Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma.

BACKGROUND: EGFR and/or HER2 expression in pancreatic cancers is correlated with poor prognoses. We generated homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (T-BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors. METHODS: EGFR and HER2 T-BsAbs were constructed using the 2 + 2 IgG-[L]-scFv T-BsAbs format bearing two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. A Fab arm exchange strategy was used to generate EGFRxHER2 heterodimeric T-BsAb carrying one Fab specific for EGFR and one for HER2. EGFR and HER2 T-BsAbs were also heterodimerized with a CD33 control T-BsAb to generate 'tumor-monovalent' EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence. RESULTS: The EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (∼150-fold and ∼6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost. CONCLUSION: EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging both targets, EGFRxHER2 heterodimeric T-BsAb exhibited potent anti-tumor effects if CDX or PDX were EGFR+HER2+ double-positive with the potential to spare single-positive normal tissue.

An improved reporter identifies ruxolitinib as a potent and cardioprotective CaMKII inhibitor.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) hyperactivity causes cardiac arrhythmias, a major source of morbidity and mortality worldwide. Despite proven benefits of CaMKII inhibition in numerous preclinical models of heart disease, translation of CaMKII antagonists into humans has been stymied by low potency, toxicity, and an enduring concern for adverse effects on cognition due to an established role of CaMKII in learning and memory. To address these challenges, we asked whether any clinically approved drugs, developed for other purposes, were potent CaMKII inhibitors. For this, we engineered an improved fluorescent reporter, CaMKAR (CaMKII activity reporter), which features superior sensitivity, kinetics, and tractability for high-throughput screening. Using this tool, we carried out a drug repurposing screen (4475 compounds in clinical use) in human cells expressing constitutively active CaMKII. This yielded five previously unrecognized CaMKII inhibitors with clinically relevant potency: ruxolitinib, baricitinib, silmitasertib, crenolanib, and abemaciclib. We found that ruxolitinib, an orally bioavailable and U.S. Food and Drug Administration-approved medication, inhibited CaMKII in cultured cardiomyocytes and in mice. Ruxolitinib abolished arrhythmogenesis in mouse and patient-derived models of CaMKII-driven arrhythmias. A 10-min pretreatment in vivo was sufficient to prevent catecholaminergic polymorphic ventricular tachycardia, a congenital source of pediatric cardiac arrest, and rescue atrial fibrillation, the most common clinical arrhythmia. At cardioprotective doses, ruxolitinib-treated mice did not show any adverse effects in established cognitive assays. Our results support further clinical investigation of ruxolitinib as a potential treatment for cardiac indications.

Experimental evidence that shear bands in metallic glasses nucleate like cracks.

Highly time-resolved mechanical measurements, modeling, and simulations show that large shear bands in bulk metallic glasses nucleate in a manner similar to cracks. When small slips reach a nucleation size, the dynamics changes and the shear band rapidly grows to span the entire sample. Smaller nucleation sizes imply lower ductility. Ductility can be increased by increasing the nucleation size relative to the maximum ("cutoff") shear band size at the upper edge of the power law scaling range of their size distribution. This can be achieved in three ways: (1) by increasing the nucleation size beyond this cutoff size of the shear bands, (2) by keeping all shear bands smaller than the nucleation size, or (3) by choosing a sample size smaller than the nucleation size. The discussed methods can also be used to rapidly order metallic glasses according to ductility.

Novel potent anti-STEAP1 bispecific antibody to redirect T cells for cancer immunotherapy.

BACKGROUND: The prognosis for metastatic Ewing sarcoma family of tumors (EFT) is still poor despite high-dose chemotherapy and radiation treatment. Immunotherapies hold promise, but cancer antigen-targeting immunotherapies have largely failed to induce effective T cell receptor-mediated antitumor response. However, T cell-engaging bispecific antibodies (T-BsAbs) have yet to be adequately explored. METHODS: Rehumanized STEAP1-IgG was used to build T-BsAb (named BC261) using the 2+2 IgG-[L]-scFv platform carrying the anti-CD3 huOKT3 scFv as the second specificity. Its binding epitope mapping, species cross-reactivity, tumor cell line staining, and in vitro cytotoxicity were investigated thoroughly. Its potency in driving tumor-infiltrating lymphocytes (TILs) was quantified using bioluminescence, correlated with in vivo antitumor response against cell line-derived or patient-derived xenografts (CDXs or PDXs) and compared with anti-STEAP1 T-BsAbs built on representative antibody platforms. RESULTS: BC261 binding epitope was mapped to its second extracellular domain of STEAP1 shared among canine and primate orthologs. BC261 induced potent cytotoxicity against panels of EFT, prostate cancer, and canine osteosarcoma cell lines despite their low antigen density. BC261 drove significantly more TILs into tumors (30-fold) and exerted superior antitumor effects compared with the other standard BsAb platforms. The antitumor efficacy of BC261 was consistent against EFT and prostate cancer CDXs and PDXs. CONCLUSIONS: BC261 was highly efficient in driving T cell infiltration and tumor ablation. Either as stand-alone therapeutics or for ex vivo armed T cells, this novel anti-STEAP1 T-BsAb BC261 has therapeutic potential.

