RF1 attenuation enables efficient non-natural amino acid incorporation for production of homogeneous antibody drug conjugates.

Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process. The RF1 mutant extract allows nnAA incorporation at previously intractable sites of an IgG1 and at multiple sites in the same polypeptide chain. Conjugation of cytotoxic agents to these nnAAs, yields homogeneous antibody drug conjugates (ADCs) that can be optimized for conjugation site, drug to antibody ratio (DAR) and linker-warheads designed for efficient tumor killing. This platform provides the means to generate therapeutic ADCs inaccessible by other methods that are efficient in their cytotoxin delivery to tumor with reduced dose-limiting toxicities and thus have the potential for better clinical impact.

Non-viral delivery of nuclear factor-kappaB decoy ameliorates murine inflammatory bowel disease and restores tissue homeostasis.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a key transcriptional regulator of inflammatory bowel disease (IBD). AIM: To investigate the therapeutic potential of a locally administered "non-viral" nuclear factor-kappaB decoy (NFkappaBD) in multiple experimental models of IBD. METHODS: A fully phosphorothioated decoy oligonucleotide with improved stability that specifically binds NF-kappaB and blocks inflammatory mediators regulated by this transcription factor without the help of viral envelope-assisted delivery was developed. The therapeutic effects of NFkappaBD were studied in the trinitrobenzene sulphonic acid, oxazolone and dextran sodium sulphate induced colitis models. RESULTS: Intracolonic administration of NFkappaBD results in the delivery of NFkappaBD to inflammatory cells and a reduction of NF-kappaB heterodimers. In the T helper cell 1-driven trinitrobenzene sulphonic acid-induced colitis model, mice receiving NFkappaBD treatment exhibit a dose-dependent reduction in disease severity and a more rapid recovery to normal body weight, similar to a clinically relevant dose of budesonide. Clinical efficacy was corroborated by considerable reductions in colitis pathology and tissue levels of several pro-inflammatory markers, including tumour necrosis factor alpha, interleukin 6, interleukin 1beta and monocyte chemotactic protein 1. NFkappaBD also mitigates disease activity in the T helper cell 2-like oxazolone colitis and epithelial injury-related acute dextran sodium sulphate colitis models. Interestingly, restoration of tissue homeostasis is observed in NFkappaBD-treated animals with the rapid re-emergence of functional goblet cells and a return to normal patterns of cell proliferation in the mucosal epithelium and smooth muscle cell layers. CONCLUSIONS: These data support the potential use of "naked" NFkappaBD as a cross-functional therapeutic in IBD, and show for the first time that it can facilitate the restoration of colon homeostasis and function.

Blockade of experimental atopic dermatitis via topical NF-kappaB decoy oligonucleotide.

Atopic dermatitis (AD) is a common chronic skin inflammatory disease. Long-term use of topical corticosteroids in skin inflammation poses risks of systemic and local side effects. The NF-kappaB transcription factor family plays a central role in the progression and maintenance of AD. This study explores the possibility of using topical NF-kappaB Decoy as a novel therapeutic alternative for targeting Th1/Th2-driven skin inflammation in experimental AD. A high-affinity, topical NF-kappaB Decoy developed for human efficacy demonstrates: (i) efficient NF-kappaB Decoy penetration in pig skin, (ii) NF-kappaB Decoy nuclear localization in keratinocytes and key immune cells, and (iii) potent "steroid-like" efficacy in a chronic dust-mite antigen skin inflammation treatment model. NF-kappaB Decoy exerts its anti-inflammatory action through the effective inhibition of essential regulators of inflammation and by induction of apoptosis of key immune cells. Unlike betamethasone valerate (BMV), long-term NF-kappaB Decoy treatment does not induce skin atrophy. Moreover, topical NF-kappaB Decoy, in contrast to BMV, restores compromised stratum corneum integrity and barrier function. Steroid withdrawal causes rapid rebound of inflammation, while the NF-kappaB Decoy therapeutic benefit was maintained for weeks. Thus, topical NF-kappaB Decoy provides a novel mechanism of reducing chronic skin inflammation with improved skin homeostasis and minimal side effects.

