Mechanism and control of recombinant murine interleukin-12-induced immunosuppression

Interleukin 12 (IL-12) is an immunomodulatory cytokine with potent antitumor, antiviral and antimicrobial effects. Its activities are attributable to its ability to induce Th1 CD4+ T cell differentiation, CD8 + T cell cytotoxicity and NK cell activation. Through its ability to induce the production of IFN-γ by T and NK cells, IL-12 indirectly activates macrophages and induces the production of nitric oxide. IFN-γ also has a variety of effects on other host cells and, relevant to its antitumor effects, IFN-γ upregulates MHC expression, slows cell proliferation and inhibits tumor angiogenesis. In our efforts to determine the mechanisms underlying rmIL-12 efficacy, we chose to study IL-12 effects during vaccination with irradiated cancer cell vaccines to avoid confounding factors of tumor growth. We identified a transient immunosuppression of antitumor responses associated with rmIL-12 treatment. rmIL-12 given to A/J mice vaccinated with irradiated SCK mammary carcinoma cells engineered to secrete GM-CSF resulted in significantly better protection from tumor challenges 28 days after vaccination but, unexpectedly, severely compromised host protection 14 days after vaccination. Immune suppression was rmIL-12 dose-dependent and manifest as reduced splenic CTL activity, stimulated cytokine release and ability to reject SCK cells. The period of suppression coincided with transiently reduced splenic T cell mitogenic responses to Con A and IL-2, suggesting that they may be causally related. We showed that suppressed mitogenic responses associated with rmIL-12 therapy were not restricted to splenocytes from SCK.GM vaccinated, rmIL-12 treated A/J mice but were also found in rmIL-12 treated, vaccinated and naive mice of multiple strains. Suppression appears to be due to impaired immune effector mechanisms rather than impaired host immunization, as evidenced by the enhanced reaction to immunogens when hosts are challenged later after rmIL-12 administration. We therefore sought to determine the mechanism of this transient immunosuppression and used both DTH (in vivo) and mitogenic (in vitro) responses in C57BL/6 mice immunized with allogeneic HKB cells. Administration of neutralizing antibodies to HKB-vaccinated C57BL/6 mice showed a role for IFN-γ, known to mediate many of IL-12 effects, but not TNFa, a cytokine implicated in IL-12 induced suppression during LCMV infection, and these results were later confirmed in IFN-γ and TNFR knockout mice. Adherent cells from the spleens of rmIL-12 treated mice were identified as the subpopulation associated with suppressed T cell mitogenic and alloproliferative responses. Further investigation revealed an IFN-γ dependent induction of macrophage derived nitric oxide. Reversion of both in vitro and in vivo suppressed responses was possible by using chemical inhibitors to NOS. Further, these inhibitors are capable of preventing suppression of vaccine efficacy measured by tumor cell rejection and reveal rmIL-12 vaccine adjuvant effects

Mechanism and control of recombinant murine interleukin-12-induced immunosuppression

