphoma therapy model. Tumor suppression was accompanied by B cell depletion, which was not dependent on one activating FcR. Surprisingly, the B-cell activating factor (BAFF) was elevated in the plasma of mice receiving anti-CD137 alone or in combination with anti-CD20, while a selective increase in some plasma cytokines was also noted and triggered by anti-CD137. These effects were independent of activating FcR. Sustained treatment of advanced lymphoma revealed increased lymphocyte infiltrates into the liver and a significant decrease in the metabolic capability of the liver in mice receiving anti-CD137. Importantly, these effects were not exacerbated in mice receiving the anti-CD20/CD137 combination, and elevations in classical liver damage markers such as alanine aminotransferase (ALT) were less than that caused by the lymphoma itself. Thus, combined anti-CD20/anti-CD137 treatment increases the therapeutic index of anti-CD20 or anti-CD137 alone. These mouse data were corroborated by ongoing clinical development studies to assess safety, tolerability and pharmacodynamic activity of human patients treated by this approach. Together, these data support the use of this sequential antibody therapeutic strategy to improve the efficacy of rituximab in B-cell lymphoma patients.

Agonistic CD40 mAb-Driven IL12 Reverses Resistance to Anti-PD1 in a T-cell-Rich Tumor.

The durability and efficacy of anti-human PD1 monoclonal antibodies (PD1 mAb) vary across different malignancies. Although an absence of tumor-infiltrating cytotoxic T lymphocytes has been identified as a cause for resistance to PD1 mAb, the presence of intratumor exhausted PD1hi T cells also contributes to insensitivity to this immune checkpoint therapy. In this study, we used mouse tumor models of PD1 mAb resistance that harbored PD1hi T cells and flow cytometry analysis of tumor-infiltrating leukocytes immediately post-therapy as a screening platform to identify agents that could resensitize T cells to PD1 blockade. We showed that an agonistic anti-CD40 mAb converted PD1hi T cells into PD1lo T cells, reversing phenotypic T-cell exhaustion and allowing the anti-PD1 refractory tumors to respond to anti-PD1 therapy. PD1 downmodulation by anti-CD40 mAb relied upon IL12 but not IL23, CD80/CD86/CD28, or CD70/CD27. Consistent with a role for regulatory T cells (Treg) in promoting T-cell exhaustion, we also showed that intratumor Treg presented with a less activated and attenuated suppressive phenotype, marked by reductions in CTLA4 and PD1. Similar to anti-CD40 mAb, anti-CTLA4 mAb also lowered intratumor T-cell PD1 expression. Our study provides a proof-of-principle framework to systematically identify immune conditioning agents able to convert PD1hi T cells to PD1lo T cells, with clinical implications in the management of anti-PD1 refractory patients. Cancer Res; 76(21); 6266-77. ©2016 AACR.

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.

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.

Co-inhibition of CD73 and A2AR Adenosine Signaling Improves Anti-tumor Immune Responses.

Preclinical studies targeting the adenosinergic pathway have gained much attention for their clinical potential in overcoming tumor-induced immunosuppression. Here, we have identified that co-blockade of the ectonucleotidase that generates adenosine CD73 and the A2A adenosine receptor (A2AR) that mediates adenosine signaling in leuokocytes, by using compound gene-targeted mice or therapeutics that target these molecules, limits tumor initiation, growth, and metastasis. This tumor control requires effector lymphocytes and interferon-γ, while antibodies targeting CD73 promote an optimal therapeutic response in vivo when engaging activating Fc receptors. In a two-way mixed leukocyte reaction using a fully human anti-CD73, we demonstrated that Fc receptor binding augmented the production of proinflammatory cytokines.