Cholix (Chx) is expressed by the intestinal pathogen Vibrio cholerae as a single chain of 634 amino acids (~70.7 kDa protein) that folds into three distinct domains, with elements of the second and third domains being involved in accessing the cytoplasm of nonpolarized cells and inciting cell death via ADP-ribosylation of elongation factor 2, respectively. In order to reach nonpolarized cells within the intestinal lamina propria, however, Chx must cross the polarized epithelial barrier in an intact form. Here, we provide in vitro and in vivo demonstrations that a nontoxic Chx transports across intestinal epithelium via a vesicular trafficking pathway that rapidly achieves vesicular apical to basal (A→B) transcytosis and avoids routing to lysosomes. Specifically, Chx traffics in apical endocytic Rab7 + vesicles and in basal exocytic Rab11 + vesicles with a transition between these domains occurring in the ER-Golgi intermediate compartment (ERGIC) through interactions with the lectin mannose-binding protein 1 (LMAN1) protein that undergoes an intracellular redistribution that coincides with the reorganization of COPI + and COPII + vesicular structures. Truncation studies demonstrated that domain I of Chx alone was sufficient to efficiently complete A→B transcytosis and capable of ferrying genetically conjoined human growth hormone (hGH). These studies provide evidence for a pathophysiological strategy where native Chx exotoxin secreted in the intestinal lumen by nonpandemic V. cholerae can reach nonpolarized cells within the lamina propria in an intact form by using a nondestructive pathway to cross in the intestinal epithelial that appears useful for oral delivery of biopharmaceuticals. One-Sentence Summary: Elements within the first domain of the Cholix exotoxin protein are essential and sufficient for the apical to basal transcytosis of this Vibrio cholerae-derived virulence factor across polarized intestinal epithelial cells.

The intestinal epithelium functions to effectively restrict the causal uptake of luminal contents but has been demonstrated to transiently increase paracellular permeability properties to provide an additional entry route for dietary macromolecules. We have examined a method to emulate this endogenous mechanism as a means of enhancing the oral uptake of insulin. Two sets of stable Permeant Inhibitor of Phosphatase (PIP) peptides were rationally designed to stimulate phosphorylation of intracellular epithelial myosin light chain (MLC) and screened using Caco-2 monolayers in vitro. Apical application of PIP peptide 640, designed to disrupt protein–protein interactions between protein phosphatase 1 (PP1) and its regulator CPI-17, resulted in a reversible and non-toxic transient reduction in Caco-2 monolayer trans-epithelial electric resistance (TEER) and opening of the paracellular route to 4 kDa fluorescent dextran but not 70 kDa dextran in vitro. Apical application of PIP peptide 250, designed to impede MYPT1-mediated regulation of PP1, also decreased TEER in a reversible and non-toxic manner but transiently opened the paracellular route to both 4 and 70 kDa fluorescent dextrans. Direct injection of PIP peptides 640 or 250 with human insulin into the lumen of rat jejunum caused a decrease in blood glucose levels that was PIP peptide and insulin dose-dependent and correlated with increased pMLC levels. Systemic levels of insulin suggested approximately 3–4% of the dose injected into the intestinal lumen was absorbed, relative to a subcutaneous injection. Measurement of insulin levels in the portal vein showed a time window of absorption that was consistent with systemic concentration-time profiles and approximately 50% first-pass clearance by the liver. Monitoring the uptake of a fluorescent form of insulin suggested its uptake occurred via the paracellular route. Together, these studies add validation to the presence of an endogenous mechanism used by the intestinal epithelium to dynamically regulate its paracellular permeability properties and better define the potential to enhance the oral delivery of biopharmaceuticals via a transient regulation of an endogenous mechanism controlling the intestinal paracellular barrier.

The low permeability of nanoparticles (NPs) across the intestinal epithelium remains a major challenge for their application of delivering macromolecular therapeutic agents via the oral route. Previous studies have demonstrated the epithelial transcytosis capacity of a non-toxic version of Pseudomonas aeruginosa exotoxin A (ntPE). Here, we show that ntPE can be used to deliver the protein cargo green fluorescent protein (GFP) or human growth hormone (hGH), as genetic fusions, across intact rat jejunum in a model where the material is administered by direct intra-luminal injection (ILI) in vivo in a transcytosis process that required less than 15 min. Next, ntPE chemically coupled onto biodegradable alginate/chitosan condensate nanoparticles (AC NPs-ntPE) were shown to transport similarly to ntPE-GFP and ntPE-hGH across rat jejunum. Finally, AC NPs-ntPE loaded with GFP as a model cargo were demonstrated to undergo a similar transcytosis process that resulted in GFP being colocalized with CD11c+ cells in the lamina propria after 30 min. Control NP preparations, not decorated with ntPE, were not observed within polarized epithelial cells or within the cells of the lamina propria. These studies demonstrate the capacity of ntPE to facilitate the transcytosis of a covalently associated protein cargo as well as a biodegradable NP that can undergo transcytosis across the intestinal epithelium to deliver a noncovalently associated protein cargo. In sum, these studies support the potential applications of ntPE to facilitate the oral delivery of macromolecular therapeutics under conditions of covalent or non-covalent association.

