John B. Mumm

The Protein Product of the Tumor Suppressor Gene, Melanoma Differentiation-Associated Gene 7, Exhibits Immunostimulatory Activity and Is Designated IL-24

The melanoma differentiation-associated gene 7 (mda-7) has been studied primarily in the context of its tumor suppressor activity. Although mda-7 has been designated as IL-24 based on its gene location in the IL-10 locus and its mRNA expression in leukocytes, no functional evidence supporting this cytokine designation exists. To further characterize MDA-7/IL-24 expression patterns in the human immune system, MDA-7/IL-24 protein levels were examined in human PBMC. MDA-7/IL-24 was detected in PHA- and LPS-stimulated whole PBMC lysate by Western blot and in PHA-activated CD56 and CD19 subsets by immunohistochemistry. The biological function of MDA-7/IL-24, secreted from Ad-MDA7-transfected HEK 293 cells, was assessed by examining the effect of MDA-7/IL-24 on the cytokine secretion profile of PBMC. Within 48 h MDA-7/IL-24 induced secretion of high levels of IL-6, TNF-α, and IFN-γ and low levels of IL-1β, IL-12, and GM-CSF from human PBMC as measured by ELISA. The MDA-7/IL-24-mediated induction of these Th1-type cytokines was inhibited by the addition of IL-10 to the PBMC cultures, suggesting that these two related protein family members may provide antagonistic functions. Therefore, because human blood leukocytes can be stimulated to produce MDA-7/IL-24, as well as respond to MDA-7/IL-24 by expressing secondary cytokines, MDA-7/IL-24 has the expression profile and major functional attributes that justify its designation as an IL.

IL-33 Induces IL-13–Dependent Cutaneous Fibrosis

IL-33 is constitutively expressed in epithelial barrier tissues, such as skin. Although increased expression of IL-33/IL-33R has been correlated with fibrotic disorders, such as scleroderma and progressive systemic sclerosis, the direct consequences of IL-33 release in skin has not been reported. To determine the effects of dysregulated IL-33 signaling in skin, we administered IL-33 s.c. and monitored its effects at the injection site. Administration of IL-33 resulted in IL-33R–dependent accumulation of eosinophils, CD3+ lymphocytes, F4/80+ mononuclear cells, increased expression of IL-13 mRNA, and the development of cutaneous fibrosis. Consistent with extensive cutaneous tissue remodeling, IL-33 resulted in significant modulation of a number of extracellular matrix-associated genes, including collagen VI, collagen III, and tissue inhibitor of metalloproteases-1. We establish that IL-33–induced fibrosis requires IL-13 using IL-13 knockout mice and eosinophils using ΔdblGATA mice. We show that bone marrow-derived eosinophils secrete IL-13 in response to IL-33 stimulation, suggesting that eosinophil-derived IL-13 may promote IL-33–induced cutaneous fibrosis. Collectively, our results identify IL-33 as a previously unrecognized profibrotic mediator in skin and highlight the cellular and molecular pathways by which this pathology develops.

IL-10 Directly Activates and Expands Tumor-Resident CD8+ T Cells without De Novo Infiltration from Secondary Lymphoid Organs

The presence of activated intratumoral T cells correlates clinically with better prognosis in patients with cancer. Although tumor vaccines can increase the number of tumor-specific CD8(+) T cells in systemic circulation, they frequently fail to increase the number of active and tumor reactive T cells within the tumor. Here we show that treatment with the pleiotropic cytokine interleukin-10 (IL-10) induces specific activation of tumor-resident CD8(+) T cells as well as their intratumoral expansion in several mouse tumor models. We found that inhibition of T-cell trafficking from lymphoid organs did not impair IL-10-induced tumor rejection or the activation of tumor-resident CD8(+) T cells. Tumor-resident CD8(+) T cells expressed elevated levels of the IL-10 receptor and were directly activated by IL-10, resulting in prominent phosphorylation of STAT3 and STAT1. Although CD4(+) T cells, regulatory T cells, NK cells, and dendritic cells have been reported as prominent targets of IL-10 in the tumor microenvironment, we found that expression of the IL-10R was required only on CD8(+) T cells to facilitate IL-10-induced tumor rejection as well as in situ expansion and proliferation of tumor-resident CD8 T cells. Together, our findings indicate that IL-10 activates CD8(+) T-cell-mediated tumor control and suggest that IL-10 may represent a potential tumor immunotherapy in human patients with cancer.