Microarray analysis of primary endothelial cells challenged with different inflammatory and immune cytokines.

To investigate the potential molecular mediators of tissue-specific recruitment, we explored the influence of different cytokine challenges on gene expression regulation in five primary endothelial cells (ECs), representing two different phenotypes: iliac artery and aortic (macrovascular); lung, colon and dermal (microvascular). We challenged ECs with cytokines that elicit different patterns of inflammatory and immune responses in immune cells: tumor necrosis factor (TNF-alpha), interferon-gamma (IFN-gamma) or interleukin-4 (IL-4), and used microarrays containing approximately 40,000 unique cDNAs, to assess changes in differential gene expression relative to untreated cells. Five hundred and sixty three sequences changed by at least 2.5 fold in one or more of the 15 possible EC /cytokine combinations. The list included highly regulated adhesion molecules, chemokines, cytokines, metalloproteases, and IFN-gamma-induced genes. Overall, IFN-gamma caused the largest number of gene expression changes and its profile was least correlated with IL-4. In addition to clusters that were predominantly EC/cytokine specific, we also observed several clusters that were regulated by more than one cytokine across several ECs. Furthermore, we identified genes that were reciprocally expressed in response to different cytokines that could serve as markers of inflammatory and immune expression. These results confirm the importance of microenvironment in primary ECs that could have important applications in developing targeted therapies for vascular diseases.

Lectin-like oxidized LDL receptor-1 is a cell-adhesion molecule involved in endotoxin-induced inflammation.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a major endothelial receptor for oxidized low-density lipoprotein, and is assumed to play a proatherogenic role in atherosclerosis. LOX-1 expression is induced by inflammatory cytokines as well as by proatherogenic stimuli. LOX-1 protein binds agedapoptotic cells, activated platelets, and bacteria, suggesting that it may have diverse activities in vivo. Here, we reveal a role for LOX-1 in endotoxin-induced inflammation. In a model of endotoxemia, injection of a high dose of endotoxin into rats induced leukopenia within 1 h and death of the animals within 24 h. Preadministration of anti-LOX-1 antibody reduced the degree of leukopenia and completely rescued the animals, whereas control IgG did not. In a model of low-dose endotoxin-induced uveitis, anti-LOX-1 antibody efficiently suppressed leukocyte infiltration and protein exudation. In situ videomicroscopic analyses of leukocyte interactions with retinal veins revealed that anti-LOX-1 antibody reduced the number of rolling leukocytes and increased the velocity of rolling, suggesting that LOX-1 functions as a vascular tethering ligand. The ability of LOX-1 to capture leukocytes under physiologic shear was confirmed in an in vitro flow model. Thus, LOX-1 is an adhesion molecule involved in leukocyte recruitment and may represent an attractive target for modulation of endotoxin-induced inflammation.

Immune surveillance and effector functions of CCR10(+) skin homing T cells.

Skin homing T cells carry memory for cutaneous Ags and play an important sentinel and effector role in host defense against pathogens that enter via the skin. CCR10 is a chemokine receptor that is preferentially expressed among blood leukocytes by a subset of memory CD4 and CD8 T cells that coexpress the skin-homing receptor cutaneous lymphocyte Ag (CLA), but not the gut-homing receptor alpha(4)beta(7). Homing and chemokine receptor coexpression studies detailed in this study suggest that the CLA(+)/CCR10(+) memory CD4 T cell population contains members that have access to both secondary lymphoid organ and skin compartments; and therefore, can act as both "central" and "effector" memory T cells. Consistent with this effector phenotype, CLA(+)/CCR10(+) memory CD4 T cells from normal donors secrete TNF and IFN-gamma but minimal IL-4 and IL-10 following in vitro stimulation. Interactions of CCR10 and its skin-associated ligand CC ligand 27 may play an important role in facilitating memory T cell entry into cutaneous sites during times of inflammation.