XTX301, a Tumor-Activated Interleukin-12 Has the Potential to Widen the Therapeutic Index of IL12 Treatment for Solid Tumors as Evidenced by Preclinical Studies.

IL12 is a proinflammatory cytokine, that has shown promising antitumor activity in humans by promoting the recruitment and activation of immune cells in tumors. However, the systemic administration of IL12 has been accompanied by considerable toxicity, prompting interest in researching alternatives to drive preferential IL12 bioactivity in the tumor. Here, we have generated XTX301, a tumor-activated IL12 linked to the human Fc protein via a protease cleavable linker that is pharmacologically inactivated by an IL12 receptor subunit beta 2 masking domain. In vitro characterization demonstrates multiple matrix metalloproteases, as well as human primary tumors cultured as cell suspensions, can effectively activate XTX301. Intravenous administration of a mouse surrogate mXTX301 demonstrated significant tumor growth inhibition (TGI) in inflamed and non-inflamed mouse models without causing systemic toxicities. The superiority of mXTX301 in mediating TGI compared with non-activatable control molecules and the greater percentage of active mXTX301 in tumors versus other organs further confirms activation by the tumor microenvironment-associated proteases in vivo. Pharmacodynamic characterization shows tumor selective increases in inflammation and upregulation of immune-related genes involved in IFNγ cell signaling, antigen processing, presentation, and adaptive immune response. XTX301 was tolerated following four repeat doses up to 2.0 mg/kg in a nonhuman primate study; XTX301 exposures were substantially higher than those at the minimally efficacious dose in mice. Thus, XTX301 has the potential to achieve potent antitumor activity while widening the therapeutic index of IL12 treatment and is currently being evaluated in a phase I clinical trial.

Novel Arginase Inhibitor, AZD0011, Demonstrates Immune Cell Stimulation and Antitumor Efficacy with Diverse Combination Partners.

Antitumor immunity can be hampered by immunosuppressive mechanisms in the tumor microenvironment, including recruitment of arginase (ARG) expressing myeloid cells that deplete l-arginine essential for optimal T-cell and natural killer cell function. Hence, ARG inhibition can reverse immunosuppression enhancing antitumor immunity. We describe AZD0011, a novel peptidic boronic acid prodrug to deliver an orally available, highly potent, ARG inhibitor payload (AZD0011-PL). We demonstrate that AZD0011-PL is unable to permeate cells, suggesting that this compound will only inhibit extracellular ARG. In vivo, AZD0011 monotherapy leads to arginine increases, immune cell activation, and tumor growth inhibition in various syngeneic models. Antitumor responses increase when AZD0011 is combined with anti-PD-L1 treatment, correlating with increases in multiple tumor immune cell populations. We demonstrate a novel triple combination of AZD0011, anti-PD-L1, and anti-NKG2A, and combination benefits with type I IFN inducers, including polyI:C and radiotherapy. Our preclinical data demonstrate AZD0011's ability to reverse tumor immunosuppression and enhance immune stimulation and antitumor responses with diverse combination partners providing potential strategies to increase immuno-oncology therapies clinically.

Discovery of CC-99677, a selective targeted covalent MAPKAPK2 (MK2) inhibitor for autoimmune disorders.

As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (SNAr) mechanism. This irreversible mechanism translates biochemical potency to cells shown by potent inhibition of heat shock protein 27 (HSP27) phosphorylation in LPS-activated monocytic THP-1 cells. The cytokine inhibitory profile of CC-99677 differentiates it from known p38 inhibitors, potentially suppressing a p38 pathway inflammatory response while avoiding tachyphylaxis. Dosed orally, CC-99677 is efficacious in a rat model of ankylosing spondylitis. Single doses, 3 to 400 mg, in healthy human volunteers show linear pharmacokinetics and apparent sustained tumor necrosis factor-α inhibition, with a favorable safety profile. These results support further development of CC-99677 for autoimmune diseases like ankylosing spondylitis.

Systemic nano-delivery of low-dose STING agonist targeted to CD103+ dendritic cells for cancer immunotherapy.