A Bidirectional Permeability Assay for beyond Rule of 5 Compounds.

Bidirectional permeability measurement with cellular models grown on Transwell inserts is widely used in pharmaceutical research since it not only provides information about the passive permeability of a drug, but also about transport proteins involved in the active transport of drug substances across physiological barriers. With the increasing number of investigative drugs coming from chemical space beyond Lipinski's Rule of 5, it becomes more and more challenging to provide meaningful data with the standard permeability assay. This is exemplified here by the difficulties we encountered with the cyclic depsipeptides emodepside and its close analogs with molecular weight beyond 1000 daltons and cLogP beyond 5. The aim of this study is to identify potential reasons for these challenges and modify the permeability assays accordingly. With the modified assay, intrinsic permeability and in vitro efflux of depsipeptides could be measured reliably. The improved correlation to in vivo bioavailability and tissue distribution data indicated the usefulness of the modified permeability assay for the in vitro screening of compounds beyond the Rule of 5.

Development of Spherical Nucleic Acids for Prostate Cancer Immunotherapy.

Although the strategy of therapeutic vaccination for the treatment of prostate cancer has advanced to and is available in the clinic (Sipuleucel-T), the efficacy of such therapy remains limited. Here, we develop Immunostimulatory Spherical Nucleic Acid (IS-SNA) nanostructures comprised of CpG oligonucleotides as adjuvant and prostate cancer peptide antigens, and evaluate their antitumor efficacy in syngeneic mouse models of prostate cancer. IS-SNAs with the specific structural feature of presenting both antigen and adjuvant CpG on the surface (hybridized model (HM) SNAs) induce stronger cytotoxic T lymphocyte (CTL) mediated antigen-specific killing of target cells than that for IS-SNAs with CpG on the surface and antigen encapsulated within the core (encapsulated model (EM) SNAs). Mechanistically, HM SNAs increase the co-delivery of CpG and antigen to dendritic cells over that for EM SNAs or admixtures of linear CpG and peptide, thereby improving cross-priming of antitumor CD8+ T cells. As a result, vaccination with HM SNAs leads to more effective antitumor immune responses in two prostate cancer models. These data demonstrate the importance of the structural positioning of peptide antigens together with adjuvants within IS-SNAs to the efficacy of IS-SNA-based cancer immunotherapy.

Heavy Cannabis Use Associated with Wernicke's Encephalopathy.

Cannabis use accounts for more than 149,000 hospital visits annually. As more states legalize recreational Cannabis, side effects that are currently rare or unknown will become increasingly more common. Here, we present one such rare case of Cannabis-induced hyperemesis causing Wernicke's encephalopathy. This is an investigational case report utilizing retrospective data from electronic medical records. A 41-year-old patient presented to the hospital in status epilepticus secondary to severe vomiting and hyponatremia. He was given one dose of thiamine, glucose and folate and admitted to the medical ICU. His history was significant for remote alcohol use (1-2 beers/week about 20 years ago) and heavy marijuana use from strains grown in the patient's own backyard. A diagnosis of Cannabis Hyperemesis Syndrome was made. Seizures resolved after correction of electrolytes, and he became awake and alert with no focal deficits. His neurological exam after he was clinically stable showed memory deficits including confabulations (e.g., incorrectly listing occupation) and delusions (e.g., praying to a queen bee). An extensive workup including routine laboratory testing, infectious panels, and autoimmune studies was entirely negative. On the day of admission, brain magnetic resonance imaging (MRI) was performed showing bilateral thalamic hyperintensities on T2 FLAIR MRI. Wernicke's encephalopathy (WE) remained most likely and intravenous thiamine led to a gradual improvement in the patient's symptoms. He is now two months into rehabilitation and continues to make progress in recalling life events. Alcohol abuse is empirically treated with thiamine whereas Cannabis, unlike alcohol, is presumed to induce hyperphagia and nutritional supplements are often not initiated. However, foods ingested by Cannabis users are nutritionally deficient due to underline malabsorption. In addition, Cannabis-induced vomiting can further cause malnutrition. Complications, like Wernicke's encephalopathy, can be prevented by supplementing thiamine early in Cannabis intoxication.

