Rikke B. Holmgaard

Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4

Indoleamine 2,3-dioxygenase suppresses infiltration and accumulation of tumor-reactive T cells in the context of anti–CTLA-4 immunotherapy and attenuates the anti-tumor efficacy.

Localized Oncolytic Virotherapy Overcomes Systemic Tumor Resistance to Immune Checkpoint Blockade Immunotherapy

Intratumoral treatment with an oncolytic virus results in inflammatory infiltrates in distant tumors and sensitizes the tumors to immune-mediated rejection in the setting of immunotherapy with CTLA-4 blocking antibodies. Immune Cells and a Virus Teaming Up to Fight Cancer Immunotherapy, or harnessing the patient’s own immune system to help fight cancer, is becoming increasingly popular as researchers discover newer and more successful approaches focused on different aspects of the immune system. Two of these approaches include oncolytic viruses and therapies that block immune checkpoints and thus stimulate the antitumor activity of T cells. One virus that is known to have oncolytic activity is Newcastle disease virus (NDV), an avian virus that is not pathogenic in humans. Although previous studies have successfully demonstrated the antitumor effects of NDV, these were thought to require direct injection of this virus into every tumor, thus greatly limiting its effectiveness against metastatic disease. Now, Zamarin and coauthors used mouse models of cancer with multiple tumor sites to demonstrate that NDV can be used even in the setting of distant spread of disease. Although the virus had to be injected into a tumor to have any effect, the subsequent tumor killing was not limited to the injected tumor. Additional masses that were distant in space and time, such as tumors implanted at different body sites and at later time points, were targeted by the immune system in animals that had been treated with NDV injection into the primary tumor. This immunostimulatory effect of NDV was particularly pronounced when the virotherapy was combined with immune checkpoint blockade by an anti–CTLA-4 antibody. The current study was performed in mice bearing tumors derived from established cell lines, and the findings will need to be confirmed in the setting of spontaneously arising metastatic tumors. Nevertheless, the results of this work raise the tantalizing possibility that two therapies known to be safe for human use could be combined and work with the immune system to eradicate tumors throughout the body. Preexisting lymphocytic infiltration of tumors is associated with superior prognostic outcomes in a variety of cancers. Recent studies also suggest that lymphocytic responses may identify patients more likely to benefit from therapies targeting immune checkpoints, suggesting that therapeutic efficacy of immune checkpoint blockade can be enhanced through strategies that induce tumor inflammation. To achieve this effect, we explored the immunotherapeutic potential of oncolytic Newcastle disease virus (NDV). We find that localized intratumoral therapy of B16 melanoma with NDV induces inflammatory responses, leading to lymphocytic infiltrates and antitumor effect in distant (nonvirally injected) tumors without distant virus spread. The inflammatory effect coincided with distant tumor infiltration with tumor-specific CD4+ and CD8+ T cells, which was dependent on the identity of the virus-injected tumor. Combination therapy with localized NDV and systemic CTLA-4 blockade led to rejection of preestablished distant tumors and protection from tumor rechallenge in poorly immunogenic tumor models, irrespective of tumor cell line sensitivity to NDV-mediated lysis. Therapeutic effect was associated with marked distant tumor infiltration with activated CD8+ and CD4+ effector but not regulatory T cells, and was dependent on CD8+ cells, natural killer cells, and type I interferon. Our findings demonstrate that localized therapy with oncolytic NDV induces inflammatory immune infiltrates in distant tumors, making them susceptible to systemic therapy with immunomodulatory antibodies, which provides a strong rationale for investigation of such combination therapies in the clinic.