Pegylated IL-10 induces cancer immunity

Recently, the development of several strategies based on immunotherapy has raised hopes for a more promising way to treat cancer patients. Here, we describe how interleukin (IL)-10, a seemingly unlikely candidate, stimulates the immune system in a particularly efficacious way. IL-10, an omnipotent anti-inflammatory cytokine, delivers an equally potent immune stimulation in the context of CD8(+) T cells and tumor immunity. By activation of tumor-resident, tumor-specific CD8(+) T cells, pegylated IL-10 can induce rejection of large and metastasizing tumors in mice. Here, we summarize the mechanisms of action of IL-10, the reasons why the mechanisms may be crucial for the treatment of cancer patients, and the rationale for applying pegylated IL-10 in the clinic.

PEG-rIL-10 treatment decreases FoxP3+ Tregs despite upregulation of intratumoral IDO

ABSTRACT IL-10 has been classically defined as a broad-spectrum immunosuppressant and is thought to facilitate the development of regulatory CD4+ T cells. IL-10 is believed to represent one of the major suppressive factors secreted by IDO+FoxP3+CD4+ Tregs. Contrary to this view, we have previously reported that PEGylated recombinant IL-10 (PEG-rIL-10) treatment of mice induces potent IFNγ and CD8+ T-cell-dependent antitumor immunity. This hypothesis is currently being tested in clinical trials and we have reported that treatment of cancer patients with PEG-rHuIL-10 results in inhibition and regression of tumor growth as well as increased serum IFNγ. We have continued to assess PEG-rIL-10s pleiotropic effects and report that treatment of tumor-bearing mice and humans with PEG-rIL-10 increases intratumoral indoleamine 2, 3-dioxygenase (IDO) in an IFNγ-dependent manner. This should result in an increase in Tregs, but paradoxically our data illustrate that PEG-rIL-10 treatment of mice reduces intratumoral FoxP3+CD4+ T cells in an IDO-independent manner. Additional investigation indicates that PEG-rIL-10 inhibits TGFβ/IL-2-dependent in vitro polarization of FoxP3+CD4+ Tregs and potentiates IFNγ+T-bet+CD4+ T cells. These data suggest that rather than acting as an immunosuppressant, PEG-rIL-10 may counteract the FoxP3+CD4+ Treg suppressive milieu in tumor-bearing mice and humans, thereby further facilitating PEG-rIL-10s potent antitumor immunity.

PEGylated IL-10 Activates Kupffer Cells to Control Hypercholesterolemia.

Interleukin-10 (IL-10) is a multifunctional cytokine that exerts potent context specific immunostimulatory and immunosuppressive effects. We have investigated the mechanism by which PEGylated rIL-10 regulates plasma cholesterol in mice and humans. In agreement with previous work on rIL-10, we report that PEGylated rIL-10 harnesses the myeloid immune system to control total plasma cholesterol levels. We have discovered that PEG-rMuIL-10’s dramatic lowering of plasma cholesterol is dependent on phagocytotic cells. In particular, PEG-rHuIL-10 enhances cholesterol uptake by Kupffer cells. In addition, removal of phagocytotic cells dramatically increases plasma cholesterol levels, suggesting for the first time that immunological cells are implicitly involved in regulating total cholesterol levels. These data suggest that treatment with PEG-rIL-10 potentiates endogenous cholesterol regulating cell populations not currently targeted by standard of care therapeutics. Furthermore, we show that IL-10’s increase of Kupffer cell cholesterol phagocytosis is concomitant with decreases in liver cholesterol and triglycerides. This leads to the reversal of early periportal liver fibrosis and facilitates the restoration of liver health. These data recommend PEG-rIL-10 for evaluation in the treatment of fatty liver disease and preventing its progression to non-alcoholic steatohepatitis. In direct confirmation of our in vivo findings in the treatment of hypercholesterolemic mice with PEG-rMuIL-10, we report that treatment of hypercholesterolemic cancer patients with PEG-rHuIL-10 lowers total plasma cholesterol by up to 50%. Taken together these data suggest that PEG-rIL-10’s cholesterol regulating biology is consistent between mice and humans.

Autochthonous T cells to the rescue IL-10 directly activates tumor-resident CD8 + T cells

Successful cancer immunotherapy is thought to require de novo priming of tumor specific CD8+ T cells in lymphatic organs. Contrasting these beliefs, cancer therapy based on interleukin-10 (IL-10) results in tumor rejection without a requirement for T-cell trafficking from lymphatic organs. Rather, IL-10 directly activates autochthonous, tumor-resident CD8+ T cells.