CD73 expression on effector T cells sustained by TGF-ß facilitates tumor resistance to anti-4-1BB/CD137 therapy.

Agonist antibodies (Ab) directed against costimulatory molecules on the surface of antigen-primed T cells are in various stages of pre-clinical and clinical trials, albeit with limited therapeutic benefit as single agents. The underlying mechanisms of action remain incompletely understood. Here, we demonstrate an inhibitory role of ecto-enzyme CD73 for agonistic anti-4-1BB/CD137 Ab therapy. In particular, anti-4-1BB treatment preferentially drives CD73- effector T cell response for tumor inhibition. Anti-CD73 neutralizing Ab further improves anti-4-1BB therapy associated with enhanced anti-tumor T cell immunity. However, the TGF-ß-rich tumor milieu confers resistance to anti-4-1BB therapy by sustaining CD73 expression primarily on infiltrating CD8+ T cells across several tumor models. TGF-ß blockade results in downregulation of CD73 expression on infiltrating T cells and sensitizes resistant tumors to agonistic anti-4-1BB therapy. Thus, our findings identify a mechanism of action for more effective clinical targeting of 4-1BB or likely other costimulatory molecules.

Type 2 Innate Lymphoid Cells Impede IL-33-Mediated Tumor Suppression.

Although a number of studies have recently explored the contribution of the adaptive immunity in IL-33-mediated antitumor effects, innate immune involvement has been poorly characterized. Utilizing Rag1-/- mice (lacking T and B lymphocytes), we show in this study that either systemic administration of recombinant IL-33 or ectopic expression of IL-33 in melanoma cells is sufficient to inhibit tumor growth independent of adaptive antitumor immunity. We have demonstrated that IL-33-mediated antitumor effects depend on expansion and activation of NK cells. Interestingly, IL-33 also promoted the expansion of active type 2 innate lymphoid cells (ILC2s) via its receptor, ST2, which in turn inhibited NK activation and cytotoxicity. This IL-33-induced ILC2 activity coincided with greater expression of the immunosuppressive ectoenzyme CD73. Removal of CD73 from ILC2s in culture with NK cells resulted in markedly increased activation levels in NK cells, offering a potential mechanism by which ILC2s might suppress NK cell-mediated tumor killing. Thus, our data reveal an important contribution of IL-33-induced ILC2 to tumor growth by weakening NK cell activation and tumor killing, regardless of adaptive immunity.

Erratum: WEE1 inhibition by MK1775 as a single-agent therapy inhibits ovarian cancer viability.

[This corrects the article DOI: 10.3892/ol.2017.6584.].

WEE1 inhibition by MK1775 as a single-agent therapy inhibits ovarian cancer viability.

Wee1-like protein kinase (WEE1) contributes to the upstream regulation of the cyclin-dependent kinase (CDK) complexes by mediating the inactivation of CDK1 [corrected]. Increased expression of WEE1 has been associated with the poor prognosis of patients with ovarian cancer. The present study aimed at examining the in vitro and in vivo antitumor activity of MK1775, a potent pharmacological inhibitor of WEE1, as a single agent against ovarian cancer cells. The cytotoxicity of MK1775 was examined in a panel of tumor cells using MTT in vitro. Subsequently, a cell apoptosis assay was performed in ovarian cancer SKOV3 and ID8 cells to characterize the function of MK1775 in tumor cell apoptosis, under either wild-type tumor protein 53 (p53) or null p53 status. In addition, cell cycle analysis and a western blot analysis were performed to validate the effect of MK1775 on cell cycle progression and to elucidate the underlying molecular mechanism of cell death. Finally, the in vivo antitumor efficacy of MK1775 as a single agent at a clinical well-tolerated dose was determined. A dose-dependent inhibitory effect of MK1775 on tumor cell viability was determined in distinct cell lines, including B16F10, LLC1, BPS1, EG7, ID8 and SKOV3. Results from the cell cycle analysis and western blotting indicated that MK1775 abrogated the G2/M checkpoint through inhibiting the phosphorylation of CDK1 and inducing the apoptosis of ovarian cancer cells that lacked mutations in p53 and breast cancer 1 (BRCA1). Additionally, a significant antitumor effect of MK1775 was observed in C57BL/6 mice bearing syngeneic ID8 ovarian tumors. The results of the present study supported the use of MK1775 as a monotherapy agent in ovarian cancer. MK1775 was effective at inducing mitotic catastrophe, independent of p53 and BRCA1 mutations. Therefore, WEE1 inhibition by MK1775 requires additional investigation to identify novel combination approaches in ovarian cancer therapy with the current DNA damaging agents, including irradiation treatment and cell cycle checkpoint inhibitors.

