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Type 2 diabetes (T2D) is a complex and progressive disease requiring polypharmacy to manage hyperglycaemia and cardiovascular risk factors. However, most patients do not achieve combined treatment goals. To address this therapeutic gap, we have developed MEDI4166, a novel glucagon-like peptide-1 (GLP-1) receptor agonist peptide fused to a proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralising antibody that allows for glycaemic control and low-density lipoprotein cholesterol (LDL-C) lowering in a single molecule. The fusion has been engineered to deliver sustained peptide activity in vivo in combination with reduced potency, to manage GLP-1 driven adverse effects at high dose, and a favourable manufacturability profile. MEDI4166 showed robust and sustained LDL-C lowering in cynomolgus monkeys and exhibited the anticipated GLP-1 effects in T2D mouse models. We believe MEDI4166 is a novel molecule combining long acting agonist peptide and neutralising antibody activities to deliver a unique pharmacology profile for the management of T2D.

Antibody-drug conjugate (ADC) which delivers cytotoxic drugs specifically into targeted cells through internalization and lysosomal trafficking has emerged as an effective cancer therapy. We show that a bivalent biparatopic antibody targeting two non-overlapping epitopes on HER2 can induce HER2 receptor clustering, which in turn promotes robust internalization, lysosomal trafficking, and degradation. When conjugated with a tubulysin-based microtubule inhibitor, the biparatopic ADC demonstrates superior anti-tumor activity over ado-trastuzumab emtansine (T-DM1) in tumor models representing various patient subpopulations, including T-DM1 eligible, T-DM1 ineligible, and T-DM1 relapsed/refractory. Our findings indicate that this biparatopic ADC has promising potential as an effective therapy for metastatic breast cancer and a broader patient population may benefit from this unique HER2-targeting ADC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The neurotrophin receptor p75(NTR) is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75(NTR) antibody or phage scFv library pre-panned against p75(NTR) are internalized by neurons expressing p75(NTR); (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75(NTR) antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75(NTR) expression is upregulated in motor neurons in response to injury and in disease, the p75(NTR) antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.

The seven trans-membrane chemokine receptor CCR-5 is a coreceptor for macrophage tropic HIV-1 strains. CCR-5 responds to a number of chemokines, including macrophage inflammatory protein (MIP)-1 alpha. We describe the use of MIP-1 alpha in a biotin tyramine-mediated proximity selection to guide the selection of CCR-5-specific phage antibodies from a large phage display human library. Proximity based selections resulted in a population of antibodies recognizing CCR-5 on primary CD4+ lymphocytes, none of which blocked MIP-1 alpha binding to cells. The selected population of phage antibodies were subsequently used as guide molecules for a second phase of selection that was carried out in the absence of MIP-1 alpha. This generated a panel of CCR-5-binding antibodies, of which around 20% inhibited MIP-1 alpha binding to CD4+. The single chain Fvs (scFv) generated by this step-back selection procedure also inhibited MIP-1 alpha-mediated calcium signaling.

Monoclonal antibodies (Mabs) have long been considered a good class of natural drugs, both because they mimic their natural role in the body and because they have no inherent toxicity. Although rodent Mabs are readily generated, their widespread use as therapeutic agents has been hampered because they are recognized as foreign by the patient. Evidently, clinical Mabs should be as human as possible and results with some of the more recently developed chimerized and humanized Mabs are testimony to this. Mabs that are entirely human are now being produced from phage display and transgenic mice. The first fully human Mabs generated by phage display have already entered clinical trials, and together with recent advances in these technologies, may finally realize the full potential of antibodies.

Using differential cDNA screening, we demonstrated that the bone-associated glycoprotein osteoglycin was highly expressed in differentiated adult rat vascular smooth muscle cells (VSMCs) but downregulated in VSMCs that had undergone proliferation in vitro. Further experiments in vitro revealed that osteoglycin gene expression was downregulated by a number of cytokines expressed in vivo (often in association with vascular injury) including basic fibroblast growth factor, transforming growth factor-beta, platelet-derived growth factor, and angiotensin II. In the normal adult rat carotid artery, osteoglycin was expressed in both the media and adventitia. However, osteoglycin mRNA expression was substantially increased in the adventitia and neointima 14 days after balloon injury, implying a role for this protein in vessel remodeling. Northern analysis of mRNA from neonatal rat aortas demonstrated upregulation of osteoglycin mRNA at week 2, after VSMC proliferation had ceased and when matrix modeling was maximal. In situ hybridization studies in human coronary arteries showed that osteoglycin mRNA was expressed by normal medial VSMCs but was downregulated in a subset of intimal VSMCs. Osteoglycin was not expressed in the VSMCs of adventitial vessels but was expressed in a subset of adventitial cells. This expression pattern contrasted with that of SM22 alpha, a contractile protein marker of VSMC differentiation, which was highly expressed in the media of all vessels. These data indicate that osteoglycin is a new marker of differentiated VSMCs and may be an essential component of the normal vascular matrix.

To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

We have cloned and sequenced a 1.9 kb fragment of the 5'-upstream sequence of the smooth-muscle-specific gene SM22 alpha. The region cloned consisted of the SM22 alpha promoter, a 65 bp exon containing most of the 5'-untranslated region and 307 bp of the first intron. A 1.5 kb fragment at the 5' end of this sequence was able to drive the expression of a reporter chloramphenicol acetyltransferase (CAT) gene in both vascular smooth-muscle cells and Rat-1 fibroblasts. This promoter region did not contain a consensus TATAA box but contained the sequence TTTAAA 25 bp from the major start site identified by primer extension. Deletion analysis showed that a fragment of the promoter from +65 to -303 was more active in both cell types than the 1.5 kb fragment suggesting that there are silencer sequences in the region 5' to the core promoter. CAT activity was also observed with fragments containing bases +65 to -193 and +65 to -117 in smooth-muscle cells. In contrast with the smooth-muscle cells, no CAT activity was detected in Rat-1 fibroblasts with the smallest two fragments. The residual promoter activity in the smallest fragment of the SM22 alpha promoter tested suggested that, unlike the smooth-muscle alpha-actin promoter, transcription from the SM22 alpha promoter can occur in smooth-muscle cells in the absence of factors binding to CC(A/Trich)6GG (CArG box) or CANNTG (E box) motifs.

SM22 alpha is a 22-kDa protein of unknown function, the mRNA of which is highly and specifically expressed in smooth muscle cells (SMC). The 5' untranslated leader sequence of the rat SM22 alpha gene was found to contain two introns of 3.6 and 2.9 kb. Two transcripts of SM22 alpha exist in all SMC types examined, and genomic mapping of the gene suggests these transcripts result from different 5' transcription start points, split by the 2.9-kb intron. A small intron (102 bp), which contains an E-box consensus sequence, was found within the coding region 178 bp from the ATG start codon. The 3.6-kb intron contains 82 bp which show 98% homology at the RNA level with the rat identifier sequence (ID). Transient reporter gene assays demonstrate that a 576-bp fragment, including the ID, contains a regulatory element which may contribute to the SMC-specific expression of SM22 alpha.