CA2238940A1 - Luminal cholecystokinin-releasing factor - Google Patents

Abstract

Luminal cholecystokinin-releasing factor (LCRF) is a cholecystokinin (CCK) releasing protein isolated from rat intestinal secretion. Purified LCRF was characterized by molecular weight, partial amino acid sequence and CCK releasing activity as shown in in vivo studies of anti-LCRF antibodies in blocking the CCK releasing effect of LCRF. Binding studies demonstrated localization in the duodenum, pancreas and in nerve fibers throughout the pancreas, sensory fibers and cell bodies of the nodose ganglia as well as in sympathetic nerve fibers in the adrenal medulla. LCRF appears to be a neuropeptide present in the enteric, parasympathetic and sympathetic nervous systems, but not in the brain. LCRF-IR is also present in enterocytes at the tips of small intestinal villi. Taken together, the studies indicate that LCRF is a neuropeptide that may have several functions in the gastrointestinal systems and other systems. Immunoaffinity studies using antibodies raised to synthetic LCRF1-6 and small intestinal lumen infusion studies indicate LCRF may be the CCK-releasing peptide present in intestinal secretion that mediates negative feedback regulation of pancreatic enzyme secretion and CCK release. LCRF and functionally related species have potential for development for treatment of insulin secretion, gastric and gallbladder emptying and regimens requiring appetite control or suppression.

Description

CA 02238940 l998-04-22 W O 97tl5671 PCTAJS96/179g8 nF,~CRTPTION

T,UMTl~AT, CHOT,l~CYSTOKTl~TN-RFT,F~SING FACTOl;~
., 5This is a continll~tion-in-part of provisional patent application SN 60/005,872filed October 26, 1995.

The United States gov~rnment has rights to use of the present invention relative to research support provided by NIH grants R01 DK-37482, R01 DK-38626 and R01 DK 33850.

1.0 BACKGROUND OF T~; INVENTION

1.1 Field of the Invention The invention relates generally to the field of molecular biology and more particularly to novel polypeptides and compositions comprising novel cholecystokinin-releasing peptides (LCRF) and the genes encoding the peptides. In certain embo-1iment~ the invention concerns the use of LCRF and nucleic acid sequences encoding the peptides for producing stim~ ti- n of an irnmune response, 20 for appetite ~u~ ssion, inhibition of gastric emptying, and for stim~ tion of insulin secretion.

1.2 Description of the Related Art Cholecystokinin (CCK) is a peptide hormone located in discrete cells of the 25 upper small intestine and secreted into the blood in response to eating. CCK plays a central role in the physiologic regulation of gallbladder conkaction and pancreatic secretion and modulates gaskic emptying, intestin~l motility and appetite (Liddle, 1989). Because ofthe central role of CCK in digestion, the meeh~ni~m~ regulatingthe release of CCK from discrete endocrine cells in the p~ illlal small intestinc have 30 been the subject of considerable investigation, reviewed by I,iddle (1995).

W O 97/15671 PCT~US96/i7998 A large body of evidence indicates that CCK is a natural satiety agent in ~nim~l e and hllm~n.e Part of the "full", pleasant feeling after a meal, termed "satiety", is clearly related to increased CCK release, and has been demonstrated to occur in "
5 many hurnan and animal ~x~ ..llents. Unfortunately, CC~K acts within int~rn~l organs and nerves to cause these effects, and therefore CCK must be ?~-lminietered intravenously or hl~ luscularly, or possibly by hl~ al ~lmini~etration. Moreover, CCK is not effective orally, since it is subject to digestive processes, and secondly, it would still have to be absorbed intact from the i ~ l tract, a complicated event, 10 even if it did survive digestive processes Dietary pl~ ~eh~s or protein digests fail to stiml~l~te CCK release from isolated 1 mucosal cells, and it has been suggested that other factors are n~ces~. y for regulation of CCK secretion (Sharara et al., 1993). In conscious rats and man, CCK
15 release and panc~ tic exocrine secretion are inhibited by trypsin, chym~lly~:jin or elastase in the proximal small intestin~ This has led to the notion that CCK release may be me~ te~l by a protease-sensitive me--hs~ni.em (Folsch et al, 1987; Slaff et al, 1984; Owyang, et al, 1986). Based on the potent stimulation of CCK release by diversion of pancreatic juice and bile from the small intestine, Miyasaka and Green 20 (1983) proposed that an intralllmin~lly secreted, trypsin sensitive intestin~l factor mediates this response. Such a substance could act as an important fee~lb~ck regulator of pancreatic el.~yl~.c secretion by stim~ ting CCK release when intt~.stin~l free (uncomplexed or uninhibited) protease activity is low, but would be rendered inactive as int~stin~l free protease activity rises (Green, et al, 1972). Subsequently, 25 researchers obtained evidence for an active factor in in~estin~l washes whichstimlll;qted CCK release and pancreatic enzyme secretion in conscious rats (Miyasaka et al., 1989) and in ~nestheti7.~rl rats (Lu et al.).