Magnesium, hemostasis, and outcomes in patients with intracerebral hemorrhage.

OBJECTIVE: We tested the hypothesis that admission serum magnesium levels are associated with hematoma volume, hematoma growth, and functional outcomes in patients with intracerebral hemorrhage (ICH). METHODS: Patients presenting with spontaneous ICH were enrolled in an observational cohort study that prospectively collected demographic, clinical, laboratory, radiographic, and outcome data. We performed univariate and adjusted multivariate analyses to assess for associations between serum magnesium levels and initial hematoma volume, final hematoma volume, and in-hospital hematoma growth as radiographic measures of hemostasis, and functional outcome measured by the modified Rankin Scale (mRS) at 3 months. RESULTS: We included 290 patients for analysis. Admission serum magnesium was 2.0 ± 0.3 mg/dL. Lower admission magnesium levels were associated with larger initial hematoma volumes on univariate (p = 0.02), parsimoniously adjusted (p = 0.002), and fully adjusted models (p = 0.006), as well as greater hematoma growth (p = 0.004, p = 0.005, and p = 0.008, respectively) and larger final hematoma volumes (p = 0.02, p = 0.001, and p = 0.002, respectively). Lower admission magnesium level was associated with worse functional outcomes at 3 months (i.e., higher mRS; odds ratio 0.14, 95% confidence interval 0.03-0.64, p = 0.011) after adjustment for age, admission Glasgow Coma Scale score, initial hematoma volume, time from symptom onset to initial CT, and hematoma growth, with evidence that the effect of magnesium is mediated through hematoma growth. CONCLUSIONS: These data support the hypothesis that magnesium exerts a clinically meaningful influence on hemostasis in patients with ICH.

Exogenous IL-33 Restores Dendritic Cell Activation and Maturation in Established Cancer.

The role of IL-33, particularly in tumor growth and tumor immunity, remains ill-defined. We show that exogenous IL-33 can induce robust antitumor effect through a CD8+ T cell-dependent mechanism. Systemic administration of rIL-33 alone was sufficient to inhibit growth of established tumors in transplant and de novo melanoma tumorigenesis models. Notably, in addition to a direct action on CD8+ T cell expansion and IFN-γ production, rIL-33 therapy activated myeloid dendritic cells (mDCs) in tumor-bearing mice, restored antitumor T cell activity, and increased Ag cross-presentation within the tumor microenvironment. Furthermore, combination therapy consisting of rIL-33 and agonistic anti-CD40 Abs demonstrated synergistic antitumor activity. Specifically, MyD88, an essential component of the IL-33 signaling pathway, was required for the IL-33-mediated increase in mDC number and upregulation in expression of costimulatory molecules. Importantly, we identified that the IL-33 receptor ST2, MyD88, and STAT1 cooperate to induce costimulatory molecule expression on mDCs in response to rIL-33. Thus, our study revealed a novel IL-33-ST2-MyD88-STAT1 axis that restores mDC activation and maturation in established cancer and, thereby, the magnitude of antitumor immune responses, suggesting a potential use of rIL-33 as a new immunotherapy option to treat established cancer.

Exogenous IL-33 overcomes T cell tolerance in murine acute myeloid leukemia.

