Oral Monoclonal Antibody Intervention in Gut Peptide Signaling p. 5
A. Summary
Interactions of the gastrointestinal luminal contents and the innermost mucosa lining of the gut maintain organism homeostasis. In response to luminal contents, specialized epithelial cells in the mucosa produce peptides and express receptors that are the body’s ‘sixth sense’ to transduce signals about the internal environment and modify immune, digestive and extra-intestinal (non-digestive) behavior (1). Data in section (B) support the novel finding that a cysteine-rich secreted protein, prokineticin 1 (PROK1) is produced in the stomach and acts via its receptor (PK-R1) on small intestinal mucosa to provoke a profound secretion(2, 3). Prokineticins have been shown to have cytokine, pro-proliferative, developmental, angiogenic, CNS and nociceptive properties via multiple signaling pathways in diverse tissues. However, PK-IR mediated signaling in gut and its role in inflammatory homeostasis is not known. The first goal for this two-year post-doctoral fellowship application is to investigate PROK1 signaling related to inflammation in intestinal epithelial cells.
The interface of lumen and mucosa is an emerging platform for therapeutic modulation by large molecules such as monoclonal antibodies (mAbs). For example, oral administration of anti-CD3 mAb induces a regulatory T-cell response that suppresses autoimmunity in several disease models(4). However, oral efficacy of a mAb that immunoneutralizes gut-derived peptides has not been investigated, to our knowledge. Thus, the second goal is to determine whether oral delivery of a PROK mAb abrogates pathology and disease score in rodent models of gut inflammation.
Pamela Hornby (Senior Research Fellow, Centocor J&J) and Juanita Merchant (H. Marvin Pollard Professor, U. Michigan) are experienced mentors who have strong track records of research in peptide gut signaling. They bring different and complementary expertise to the collaboration for a productive post-doctoral training experience. Experimental design and data collection is optimized so that it can be analyzed concurrently by complementary techniques between both labs. Successful completion of the aims provides new information on PROK1 signaling in gut epithelial cells and early Proof of Concept for a mAb platform that targets peptide signaling at the luminal-mucosal interface.
B. Background and preliminary data
The mucosa that lines the innermost wall of the gut has evolved to maintain organism homeostasis in response to the gastrointestinal contents, which includes dietary constituents, non-digestible matter, microorganisms and toxins. In response to luminal contents, specialized epithelial cells in the mucosa release peptides and express receptors that transduce signals about the internal environment to modify innate and adaptive immunity, digestive and extra-intestinal (non-digestive) behavior (1). Therapeutic exploitation of peptides produced by the gut epithelial enteroendocrine cell is evidenced treatment of Type 2 Diabetes by daily injections of Glucagon-Like Peptide 1 or its analogues (e.g. exenatide). The finding that gut-derived serotonin negatively controls bone mass provides a gut-bone link to osteoporosis(5) and is just one example of the wide-ranging impact of gut peptides in extra-intestinal systems.
Many peptides that are secreted by the gut mucosa signal in a paracrine fashion via their receptors on smooth muscle, nerves and blood vessels or cross-talk with immune cells in the subjacent lamina propria. The preliminary data in this section describe a novel protein, a prokineticin, that is secreted by the stomach and acts on its receptor downstream in the small bowel. The peptide is active when given orally and is a potent intestinal secretogogue(2).
Prokineticins are two secreted proteins (PROK1 and PROK2) that are the mammalian orthologs of snake toxin (MIT-1) (6) and a related protein in frog skin (Bv8)(7). Their cognate G-protein coupled receptors (PK-R1 & PK-R2)(8) were de-orphaned about 10 years ago(6, 9). Since then, prokineticins have been shown to have cytokine-like properties and are expressed in immune cell lineages (10, 11); have CNS activity(12) and are nociceptive(13); are pro-proliferative in endocrine glands(14); participate in growth and development(15), and are angiogenic(16).
Prokineticins were so named for their effect in gut muscle(6), and PK-1R is expressed in colonic neurons(17); however, little else was known about their localization and effects on the gut. PROK1 mRNA is highly expressed in the stomach (Fig. 1; clear bars), whereas its receptor PK-R1 (Fig. 1; stippled bar) is expressed most highly in the distal small intestine. In distal intestine (ileum) PK-R1 immunoreactivity is localized in submucosal and myenteric neurons (Fig. 2A & B) and, interestingly, in epithelial cells within the crypts (Fig. 2 C&D). Importantly, oral administration of PROK1 to rats evokes a profound secretory response in the small intestine that is similar to a known secretogogue prostaglandin E2 (PGE2) (Fig. 3). The PROK1 mediated secretory response is not observed in ileal tissue from PK-1R knock-out mice whereas a robust response is noted in tissue from wild type controls (Fig. 4). Our interpretation of these data is that PROK1 is synthesized in the stomach and evokes a secretory response in the intestines. PK-R1 neuronal expression suggests PROK mediated neurotransmission integrating intestinal and extra-intestinal effects.
