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Androgen deprivation (AD) therapy failure leads to terminal and incurable castration-resistant prostate cancer (CRPC). We show that the redox-protective protein thioredoxin-1 (TRX1) increases with prostate cancer progression and in androgen-deprived CRPC cells, suggesting that CRPC possesses an enhanced dependency on TRX1. TRX1 inhibition via shRNA or a phase I-approved inhibitor, PX-12 (untested in prostate cancer), impedes the growth of CRPC cells to a greater extent than their androgen-dependent counterparts. TRX1 inhibition elevates reactive oxygen species (ROS), p53 levels and cell death in androgen-deprived CRPC cells. Unexpectedly, TRX1 inhibition also elevates androgen receptor (AR) levels under AD, and AR depletion mitigates both TRX1 inhibition-mediated ROS production and cell death, suggesting that AD-resistant AR expression in CRPC induces redox vulnerability. In vivo TRX1 inhibition via shRNA or PX-12 reverses the castration-resistant phenotype of CRPC cells, significantly inhibiting tumor formation under systemic AD. Thus, TRX1 is an actionable CRPC therapeutic target through its protection against AR-induced redox stress. Identifying actionable components in castration–resistant prostate cancer (CRPC) is critical for the development of effective treatments. Here, the authors show that the inhibition of the redox-protective protein TRX1 decreases the growth of CRPC cells through the regulation of ROS levels, p53 and androgen receptor expression.

P2X 3 and P2X 2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X 3 and P2X 2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X 3 and P2X 2/3 receptor-mediated calcium flux ( K i = 22–92 nM) and was highly selective (IC 50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X 3 and P2X 2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X 3 containing channels was stereospecific because the R -enantiomer (A-317344) of A-317491 was significantly less active at P2X 3 and P2X 2/3 receptors. A-317491 dose-dependently (ED 50 = 30 μmol/kg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED 50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R -enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED 50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X 3 and P2X 2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X 3 and P2X 2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

BackgroundHCW9218 is a bifunctional protein complex comprising dimeric extracellular domains of human transforming growth factor beta (TGF-β) receptor II and interleukin-15 (IL-15). HCW9218 1) stimulates immune effector cells and 2) sequesters soluble immunosuppressive TGF-β.1 2 The objectives of our pre-clinical and first-in-human studies are to explore the antitumor mechanism of action (MOA) and determine the RP2D.MethodsTwo syngeneic mouse tumor models were used to explore the MOA of HCW9218. A phase I dose escalation study (NCT05322408) of HCW9218 monotherapy (Q3W, subcutaneous) has been completed in adult patients (n=12) with refractory advanced/metastatic solid tumors and is expanding at the highest dose level (1.2mg/kg).3 Correlative analyses included pharmacokinetics, serum cytokines, blood lymphocytes, and cellular/molecular profiling of tumors.ResultsIn B16F10 melanoma and 4T1 breast cancer murine models, HCW9218 promotes proliferation and activation of progenitor exhausted (Tpex) and exhausted transitory effector (Tem) CD8+ T cells in draining lymph nodes, and CD8+ memory T cells and NK cells in peripheral blood. HCW9218 increases tumor infiltrating Tpex, Tem, and memory CD8+ T cells and enhances antitumor potency of immune checkpoint blockade therapy, likely due to stimulation of Tpex and Tem cells. HCW9218 protein localizes in tumors and significantly lowers TGF-β levels and increases proinflammatory cytokines. In the first-in-human clinical trial, there were no dose limiting toxicities. The most common adverse events are grade 1–2 injection site reactions and transient lymphopenia. Patients receiving ≥0.25 mg/kg HCW9218 exhibit robust NK cell and CD8+ T cell proliferation through day 15 and recurred with each treatment cycle, a biologic effect beyond what has been previously observed with other IL-15 agonists. At ≥0.5 mg/kg HCW9218, serum TGF-β1 levels decrease to baseline through day 8. Immunofluorescent staining of patients’ tumors show that HCW9218 increases CD8+ T cell infiltration with elevated tumor Tpex and/or fully differentiated memory CD8+ T cells correlating with disease stabilization. Single-cell RNA-seq tumor analysis demonstrates that HCW9218 reduces expression of genes associated with tumor invasion, immunosuppression, and inflammation and upregulates genes involved in differentiation of Tpex, TCR signaling, and inflammatory response, while downregulating proinflammatory response genes of tumor lymphocytes. HCW9218 has a serum half-life of ~25h in patients.ConclusionsRepeated HCW9218 administration at ≥0.5 mg/kg in heavily pretreated advanced solid tumor patients resulted in immune cell activation, proliferation, and infiltration into the tumor microenvironment without causing unacceptable toxicity. HCW treatment presents a promising approach to enhancing the antitumor activity of immune checkpoint inhibitors in patients with solid tumors.Trial RegistrationNCT05322408ReferencesLiu B, Zhu X, Kong L, et al. Bifunctional TGF-beta trap/IL-15 protein complex elicits potent NK cell and CD8(+) T cell immunity against solid tumors. Mol Ther 2021;29:2949–62.Chaturvedi P, George V, Shrestha N, et al. Immunotherapeutic HCW9218 augments anti-tumor activity of chemotherapy via NK cell-mediated reduction of therapy-induced senescent cells. Mol Ther 2022;30:1171–87.Geller M, Patel M, Wong H, et al. A phase I study of HCW9218, a bifunctional TGF-β antagonist/IL-15 protein complex, in advanced solid tumors. J Immunother Cancer 2022;10. doi: 10.1136/jitc-2022-SITC2022.0724Ethics ApprovalThis study was approved by the University of Minnesota’s Institutional Review Board; approval number: 00015102.ConsentWritten informed consent was obtained from the patients included in the clinical trial for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming (“Prime” step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 (“Expand” step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting “Prime and Expand” ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the “Prime and Expand” strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