CCK is produced in discrete endocrine cells in the proximal small int~stin~
30 and is released into the blood stream following a meal. Ingested fats, proteins, and to W O Y7/15671 PCT~US96/17998 a lesser degree, carbohydrates, stim~ te CCK release (Marx et al.; Fried et al.), but the me~ h~ni~m~ underlying the CCK releasing activity of these compounds is ..
unknown.

Studies in rats have demonstrated that diversion of biliary-pancreatic secretions away from the small intestine or infusion of trypsin inhibitors or intact protein into the srnall int~stin~ strongly stimlll~tPs pd~ GdliC enzyme secretion, and this phenomenon is termed "feedback regulation of pancreatic enzyme secretion"
(Green et al., 1972; Green et al., 1973). These and later studies show that pancreatic enzyrne secretion and CCK release in rats and hl-m~nc is inhibited by trypsin, chym~LL~sin, and el~et~e in the proximal small intestine (Schneeman et al.; Green et al., 1985; Louie et al.; Folsch et al.; Slaff et al.; Owyang et al., 1986).

The hypothesis that protease-dependent feeAb~c~ regulation of pancreatic enzyme secretion is mediated by an endogenous, intraluminally secreted int~stin~l peptide was spurred by earlier reports that gastrointestin~l peptides appeared in the gut lumen in sigIuficant amounts (IJvnas-Wallensten; Lake-Bakaar et al.; Chang et al.).
The origin of luminal peptides was controversial. Some investigators reported that the gut cleared circulating peptides by secreting them into the lumen (Jordan et al.;
Ayalon et al.). On the other hand, Uvnas-Wallensten argued that the immediate source of luminal GI peptides was the corresponding gut endocrine cell (Uvnas-Wallensten), which was described as secreting bi-directionally, i.e., into the lumen and into the circulation via diffusion -from the h~ lilial fluid adjacent to basal and lateral parts of the endocrine cell surface.
Fee-lh~ regulation of CCK release mill.ir~~ecl by dietary protease inhibitors or intact protein (but not by diversion of pancreatic juice) was proposed to be mediated by a cholecystokinin-releasing peptide, monitor peptide (Iwai et al.; Fushiki et al.), which has been purified from pancreatic juice. Monitor peptide, also known as pancreatic secretory trypsin inhibitor-61 (PSTI-61), is a~ ly not present in .

W O 97/lS671 PCT~US96/17998 intestin~l secretion (Guan et al.). However, two peptides with sequence simil~rity or identity with monitor peptide have been isolated from pig intestine, although it is not known whether these peptides stim~ te CCK release or are secreted intraluminally(Agerbeth et al. 1991, Agerbeth et al. 198~
s Additionally, Owyang and coworkers (Owyang et al. 1990; Herzig et al. 1995) have described the pnrifi(~tion of a cholecystokinin releasing peptide from porcine intçstin~l mucosa which stimulates CCK release when infused into the rat intt?stine This peptide has been i(lentified as identical to the previously reported peptide 10 diazepam binding inhibitor (DBI).

2. 0 Summaly of the Invention The present invention seeks to address these and other drawbacks inherent in 15 the prior art by providing purified cholecystokinin-rele~cing polypeptide compositions and methods for tre~trnent of various conditions related to lack of or insufficient regulation of CCK release. The invention relates in particular to a novel polypeptide hormone-like compound, luminal cholecystokinin-releasing factor(LCRF), which waspurified from rat intestin~l secretions. Tmml-no~ffinity studies using antibodies raised 20 to synthetic LCRF indicate that the polypeptide product isolated and characterized is a CCK-releasing peptide present in i.,le~li,.~t secretion. The properties ofthe peptide indicate that it me~ti~tes "negative fee(lb~çk regulation" of ~ cleatic enzyme secretion and CCK release.
LCRF represents one of a new class of regulatory peptides that are secreted 25 intraluminally in the gut and serve an important physiological function in the regulation of metabolic functions that depend on CCK stimulation.

W O 97115671 PCT~US96tl7998 2.1 Novel CCK releasing polypeptides In an important aspect therefore, the present invention relates to the discoveryof a novel CCK-releasing polypeptide isolated from luminal int~stin~l secretions. The 5 new peptide differs from other known CCK-releasing factors. The partial peptide sequence (SEQ ID NO:l) has little homology with diazepam binding inhibitor (DBI)or other 11~t~b~e deposited protein sequences available at the time of the invention.

2.2 LCRF Pharmaceutical Compositions Another aspect of the present invention includes novel compositions comprising isolated and purified LCRF protein or nucleic acids which encode LCRF protein. It will, of course, be lm~l~r~tood that one or more than one CCK-releasing factor gene may be used in the methods and compositions of the invention. The nucleic acid delivery 15 methods may thus entail the ~-lmini~tration of one, two, three, or more, homologous genes. The m~x;,~....~, number of genes that may be applied is limited only by practical considerations, such as the effort involved in ~imlllt~npously plep~ g a large nurnber of gene constn~cts or even the possibility of eliciting an adverse ~;yloLo~ic effect.
-The compositions will contain a biologically effective amount of the novel peptide or peptides. As used herein a "biologically effective arnount" of a peptide or composition refers to an amount effective to stim~ te CCK release. As disclosed herein, di~ peptide amounts are effective, as shown in vi~ro and in vivo such asthose between about 6 to about 11 mg/kg.
Clinical doses will of course be determin~d by the n~ltrition~l status, age, weight and health of the patient. The ~lu~llily and volume of the peptide composition s~t1mini~t~red will depend on the subject and the route of ~lmini~tr~tion. The precise amounts of active peptide required will depend on the j~lclgm~nt of the practitioner and W O 97/1~671 PCT~US96/17998 may be peculiar to each individual. However, in light of the data ~ senLed herein, the .1~* .,1.;..~;on of a suitable dosage range for use in hlml~n~ will be strai~hlr l~v~d.