Interleukin 12 (IL-12) is an immunomodulatory cytokine with potent antitumor, antiviral and antimicrobial effects. Its activities are attributable to its ability to induce Th1 CD4+ T cell differentiation, CD8 + T cell cytotoxicity and NK cell activation. Through its ability to induce the production of IFN-γ by T and NK cells, IL-12 indirectly activates macrophages and induces the production of nitric oxide. IFN-γ also has a variety of effects on other host cells and, relevant to its antitumor effects, IFN-γ upregulates MHC expression, slows cell proliferation and inhibits tumor angiogenesis. In our efforts to determine the mechanisms underlying rmIL-12 efficacy, we chose to study IL-12 effects during vaccination with irradiated cancer cell vaccines to avoid confounding factors of tumor growth. We identified a transient immunosuppression of antitumor responses associated with rmIL-12 treatment. rmIL-12 given to A/J mice vaccinated with irradiated SCK mammary carcinoma cells engineered to secrete GM-CSF resulted in significantly better protection from tumor challenges 28 days after vaccination but, unexpectedly, severely compromised host protection 14 days after vaccination. Immune suppression was rmIL-12 dose-dependent and manifest as reduced splenic CTL activity, stimulated cytokine release and ability to reject SCK cells. The period of suppression coincided with transiently reduced splenic T cell mitogenic responses to Con A and IL-2, suggesting that they may be causally related. We showed that suppressed mitogenic responses associated with rmIL-12 therapy were not restricted to splenocytes from SCK.GM vaccinated, rmIL-12 treated A/J mice but were also found in rmIL-12 treated, vaccinated and naive mice of multiple strains. Suppression appears to be due to impaired immune effector mechanisms rather than impaired host immunization, as evidenced by the enhanced reaction to immunogens when hosts are challenged later after rmIL-12 administration. We therefore sought to determine the mechanism of this transient immunosuppression and used both DTH (in vivo) and mitogenic (in vitro) responses in C57BL/6 mice immunized with allogeneic HKB cells. Administration of neutralizing antibodies to HKB-vaccinated C57BL/6 mice showed a role for IFN-γ, known to mediate many of IL-12 effects, but not TNFa, a cytokine implicated in IL-12 induced suppression during LCMV infection, and these results were later confirmed in IFN-γ and TNFR knockout mice. Adherent cells from the spleens of rmIL-12 treated mice were identified as the subpopulation associated with suppressed T cell mitogenic and alloproliferative responses. Further investigation revealed an IFN-γ dependent induction of macrophage derived nitric oxide. Reversion of both in vitro and in vivo suppressed responses was possible by using chemical inhibitors to NOS. Further, these inhibitors are capable of preventing suppression of vaccine efficacy measured by tumor cell rejection and reveal rmIL-12 vaccine adjuvant effects

Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8+ T cells directly within the tumor microenvironment

BACKGROUND: Blockade of immune inhibitory pathways is emerging as an important therapeutic modality for the treatment of cancer. Single agent treatments have partial anti-tumor activity in preclinical models and in human cancer patients. Inasmuch as the tumor microenvironment shows evidence of multiple immune inhibitory mechanisms present concurrently, it has been reasoned that combination therapies may be required for optimal therapeutic effect. METHODS: To test this notion, we utilized permutations of anti-CTLA-4 mAb, anti-PD-L1 mAb, and/or the IDO inhibitor INCB23843 in the murine B16.SIY melanoma model. RESULTS: All three combinations showed markedly improved tumor control over single treatments, with many mice achieving complete tumor rejection. This effect was seen in the absence of vaccination or adoptive T cell therapy. The mechanism of synergy was investigated to examine the priming versus effector phase of the anti-tumor immune response. Only a minimal increase in priming of anti-tumor T cells was observed at early time points in the tumor-draining lymph nodes (TdLN). In contrast, as early as three days after therapy initiation, a marked increase in the capacity of tumor-infiltrating CD8(+) T cells to produce IL-2 and to proliferate was found in all groups treated with the effective combinations. Treatment of mice with FTY720 to block new T cell trafficking from secondary lymphoid structures still enabled restoration of IL-2 production and proliferation by intratumoral T cells, and also retained most of the tumor growth control. CONCLUSIONS: Our data suggest that the therapeutic effect of these immunotherapies was mainly mediated through direct reactivation of T cells in situ. These three combinations are attractive to pursue clinically, and the ability of intratumoral CD8(+) T cells to produce IL-2 and to proliferate could be an important biomarker to integrate into clinical studies

INCB054828 (pemigatinib), a potent and selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays activity against genetically defined tumor models.