Immune checkpoint therapy, which can target regulatory pathways of T cells to enhance antitumor immune responses, has led to important clinical advances and provides novel strategies for combination immunotherapies against cancer. ALT-803 is an IL-15 superagonist complex capable of stimulating T cell and NK cell responses without triggering Treg activity. ALT-803 exhibits improved pharmacokinetics and lymphoid organ biodistribution compared to native IL-15, allowing this complex to have more potent efficacy against various hematologic and solid tumor models. Antibodies that block checkpoint inhibitor interactions, such as PD-1/PD-L1 or CTLA-4, have shown clinical efficacy against both melanoma and lung cancer, and may also be active in other tumor types, including bladder cancer. Here, we report that the combination of immune checkpoint blockers with ALT-803 has potent anti-tumor activity in C57BL/6 mice bearing syngenic orthotopic MB49-luc bladder tumors. These tumor cells were found to express CTLA-4 ligands and PD-L1 on the cell surface; thus, blockade of both CTLA-4 and PD-1/PD-L1 pathways were evaluated. Treatment of mice bearing established MB49luc bladder tumors with ALT-803 monotherapy exhibited a statistically significant increase in survival compared to controls (P < 0.05). However, the combination of ALT-803 with anti-PD-L1 and anti-CTLA-4 Abs further prolonged survival compared to control or related monotherapies. This effect was also seen with combination therapy of ALT-803+anti-PD-1 and ALT-803+anti-PD-1/anti-CTLA-4 Abs. Additionally, mice that were cured of MB49-luc tumors by ALT-803 plus anti-PD-L1/anti-CTLA-4 Ab therapy were resistant from bladder tumor rechallenge without further drug treatment whereas age-matched treatment-naïve mice developed tumors and die following MB49-luc tumor cell instillation. These results demonstrate that a short course of ALT-803 plus immune checkpoint inhibitor treatment not only has potent efficacy against established MB49luc tumor, but is also capable of inducing long-lasting protective immunologic memory against subsequent tumor cell rechallenge. Characterization of immune cell activity responsible for improved antitumor efficacy is underway. Overall, these results confirm that enhanced antitumor responses can be achieved by combining immune checkpoint blockers with the immunostimulatory agonist ALT-803, warranting evaluation of these strategies in the clinical setting.

One subtype of ATP-gated ion channel, the P2X(3) receptor, is expressed primarily on peripheral sensory neurons. While it is known that P2X(3) receptors can participate in certain forms of nociceptive signaling, their involvement in neuropathic pain transmission is not known. We have examined the expression and function of P2X(3) receptors in a rat spinal nerve ligation model of neuropathic pain. Fourteen days following L5/L6 spinal nerve ligation, the corresponding dorsal root ganglia (DRG) were removed from animals exhibiting mechanical allodynia, and these were studied using immunohistochemical and electrophysiological techniques. Using a polyclonal antibody to label the P2X(3) receptor, a significant reduction in neuronal P2X(3) immunoreactivity was observed in the ipsilateral (injured) L5 and L6 DRG following nerve ligation. In vitro electrophysiological analysis of acutely isolated DRG neurons revealed a similar decrease in functional P2X(3)-containing receptors. In small diameter (22-25 micro m) neurons, a significant reduction in the number of cells exhibiting a response to alpha,beta-meATP was observed. However, a subset of small diameter neurons retained P2X(3) responses of equal amplitude to those recorded from naive and sham control DRG neurons. Interestingly, P2X(3) immunoreactivity and P2X(3)-like responses were also detected in a subset of larger diameter (50 micro m) neurons and the number and amplitude of these responses were unchanged after spinal nerve ligation. These results suggest that, while there appears to be a decrease in fast desensitizing P2X(3) receptors following L5/L6 nerve ligation injury, certain subsets of small and large DRG neurons maintain normal P2X(3) receptor expression and function. These remaining receptors may provide a P2X(3) receptor-mediated component to neuropathic pain.

P2X 3 and P2X 2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X 3 and P2X 2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X 3 and P2X 2/3 receptor-mediated calcium flux ( K i = 22–92 nM) and was highly selective (IC 50 >10 μM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X 3 and P2X 2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X 3 containing channels was stereospecific because the R -enantiomer (A-317344) of A-317491 was significantly less active at P2X 3 and P2X 2/3 receptors. A-317491 dose-dependently (ED 50 = 30 μmol/kg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED 50 = 10–15 μmol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R -enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED 50 >100 μmol/kg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X 3 and P2X 2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X 3 and P2X 2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

P2X3and P2X2/3receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3and P2X2/3receptor activation. A-317491 potently blocked recombinant human and rat P2X3and P2X2/3receptor-mediated calcium flux (Ki= 22-92 nM) and was highly selective$(IC_{50} > 10\\;\\mu M)$over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3and P2X2/3receptors in rat dorsal root ganglion neurons. Blockade of P2X3containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3and P2X2/3receptors. A-317491 dose-dependently (ED50= 30 μ mol/kg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50= 10-15 μ mol/kg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective$(ED_{50} > 100\\;\\mu mol/kg\\;s.c.)$in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3and P2X2/3receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3and P2X2/3receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.