The compositions for use in 5timlll~ting CCK release in accordance with the 5 present invention will be compositions that contain the full length peptide which has about 70-75 amino acid residues and a molecular weight of about 8136 daltons or functional ~gm~nts and variants t_ereof such as the sequences ~ SellL by SEQ ID
NO: 1, SEQ ID NO:3 amino acid positions 1-6, 7-23, or 22-37 of SEQ ID NO:1. The term "a peptide" or "a polypeptide" in this sense means at least one peptide or 10 polypeptide which includes a sequence of any of the aforementioned ~ u~ es orvariants thereof. The terms peptide and polypeptide are used i~ angeably.

In addition to including an amino acid sequence in accordance with SEQ ID
NO:l, the peptides may include various other shorter or longer ~Agmente or other short 15 peptidyl sequences of various amino acids. In certain embo~1im~nt~, the peptides may include a repeat of shorter sequences, for example, SEQ ID NO:3, or additional sequences such as short targeting sequences, tags, labelled rç~icllle~, amino acids c~ ell~lated to increase the half life or stability of the peptide or any additional residue - for a ~le~i n~t~1 purpose, so long as the peptide still functions as a CCK rell~cin~ agent.
Such functionality may be readily det~rmin~?~l by assays such as those described herein.

Any of the comm~ nly oCcllrring arnino acids may be incorporated into the peptides, including ~l~nine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, gl~ , glycine, hi~ti~linP, isoleucine, leucine, lysine, methinnin~, phenyl~l~nin~, proline, serine, threonine, tryptophan, tyrosine and valine. Likewise, any of the so-called rare or modified arnino acids may also be incorporated into a peptide of the invention, inclu&g: 2-~mino~rlipic acid, 3-~mino~-liric acid, beta-Al~nine (beta-Aminopropionic acid), 2-Aminobutyric acid, 4-Aminobutyric acid (piperidiriic acid), 6-Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Arninoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Decmosin~7 2,2'-WO 97/15671 PCTrUS96/17998 Diaminopimelic acid, 2,3-Diarninopropionic acid, N-Ethylglycine, N-Ethylasparagine, Hy~xyly~ine, allo-Hy~xylysine, 3-Hy~Lo~y~luline, 4-Hydroxyproline, Isoeesmosine, allo-Isoleucine, N-Methylglycine ~ ;o~ille), N-Methylisoleucine, N-Methylvaline, Norvaline, Norleucine and Ornithine.
s The inhibitory compositions of the invention may include a peptide modified to render it biologically protected. Biologically protected peptides have certain advantages over ullplotecl~d peptides when ~-1mini~tered to human subJects and, as disclosed in U.S. patent 5,028,592, incol~o.d~d herein by rert-t;~lce, ~,rolGct~d peptides often exhibit 10 il.~ ed ph~rrn~cological activity.

Compositions for use in the present invention may also co~ lise peptides which include all L-amino acids, all D-amino acids or a n~,xLulG thereo~ The use of D-amino acids may confer ~d~1itinn~1 r~si~t~nre to proteases naturally found within the human 15 body and are less immlm-)genic and can therefore be expected to have longer biological half lives.

Likewise, compositions that make use of CCK-releasing factor encoding genes are also colllGllll.lated. The particular combination of genes may be two or more 20 variants of LCRF genes; or it may be such that a CCK-rele:~in~ factor gene is combined with another gene andlor another protein such as a cytoskeletal protein, cofactor or other biomolecule, a hormone or growth factor gene may even be combined with a gene encoding a cell surface lGce~ capable of inl~r~ctin~ with the polypeptide product of the first gene.
In using multiple genes, they may be combined on a single genetic construct under control of one or more promoters, or they may be p~ d as ~dLG constructs of the same or difLre~L types. Thus, an almost endless combination of ~ lll genes and genetic constructs may be employed. Certain gene combinations may be ~le~i n~d 30 to, or their use may otherwise result in, achieving synergistic effects on cell grow~

W O 97/15671 PCTrUS96/17998 and/or stim11k7tir)n of an ;mm11nr- response. Any and all such combinations are intr~nrlr~d to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scir~n77fic li~ ule, so that one of oldil~y skill in the art would readily be able to identify likely synergistic gene combin~tir~nc~ or even gene-protein combinations.

