842 A novel agonistic anti-CD40 targeting strategy with an affinity peptide binding feature for antigen cargo functionality: improving peptide stability and T cell proliferation

BackgroundTo induce a prominent anti-tumor T-cell response, a viral or tumor derived antigen epitope imbedded in a longer synthetic peptide (SLP) can be used, which also requires internalization and processing by antigen presenting cells (APCs) to enable T cell priming. Herein we present the design and evaluation of a CD40 targeting tetravalent bispecific antibody, binding peptides through affinity as an antibody-drug conjugate. APC activation as well as in vitro and in vivo T-cell proliferation studies demonstrate retained agonistic activity as well as improved T cell proliferation/expansion in vitro and in vivo, compared to non-linked peptide/antibody mixes.MethodsT-cell priming was evaluated with B3Z assay or a cytomegalovirus (CMV) model and displayed superior uptake to non-bound peptide in the co-stimulatory independent B3Z assay. In addition, intracellular peptide release in APCs was analysed using a unique quenching strategy displaying peptide release after around 4-6 hour post antigen.ResultsPeptide stability in vitro, when bound to the antibody, was analysed by mass spectrometry and displayed prolonged peptide stability in serum, increasing the peptide half-life by 15 times in vitro (ConclusionsData support that the novel delivery system can improve antigen targeting to dendritic cells, but can also provide a prolonged peptide half-life as well as a peptide delivery to APCs. Combined this improves the efficiency of both antigen delivery and CD40 agonistic functionality.

The Effects of Lipidation on a TAT-Containing Peptide-Based Inhibitor of PSD-95

Stability and cell permeability are critical parameters in the development of peptide therapeutics. Conjugation to fatty acids and cell-penetrating peptides, such as TAT (YGRKKRRQRRR), are established strategies to increase peptide stability and permeation, respectively. Here, we prepared lipidated analogues of a potent TAT-containing dimeric peptide-based inhibitor of the intracellular scaffolding protein PSD-95, an emerging drug target in ischaemic stroke. Lipidation increased peptide stability in vitro and in vivo. Combining both lipidation and conjugation to TAT improved brain/plasma ratios, but caused acute toxic effects due to the potent haemolytic activity of the TAT-lipid moiety.

The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR’s protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit β-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.

The Meta-Position of Phe4 in Leu-enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide [D-Ala2, D-Leu5]-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR’s protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and μ-opioid receptors (μORs) and potency to inhibit cAMP signaling and to recruit β-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and μOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.Abstract FigureTOC FIGURE

Efficacy of Rhesus Theta-Defensin-1 in Experimental Models of Pseudomonas aeruginosa Lung Infection and Inflammation

ABSTRACT Chronic airway infection and inflammation contribute to the progressive loss of lung function and shortened survival of patients with cystic fibrosis (CF). Rhesus theta defensin-1 (RTD-1) is a macrocyclic host defense peptide with antimicrobial and immunomodulatory activities. Combined with favorable preclinical safety and peptide stability data, RTD-1 warrants investigation to determine its therapeutic potential for treatment of CF lung disease. We sought to evaluate the therapeutic potential of RTD-1 for CF airway infection and inflammation using in vitro, ex vivo, and in vivo models. We evaluated RTD-1's effects on basal and Pseudomonas aeruginosa-induced inflammation in CF sputum leukocytes and CF bronchial epithelial cells. Peptide stability was evaluated by incubation with CF sputum. Airway pharmacokinetics, safety, and tolerance studies were performed in naive mice. Aerosolized RTD-1 treatment effects were assessed by analyzing lung bacterial burdens and airway inflammation using an established model of chronic P. aeruginosa endobronchial infection in CF (ΔF508) mice. RTD-1 directly reduces metalloprotease activity, as well as inflammatory cytokine secretion from CF airway leukocyte and bronchial epithelial cells. Intrapulmonary safety, tolerability, and stability data support the aerosol administration route. RTD-1 reduced the bacterial lung burden, airway neutrophils, and inflammatory cytokines in CF mice with chronic P. aeruginosa lung infection. Collectively, these studies support further development of RTD-1 for treatment of CF airway disease.