Peptide Binder to Glypican-3 as a Theranostic Agent for Hepatocellular Carcinoma.

Glypican-3 (GPC3) is a membrane-associated glycoprotein that is significantly upregulated in hepatocellular carcinomas (HCC) with minimal to no expression in normal tissues. The differential expression of GPC3 between tumor and normal tissues provides an opportunity for targeted radiopharmaceutical therapy to treat HCC, a leading cause of cancer-related deaths worldwide. Methods: DOTA-RYZ-GPC3 (RAYZ-8009) comprises a novel macrocyclic peptide binder to GPC3, a linker, and a chelator that can be complexed with different radioisotopes. The binding affinity was determined by surface plasma resonance and radioligand binding assays. Target-mediated cellular internalization was radiometrically measured at multiple time points. In vivo biodistribution, monotherapy, and combination treatments with 177Lu or 225Ac were performed on HCC xenografts. Results: RAYZ-8009 showed high binding affinity to GPC3 protein of human, mouse, canine, and cynomolgus monkey origins and no binding to other glypican family members. Potent cellular binding was confirmed in GPC3-positive HepG2 cells and was not affected by isotope switching. RAYZ-8009 achieved efficient internalization on binding to HepG2 cells. Biodistribution study of 177Lu-RAYZ-8009 showed sustained tumor uptake and fast renal clearance, with minimal or no uptake in other normal tissues. Tumor-specific uptake was also demonstrated in orthotopic HCC tumors, with no uptake in surrounding liver tissue. Therapeutically, significant and durable tumor regression and survival benefit were achieved with 177Lu- and 225Ac-labeled RAYZ-8009, as single agents and in combination with lenvatinib, in GPC3-positive HCC xenografts. Conclusion: Preclinical in vitro and in vivo data demonstrate the potential of RAYZ-8009 as a theranostic agent for the treatment of patients with GPC3-positive HCC.

Metabolic screening in primary care for patients with schizophrenia or schizoaffective disorder and taking antipsychotic medication.

INTRODUCTION Life expectancy in patients with schizophrenia is 15-20 years less than the general population. A dominant cause of morbidity and mortality in these patients is cardiovascular disease. Adverse consequences of modifiable cardiovascular risk factors can be reduced by regular monitoring of metabolic outcomes and intervention if required. AIM To evaluate the metabolic screening in primary care for patients with schizoaffective disorders managed in primary care. To show the usefulness of combining simple practice audits in evaluating such areas of clinical practice. METHODS An audit was undertaken in eight general practices in the Waikato and Bay of Plenty regions of New Zealand. Specifically, the monitoring of patients with schizophrenia or schizoaffective disorder whose antipsychotic medication was prescribed by primary care doctors was audited. Patient monitoring was compared to the guideline recommendation of the Royal Australian and New Zealand College of Psychiatrists (RANZCP) and the Best Practice Advisory Centre (BPAC). RESULTS In total, 117 patients were included in the audit and none were fully monitored, as recommended by the RANZCP guidelines. Although two-thirds of patients had been evaluated for glycosylated haemoglobin (HbA1c), lipids, blood pressure, complete blood count and weight, <10% of patients had had prolactin, waist circumference or electrocardiogram measurements recorded. The proportion of patients having a HbA1c measured was also significantly higher in younger patients and patients who were non-Maori or enrolled with an urban practice (all P<0.05). When using the simplified BPAC guidelines, half of all patients were correctly monitored. DISCUSSION These findings show there is room for improvement in the monitoring of patients receiving antipsychotic medication in primary care. This may indicate the need for clear guidance and general practitioner education around the monitoring requirements of these patients. Alternatively, a more simplified monitoring protocol may need to be developed. This audit has also shown that there is value in several practices completing the same audit and providing a larger cohort of patients for pooled data analysis.

Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide.

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a ß-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.

Enhancing antisense efficacy with multimers and multi-targeting oligonucleotides (MTOs) using cleavable linkers.

The in vivo potency of antisense oligonucleotides (ASO) has been significantly increased by reducing their length to 8-15 nucleotides and by the incorporation of high affinity RNA binders such as 2', 4'-bridged nucleic acids (also known as locked nucleic acid or LNA, and 2',4'-constrained ethyl [cET]). We now report the development of a novel ASO design in which such short ASO monomers to one or more targets are co-synthesized as homo- or heterodimers or multimers via phosphodiester linkers that are stable in plasma, but cleaved inside cells, releasing the active ASO monomers. Compared to current ASOs, these multimers and multi-targeting oligonucleotides (MTOs) provide increased plasma protein binding and biodistribution to liver, and increased in vivo efficacy against single or multiple targets with a single construct. In vivo, MTOs synthesized in both RNase H-activating and steric-blocking oligonucleotide designs provide ≈4-5-fold increased potency and ≈2-fold increased efficacy, suggesting broad therapeutic applications.