It will also be understood that, if desired, the nucleic acid segm-~nt or gene encoding a LCRF polypeptide could be ~7~7mini~tr-red in combination with further agents, such as, e.g, proteins or polypeptides or various rh~7rm~entically active agents. So long as the composition comprises a LCRF gene, there is vi tually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The nucleic acids may thus be delivered along with various other agents as required in the particular in~t~7nr.e ph~77m~7re11tical compositions ~l~,,J~.,d in accordance with the present invention find use in several applications, incl11~7ing appetite :~u~ ion, stim~ tinn of insulin release and ~u~le3:~ion of gastric or gall bladder ~;;Lllplying. Such methods generally - involve ~ k~ to a m~7mm~71 a ph~7nn~7r~e17tir~71 composition comprising an immunologically effective amount of a LCRF composition. This composition may include an immlmr~logically-effective amount of either a LC~ peptide or a LCRF-encoding nucleic acid composition. Such compositions may also be used to generate an immllne lc~ ollse in a m~7mm~1 Therapeutic kits comprising LCRF peptides or LCRF-encoding nucleic acid segmr-ntc cr mpri~e another aspect of the present invention. Such kits will generally contain, in suitable cf~ means, a ph,77m~7r~e71tically acceptable f~7~711k7tir)n of LCRF peptide or a LCRF-encoding nucleic acid composition. The kit may have a single container means that contains the LCRF composition or it may have distinct CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 cont~iner means for the LCRF composition and other reagents which may be included within such kits.
The coll~ollents of the kit may be provided as liquid solution(s), or as dried - powder(s). When the co.l~onents are provided in a liquid solution, the liquid solution is S an aqueous solution, with a sterile aqueous solution being particularly preferred. When reagents or colll~onell~ are provided as a dry powder, the powder can be recon~t~ te~l by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another c.~ means.

10In related embo-liment~, the present invention c~ tPS the p~ ion of diagnostic kits that may be employed to detect the presence of LCRF proteins or peptides and/or antibodies in a sample. Generally spç~king, kits in accordance with the present invention will include a suitable LCRF protein or peptide or antibody directed against such a protein or peptide, together with an immlm~detecti~n reagent and a 15 means for C~J~ llg the antibody or antigen and reagent. The components of thediagnostic kits may be packaged either in aqueous media or in lyophili7~cl form.
:
The immlmndetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands 20 might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or ~ dvidin) ligand having an ~c~oçi~tPcl label. Of course, as noted above, a number of Pl-en~pl~ry labels are known in the art and all such labels may be employed in c~l"~e~;lion with the present invention. The kits may contain antibody-label conjugates either in fully conju~,d~ed form, irl the form of int~rmP~i~tes, or as sep~dl~:
25 moieties to be conjugated by the user of the kit.

The cnnt~iner means will gen~r~lly include at least one vial, test tube, flask, bottle, syringe or other cr.nt~iner means, into which the antigen or antibody may be placed, and preferably suitably aliquoted. Where a second binding ligand is provided, 30 the kit will also generally contain a second vial or other col~ into which this ligand W O 97/15671 PCTnUS96/17998 or antibody may be placed. The kits of the present invention will also typically include a means for co~ ;..g the antibody, ~nhgen, and reagent co..l~ in close confinement for commercial sale. Such Cu..l;.il..,~ may include injection or blow-mol(le~l plastic c~ e~ i into which the desired vials are retained.
s 2.3 LCRF Antibodies In another aspect, the present invention c~ tes an antibody that is Ga~iLive with a polypeptide of the invention. An antibody can be a polyclonal 10 or a monoclonal antibody. In a pler~Gd embodiment, an antibody is a monoclonal antibody. Means for p~ g and char~cteri~ing antibodies are well known in the art (See, e.g, Howell and Lane, 1988).

Briefly, a polyclonal antibody is plG~aled by i~ an animal with an 15 immlm( gen cul"~ g a polypeptide of the present invention and collecting ~nti~P~
from that ;.. ;,~ animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a T-~..xlrl or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a plGr~llGd choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for LCRF may be prepared using c~"lvGlllional i~ l;on techniques, as will be generally known to those of skill in the art. A composition cr...li~;..;..g antigenic epitopes of LCRF can be used to ;-----,--";,~ one or more G2c~ ent~ nim~l~, such as a rabbit or mouse, which 25 will then proceed to produce specific antibodies against LCRF. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and p,~",g serum samples from the whole blood.

To obtain monoclonal antibodies, one would also initially ;...I~u.~ an ~ nt~l animal, often preferably a mouse, with a LCRF composition. One would W O 97/15671 PCT~US96/17998 then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hyhriclom~e These hybridomas may be isolated to obtain individual clones which can S then be screened for production of antibody to the desired LCRF peptide.

Following ;.. ,.. i,i1lion, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hyhri<lom~ secreting monoclonal antibodies against LCRF. Hybridomas which produce monoclonal antibodies to the 10 selected antigens are i~lentified using standard techniques, such as ELISA and Western blot methods. ~yhritlom~ clones can then be cultured in liquid media and the culture sUp~rn~t~nte purified to provide the LCRF-specif c monoclonal antibodies.

It is proposed that the monoclonal antibodies of the present invention will find15 useful application in standard immllnochemical procedures, such as ELISA and Western blot methods, as well as other procedures which may utilize antibody specific to LCRF
~i~o~es.