Current methods of STING activation based on intra-tumoral injections of cyclic dinucleotides (CDNs) are not suitable for addressing tumor heterogeneity or for inaccessible, metastatic and abscopal tumors. In this study, we developed systemically administered CD103+ dendritic cell (DCs) targeted liposomal formulations and evaluated the anti-tumor efficacy with low dose. Liposomal CDN formulations were prepared using Clec9a targeting peptide and evaluated therapeutic efficacy in vitro and in vivo in subcutaneous MC38 and B16F10 tumor models. Targeted delivery of CDNs is expected to enhance anti-tumor immune response as well as reduce off-target toxicities. With intravenous 0.1 mg/kg systemic CDN dose of the targeted liposomal formulation, our results showed robust immune response with significant antitumor efficacy both as a monotherapy and in combination with anti-PD-L1 antibody. These results show that a CD103+ DC targeted CDN formulation can lead to potent immune stimulation upon systemic administration even in relatively "cold" tumors such as B16F10.

SMaSh: A Streptavidin Mass Shift Assay for Rapidly Quantifying Target Occupancy by Irreversible Inhibitors.

The streptavidin mass shift (SMaSh) assay is a robust and fast approach for quantifying target protein occupancy by a covalent inhibitor or ligand. It exploits the biotin-streptavidin bond using the Simple Western platform. One measurement on a single sample determines both total and occupied target protein simultaneously and is, therefore, self-normalizing. The approach works in diverse and complex biological matrices and, with no need for matched vehicle-treated controls, readily applies to tissues from animal pharmacology models. Assessing occupancy is critical in the development of targeted covalent drugs. We demonstrate its use by characterizing and validating a variety of chemical probes for Bruton's tyrosine kinase (BTK, UniprotKB Q10607) and mitogen-activated protein kinase (ERK1/2/MAPK1/2, UniprotKB P28482 and P27361) and determining target engagement of covalent inhibitors for both targets and off-target engagement for ERK. We demonstrated that it works in cell lysates, tissues, and human peripheral blood mononuclear cells. The SMaSh assay is superior to traditional methods and broadly useful as a tool in assessing covalent biological probes or targeted covalent inhibitors.

STAT3 Antisense Oligonucleotide Remodels the Suppressive Tumor Microenvironment to Enhance Immune Activation in Combination with Anti-PD-L1.

PURPOSE: Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action. EXPERIMENTAL DESIGN: Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen. RESULTS: Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti-PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti-PD-L1. CONCLUSIONS: STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice.

BACKGROUND: Forty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects. HC-070 IN VITRO: To assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases. HC-070 IN VIVO: Upon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC50) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.

Design, synthesis, and biological evaluation of peptidomimetic inhibitors of factor XIa as novel anticoagulants.

Human coagulation factor XIa (FXIa), a serine protease activated by site-specific cleavage of factor XI by thrombin, FXIIa, or autoactivation, is a critical enzyme in the amplification phase of the coagulation cascade. To investigate the potential of FXIa inhibitors as safe anticoagulants, a series of potent, selective peptidomimetic inhibitors of FXIa were designed and synthesized. Some of these inhibitors showed low nanomolar FXIa inhibitory activity with >1000-fold FXa selectivity and >100-fold thrombin selectivity. The X-ray structure of one of these inhibitors, 36, demonstrates its unique binding interactions with FXIa. Compound 32 caused a doubling of the activated partial thromboplastin time in human plasma at 2.4 microM and was efficacious in a rat model of venous thrombosis. These data suggest that factor XIa plays a significant role in venous thrombosis and may be a suitable target for the development of antithrombotic therapy.

The three-dimensional structure of the ZAP-70 kinase domain in complex with staurosporine: implications for the design of selective inhibitors.

The ZAP-70 tyrosine kinase plays a critical role in T cell activation and the immune response and therefore is a logical target for immunomodulatory therapies. Although the crystal structure of the tandem Src homology-2 domains of human ZAP-70 in complex with a peptide derived from the zeta subunit of the T cell receptor has been reported (Hatada, M. H., Lu, X., Laird, E. R., Green, J., Morgenstern, J. P., Lou, M., Marr, C. S., Phillips, T. B., Ram, M. K., Theriault, K., Zoller, M. J., and Karas, J. L. (1995) Nature 377, 32-38), the structure of the kinase domain has been elusive to date. We crystallized and determined the three-dimensional structure of the catalytic subunit of ZAP-70 as a complex with staurosporine to 2.3 A resolution, utilizing an active kinase domain containing residues 327-606 identified by systematic N- and C-terminal truncations. The crystal structure shows that this ZAP-70 kinase domain is in an active-like conformation despite the lack of tyrosine phosphorylation in the activation loop. The unique features of the ATP-binding site, identified by structural and sequence comparison with other kinases, will be useful in the design of ZAP-70-selective inhibitors.