Lead discovery and optimization strategies for peptide macrocycles.

Peptide macrocycles represent a chemical space where the best of biological tools can synergize with the best of chemical approaches in the quest for leads against undruggable targets. Peptide macrocycles offer some key advantages in both lead discovery and lead optimization phases of drug discovery when compared to natural product and synthetic macrocycles. In addition, they are uniquely positioned to capitalize on the therapeutic potential of peptides because cyclization can help drive selectivity, potency and overcome the common limitations of metabolic instability of peptides.

Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes.

It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody-drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C-C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

Parallel synthesis and screening of peptide conjugates.

Peptide conjugates represent an emerging class of therapeutics. However, in contrast to that of small molecules and peptides, the discovery and optimization of peptide conjugates is low in throughput, resource intensive, time-consuming, and based on educated decisions rather than screening. A strategy for the parallel synthesis and screening of peptide conjugates is presented that (1) reduces variability in the conjugation steps; (2) provides a new method to rapidly and quantitatively measure conversion in crude conjugation mixtures; (3) introduces a purification step using an immobilized chemical scavenger that does not rely on protein-specific binding; and (4) is supported by robust analytical methods to characterize the large number of end products. Copper-free click chemistry is used as the chemoselective ligation method for conjugation and purification. The productivity in the generation and screening of peptide conjugates is significantly improved by applying this strategy as is demonstrated by the optimization of the anti-Angiopoietin-2 (Ang2) CovX-body, CVX-060, a peptide-antibody scaffold conjugate that has advanced in clinical trials for oncology indications.

Development of a long acting human growth hormone analog suitable for once a week dosing.

Human growth hormone was conjugated to a carrier aldolase antibody, using a novel linker by connecting a disulphide bond in growth hormone to a lysine-94 amine located on the Fab arm of the antibody. The resulting CovX body showed reduced affinity towards human growth hormone receptor, reduced cell-based activity, but improved pharmacodynamic properties. We have demonstrated that this CovX-body, given once a week, showed comparable activity as growth hormone given daily in an in vivo hypophysectomized rat model.

Development of novel linkers to conjugate pharmacophores to a carrier antibody.

We have developed modified maleimide novel linkers with improved chemical stability that could potentially be used in conjugating various pharmacophores such as oligo nucleotides, peptides, and proteins to antibodies to afford novel biologics with well-defined therapeutic benefits and improved pharmacokinetic properties. These linkers expand the array of tools available for bioconjugation of pharmacophores to antibodies.

Kappa agonist CovX-Bodies.

Small peptidic kappa agonists were covalently linked to the reactive lysine of the CovX antibody to create compounds having potent activity at the kappa receptor with greatly extended half-life when compared to the parent peptide as exemplified by compound 20.

Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats.

Calorie restriction [CR; ∼40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.

Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats.

Calorie restriction [CR; ~65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[(3)H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr(308) and Ser(473)) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser(588) and Thr(642)) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.

Specifically targeting angiopoietin-2 inhibits angiogenesis, Tie2-expressing monocyte infiltration, and tumor growth.

PURPOSE: Angiopoietin-1 (Ang1) plays a key role in maintaining stable vasculature, whereas in a tumor Ang2 antagonizes Ang1's function and promotes the initiation of the angiogenic switch. Specifically targeting Ang2 is a promising anticancer strategy. Here we describe the development and characterization of a new class of biotherapeutics referred to as CovX-Bodies, which are created by chemical fusion of a peptide and a carrier antibody scaffold. EXPERIMENTAL DESIGN: Various linker tethering sites on peptides were examined for their effect on CovX-Body in vitro potency and pharmacokinetics. Ang2 CovX-Bodies with low nmol/L IC(50)s and significantly improved pharmacokinetics were tested in tumor xenograft studies alone or in combination with standard of care agents. Tumor samples were analyzed for target engagement, via Ang2 protein level, CD31-positive tumor vasculature, and Tie2 expressing monocyte penetration. RESULTS: Bivalent Ang2 CovX-Bodies selectively block the Ang2-Tie2 interaction (IC(50) < 1 nmol/L) with dramatically improved pharmacokinetics (T(½) > 100 hours). Using a staged Colo-205 xenograft model, significant tumor growth inhibition (TGI) was observed (40%-63%, P < 0.01). Ang2 protein levels were reduced by approximately 50% inside tumors (P < 0.01), whereas tumor microvessel density (P < 0.01) and intratumor proangiogenic Tie2(+)CD11b(+) cells (P < 0.05) were significantly reduced. When combined with sunitinib, sorafenib, bevacizumab, irinotecan, or docetaxel, Ang2 CovX-Bodies produced even greater efficacy (∼80% TGI, P < 0.01). CONCLUSION: CovX-Bodies provide an elegant solution to overcome the pharmacokinetic-pharmacodynamic problems of peptides. Long-acting Ang2 specific CovX-Bodies will be useful as single agents and in combination with standard-of-care agents.