Programmed Cell Death-Ligand 1 (PD-L1) binds to its receptor, Programmed Cell Death protein (PD-1) and is expressed on a range of activated immune cells and is upregulated by tumor cells and by immune cells in the tumor microenvironment. PD-L1 immunotherapies have emerged as effective treatments both as monotherapies and in combinations, providing long term responses in a subset of patients. Phage display selections have identified a range of potent Affimer biotherapeutic antagonists to both human and mouse programmed death-ligand 1 (PD-L1). We have demonstrated when formatted as bivalent molecules they have improved sub nM affinity, half-life extension in a mouse PK study, blocking activity in vitro and in vivoefficacy in syngeneic or xenograph mouse models. Affimer biotherapeutics are a 14kDa monomeric scaffold protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. Large diverse phage display libraries have been created by engineering in two nine amino acid peptide loops into the scaffold backbone. We have identified a range of affinity competitive binders to mouse or human PD-L1 confirmed by performing competition ELISA and SPR using Fc formatted antigen. Half-life extension of lead Affimer proteins was achieved by fusing to human IgG1Fc or as an in-line fusion (ILF) fused to serum albumin binding Affimers. We demonstrated avidity for binding to target antigen, binding to cells in vitro and blockade of the PD-1L/PD-1 interaction using Promega cell based assay. The PK properties of the formatted Affimers in mouse showed half-life extension from 40-120 hours depending on the clone and half-life extension technology used. We have demonstrated that the Affimer scaffold has the necessary properties to be developed as an immunotherapy. The anti-PD-L1 Affimer scaffold proteins were formatted as Fc fusion and in-line fusion formats which showed in vivo efficacy. These molecules will be developed further for both bispecific and drug conjugates to generate new novel biotheraputics that will give greater efficacy in the clinic. Citation Format: Amrik Basran, Emma Jenkins, Estelle Adam, Floriane Laurent, Michele Writer, Assa Oumie, Jyrki Sivula, Maureen West, Emma Stanley, Jennifer Hillman, Viviana Robles-munoz. Preclinical evaluation of half-life extended Affimer® biotherapeutics targeting the PD-L1 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4108.

Programmed Cell Death-Ligand 1 (PD-L1) is part of the immune checkpoint system involved in preventing autoimmunity. PD-L1 is upregulated on tumors cells and binds to its receptor, PD-1, expressed by immune cells in the tumor microenvironment. Anti-PD-L1 immunotherapies have shown to be effective treatments both as monotherapies and in combinations with chemotherapies and radiotherapies, providing long term responses in a subset of patients. We have developed a PD-L1 Affimer antagonist that could differentiate itself from the current clinically approved mAbs due to its smaller size, improved tissue penetration and alternative routes of delivery such as a subcutaneous injection. It also opens up the possibility of generating additional PD-L1 antagonist therapeutics such as bispecifics and cytokine fusions. The Affimer biotherapeutic scaffold is a 14kDa monomeric protein based on the human protease inhibitor Stefin A, lacking post-translational modifications such as disulfide bonds and glycosylation. We have identified a range of high-affinity competitive binders to human PD-L1 confirmed by SPR using Fc formatted antigen, competition ELISA and blockade of PD-L1/PD-1 axis using various cell-based assays. We demonstrated efficacy in a humanized PD-L1 MC38 tumor growth inhibition model and compared it to approved PD-L1 antibody therapies. Pharmacokinetics (PK) and biodistribution are key parameters that influence the efficacy and safety of therapeutic proteins. Half-life extension of lead Affimer proteins was achieved by fusing to a human Fc region of an IgG (designated AVA004).Pharmacokinetic analysis of AVA004 Fc molecules were performed in C57Bl/6, humanized FcRn/HSA mice and cynomolgus after single intravenous administration. Tissue distribution following intravenous administration of AVA004 Fc to humanized NOG mice bearing orthotopic MDA-MB-231 tumor cells was evaluated using radiolabeled 125I- AVA004 Fc. Biodistribution imaging studies were conducted in mice bearing A375 tumor cells using IRDye800 conjugated to AVA004 Fc followed by ex vivo imaging. A more quantitative biodistribution study was conducted in the same model using SPECT imaging DOTAGA bioconjugated AVA004 Fc molecules radiolabeled with 111Indium. The imaging biodistribution allowed us to select the most favorable molecule based on its tumor/plasma and tumor/liver ratios. Citation Format: Estelle Adam, Emma Jenkins, Floriane Laurent, Jennifer Hillman, Maureen West, Emma Stanley, David Sanford, Agnieszka Kownacka, Céline Mothes, Alexandra Oudot, Vincent Matthew, David Gilfoyle, Barry Jones, William Bachovchin, Amrik Basran, Cyril Berthet. Pharmacokinetics and biodistribution of formatted Affimer® biotherapeutics targeting PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1866.