719 XTX301, a protein-engineered IL-12, exhibits tumor-selective activity in mice without peripheral toxicities and is well tolerated in non-human primates

BackgroundInterleukin-12 (IL-12) is a proinflammatory cytokine which bridges innate and adaptive immunity via induction of T helper 1 differentiation and promoting cytolytic activity of natural killer and T cells. IL-12 has demonstrated potent antitumor activity in syngeneic mouse models and promising anti-tumor efficacy in humans. However, development of IL-12 has been limited by severe systemic toxicities. To overcome toxicity and improve the therapeutic index of IL-12, we employed protein engineering to generate XTX301, a highly potent, half-life extended and masked IL-12. The masking domain of XTX301 is designed to pharmacologically inactivate IL-12 systemically and render an active IL-12 moiety upon cleavage by proteases that are enriched in the tumor microenvironment.MethodsWe conducted experiments to assess the binding, bioactivity, safety, and anti-tumor efficacy of XTX301. Binding interactions were measured via SPR, bioactivity was measured using STAT-4 phosphorylation in a reporter cell line, and IFN-g production was assessed in human PBMCs via ELISA. Anti-tumor efficacy and pharmacodynamics were assessed in MC38 and B16F10 syngeneic tumor mouse models using a XTX301 murine surrogate, mXTX301. Safety and pharmacokinetics of XTX301 were evaluated in non-human primates (NHP).ResultsXTX301 showed no detectable binding to the high affinity IL12RB2 demonstrating that the masking domain indeed prevents interaction with the receptor. Upon cleavage of the masking domain by relevant proteases, binding was observed and was comparable to XTX300 unmasked control. Likewise, restoration of activity upon proteolytic cleavage was observed in an IL-12-dependent reporter gene assay and in primary human PBMCs. Human IL-12 does not cross react with mouse IL-12 receptors; hence a murine surrogate (mXTX301) was created for in vivo anti-tumor efficacy evaluation. A single dose of mXTX301 demonstrated up to 90% tumor growth inhibition in an inflamed MC38 and non-inflamed B16F10 syngeneic mouse models. mXTX301 induced a ~3 fold increase in IFN-g in tumors compared to vehicle control and ~150 fold less peripheral IFN-g compared to mXTX300. XTX301 exhibits minimal elevation in liver enzymes and a 50-fold improvement in tolerability compared to XTX300, in a repeat dose NHP safety study.ConclusionsOur data demonstrates that both XTX301 and mXTX301 are inactive when in masked form and become activated upon proteolytic cleavage to exert bioactivity comparable to recombinant IL-12. For efficacy, mXTX301 demonstrated tumor selective activity in syngeneic mouse models. XTX301 was well tolerated in repeat dose NHP safety study. In conclusion, XTX301 has potential for exerting potent anti-tumor activity with a favorable tolerability profile.

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Tumor-selective activity of XTX202, a protein-engineered IL-2, in mice without peripheral toxicities in nonhuman primates.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.2563 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 2563-2563

Author(s):

Jennifer O'Neil ◽

Wilson Guzman ◽

Oleg Yerov ◽

Parker Johnson ◽

Manoussa Fanny ◽

...

Keyword(s):