The PTEN pathway in Tregs is a critical driver of the suppressive tumor microenvironment

Tumors depend on a specialized pathway of regulatory T cell activation to create their immunosuppressive microenvironment, which can be blocked by inhibiting PTEN phosphatase. The tumor microenvironment is profoundly immunosuppressive. We show that multiple tumor types create intratumoral immune suppression driven by a specialized form of regulatory T cell (Treg) activation dependent on the PTEN (phosphatase and tensin homolog) lipid phosphatase. PTEN acted to stabilize Tregs in tumors, preventing them from reprogramming into inflammatory effector cells. In mice with a Treg-specific deletion of PTEN, tumors grew slowly, were inflamed, and could not create an immunosuppressive tumor microenvironment. In normal mice, exposure to apoptotic tumor cells rapidly elicited PTEN-expressing Tregs, and PTEN-deficient mice were unable to maintain tolerance to apoptotic cells. In wild-type mice with large established tumors, pharmacologic inhibition of PTEN after chemotherapy or immunotherapy profoundly reconfigured the tumor microenvironment, changing it from a suppressive to an inflammatory milieu, and tumors underwent rapid regression. Thus, the immunosuppressive milieu in tumors must be actively maintained, and tumors become susceptible to immune attack if the PTEN pathway in Tregs is disrupted.

Intratumoral modulation of the inducible co-stimulator ICOS by recombinant oncolytic virus promotes systemic anti-tumour immunity

Emerging data suggest that locoregional cancer therapeutic approaches with oncolytic viruses can lead to systemic anti-tumour immunity, although the appropriate targets for intratumoral immunomodulation using this strategy are not known. Here we find that intratumoral therapy with Newcastle disease virus (NDV), in addition to the activation of innate immunity, upregulates the expression of T-cell co-stimulatory receptors, with the inducible co-stimulator (ICOS) being most notable. To explore ICOS as a direct target in the tumour, we engineered a recombinant NDV-expressing ICOS ligand (NDV-ICOSL). In the bilateral flank tumour models, intratumoral administration of NDV-ICOSL results in enhanced infiltration with activated T cells in both virus-injected and distant tumours, and leads to effective rejection of both tumours when used in combination with systemic CTLA-4 blockade. These findings highlight that intratumoral immunomodulation with an oncolytic virus expressing a rationally selected ligand can be an effective strategy to drive systemic efficacy of immune checkpoint blockade.

Targeting myeloid-derived suppressor cells with colony stimulating factor-1 receptor blockade can reverse immune resistance to immunotherapy in indoleamine 2,3-dioxygenase-expressing tumors

Tumor indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression by direct action on effector T cells and Tregs and through recruitment, expansion and activation of myeloid-derived suppressor cells (MDSCs). Targeting of MDSCs is clinically being explored as a therapeutic strategy, though optimal targeting strategies and biomarkers predictive of response are presently unknown. Maturation and tumor recruitment of MDSCs are dependent on signaling through the receptor tyrosine kinase CSF-1R on myeloid cells. Here, we show that MDSCs are the critical cell population in IDO-expressing B16 tumors in mediating accelerated tumor outgrowth and resistance to immunotherapy. Using a clinically relevant drug, we show that inhibition of CSF-1R signaling can functionally block tumor-infiltrating MDSCs and enhance anti-tumor T cell responses. Furthermore, inhibition of CSF-1R sensitizes IDO-expressing tumors to immunotherapy with T cell checkpoint blockade, and combination of CSF-1R blockade with IDO inhibitors potently elicits tumor regression. These findings provide evidence for a critical and functional role for MDSCs on the in vivo outcome of IDO-expressing tumors.

The New Era of Cancer Immunotherapy: Manipulating T-Cell Activity to Overcome Malignancy.

Using the immune system to control cancer has been investigated for over a century. Yet it is only over the last several years that therapeutic agents acting directly on the immune system have demonstrated improved overall survival for cancer patients in phase III clinical trials. Furthermore, it appears that some patients treated with such agents have been cured of metastatic cancer. This has led to increased interest and acceleration in the rate of progress in cancer immunotherapy. Most of the current immunotherapeutic success in cancer treatment is based on the use of immune-modulating antibodies targeting critical checkpoints (CTLA-4 and PD-1/PD-L1). Several other immune-modulating molecules targeting inhibitory or stimulatory pathways are being developed. The combined use of these medicines is the subject of intense investigation and holds important promise. Combination regimens include those that incorporate targeted therapies that act on growth signaling pathways, as well as standard chemotherapy and radiation therapy. In fact, these standard therapies have intrinsic immune-modulating properties that can support antitumor immunity. In the years ahead, adoptive T-cell therapy will also be an important part of treatment for some cancer patients. Other areas which are regaining interest are the use of oncolytic viruses that immunize patients against their own tumors and the use of vaccines against tumor antigens. Immunotherapy has demonstrated unprecedented durability in controlling multiple types of cancer and we expect its use to continue expanding rapidly.