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It has been shown that prokineticins signal via multiple messengers including phosphorylation of c-Src, epidermal growth factor receptor, ERK 1/2 (18), Akt (protein kinase) to induce the proliferation(19) as well as Gai signaling and i[Ca2+]mobilization (20). A majority of the information on PROK1 signaling is known in steroidogenic tissue (where PROK1 is known as Endocrine Gland (EG)-VEGF) but not other tissues expressing the protein, such as intestinal epithelial cells. A response to PROK1 in monocytes is secretion of chemokines that are geared toward a pro-inflammatory response, such as CCL4, CXCL1 and CXCL8 for leukocyte recruitment, extravasation and tissue invasion(21). Not known are the PROK1/PK-R1 mediated phenotypic responses in intestinal epithelial cells. Therefore, the first goal is to investigate PROK1 signaling related to inflammation and proliferation in human intestinal epithelial cell lines and tissue derived from PK-R1 knock-out mice by using pharmacological approaches, shRNA silencing or PROK1 blocking mAbs.
Dr. Merchant has long standing interest in gut immune peptide interactions and transcriptional regulation(1, 22-24). She recently demonstrated that inflammatory homeostasis in the gut is driven by dietary fiber and colonizing bacteria to maintain transcriptional regulation within colonic epithelial cells. Butyrate is produced by bacterial fermentation from undigested carbohydrates and fiber. Her work uncovered the mechanisms by which butyrate, a histone deacetylase inhibitor, limits mucosal damage and averts inflammation. Specifically, butyrate activates ATM kinase (a key protein that transduces DNA damage signals, promotes cell cycle arrest and DNA repair) and interacts with a zinc finger binding protein (ZBP-89) to improve colitis in mice given dextran sodium sulfate (DSS) (25, 26). In reproductive tissue, the PROK-1 gene promoter has a potential binding site for steroidogenic factor (27) and we plan to investigate PROK transcriptional regulation in intestinal epithelial cells. The role of PROK1 in intestinal inflammatory homeostasis is not known; however, PROK mRNA is rapidly upregulated in intestinal tissue from mice with colitis(28). Therefore, we will investigate regulation whether PK-R1 activation modulates disease severity in several models of intestinal inflammation, such as DSS colitis and post-operative ileus.
The interface of lumen and mucosa is an emerging platform for therapeutic modulation by large molecules such as monoclonal antibodies (mAbs). It has been reported that oral anti-CD3 mAb remains biologically active in the gut and induces CD4+CD25+ regulatory T-cell. This suppresses autoimmunity in several disease models, including experimental autoimmune encephalitis and autoimmune diabetes(4). However, oral efficacy of a mAb that immunoneutralizes gut-derived peptides has not been investigated, to our knowledge. Thus, a goal is to determine whether oral delivery of a PROK mAb abrogates pathology and disease score in rodent models of gut inflammation.
C. Specific objectives and timelines
Two Specific Aims are defined with associated experimental approaches that could be used to test these aims. There is an intentional diversity of molecular, cellular and in vivo techniques that allows the research plan to be customized based on the interests and previous training of the post-doctoral fellow. This provides flexibility to focus on positive findings, with alternative strategies as necessary.
A. Test the hypothesis that PROK1 activates PK-1R on intestinal epithelial cells and activates signaling pathways involved in proliferation or inflammation.
1) Immortalized human epithelial (T-84, HT-29 or Caco-2) enterocytes or endocrine cells(22, 29) will be selected for study based on expression of PROK1&2 and PK-1R&2R mRNA by RT-PCR and protein by IHC using polyclonal antibodies (Bethyl Labs). PROK1 mediated signaling pathways (previously demonstrated in other tissues) will be assessed in selected cell type for detection of changes in phosphorylation state or i[Ca++]. Standard pharmacological blockade or shRNA silencing will be used to PROK1 pathway selectivity. Inflammatory responses will be determined concurrently at J&J using ELISA/bead based cytokine detection in supernatants from same experiments. Routine methods will be used to determine whether there PROK transcriptional regulation occurs in response to PROK in these cells(24). Pending evidence for a contribution to regulation of cell cycle and growth of crypt cells (see also # 3), endocrine cell differentiation and growth in response to PROK1 could be investigated. These experiments will be done at UM in 0-12 month period.
2) Mice will be immunized and hybridomas made for PRK1 and PK-R1 mAbs; mAbs selected by binding affinity (BIAcore) and IC50 and Ki to inhibit PRK1-evoked i[Ca++] in PK-1R expressing HEK293 cells. Generation of mAb is initiated after and overlap with experiments #1 above (see Risks and Mitigation) and will be done using resources available at J&J in 6-12 month period.