Additionally, it is proposed that monoclonal antibodies specific to the particular 20 chemokine may be utilized in other useful applications. For example, their use in immlmo~hsorbent protocols may be useful in puliryillg native or recombinant LCRFspecies or variants thereof.

In general, both poly- and monoclonal antibodies against LCRF may be used in 25 a variety of embo~liment~ For ex~n~rle, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding LCRl; or related proteins. They may also be used in inhibition studies to analyze the effects of LCRF in cells or ~nim~l~ Anti-LCRF antibodies will also be useful in immlm~ loc~li7~tinn studies to analyze the distribution of LCRF during various cellular events, for example, to ~let~rrnine the 30 cellular or tissue-specific distribution of the LCRF peptide under different physiological CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 conditions. A particularly useful applic~til~n of such antibodies is in ~ul;rying native or - recombinant LCRF, for example, using an antibody affinity column. The operation of all such immlln~logical techniques will be known to those of skill in the art in light of the present disclosure.
s 2.4 LCRF Compositions and Appetite Suppression LCRF has distinct advantages as an appetite :iu~ ;s~ and thus as a 10 potential tool in the arsenal of weight management. Unlike CCK, LCRF may be ~flmin;~tered orally, thus providing a simple method of keating patients with minim~i inconvenience or discomfort.

Effects on gastric c~ yillg may also be an important contributor to satiety 15 and part of the effect of LCRF on satiety may be through its effects to delay gastric Lyil~g.

., Once the peptide agent reaches the duodenum, it is subject to digestion by the pancreatic digestive enzymes. LCRF is norrnally secreted into the lumen of the 20 duodenum and survives intact, if food protein or dietary protease inhibitors are present to protect the peptide from pancreatic digestive enzymes. Orally effective formulations of LCRF could best be taken with meals, and the meal protein would further protect the peptide agent in the int~stin~ Similarly, a forrnulation CO~ p a protease inhibitor, such, for example, as potato protease inhibitor II (POT II) or 25 soybean protease inhibitor, along with the peptide agent, may be added to increase the survival of the peptide agent and thus effectiveness in the intestin~ For example, oral 2q-1mini~tration of the peptide horrnone, vaso~-,s~in, accompanied with a protease inhibitor, Trasylol, resulted in sufficient hormone surviving intes~in~l digestion to be absorbed in effective amounts (Franco-Saenz et al., 1979).

Since LCRF is active from the luminal side of the intestin~, it is believed nPcf~s~ry only to deliver it safely to the duodenal lumen; it is not n~cess~ry to f~ ilit~te its absorption. Thus oral ~ ions ~,-vill be preferable in most cases.

Orally ~ ini~t~qred LCRF may be used to stimulate CCK secretion. Should the LCRF be pepsin-sensitive, it may be ~lmini~t~red in enterically protected f~rmul~tions so that it is freed in the small intestin~ I;vely~ it may be ~rlmini~tered with pepsin inhibitors, inhibitors of ~Lolliach acid secretion or antacids of traditional types. LCRF may be made more resistant to digestion by modifying its amino acids, for example, by substituting horno~rginine for arginine or replacing one or both lysines. Because LCRF is trypsin-sensitive, fr~gm~-nts of LCR~ in the vicinity of one of the lysines or the arginine should retain biological cholecystokinin-releasing or other activities. Amino acid modifications or ~ub~Lilulions with whole or fr~gment~cl LCRF are expected to provide more easily prepared and/or digestion-resistant substances.

2.5 LCRF Compositions and Insulin Secretion LCRF compositions are contemplated to be useful for the stimnl~tion of insulin secretion. CCK has been demonstrated to potentiate amino acid-in~ cerl insulin secretion. Therefore, in conditions in which insulin secretion is deficient, such as type I or II diabetes mellitus, CCK may be useful, and therefore a CCK-releasing peptide that is orally active, such as LCRF, will be valuable. In addition, CCK can reduce elevated blood sugar levels after eating a meal by delaying gastric t;lllplymg, and can increase small and large int~stin~l motility. When the above uses for LCR~
are described, it is understood that this may involve LCR~ fragments, derivatives or analogs that retain the desired biological activities.

LCRF is also useful to regulate stomach ~lllplyillg, a condition that has been shown to be associated with some types of diabetes. CCK is well-established as a -W O 97/15671 PCT~US96/17998 physiological regulator of stom~ch c.ll~lying; specifically, CCK inhibits stomach ~ll~lyhlg. Clinical problems with stomach e~ yhlg involve both delayed and accelerated stc-rn~ Gl~l~3ly Ulg. Early stage diabetes of both type I (insulin-dependent) and tvpe II (non-insulin-dependent, or "adult onset"), involve accelerated stomach 5 e~ yillg, which may later change to delayed stomach Glll~Ly;llg when the nervous system is damaged by the tli~e~e. Deficient CCK release has been implicated in accelerated stomach e~ yillg in type II diabetes (Rushakoff et al., 1993). LCRF, as an oral agent that releases CCK, will be useful to overcome this defect in early stage diabetes to slow the progression of the disease. There is a significant need for this 10 application because of the large number of people with type II diabetes, especially as the Hispanic and Asiatic populations of the United States increase, as they are particularly susceptible to type II diabetes, particularly when they adopt a more calorie-dense, wGsl~ type diet.