Abstract PR08: Antitumor activity and immune correlates of PEGylated human IL-10 (AM0010) alone or in combination with anti-PD-1

Background: The success of and the durability of immune therapy of cancer is thought to depend on the activation and expansion of tumor reactive and infiltrating CD8+ T cells. The response to immune checkpoint blockade, depends on a pre-existing, CD8 T cell-rich tumor microenvironment. IL-10 stimulates the antigen mediated cytotoxicity, survival and proliferation of intra-tumoral CD8+ T cells and simultaneously dampens chronic inflammation. T cell receptor mediated activation of CD8 T cells induces the expression of IL-10 receptors on these cells. IL-10 activates with STAT3 an essential survival and proliferation signal in antigen activated CD8 T cells. This also provides a mechanistic rationale for combining AM0010 and anti-PD1 in the clinic. To evaluate the clinical activity, tolerability and anti-tumor activity of AM0010 alone or in combination with chemotherapy or immune checkpoint inhibitors a multi-basket phase 1 study was conducted. Additional disease specific expansion cohorts for the combination of AM0010 with FOLFOX in pancreatic cancer or with nivolumab in RCC or NSCLC are currently evaluated Results: Tolerability and anti-tumor activity of AM0010 alone or in combination with chemotherapy or immune checkpoint inhibitors was established in this multi-basket phase 1 study. In monotherapy, objective responses were observed in pts with uveal melanoma, cutaneous T cell lymphoma and in 4 of 15 pts with RCC. Patients with advanced melanoma, RCC or NSCLC were also treated with AM0010 (daily SC) in combination with anti-PD-1 immune checkpoint blockade. Tumor responses were monitored following irRC. Immune responses were measured by analysis of serum cytokines, activation of blood derived T cells, peripheral T cell clonality and immunohistochemistry of tumor infiltrating CD8 T cells. In 19 pts, AMO010 10 μg/kg (n = 13) or 20 μg/kg (n = 6) in combination with anti-PD1 - pembrolizumab (2mg/kg) was well tolerated (observation period 10-15 months). Immune related TrAE occured in the frequency and severity as expected from pembrolizumab montherapy. The combination of AM0010 with pembrolizumab achieved objective responses (PR/CR) in 4 of 8 RCC pts, 2 of 5 NSCLC pts and 2 of 6 melanoma pts. 2 additional melanoma pts had tumor increase followed by decrease (pseudoprogression). Independent of the combination with either chemotherapy or anti-PD-1, AM0010 increased Th1 cytokines (IL-18, IFNγ, IL-7) in a dose dependent fashion. FasL and lymphotoxin beta - products of cytotoxic T cells - were also increased in the serum of AM0010 treated patients. In contrast, mediators of chronic inflammation, such as IL-23 and IL-17 and the immune suppressive cytokine TGFbeta were reduced in the serum of patients. AM0010 increased the number and proliferation of PD1+ activated CD8 T cells while decreasing the proliferation of FoxP3+ Tregs and TGFβ in the blood. AM0010 induced de-novo oligoclonal expansion of T cell clones in the blood of patients without affecting total lymphocyte counts. This clonal expansion appeared enhanced and accelerated in patients treated with a AM0010 anti PD-1 combination, but was also seen in patients which received a AM0010 - chemotherapy combination. AM0010 also increased the number of tumor infiltrating Phospho-STAT3+ CD8 T cells in tumors and the number of Granzyme+ PD1+ CD8+ T cells in tumor biopsies of treated patients. Conclusion: AM0010 alone or in combination with anti-PD1 is well-tolerated. The clinical activity and the observed CD8 T cell activation encourages the phase 2/3 studies of AM0010 in combination with anti-PD1 planned for later in 2016. Trial registration: www.clinicaltrials.govNCT02009449. Citation Format: Aung Naing, Jeffrey R. Infante, Kyriakos P. Papadopoulos, Deborah J. Wong, Karen A. Autio, Gerald S. Falchook, Manish Patel, Shubham Pant, Amita Patnaik, Melinda Whiteside, Johanna C. Bendell, John Mumm, Ivan H. Chan, Gail L. Brown, Peter VanVlasselaer, J. R. Hecht, David S. Hong, Nizar M. Tannir, Martin Oft. Antitumor activity and immune correlates of PEGylated human IL-10 (AM0010) alone or in combination with anti-PD-1 [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR08.

