Preclinical characterization and clinical translation of pharmacodynamic markers for MK-5890: a human CD27 activating antibody for cancer immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI therapy. CD27 is a member of the tumor necrosis factor receptor superfamily that plays a critical role in promoting responses of T cells, B cells and NK cells. METHODS: Anti-CD27 antibodies were generated and selected for agonist activity using NF-кB luciferase reporter assays. Antibodies were humanized and characterized for agonism using in vitro T-cell proliferation assays. The epitope recognized on CD27 by MK-5890 was established by X-ray crystallography. Anti-tumor activity was evaluated in a human CD27 knock-in mouse. Preclinical safety was tested in rhesus monkeys. Pharmacodynamic properties were examined in mouse, rhesus monkeys and a phase 1 dose escalation clinical study in patients with cancer. RESULTS: Humanized anti-CD27 antibody MK-5890 (hIgG1) was shown to bind human CD27 on the cell surface with sub-nanomolar potency and to partially block binding to its ligand, CD70. Crystallization studies revealed that MK-5890 binds to a unique epitope in the cysteine-rich domain 1 (CRD1). MK-5890 activated CD27 expressed on 293T NF-κB luciferase reporter cells and, conditional on CD3 stimulation, in purified CD8+ T cells without the requirement of crosslinking. Functional Fc-receptor interaction was required to activate CD8+ T cells in an ex vivo tumor explant system and to induce antitumor efficacy in syngeneic murine subcutaneous tumor models. MK-5890 had monotherapy efficacy in these models and enhanced efficacy of PD-1 blockade. MK-5890 reduced in an isotype-dependent and dose-dependent manner circulating, but not tumor-infiltrating T-cell numbers in these mouse models. In rhesus monkey and human patients, reduction in circulating T cells was transient and less pronounced than in mouse. MK-5890 induced transient elevation of chemokines MCP-1, MIP-1α, and MIP-1ß in the serum of mice, rhesus monkeys and patients with cancer. MK-5890 was well tolerated in rhesus monkeys and systemic exposure to MK-5890 was associated with CD27 occupancy at all doses. CONCLUSIONS: MK-5890 is a novel CD27 agonistic antibody with the potential to complement the activity of PD-1 checkpoint inhibition in cancer immunotherapy and is currently undergoing clinical evaluation.

Hobit and Blimp-1 regulate TRM abundance after LCMV infection by suppressing tissue exit pathways of TRM precursors.

Tissue-resident memory T cells (Trm) are retained in peripheral tissues after infection for enhanced protection against secondary encounter with the same pathogen. We have previously shown that the transcription factor Hobit and its homolog Blimp-1 drive Trm development after viral infection, but how and when these transcription factors mediate Trm formation remains poorly understood. In particular, the major impact of Blimp-1 in regulating several aspects of effector T-cell differentiation impairs study of its specific role in Trm development. Here, we used the restricted expression of Hobit in the Trm lineage to develop mice with a conditional deletion of Blimp-1 in Trm, allowing us to specifically investigate the role of both transcription factors in Trm differentiation. We found that Hobit and Blimp-1 were required for the upregulation of CD69 and suppression of CCR7 and S1PR1 on virus-specific Trm precursors after LCMV infection, underlining a role in their retention within tissues. The early impact of Hobit and Blimp-1 favored Trm formation and prevented the development of circulating memory T cells. Thus, our findings highlight a role of Hobit and Blimp-1 at the branching point of circulating and resident memory lineages by suppressing tissue egress of Trm precursors early during infection.

Murine iNKT cells are depleted by liver damage via activation of P2RX7.

Invariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells induces acute immune-mediated liver injury. However, how tissue damage events regulate iNKT cell function and homeostasis remains unclear. We found that specifically tissue-resident iNKT cells in liver and spleen express the tissue-damage receptor P2RX7 and the P2RX7-activating ectoenzyme ARTC2. P2RX7 expression restricted formation of iNKT cells in the liver suggesting that liver iNKT cells are actively restrained under homeostatic conditions. Deliberate activation of P2RX7 in vivo by exogenous NAD resulted in a nearly complete iNKT cell ablation in liver and spleen in a P2RX7-dependent manner. Tissue damage generated by acetaminophen-induced liver injury reduced the number of iNKT cells in the liver. The tissue-damage-induced iNKT cell depletion was driven by P2RX7 and localized to the site of injury, as iNKT cells in the spleen remained intact. The depleted liver iNKT cells reconstituted only slowly compared to other lymphocytes such as regulatory T cells. These findings suggest that tissue-damage-mediated depletion of iNKT cells acts as a feedback mechanism to limit iNKT cell-induced pathology resulting in the establishment of a tolerogenic environment.