Chemical generation of bispecific antibodies.

Bispecific antibodies (BsAbs) are regarded as promising therapeutic agents due to their ability to simultaneously bind two different antigens. Several bispecific modalities have been developed, but their utility is limited due to problems with stability and manufacturing complexity. Here we report a versatile technology, based on a scaffold antibody and pharmacophore peptide heterodimers, that enables rapid generation and chemical optimization of bispecific antibodies, which are termed bispecific CovX-Bodies. Two different peptides are joined together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. Whereas the pharmacophores are responsible for functional activities, the antibody scaffold imparts long half-life and Ig-like distribution. The pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. As a prototype, we developed a bispecific antibody that binds both vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) simultaneously, inhibits their function, shows efficacy in tumor xenograft studies, and greatly augments the antitumor effects of standard chemotherapy. This unique antiangiogenic bispecific antibody is in phase-1 clinical trials.

Targeting the ANGPT-TIE2 pathway in malignancy.

Angiopoietins (ANGPTs) are ligands of the endothelial cell receptor TIE2 and have crucial roles in the tumour angiogenic switch. Increased expression of ANGPT2 relative to ANGPT1 in tumours correlates with poor prognosis. The biological effects of the ANGPT-TIE system are context dependent, which brings into question what the best strategy is to target this pathway. This Review presents an encompassing picture of what we know about this important axis in tumour biology. The various options for therapeutic intervention are discussed to identify the best path forwards.

Insulin resistance for glucose uptake and Akt2 phosphorylation in the soleus, but not epitrochlearis, muscles of old vs. adult rats.

The slow-twitch soleus, but not fast-twitch muscle, of old vs. adult rats has previously been demonstrated to become insulin resistant for in vivo glucose uptake. We probed cellular mechanisms for the age effect by assessing whether insulin resistance for glucose uptake was an intrinsic characteristic of the muscle ex vivo and by analyzing key insulin signaling steps. We hypothesized that isolated soleus and epitrochlearis (fast-twitch) muscles from old (25 mo) vs. adult (9 mo) male Fisher-344 x Brown Norway rats would have insulin resistance for Akt2 Thr308 phosphorylation (pAkt2Thr308), AS160 phosphorylation Thr642 (pAS160Thr642), and atypical PKC (aPKCzeta/lambda) activity corresponding in magnitude to the extent of insulin resistance for [3H]-2-deoxyglucose (2-DG) uptake. Epitrochlearis insulin-stimulated 2-DG uptake above basal values was unaltered by age, and epitrochlearis pAkt2Thr308, pAS160Thr642, and aPKCzeta/lambda activity were not significantly different in adult vs. old rats. Conversely, insulin-stimulated 2-DG uptake by the soleus of old vs. adult rats was reduced with 1.2 nM (42%) and 30 nM (28%) insulin concomitant with an age-related decline in pAkt2Thr308 of the insulin-stimulated soleus. There were no age effects on pAS160Thr642 or aPKCzeta/lambda activity or abundance of Akt2, AS160, GLUT4 or Appl1 protein in either muscle. The results suggest the possibility that an age-related decline in pAkt2Thr308, acting by a mechanism other than reduced pAS160Thr642, may play a role in the insulin resistance in the soleus of old rats. Skeletal muscle insulin resistance in old age is distinctive compared with other insulin-resistant rodent models that are not selective for greater insulin resistance in the soleus vs. the epitrochlearis.

Pyrrolopyrazinedione-based inhibitors of human hormone-sensitive lipase.

The regulation of lipid metabolism and it's effect on glucose control and diabetes has received intense interest. Hormone-sensitive lipase (HSL) is a vital enzyme in lipid metabolism. A series of novel pyrrolopyrazinediones has been discovered that demonstrate submicromolar activity both in the enzyme assay and in a (14)C-emulsion assay employing cholesteryl oleate as a substrate as a secondary measure of HSL activity. These compounds represent novel inhibitors of the human HSL enzyme.

Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant Gp120.

Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.