Tumor-associated PD-L1 expression is predictive of clinical response to PD-1-directed immunotherapy. However, PD-L1-negative patients may also respond to PD-1 checkpoint blockade, suggesting that other PD-1 ligands may be relevant to the clinical activity of these therapies. The prevalence of PD-L2, the other known ligand of PD-1, and its relationship to response to anti-PD-1 therapy were evaluated. PD-L2 expression was assessed in archival tumor tissue from seven indications using a novel immunohistochemical assay. In addition, relationships between clinical response and PD-L2 status were evaluated in tumor tissues from patients with head and neck squamous cell carcinoma (HNSCC) with recurrent or metastatic disease, treated with pembrolizumab. PD-L2 expression was observed in all tumor types and present in stromal, tumor, and endothelial cells. The prevalence and distribution of PD-L2 correlated significantly with PD-L1 ( = 0.0012-<0.0001); however, PD-L2 was detected in the absence of PD-L1 in some tumor types. Both PD-L1 and PD-L2 positivity significantly predicted clinical response to pembrolizumab on combined tumor, stromal and immune cells, with PD-L2 predictive independent of PD-L1. Response was greater in patients positive for both PD-L1 and PD-L2 (27.5%) than those positive only for PD-L1 (11.4%). PD-L2 status was also a significant predictor of progression-free survival (PFS) with pembrolizumab independent of PD-L1 status. Longer median times for PFS and overall survival were observed for PD-L2-positive than PD-L2-negative patients. Clinical response to pembrolizumab in patients with HNSCC may be related partly to blockade of PD-1/PD-L2 interactions. Therapy targeting both PD-1 ligands may provide clinical benefit in these patients. .

No abstract

IL-33 is constitutively expressed in epithelial barrier tissues, such as skin. Although increased expression of IL-33/IL-33R has been correlated with fibrotic disorders, such as scleroderma and progressive systemic sclerosis, the direct consequences of IL-33 release in skin has not been reported. To determine the effects of dysregulated IL-33 signaling in skin, we administered IL-33 s.c. and monitored its effects at the injection site. Administration of IL-33 resulted in IL-33R–dependent accumulation of eosinophils, CD3+ lymphocytes, F4/80+ mononuclear cells, increased expression of IL-13 mRNA, and the development of cutaneous fibrosis. Consistent with extensive cutaneous tissue remodeling, IL-33 resulted in significant modulation of a number of extracellular matrix-associated genes, including collagen VI, collagen III, and tissue inhibitor of metalloproteases-1. We establish that IL-33–induced fibrosis requires IL-13 using IL-13 knockout mice and eosinophils using ΔdblGATA mice. We show that bone marrow-derived eosinophils secrete IL-13 in response to IL-33 stimulation, suggesting that eosinophil-derived IL-13 may promote IL-33–induced cutaneous fibrosis. Collectively, our results identify IL-33 as a previously unrecognized profibrotic mediator in skin and highlight the cellular and molecular pathways by which this pathology develops.

Purpose: S-CKD602 is a STEALTH liposomal formulation of CKD-602, a camptothecin analogue. The cytotoxicity of camptothecin analogues is related to the duration of exposure in the tumor. STEALTH liposomal formulations contain lipid conjugated to methoxypolyethylene glycol and have been designed to prolong drug circulation time, increase tumor delivery, and improve the therapeutic index. For STEALTH liposomal formulations of anticancer agents to achieve antitumor effects, the active drug must be released into the tumor extracellular fluid (ECF). Experimental Design: S-CKD602 at 1 mg/kg or nonliposomal CKD-602 at 30 mg/kg was administered once via tail vein to mice bearing A375 human melanoma xenografts. Mice (n = 3 per time point) were euthanized at 0.083 to 24 h, 48 h, and 72 h after S-CKD02 and from 0.083 to 24 h after nonliposomal CKD-602. Plasma samples were processed to measure encapsulated, released, and sum total (encapsulated plus released) CKD-602, and tumor and tissue samples were processed to measure sum total CKD-602. Microdialysis samples of tumor ECF were obtained from 0 to 2 h, 4 to 7 h, and 20 to 24 h after nonliposomal CKD-602 and from 0 to 2 h, 24 to 27 h, 48 to 51 h, and 72 to 75 h after S-CKD602. A liquid chromatographymass spectrometry assay was used to measure the total (sum of lactone and hydroxyl acid) CKD-602. The area under the concentration-versus-time curves (AUC) from 0 to infinity and time >1ng/mL in tumor were estimated.Results: For S-CKD602, the CKD-602 sum total AUC in plasma and tumor and the CKD-602 AUC in tumor ECF were 201,929, 13,194, and 187 ng/mL h, respectively. For S-CKD602, 82% of CKD-602 remains encapsulated in plasma. For nonliposomal CKD-602, the CKD-602 AUC in plasma and tumor and the CKD-602 AUC in tumor ECF were 9,117, 11,661, and 639 ng/mL Á h, respectively. The duration of time the CKD-602 concentration was >1ng/mL in tumor ECF after S-CKD602 and nonliposomal CKD-602 was >72 and f20 h, respectively. For S-CKD602, the CKD-602 sum total exposure was 1.3-fold higher in fat as compared with muscle. The ratio of CKD-602 sum total exposure in fat to muscle was 3.8-fold higher after administration of S-CKD602 compared with nonliposomal CKD-602. Conclusion: S-CKD602 provides pharmacokinetic advantages in plasma, tumor, and tumor ECF compared with nonliposomal CKD-602 at 1/30th of the dose, which is consistent with the improved antitumor efficacy of S-CKD602 in preclinical studies. The distribution of S-CKD602 is greater in fat compared with muscle whereas the distribution of nonliposomal CKD-602 is greater in muscle compared with fat.These results suggest that the body composition of a patient may affect the disposition of S-CKD602 and released CKD-602.