15 2.6 LCRF Co~.c~ilions and Gallbladder E, .I~Iy;~.g .

LCRF may also be used as part of a trç~tment for gallbladder dise~e, particularly gall~ton~s. The need for such a medication is quite large, especially among women, Hispanic-Americans, native Americans, and people undergoing very 20 low calorie weight loss programs. Gallstones occur with va~ying degrees of frequency in North American populations, depending upon gender, age, diet, socioeconomic status, and ethnicity. The risk is several fold higher in women than men (15~0%
after age 50 in C~ n females), and is increased with obesity. Gallstones occur with dramatic frequency during rapid weight loss, as well as in patients on total 25 parenteral nutrition (IPN). In Hispanic-American females over age 60, the incidence is as high as 44%. The highest reported rate in a defined population is 70% in adult female Pima Indians of the ~mericz~n SUULhW~

Although the cause of gallstone formation is complex, a common thread is 30 believed to be reduced motility of the gallbladder, rPsnltin~ in less frequent and less CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 complete enlL,Lyillg. Even if small g~ll.ctcnes formed, regular and complete emptying would discharge them harmlessly into the duodenum before they got large enough to be clinically relevant. Since cholecystokinin is the maior factor c~ ing the gallbladder, at least some illl~ ed gallbladder c~ lyhlg is due to insufficient release 5 of CCK to completely empty the gallbladder. The enhanced release of CCK, as byorally ~lmini~t~red LCRF, will improve gallbladder t;l~Lyillg in gallstone-pronepeople, reduce the inc~ nce of gallbladder disease and thus the need for costly clinical intervention.

10 2.7 Recombinant LCRF Polypeptides Recombinant versions of a protein or polypeptide are deemed as part of the present invention. Thus one may, using techniques f~mili~r to those skilled in the art, express a recombinant version of the polypeptide in a recombinant cell to obtain the 15 polypeptide from such cells. The techniques are based on cloning of a DNA molecule encoding the polypeptide from a DNA library, that is, on obtaining a specific DNA
molecule distinct from other DNAs. One may, for example, clone a cDNA molecule, or clone genomic DNA. Techniques such as these would also be ~pr~pl;ate for the - production of the mutacin polypeptides in accordance ~,vith the present invention.
2.8 LCRF Genes As known to those of skill in the art, the original source of a recombinant geneor DNA segmPnt to be used in a th~;la~. lic regimen need not be of the same species as 25 the animal to be treated. In this regard, it is contemplated that any recombinant LCRF
gene may be employed in the methods ~i~closecl herein such as the identification of cells ~ C~ g DNA encoding LCRF or v~;~lL~ of LCRF.

Particularly l,l.,f~ d genes are those isolated from hllm~n~ However, since the 30 sequence homology for genes encoding LCRF polypeptides is t;~e~;L~d to be conserved W O 97/15671 PCTnJS96/17998 across species lines, equine, mlTrinP, and bovine species may also be colllclll~lated as sources, in that such genes and DNA segments are readily available, with the human or murine forms of the gene being most pler~ d for use in human trÇ~ttnent regim~nc.
Recombinant proteins and polypeptides encoded by isolated DNA segm~ntc and genesS are often referred to w~th the prefix "r" for recombinant and "rh" for recombinant human. As such, DNA segm~nts encoding rLCRFs, or rLCRF-related genes, etc. are colllc.,lplated to be particularly useful in conn~ctit~n with this invention. Any recombinant LCRF gene would likewise be very useful with the methods of the 3nventl0n.

Isolation of the DNA encoding LCRF polypeptides allows one to use methods well known to those of skill in the art and as herein described to make changes in the codons for specific amino acids such that the codons are ' ~l~r. ~ed usage" codons for a given species. Thus for example, pler.,l.~d codons will vary significantly for b~ctPri~l 15 species as colll~,d with m~mm~ n species; however, there are l,.cr..e.lces even arnong related species. Shown below are ~..,r~ d codon usage tables for rat and human. Isolation of rat DNA encoding LCRF will allow ~ub~Ltulions for ~erc~,d human codons, although ~ .c~ed polypeptide product from human DNA is t;~e-;Lcd to be highly homologous to m~mm~ n LCRF and so would be e~pecte~l to be 20 structurally and functionally equivalent to LCRF isolated from rat.