Tumor Growth ◽

Immune Activation ◽

Half Life ◽

Second Generation ◽

Therapeutic Index ◽

Repeat Dose ◽

Third Generation ◽

Proteolytic Activation ◽

Tumor Selective ◽

Selective Activity

2563 Background: High-dose recombinant human interleukin-2 (aldesleukin) elicits anti-tumor immunity and is approved for the treatment of renal cell carcinoma and melanoma based on durable complete remissions. However, use of aldesleukin is limited due to treatment-related life-threatening toxicities. Recent second-generation efforts to alleviate toxicities have largely focused on eliminating binding to IL-2Rα, often with half-life extension. We have determined that mice and non-human primates (NHPs) treated with a second generation IL-2 surrogate still experience characteristic dose-limiting toxicities, including vascular leak syndrome. To overcome these toxicities and improve the therapeutic index (TI) of IL-2 as an anti-tumor immunotherapy, we employed protein engineering to generate XTX202, a highly potent third generation masked IL-2. XTX202 is unmasked in the tumor microenvironment by proteolytic activation resulting in full restoration of binding to IL-2Rβ without binding to IL-2Rα. The current study characterizes the therapeutic index of XTX202 versus aldesleukin and a second generation IL-2 surrogate. Methods: XTX202 bioactivity was measured using STAT-5 phosphorylation in human PBMCs and reporter cell lines. Anti-tumor efficacy and peripheral immune activation were evaluated in mice bearing syngeneic tumor models. Safety was evaluated in rodents and Cynomolgus monkeys. XTX200, an unmasked half-life extended IL-2 that does not bind to IL-2Rα, was used as a surrogate second generation IL-2. Results: Masked XTX202 showed limited IL-2R-dependent STAT-5 signaling in vitro. Proteolytic activation of XTX202 resulted in CD8+ T and NK cell activation and over 1000-fold reduction in Treg activation as compared to WT IL-2. XTX202 achieved potent tumor growth inhibition in syngeneic mouse models as a single agent with no evidence of toxicity or peripheral immune activation, thus demonstrating tumor selective activity. XTX202 efficacy in mice at 2 mg/kg dose was equivalent to that achieved with the MTD dose of 0.5 mg/kg of a second generation IL-2 surrogate. XTX202 was well tolerated in NHPs in a 4-week repeat dose study at doses up to 30 mg/kg QW whereas a second generation IL-2 surrogate was not tolerated beyond 0.7 mg/kg QW. Based on these data, XTX202 has a 10 fold improvement in TI vs second generation IL-2. Based on comparative efficacy studies with aldesleukin and literature NHP tolerability data, XTX202 is projected to have a ≥150 fold greater TI than aldesleukin. Conclusions: XTX202, a third generation, tumor-selective IL-2, inhibits tumor growth and is well tolerated in repeat dose studies in NHPs at high doses. GLP toxicity studies with XTX202 are underway and first-in-human studies are expected to initiate this year. XTX202 has the potential to be a best-in-class IL-2 immunotherapy by expanding the curative anti-tumor activity of IL-2 while minimizing dose-limiting toxicities.

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Faculty Opinions recommendation of Evaluation of systemic targeting of RET oncogene-based MTC with tumor-selective peptide-tagged Ad vectors in clinical mouse models.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.8359956.8805055 ◽

2011 ◽

Author(s):

Gilbert Cote

Keyword(s):

Mouse Models ◽

Ret Oncogene ◽

Tumor Selective

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microRNA-130a-5p suppresses myocardial ischemia reperfusion injury by downregulating the HMGB2/NF-κB axis

BMC Cardiovascular Disorders ◽

10.1186/s12872-020-01742-4 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yong Li ◽

Hongbo Zhang ◽

Zhanhu Li ◽

Xiaoju Yan ◽

Yuan Li ◽

...

Keyword(s):

Oxidative Stress ◽

Myocardial Ischemia ◽

Reperfusion Injury ◽

Mouse Models ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Luciferase Reporter ◽

Gene Assay ◽

Myocardial Ischemia Reperfusion Injury ◽

Myocardial Ischemia Reperfusion

Abstract Background Myocardial ischemia reperfusion injury (MIRI) is defined as tissue injury in the pathological process of progressive aggravation in ischemic myocardium after the occurrence of acute coronary artery occlusion. Research has documented the involvement of microRNAs (miRs) in MIRI. However, there is obscure information about the role of miR-130a-5p in MIRI. Herein, this study aims to investigate the effect of miR-130a-5p on MIRI. Methods MIRI mouse models were established. Then, the cardiac function and hemodynamics were detected using ultrasonography and multiconductive physiological recorder. Functional assays in miR-130a-5p were adopted to test the degrees of oxidative stress, mitochondrial functions, inflammation and apoptosis. Hematoxylin and eosin (HE) staining was performed to validate the myocardial injury in mice. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to assess the expression patterns of miR-130a-5p, high mobility group box (HMGB)2 and NF-κB. Then, dual-luciferase reporter gene assay was performed to elucidate the targeting relation between miR-130a-5p and HMGB2. Results Disrupted structural arrangement in MIRI mouse models was evident from HE staining. RT-qPCR revealed that overexpressed miR-130a-5p alleviated MIRI, MIRI-induced oxidative stress and mitochondrial disorder in the mice. Next, the targeting relation between miR-130a-5p and HMGB2 was ascertained. Overexpressed HMGB2 annulled the protective effects of miR-130a-5p in MIRI mice. Additionally, miR-130a-5p targets HMGB2 to downregulate the nuclear factor kappa-B (NF-κB) axis, mitigating the inflammatory injury induced by MIRI. Conclusion Our study demonstrated that miR-130a-5p suppresses MIRI by down-regulating the HMGB2/NF-κB axis. This investigation may provide novel insights for development of MIRI treatments.

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