The New Era of Cancer Immunotherapy

Using the immune system to control cancer has been investigated for over a century. Yet it is only over the last several years that therapeutic agents acting directly on the immune system have demonstrated improved overall survival for cancer patients in phase III clinical trials. Furthermore, it appears that some patients treated with such agents have been cured of metastatic cancer. This has led to increased interest and acceleration in the rate of progress in cancer immunotherapy. Most of the current immunotherapeutic success in cancer treatment is based on the use of immune-modulating antibodies targeting critical checkpoints (CTLA-4 and PD-1/PD-L1). Several other immune-modulating molecules targeting inhibitory or stimulatory pathways are being developed. The combined use of these medicines is the subject of intense investigation and holds important promise. Combination regimens include those that incorporate targeted therapies that act on growth signaling pathways, as well as standard chemotherapy and radiation therapy. In fact, these standard therapies have intrinsic immune-modulating properties that can support antitumor immunity. In the years ahead, adoptive T-cell therapy will also be an important part of treatment for some cancer patients. Other areas which are regaining interest are the use of oncolytic viruses that immunize patients against their own tumors and the use of vaccines against tumor antigens. Immunotherapy has demonstrated unprecedented durability in controlling multiple types of cancer and we expect its use to continue expanding rapidly.

The PTEN pathway in Tregs functions as a critical driver of the immunosuppressive tumor microenvironment and tolerance to apoptotic cells

The tumor microenvironment is profoundly immunosuppressive, but exactly how this is coordinated and maintained remains poorly understood. We show that multiple transplantable and autochthonous mouse tumors actively elicit a population of highly suppressive regulatory T cells (Tregs) expressing the lipid phosphatase PTEN. These PTEN+ Tregs co-expressed PD-1, Foxp3, and high levels of Eos (Ikzf4). PTEN signaling acted to stabilize tumor-associated Tregs, maintaining their suppressor activity and preventing conversion into pro-inflammatory effector cells (“ex-Tregs”) in the face of inflammation. Mice with a targeted deletion of PTEN in Tregs (PTEN-Treg-KO mice) were healthy and fertile when young, but gradually developed lupus-like autoimmunity as they aged. Tumors implanted in young, healthy PTEN-Treg-KO mice were unable to create the normal immunosuppressive tumor microenvironment; instead, tumors were constitutively immunogenic, chronically inflamed, and could barely grow. In wild-type mice with large, pre-established tumors, pharmacologic inhibition of PTEN during the period following chemotherapy or adoptive immunotherapy caused a profound reconfiguration of the tumor microenvironment. The normally suppressive intratumoral Tregs became destabilized, and rapidly reprogrammed into pro-inflammatory “ex-Tregs” expressing IL-2, IL-17 and CD40L. The dominant APCs in the tumor changed from tolerogenic DCs expressing high levels of PD-L1, and were replaced by inflammatory myeloid DCs expressing high levels of CD86, MHC class II, IL-6 and IL-12. CD8+ effector T cells in the tumor, which had previously been unresponsive and PD-1+ (exhausted), became activated and expressed IFNγ and GzmB, and mediated tumor regression. Pharmacologic inhibition of PTEN was highly synergistic with conventional chemotherapy, allowing a single modest, normally ineffective dose of chemotherapy to trigger rapid tumor involution. This synergy was strictly immune-mediated, and was lost in the absence of host CD8+ T cells. In mice without tumors, identical PTEN+ Tregs were physiologically elicited by exposure to apoptotic cells; and PTEN-Treg-KO mice rapidly developed lupus-like autoimmunity when repeatedly challenged with apoptotic cells. The induction of PTEN+ Tregs by apoptotic cells was driven by indoleamine 2,3-dioxygenase (IDO) in the host, and was blocked by pharmacologic inhibition of IDO. Taken together, these data identify the PTEN pathway in Tregs as a potent immunosuppressive mechanism in tumors. PTEN+ Tregs controlled the downstream activation of inflammatory DCs and effector CD8+ T cells, and were part of the fundamental mechanism of tolerance to apoptotic cells. The PTEN pathway thus represents a potent, centrally-positioned immunosuppressive mechanism in tumors, which is amenable to pharmacologic inhibition and shows synergy with both adoptive immunotherapy and conventional chemotherapy.