W O 97/15671 PCT~US96/17998 o o o ~ o~ o oo ~o oo ~ t -- oo o ooo ~ c~ o ~ o o~

o ~ v ¢ c~ ~ v 'c ~ ~ v ~ c~ ~ v ~ c~ ~' v ~¢ ~ ~ ~ g v v¢ ¢ ¢ ¢¢ ¢ ¢ c~

3 3 3 ~ V ~ V V ~ Vv ¢ ~ ~ V

3 ~ ~ V~ V ¢3 ¢3 WO 97/15671 PCTrUS96/17998 r-- ~ cr o c~ ~o o CJ~ U~ 00 00 o o .o _. .__ _ _ _ ~ _ ~V¢~ ~V¢V ~V¢V
.~ ~ VVVV VVVV VV~V
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~
~C ~~ Cd Cr~ ~ ~ -- ~ ~ ~~
o ;~, _ o ~ t~

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V ~ VVVV ¢¢¢~

~ ~ ~ cr~ o ~ e~ ~ o .D ~ t-- ~ ~ ~ O ~ O~
~ ~ t-- O e~ ~ O ~ ~D ~ -- ~ ~D
¢v~v¢v ~v¢v o v v v v v v v v c~ v v v v ~ vvvv ¢¢¢¢

-- ~D O O~ O ~ 1-- ~ ~ ~ ~ C~
~ ~ ~ -- ~ ~-- ~ ~ to-- ~ o o D ~ ~~ O In C~ ~ ~
- ~ ~V¢V~V¢V ~V¢V
~o~ ~ ~ ~ ~ ~ 3 ~
V ~ ~ ~ ;~ V V V V ¢ ¢ ¢ ¢

CA 02238940 l998-04-22 W 0'97/15671 PCT~US96/17998 _19_ X ~ ~ o ~

E-- ~ ~ o O ~ t_ ~D
~ D ~
D ~ ~ ~ _I ~

~ ~ ~ V V :~

O

~ O ~

V ~ V C~ ~ --~ O ~ ~ ~
~ O ~ ~

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~ C~ ~ V
~ V g V
V V V C~ g ~ ~: ~ O~ CJ~ O ~ O

O ~ ~D ~ ~~ V

~ V ~ ) V ~J D

W O 97/15671 PCT~US96/17998 The ~l~finiti~n of a "LCRF gene", as used herein, is a gene that hybrifli7~c, under relatively stringent hyhril1i7~tion conrli~icn~ (see, e.g, Maniatis etaL, 1982), to DNA
sequences pl~s~ ly known to include cytokine gene sequences. The definition of a"CCK-releasing factor gene", as used herein, is a gene that hybridizes, under relatively S stringent hybri~li7~til n conditions to DNA sequences ~l~s~,.lly known to include CCK-releasing factor gene sequences.

To prepare a LCRF gene segrn~nt or cDNA one may follow the tf~rhingc disclosed herein and also the t~rhings of any of patents or scientific docllm~nt.c specifically referenced herein. One may obtain a rLCRF- or ot_er CCK-r~le~ing factor-encoding DNA ~egrnentc using molecular biological techniques, such as polymerase chain reaction (PCRT~ or screening of a cDNA or genomic library, using primers or probes with sequences based on the above nucleotide sequence. Such fr~grn~ntc may be readily ~ ed by, for example, directly synth~ in~ the fr~gm~ntby chemical means, by application of nucleic acid reproduction technology, such as the PCR~M technology of U.S. Patents 4,683,195 and 4,683,202 (herein incorporated byreference). The practice of these techniques is a routine matter for those of skill in the art, as taught in various scientific texts (see e.g, Sambrook et al., 1989), incorporated herein by reference. Certain docurnents further particularly describe suitable rn~mm~ n G~lc;ssion vectors, e.g, U.S. Patent 5,168,050, incol~ol~d herein by ef~ ,ce. The LCRF genes and DNA segrnents that are particularly ~l~r~ ,d for use in certain aspects of the present methods are those encoding LCRF and LCRF-related polypeptides.

~t is also contemplated that one may clone further genes or cDNAs that encode a CCK-releasing factor peptide, protein or polypeptide. The techniques for cloning DNA
molecules, i.e., obt~ining a specific coding sequence from a DNA library that is distinct from other portions of DNA, are well known in the art. This can be achieved by, for example, S~l.~l,l,lg an ,~ liate DNA library which relates to the cloning of a 30 chemokine gene such as LCRF. The screening procedure may be based on the CA 02238940 l998-04-22 W O-97/lS671 PCT~US96/17998 hyhn~i7~tion of oligonucleotide probes, designed from a cnn~i~1er~tion of portions of the amino acid sequence of known DNA sequences encoding related cytokine proteins. The operation of such screening protocols are well known to those of skill in the art and are ~ described in detail in the scientific liLelalule, for example, see Sambrook et al., 1989.

Techniques for introducing ch~ng~ in nucleotide sequences that are designP~l to alter the functional pl~l Lies of the encoded proteins or polypeptides are well known in the art, e.g, U.S. Patent 4,518,584, incorporated herein by reference, which techniques are also described in further detail herein. Such modifications include the deletion, 1() insertion or ~ub~LiLulion of bases, and thus, changes in the amino acid sequence.
Changes may be made to hl;l~,ase the cytokine activity of a protein, to increase its biological stability or half-life, to change its glycosylation pattern, and the like. All such mo-1ific ~tions to the nucleotide sequences are encomp~ed by this invention.

15 2. 8.1 LCl~F-Encoding DNA Sc~

The present invention, in a general and overall sense, also concerns the isolation and char~rt~ri7~tion of a novel gene, Icr which encodes the novel CCK-rele~eing polypeptide, LCRF. A preferred embodiment of the present invention is a purified20 nucleic acid segment that encodes a protein that has at least a partial amino acid sequence in accordance with SEQ ID NO:l. Another embodiment of the present invention is a purified nucleic acid se~n~nt further defined as including a nucleotide sequence in accordance with SEQ ID NO:2.