3) Phenotypic responses to PROK will characterized in human epithelial intestinal cells selected in 1) grown in monolayers in 96 well plates. ACEA (Roche) is a impedence-based system for measuring phenotypic over time in live cells. Short term (<15 mins) changes have been well validated for GPCR activation but have not been reported for PROK, a Gai agonist. Long term responses (days) provide a kinetic measure of cellular proliferation. Functional blockade with selected mAbs from #2 will enable the second Aim. These experiments will be done by the post-doc at J&J in the 12-15 month period
Risks and their Mitigation: There are no known selective PK-1R antagonists available commercially but shRNA constructs are available through TRC to J&J. Controls for PK-R1 selectivity could involve primary intestinal cells isolated from PK-1R (GPR73a) knock-out and wildtype control mice. Successful completion of #1 for validation of PK-1R role in intestinal epithelial cells inflammation/proliferation will be necessary to initiate generation of blocking mAbs at J&J. If #1 is not successful and, pending the interests of the post-doctoral fellow, an alternative signaling peptide produced by the gut mucosa would be considered. For example, nerve growth factor (NGF) is present in mucosa and proNGF is detected in epithelial cell lines in vitro. The receptors trkA and p75NTR are co-expressed on submucosal neurons (30) and NGF evokes in trkA phosphorylation within myenteric neurons (31). Colonic NGF triggers and maintains long-term alterations of visceral sensitivity and gut mucosal integrity(32) consistent with a role in inflammation not yet explored. NGF blocking mAb and tools are available at J&J.
B. To test the hypothesis that oral administration of PROK1 mAb abrogates the intestinal pathophysiological in experimental rodent models of inflammation in vivo.
1) Mice will be rendered colitic by 4% DSS in drinking water and treated with PROK1 mAb or isotype control administered orally by gavage. Colonic histology and disease score will be compared at termination of experiment on day 5. An additional option is to perform mouse rigid sigmoidoscopy for timed longitudinal studies using colon biopsies from living mouse(33). A small animal imaging lab is available to do bioluminescence, CT, MRI and PET scanning on mice to track labeled mAb. Concurrently RT-PCR and low density micro array of the same intestinal tissue using probes for inflammatory, tissue repair and tight junction gene expression will be performed at J&J to elucidate MoA of PROK1. These experiments will be done by the post-doc at UM in the 15-24 month period
2) Based on initial results in #1 and the cytokine profile in response to PROK1 in A) an alternative inflammatory model of post-operative ileus may be considered (34)(35). Mice will be treated with PROK1 mAb or isotype control administered orally by gavage for two to three days following laparotomy and gentle intestinal manipulation. Myeloperoxidase histochemistry on mucosal stripped intestinal whole mounts will be performed to assess for leukocyte infiltration.
Risks and their Mitigation: Although PROK1 role in intestinal inflammation in vivo is unkown, PROK mRNA is rapidly upregulated in intestinal tissue from mice with colitis (28) suggesting that blocking PROK1:PK-1R may abrogate the inflammatory and disease responses. PK1R knock-out mice provide positive controls for the effects on inflammation in the absence of PK-1R. Human receptors are ~80% identical to the mouse orphan receptor GPR73a but at this time it is not known whether hPROK mAbs will functionally block murine PK-1R. In Aim A, if hPROK1 mAb does not block PROK1 evoked responses in mouse cells, surrogate antibodies generated to murine PROK1 would be a strategy.
D. Description of a Michigan/Centocor J&J partnership plan
Both PI’s have a proven track record in research on peptide signaling in the gut and bring complementary expertise to the collaboration. The UM techniques enable quantification and identification of early events in gut epithelial cells resulting from PK-R1 activation in vitro. These include RT-PCR, microarray, co-immunoprecipitation DNA affinity precipitation assays, immunoblots and reporter assays. J&J provides the tools to do this including immunoneutralizing PROK1 or PK-R1 monoclonal antibodies selected by binding by BIAcore, blockade of i[Ca++] in PK-R1 stably expressing HEK cells; recombinantly expressed and engineered peptides; PK-R1 knock-out mice; target silencing through shRNA constructs (The RNAi Consortium).
To determine the functional blockade of PROK in epithelial cells, J&J has impedance based phenotypic assessment of PROK-mediated (Gai coupled receptor) and proliferation responses (ACEA, Roche) at J&J. This will expose the fellow to efficient and novel techniques to record functional PK-R1 mediated responses and generate IC50 and Ki values for blocking mAbs in vitro.
In vivo inflammations models, such as DSS colitis (UM) and post-operative ileus (J&J) are routine in rodents. UM has a small animal imaging lab to do bioluminescence, CT, MRI and PET scanning on mice to track mAb. Mouse rigid sigmoidoscopy for colon biopsies from live mouse(33).