Timing of CSF-1/CSF-1R signaling blockade is critical to improving responses to CTLA-4 based immunotherapy

ABSTRACT Colony stimulating factor-1 (CSF-1) is produced by a variety of cancers and recruits myeloid cells that suppress antitumor immunity, including myeloid-derived suppressor cells (MDSCs.) Here, we show that both CSF-1 and its receptor (CSF-1R) are frequently expressed in tumors from cancer patients, and that this expression correlates with tumor-infiltration of MDSCs. Furthermore, we demonstrate that these tumor-infiltrating MDSCs are highly immunosuppressive but can be reprogrammed toward an antitumor phenotype in vitro upon CSF-1/CSF-1R signaling blockade. Supporting these findings, we show that inhibition of CSF-1/CSF-1R signaling using an anti-CSF-1R antibody can regulate both the number and the function of MDSCs in murine tumors in vivo. We further find that treatment with anti-CSF-1R antibody induces antitumor T-cell responses and tumor regression in multiple tumor models when combined with CTLA-4 blockade therapy. However, this occurs only when administered after or concurrent with CTLA-4 blockade, indicating that timing of each therapeutic intervention is critical for optimal antitumor responses. Importantly, MDSCs present within murine tumors after CTLA-4 blockade showed increased expression of CSF-1R and were capable of suppressing T cell proliferation, and CSF-1/CSF-1R expression in the human tumors was not reduced after treatment with CTLA-4 blockade immunotherapy. Taken together, our findings suggest that CSF-1R-expressing MDSCs can be targeted to modulate the tumor microenvironment and that timing of CSF-1/CSF-1R signaling blockade is critical to improving responses to checkpoint based immunotherapy. Significance: Infiltration by immunosuppressive myeloid cells contributes to tumor immune escape and can render patients resistant or less responsive to therapeutic intervention with checkpoint blocking antibodies. Our data demonstrate that blocking CSF-1/CSF-1R signaling using a monoclonal antibody directed to CSF-1R can regulate both the number and function of tumor-infiltrating immunosuppressive myeloid cells. In addition, our findings suggest that reprogramming myeloid responses may be a key in effectively enhancing cancer immunotherapy, offering several new potential combination therapies for future clinical testing. More importantly for clinical trial design, the timing of these interventions is critical to achieving improved tumor protection.

Association of a functional Indoleamine 2,3-dioxygenase 2 genotype with specific immune responses

Two frequent single-nucleotide-polymorphisms (SNPs) are present in the indoleamine 2,3-dioxygenase 2 (IDO2) gene that influence its enzymatic activity. Thus, one SNP (R248W) is associated with a reduction in IDO2 catalytic activity, whereas the other SNP (Y359stop) generates a premature stop codon abolishing activity completely. In the present study, we describe the presence of a specific cellular immune response in the periphery which correlated with the functional status of the IDO2 protein. Hence, the induction of IDO2-specific T cells in peripheral blood requires the presence of a functional IDO2 protein and, consequently, is restricted to individuals that are not homozygous for the stop codon. Furthermore, we detected stronger T-cell responses in donors with the homozygous Y wild type at position 359 when compared with the heterozygous genotype. Interestingly, we found a higher number of immune responses against IDO2 in patients homozygous for the 248W giving reduction in IDO2 activity compared with the 248R. Hence, spontaneous immune responses against IDO2 seem to be correlated with reduced enzymatic activity of IDO2. The patient IDO2 genotype may well influence the outcome of IDO2-based anti-cancer vaccination.