Oral Monoclonal Antibody Intervention in Gut Peptide Signaling
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).
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Oral Monoclonal Antibody Intervention in Gut Peptide Signaling
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.
FIG HERE
It has been shown that prokineticins signal via multiple messengers including phosphorylation of cSrc, epidermal growth factor receptor, ERK 1/2 (18), Akt (protein kinase) to induce the
proliferation(19) as well as Gi 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 proinflammatory 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.
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Oral Monoclonal Antibody Intervention in Gut Peptide Signaling
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 RTPCR 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 Gi
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
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Oral Monoclonal Antibody Intervention in Gut Peptide Signaling
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 PKR1 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 (Gi coupled receptor) and proliferation responses
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Oral Monoclonal Antibody Intervention in Gut Peptide Signaling
(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).
Dr Hornby and Merchant are experienced mentors and have previously trained 5 and X post-doctoral
fellows, respectively. Bimonthly updates by teleconferencing and LiveMeeting wil be used for
decisions on experimental design, data review and strategic planning. The post-doctoral fellow will
be expected to present their work nationally at one internal (e.g. Annual R&D Symposium) and
external (e.g. Digestive Diseases Week) meeting and publish >2 peer reviewed manuscripts per year.
E. PLANS FOR RECRUITING A POST-DOCTORAL FELLOW
Both PIs have a network of highly regarded Gastroenterologists and researchers through the
American Gastroenterology Association who can help identify US citizens who are close to, or have
already obtained, an MD/ PH.D. and are already working in this field. PH maintains ties with
Louisiana State Health Science Centers and the South, JM with Michigan network of colleges and the
Midwest serving on Ph.D. committees. Informal searches will occur as soon as notified of selection.
In addition, Dr. Merchant is a member of…… review board member of ….Minority programs.
Interviews will include a seminar and be conducted at the site most convenient for the interviewee
with participation across sites by LiveMeeting.
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Oral Monoclonal Antibody Intervention in Gut Peptide Signaling
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