Emerging studies suggest that dominant peripheral tolerance is a major mechanism of immune escape in disseminated leukemia. Using an established murine acute myeloid leukemia (AML) model, we here show that systemic administration of recombinant IL-33 dramatically inhibits the leukemia growth and prolongs the survival of leukemia-bearing mice in a CD8+ T cell dependent manner. Exogenous IL-33 treatment enhanced anti-leukemia activity by increasing the expansion and IFN-γ production of leukemia-reactive CD8+ T cells. Moreover, IL-33 promoted dendritic cell (DC) maturation and activation in favor of its cross presentation ability to evoke a vigorous anti-leukemia immune response. Finally, we found that the combination of PD-1 blockade with IL-33 further prolonged the survival, with half of the mice achieving complete regression. Our data establish a role of exogenous IL-33 in reversing T cell tolerance, and suggest its potential clinical implication into leukemia immunotherapy.

Targeting Ornithine Decarboxylase by α-Difluoromethylornithine Inhibits Tumor Growth by Impairing Myeloid-Derived Suppressor Cells.

α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1(-/-) mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8(+) T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8(+) T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.

Parallel chemistry in the 21st century.

The tool chest of techniques, methodologies, and equipment for conducting parallel chemistry is larger than ever before. Improvements in the laboratory and developments in computational chemistry have enabled compound library design at the desks of medicinal chemists. This unit includes a brief background in combinatorial/parallel synthesis chemistry, along with a discussion of evolving technologies for both solid- and solution-phase chemistry. In addition, there are discussions on designing compound libraries, acquisition/procurement of compounds and/or reagents, the chemistry and equipment used for chemical production, purification, sample handling, and data analysis.

Soil nitrate leaching in silvopastures compared with open pasture and pine plantation.

Wide acceptance of silvopasture as an alternative sustainable agricultural system in the southeastern United States will depend on an improved understanding of the tree-forage interactions and recognition of its environmental benefits. The objective of this study was to evaluate differences in soil nitrate leaching in different land-use systems, in north Florida. An 18-yr-old loblolly pine (Pinus taeda L.) plantation was thinned in the summer of 2002 to create a fifth-row thinned, nontraditional intensive pine plantation (FO), silvopastures (HE = fourth-row conventionally thinned with random tree distribution and DO = double-row sets of trees with 15-m wide alleys), and an open pasture (PA). 'Argentine' bahiagrass (Paspalum notatum Flügge.) was established as understory vegetation in HE, DO, and PA. From 2004 to 2005 soil nitrate leaching was sampled and compared in the DO, HE, PA, and FO systems at 0.3 and 1.2 m depths after fertilizer application. Significant nitrate peaks were observed at 0.3 m depth after N fertilizer application in all systems. At the 1.2 m depth, the maximum nitrate concentrations were 67, 18, and 8 mg L(-1), in the forest plantation, open pasture, and both silvopastures, respectively. In general, reduced nitrate leaching at 1.2 m depth was observed in silvopastures compared with other land-used systems. These results are not intended to have a direct bearing on traditional pine plantation management, but rather support the potential role of silvopasture systems in reducing nitrate losses from the soil.

Total synthesis of apoptolidin A.

A highly convergent, enantioselective total synthesis of the potent antitumor agent apoptolidin A has been completed. The key transformations include highly selective glycosylations to attach the C27 disaccharide and the C9 6'-deoxy-l-glucose, a cross-metathesis to incorporate the C1-C10 trienoate unit, and a Yamaguchi macrolactonization to complete the macrocycle. Twelve stereocenters in the polypropionate segments and sugar units were established through diastereoselective chlorotitanium enolate aldol reactions.

Enantioselective synthesis of apoptolidinone: exploiting the versatility of thiazolidinethione chiral auxiliaries.

An efficient, enantioselective synthesis of apoptolidinone has been completed, demonstrating the versatility of thiazolidinethione auxiliaries. Three propionate aldol additions and two asymmetric glycolate alkylations function to establish 8 of the 12 stereogenic carbon centers. A cross-metathesis reaction is utilized to assemble the C1-C10 trieneoate fragment and the C11-C28 polypropionate region of the molecule.

Enantioselective synthesis of apoptolidin sugars.

[structure: see text] The de novo synthesis of the C9 and C27 sugar subunits (2) and (3), respectively, of the potent antitumor agent, apoptolidin, has been accomplished. A titanium tetrachloride-mediated asymmetric anti glycolate aldol addition was utilized to establish the 4' and 5' stereogenic centers of each of the three monosaccharides. Elaboration of the aldol adducts efficiently provided the three sugar units. A beta-selective glycosidation completed the construction of the C27 disaccharide.