A novel method is proposed in which combined restraints derived from linear-scaling semiempirical quantum-mechanical (QM) calculations and X-ray diffraction data are combined to refine crystal structures of proteins. Its performance has been tested on a small protein molecule, bovine pancreatic trypsin inhibitor (BPTI). The refinement involves minimization of the sum of a geometric energy function and an X-ray target function based on either the least-squares residual or the maximum-likelihood formalism. For comparison, similar refinement runs have also been performed using energy restraints derived from the force field available in the Crystallography & NMR System (CNS) program. The QM refinements were carried out with weights that were varied by several orders of magnitude and the optimal weights were identified by observing the trend in the final free R values, QM heats of formation and coordinate root-mean-square deviations (r.m.s.d.s) from the crystal structure. It is found that the QM weights are typically smaller but generally on the same scale as the molecular-mechanics (MM) weights for the respective X-ray target functions. The crystallographic R, free R, real-space R values and correlation coefficients based on the structures refined with the energy restraints derived from our QM calculations and Engh and Huber parameters are comparable, suggesting that the QM restraints are capable of maintaining reasonable stereochemistry to a similar degree as the force-field parameters. A detailed inspection of the structures refined with the QM and MM energy restraints reveals that one of the common differences between them and the crystal structure is that the strained bond angles in the crystal structure are corrected after energetically restrained refinements. Systematic differences in certain bond lengths between the QM-refined structures and the statistical averages of experimental structures have also been observed and discussed.

No abstract

Abstractβ-Secretase, a.k.a. β-APP cleaving enzyme (BACE), is an aspartyl protease that has been implicated as a key target in the pathogenesis of Alzheimer's disease (AD). The identification of the protonation states of the key aspartates in β-secretase is of great interest both in understanding the reaction mechanism and in guiding the design of drugs against AD. However, the resolutions of currently available crystal structures for BACE are not sufficient to determine the hydrogen atom locations. We have assigned the protonation states of the key aspartates using a novel method, QM/MM X-ray refinement. In our approach, an energy function is introduced to the refinement where the atoms in the active site are modeled by quantum mechanics (QM) and the other atoms are represented by molecular mechanics (MM). The gradients derived from the QM/MM energy function are combined with those from the X-ray target to refine the crystal structure of a complex containing BACE and an inhibitor. A total number of 8 protonation configurations of the aspartyl dyad were considered and QM/MM X-ray refinements were performed for all of them. The relative stability of the refined structures was scored by constructing the thermodynamic cycle using the energetics calculated by fully quantum mechanical self-consistent reaction field (QM/SCRF) calculations. While all 8 refined structures fit the observed electron density about equally well, we find the mono-protonated configurations to be strongly favored energetically, especially the configuration with the inner oxygen of Asp32 protonated and the hydroxyl of the inhibitor pointing towards Asp228. It was also found that these results depend on the constraints imposed by the X-ray data. We suggest that one of the strengths of this approach is that the resulting structures are a consensus of theoretical and experimental data and remark on the significance of our results in structure based drug design and mechanistic studies.

The antitumor activity of a novel thiolytically cleavable lipid-based prodrug of mitomycin C (MMC) delivered by STEALTH liposomes (SL) was studied in drug resistant human ovarian carcinoma A2780/AD model and compared with free MMC and both free and SL forms of an established anticancer drug--doxorubicin (DOX). It was found that SL-prodrug (SL-pMMC) possessed enhanced antitumor activity when compared with the parent MMC, free DOX, and SL-DOX. An observance of the high antitumor efficiency of SL-pMMC was a result of its preferential accumulation in the tumor by the enhanced permeability and retention (EPR) effect, suppression of multidrug resistance (MDR) associated with P-glycoprotein and MRP drug efflux pumps, activation of caspase-dependent apoptosis signaling pathways and suppression of antiapoptotic cellular defense by increasing the BAX/BCL2 ratio. Consequently, the described SL-pMMC formulations can be considered good candidates for the chemotherapy of multidrug resistant tumors.