CD4+ T cell help creates memory CD8+ T cells with innate and help-independent recall capacities.

CD4+ T cell help is required for the generation of CD8+ cytotoxic T lymphocyte (CTL) memory. Here, we use genome-wide analyses to show how CD4+ T cell help delivered during priming promotes memory differentiation of CTLs. Help signals enhance IL-15-dependent maintenance of central memory T (TCM) cells. More importantly, help signals regulate the size and function of the effector memory T (TEM) cell pool. Helped TEM cells produce Granzyme B and IFNγ upon antigen-independent, innate-like recall by IL-12 and IL-18. In addition, helped memory CTLs express the effector program characteristic of helped primary CTLs upon recall with MHC class I-restricted antigens, likely due to epigenetic imprinting and sustained mRNA expression of effector genes. Our data thus indicate that during priming, CD4+ T cell help optimizes CTL memory by creating TEM cells with innate and help-independent antigen-specific recall capacities.

Subcellular Localization of Antigen in Keratinocytes Dictates Delivery of CD4+ T-cell Help for the CTL Response upon Therapeutic DNA Vaccination into the Skin.

In a mouse model of therapeutic DNA vaccination, we studied how the subcellular localization of vaccine protein impacts antigen delivery to professional antigen-presenting cells and efficiency of CTL priming. Cytosolic, membrane-bound, nuclear, and secretory versions of ZsGreen fluorescent protein, conjugated to MHC class I and II ovalbumin (OVA) epitopes, were expressed in keratinocytes by DNA vaccination into the skin. ZsGreen-OVA versions reached B cells in the skin-draining lymph node (dLN) that proved irrelevant for CTL priming. ZsGreen-OVA versions were also actively transported to the dLN by dendritic cells (DC). In the dLN, vaccine proteins localized to classical (c)DCs of the migratory XCR1+ and XCR- subtypes, and-to a lesser extent-to LN-resident cDCs. Secretory ZsGreen-OVA induced the best antitumor CTL response, even though its delivery to cDCs in the dLN was significantly less efficient than for other vaccine proteins. Secretory ZsGreen-OVA protein proved superior in CTL priming, because it led to in vivo engagement of antigen-loaded XCR1+, but not XCR1-, cDCs. Secretory ZsGreen-OVA also maximally solicited CD4+ T-cell help. The suboptimal CTL response to the other ZsGreen-OVA versions was improved by engaging costimulatory receptor CD27, which mimics CD4+ T-cell help. Thus, in therapeutic DNA vaccination into the skin, mere inclusion of helper epitopes does not ensure delivery of CD4+ T-cell help for the CTL response. Targeting of the vaccine protein to the secretory route of keratinocytes is required to engage XCR1+ cDC and CD4+ T-cell help and thus to promote CTL priming. Cancer Immunol Res; 6(7); 835-47. ©2018 AACR.

CD4+ T Cell Help Confers a Cytotoxic T Cell Effector Program Including Coinhibitory Receptor Downregulation and Increased Tissue Invasiveness.

CD4+ T cells optimize the cytotoxic T cell (CTL) response in magnitude and quality, by unknown molecular mechanisms. We here present the transcriptomic changes in CTLs resulting from CD4+ T cell help after anti-cancer vaccination or virus infection. The gene expression signatures revealed that CD4+ T cell help during priming optimized CTLs in expression of cytotoxic effector molecules and many other functions that ensured efficacy of CTLs throughout their life cycle. Key features included downregulation of PD-1 and other coinhibitory receptors that impede CTL activity, and increased motility and migration capacities. "Helped" CTLs acquired chemokine receptors that helped them reach their tumor target tissue and metalloprotease activity that enabled them to invade into tumor tissue. A very large part of the "help" program was instilled in CD8+ T cells via CD27 costimulation. The help program thus enhances specific CTL effector functions in response to vaccination or a virus infection.