Alterations in fibroblast growth factor receptor (FGFR) genes have been identified as potential driver oncogenes. Pharmacological targeting of FGFRs may therefore provide therapeutic benefit to selected cancer patients, and proof-of-concept has been established in early clinical trials of FGFR inhibitors. Here, we present the molecular structure and preclinical characterization of INCB054828 (pemigatinib), a novel, selective inhibitor of FGFR 1, 2, and 3, currently in phase 2 clinical trials. INCB054828 pharmacokinetics and pharmacodynamics were investigated using cell lines and tumor models, and the antitumor effect of oral INCB054828 was investigated using xenograft tumor models with genetic alterations in FGFR1, 2, or 3. Enzymatic assays with recombinant human FGFR kinases showed potent inhibition of FGFR1, 2, and 3 by INCB054828 (half maximal inhibitory concentration [IC50] 0.4, 0.5, and 1.0 nM, respectively) with weaker activity against FGFR4 (IC50 30 nM). INCB054828 selectively inhibited growth of tumor cell lines with activation of FGFR signaling compared with cell lines lacking FGFR aberrations. The preclinical pharmacokinetic profile suggests target inhibition is achievable by INCB054828 in vivo with low oral doses. INCB054828 suppressed the growth of xenografted tumor models with FGFR1, 2, or 3 alterations as monotherapy, and the combination of INCB054828 with cisplatin provided significant benefit over either single agent, with an acceptable tolerability. The preclinical data presented for INCB054828, together with preliminary clinical observations, support continued investigation in patients with FGFR alterations, such as fusions and activating mutations

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies

<div><p>The Proviral Integration site of Moloney murine leukemia virus (PIM) serine/threonine protein kinases are overexpressed in many hematologic and solid tumor malignancies and play central roles in intracellular signaling networks important in tumorigenesis, including the Janus kinase–signal transducer and activator of transcription (JAK/STAT) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. The three PIM kinase isozymes (PIM1, PIM2, and PIM3) share similar downstream substrates with other key oncogenic kinases and have differing but mutually compensatory functions across tumors. This supports the therapeutic potential of pan-PIM kinase inhibitors, especially in combination with other anticancer agents chosen based on their role in overlapping signaling networks. Reported here is a preclinical characterization of INCB053914, a novel, potent, and selective adenosine triphosphate-competitive pan-PIM kinase inhibitor. <i>In vitro</i>, INCB053914 inhibited proliferation and the phosphorylation of downstream substrates in cell lines from multiple hematologic malignancies. Effects were confirmed in primary bone marrow blasts from patients with acute myeloid leukemia treated <i>ex vivo</i> and in blood samples from patients receiving INCB053914 in an ongoing phase 1 dose-escalation study. <i>In vivo</i>, single-agent INCB053914 inhibited Bcl-2–associated death promoter protein phosphorylation and dose-dependently inhibited tumor growth in acute myeloid leukemia and multiple myeloma xenografts. Additive or synergistic inhibition of tumor growth was observed when INCB053914 was combined with selective PI3Kδ inhibition, selective JAK1 or JAK1/2 inhibition, or cytarabine. Based on these data, pan-PIM kinase inhibitors, including INCB053914, may have therapeutic utility in hematologic malignancies when combined with other inhibitors of oncogenic kinases or standard chemotherapeutics.</p></div

Structure of INCB053914 and IC₅₀ values for the inhibition of PIM isozymes by INCB053914 in biochemical assays.

<p>Structure of INCB053914 and IC₅₀ values for the inhibition of PIM isozymes by INCB053914 in biochemical assays.</p

INCB053914 inhibits erythroid colony formation in patients with JAK2 V617F-positive MPNs.

<p>Error bars represent standard deviation. *p < 0.05; **p < 0.01.</p

Preclinical characterization of INCB053914, a novel pan-PIM kinase inhibitor, alone and in combination with anticancer agents, in models of hematologic malignancies - Fig 2

<p><b>INCB053914 inhibits cellular proliferation in hematologic tumor cell lines (A), inhibits phosphorylation of PIM substrates (B), including pBAD (C), and increases PIM2 expression (D) in hematologic tumor cell lines.</b> For all Western blots, actin controls confirmed equivalent loading. IC₅₀ values for pBAD inhibition were determined by fitting the percent inhibition versus the log [INCB053914] data to sigmoidal dose–response (variable slope) curve. Error bars represent standard deviation. GI₅₀ values >3 μM are not shown. *pP70S6K band intensities in KMS-12-BM (MM) cells were below the limit of detection at all INCB053914 concentrations tested. HL, Hodgkin lymphoma; ND, not determined. Original Western blot images are shown in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199108#pone.0199108.s008" target="_blank">S4 File</a>.</p