Abstract CT098: Anti-tumor activity of PEGylated human IL-10 (AM0010) in patients with advanced solid tumors

Purpose: IL-10 inhibits inflammation but stimulates cytotoxic CD8 T cells. In preclinical models, PEGylated IL-10 induces rejection of tumors and establishes CD8 T cell memory. PEG-IL-10 (AM0010) activates the anti-apoptotic STAT3 in tumor infiltrating activated CD8 T cells. This leads to the expansion of tumor reactive memory CD8 T cells both in the tumor and the blood. The primary objective of this phase 1 study is to establish tolerability and anti-tumor activity of AM0010 alone and in combination with chemotherapy and anti PD-1 immunotherapy. Here we report the results of AM0010 in the monotherapy dose escalation and one expansion cohort in renal cell cancer (RCC). Procedures: 33 patients (pts) with advanced cancers including melanoma, RCC, non-small cell lung, colorectal, ovarian, prostate and pancreatic cancer were enrolled in cohorts of 3-6 pts each, followed by disease specific expansion cohorts at the recommended phase 2 dose (RP2D). AM0010 was self-administrated daily subcutaneously for the duration of treatment and responses were monitored following immune related response criteria (irRC). Immune responses were monitored through analysis of serum cytokines, activation of blood derived T cells, immunosequencing for peripheral T cell clonality and immunohistochemistry for the infiltration of tumors by CD8 T cells. Results: 33 pts were enrolled in dose escalation cohorts with AM0010 monotherapy at doses from 1 to 40 μg/kg. MTD was not established. The RP2D (20μg/kg) was determined based on tolerability, single agent anti-tumor activity and immune activation. Patients were heavily pretreated with a median of 5 prior treatments. Most treatment related adverse events (TrAE) were low grade and included anemia, thrombocytopenia, rash, injection site reaction, fatigue, increased lipase, dyslipidemia and transaminitis. G3/4 non-hematopoietic TrAEs were observed in 11 of 51 pts. G3/4 anemia or thrombocytopenia TrAEs were observed in 13 pts. Most TrAEs were transient with only one patient discontinuing treatment due to a TrAE. Immune related TrAEs such as colitis, pneumonitis and endocrine disruption were not observed. AM0010 induced a dose dependent Th1 cytokine signature (IL-18, IFNγ, IL-7) and a reduction of TGFβ in the serum of pts. AM0010 increased PD-1 positive activated CD8 T cells in the blood and the tumor and decreased proliferation of FoxP3 Tregs in the blood. AM0010 lead to an oligoclonal expansion of T cell clones in the blood without affecting the overall number of lymphocytes. Many of the expanded T cell clones were not detectable before treatment. Partial responses (PR) were observed in pts with RCC and uveal melanoma. Four of 15 RCC pts treated at RP2D (27%) had an objective response. Prolonged stable disease of 6 or more months was observed in several indications including colon cancer. Conclusion: AM0010 is well-tolerated and leads to sustained and systemic immune stimulation. The pharmacodynamics and clinical activity observed support the further exploration of AM0010 in monotherapy and in combination regimens with checkpoint inhibitors and with cytotoxic chemotherapies. Trial registration: www.clinicaltrials.govNCT02009449 Citation Format: Jeffrey R. Infante, Aung Naing, Kyriakos P. Papadopoulos, Karen A. Autio, Patrick A. Ott, Deborah J. Wong, Gerald S. Falchook, Manish Patel, Shubham Pant, Melinda Whiteside, John B. Mumm, Ivan H. Chan, Johanna C. Bendell, Todd M. Bauer, Filip Janku, Milind Javle, Rivka R. Colen, Nizar M. Tannir, Martin Oft. Anti-tumor activity of PEGylated human IL-10 (AM0010) in patients with advanced solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT098.

Killing from within

Interleukin-10 (IL-10) is considered to be an immunosuppressive cytokine. However, the continuous administration of pegylated IL-10 (PEG-IL10) leads to the rejection of large, firmly established and metastatic syngeneic tumors. PEG-IL10 therapy induces the expansion and activation of intratumoral, tumor antigen-specific CD8+ T cells, leading to interferon γ (IFNγ)-mediated Th1 like immunity and tumor rejection.