Combination of testosterone and vardenafil increases female sexual functioning in sub-primed rats.

INTRODUCTION: Hypoactive sexual desire disorder (HSDD) is a common problem in women and may have a negative impact on quality of life. A recent clinical study shows an increase in sexual drive of HSDD women after cotreatment of testosterone and vardenafil (phosphodiesterase type 5 inhibitor). AIM: In this study, we investigated the effect of testosterone and vardenafil on sexual activity in female rats. MAIN OUTCOME MEASURES: Proceptive (darts and hops), receptive (lordosis), and paced-mating (percentages after exits and contact-return latencies) behaviors were quantified. METHODS: Ovariectomized female rats, sub-primed with only estradiol and fully primed with estradiol and progesterone, were tested in a paced-mating sex test and sexual behaviors were quantified. The sub-primed rats are thought to model HSDD. The effect of testosterone (100 and 300 µg, subcutaneous [SC]) and vardenafil (10 mg/kg, per os [PO]) alone and testosterone (300 µg, SC) in combination with vardenafil (3 and 10 mg/kg, PO) were tested. We also studied the effects of testosterone (300 µg, SC) + intracerebroventricular (ICV) injections of vardenafil (25 and 50 µg) on sexual activity. RESULTS: No effect of testosterone and vardenafil alone was found, but cotreatment of testosterone and vardenafil (PO) caused a significant increase in proceptive and receptive behavior in the sub-primed female rats. Testosterone and vardenafil did not affect fully primed females. ICV administration of vardenafil combined with systemic testosterone, on the other hand, had no effect on sexual activity in both sub-primed and fully primed female rats. CONCLUSIONS: We conclude that cotreatment of subcutaneous testosterone and oral vardenafil increase sexual activity in sub-primed female rats. Our data supports the human finding that combination treatment of testosterone and vardenafil could be used as a new treatment for women with HSDD.

Serotonin transporter null mutation and sexual behavior in female rats: 5-HT1A receptor desensitization.

INTRODUCTION: Serotonin plays a key role in sexual behavior. In serotonin transporter (SERT) knockout rats (-/-), basal extracellular 5-HT levels are considerably increased, indicating a serotonergic disturbance. Heterozygous SERT(+/-) rats express 50% of SERT in comparison to wild-type rats and may therefore model the s/s phenotype of the human SERT promoter (5-HTTLPR) polymorphism. AIM: In the present study, we used both homozygote and heterozygote SERT knockout and wild-type rats (+/+) to study the putative role of the SERT in female sexual behavior. METHODS: Female rats were brought into estrous by hormonal injections before a paced mating sex test. The effects of the 5-HT(1A)/5-HT(7) receptor agonist (+/-)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (+/-8-OH-DPAT) (0.03-1 mg/kg s.c.) and the 5-HT(1A) receptor antagonist WAY-100635 (0.1-1-mg/kg i.p.) on sexual behaviors of the females were tested separately and in a selected combination of both in all three genotypes. MAIN OUTCOME MEASURES: Proceptive (darting and hopping) and receptive (lordosis) behaviors were quantified. RESULTS: Basal proceptive and receptive sexual activities were not different between SERT+/+, +/- and -/- female rats. The dose-effect curve after +/-8-OH-DPAT for these activities was clearly shifted to the right in SERT-/- animals compared to other genotypes. WAY-100635 alone had no effect on sexual behavior in any genotype, but was able to antagonize the +/-8-OH-DPAT-induced decrease in sexual activities indicating the involvement of the 5-HT(1A) receptor. CONCLUSIONS: The absence (-/-) or reduced (+/-) expression of SERT does not affect basal sexual activity in female rats in a paced mating situation. The data indicate a desensitized 5-HT1A receptor in the SERT-/-, but not in the SERT+/- females. Under normal basal conditions, desensitized 5-HT1A receptors apparently do not play a role in female sexual behavior of the SERT-/-. However, upon activation of the 5-HT1A receptor in "normal" females (SERT+/+ and SERT+/-), a hyposexual behavior is induced.