In a more pl~,r.,.led embodiment the purified nucleic acid segrnent consists e~nti~lly of the nucleotide sequence of SEQ ID NO:2 its complement and the degenerate variants thereof. As used herein, the term "nucleic acid segm~nt" and "DNA
se~n~nt" are used interchangeably and refer to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a "purified" DNA
30 or nucleic acid segn~t-nt as used herein, refers to a DNA segm~nt which c~ s a W O 97/15671 PCTrUS96/17998 LCRF coding se4.l~nce yet is isolated away from, or purified free from, total genomic DNA, for example, total cDNA or human genomic DNA. Tn~ decl within the term "DNA se~ e..l", are DNA segmPnt~ and smaller fri~_m~nt~ of such se_m~-nt~, and also recombinant vectors, including, for example, pk~micls, cosmi-le, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified Icr gene refers to aDNA sepm~nt inc,lllfling LCRF coding sequences isolated substantially away from other naturally occllrrinp- genes or protein encoding sequences. ln this respect, the term 10 "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be lmcl~nstood by those in the art, this fimctional term includes both genomic sequences, cDNA sequences or combin~tion~ thereof. "Isolated subst~nti~Tly away from other coding sequences" means that the gene of interest, in this case Icr, forms the significant part of the coding region of the DNA segm~nt, and that 15 the DNA segment does not contain large portions of naturally-occ~rring coding DNA, such as large chromosomal friqpm~nt~ or other functional genes or cDNA coding regions. Of course, this refers to the DNA segmt?nt as originally isolated, and does not exclude genes or coding regions later added to the segrnent by the hand of man.

In particular embo(lim~nt~, the invention concerns isolated DNA segm~ntc and recombinant vectors incorporating DNA sequences which encode a Icr gene, that inclll(les within its amino acid sequence an atnino acid sequence in accordance with SEQ ID NO:l. Moreover, in other particular embo~im~ntc~ the invention concerns isolated DNA segm~nt~ and recombinant vectors incol~old~ g DNA sequences which 2~ encode a gene that includes within its amino acid sequence the amino acid sequence of a Icr gene corresponding to murine Icr.

Another ~l~ r~ d embodiment of the present invention is a putified nucleic acid segmPnt that encodes a protein in accordance with SEQ ID NO:l, filrther defined as a recombinant vector. As used herein the term, "recombinant vector", refers to a vector W 0 97/15671 PCT~US96/17998 that has been modified to contain a nucleic acid segmPnt that encodes a LC~F protein, or a ~gment thereof. The recombinant vector may be further defined as an 1A~les:jion vector cnmpri~ing a promoter operatively linked to said LCRF-encoding nucleic acid segment A further ~leI~,lt;d embodiment of the present invention is a host cell, made recombinant with a recombinant vector comprising a Icr gene. The recombinant host cell may be a prokaryotic cell. In a more ~lt;r~ d embo~lim~nt the recombinant host cell is a ~k~yulic cell. As used herein, the term "engin~ered" or "recombinant" cell is 10 int.on~lP~1 to refer to a cell into which a recombinant gene, such as a gene encoding LCRF, has been introduced. Therefore, engin~oPred cells are distinguishable fromnaturally occ-lrring cells which do not contain a recoml~ lly introduced gene.
Fngine-red cells are thus cells having a gene or genes introduced through the hand of man. Recombh~lly introduced genes will either be in the form of a cDNA gene (i.e., 15 they will not contain introns), a copy of a genomic gene, or will include genes positioned ~dj~nt to a promoter not naturally associated with the particular introduced gene.

- Generally spe~king, it may be more convenient to employ as the recombinant 20 gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will g~n~r~lly be much smaller and more readily employed to tr~n~f~ct the targeted cell than will a genomic gene, which will typically be up to an order of m~gnitllrle larger than the cDNA gene. However, the UlVt;ll~Ul::i do not exclude the possibility of employing a genomic version of a particular 25 gene where desired.

In certain embo-limPnts, the invention c-nc~m~ isolated DNA segTnent.c and recombinant vectors which encode a protein or peptide that inrllldes within its amino acid sequence an amino acid se~uc;llce ~o~s~nti~lly ~ set forth in SEQ ID NO:l.
30 Naturally, where the DNA segTn~-nt or vector encodes a full length LCRF protein, or is W O 97/15671 PCT~US96/17998 int~nfl~:<l for use in t;~ s~ g the LCRF protein, the most preferred sequences are those which are ~c~nti~lly as set forth in SEQ ID NO:l. It is recognized that SEQ ID NO:1 ~resenl~ 41 of the 63-70 or so amino acids of the full length protein encoded by the Icr gene and that contemplated embo-lim~nt.c include up to the full length sequence and 5 functional variants as well.

The term "a sequence e~s~nti~lly as set forth in SEQ ID NO:l" means that the sequence ~ul~ ially corresponds to a portion of SEQ ID NO: 1 and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the 10 amino acids of SEQ ID NO:l. The term "biologically fimrtion~l equivalent" is well n~ nctood in the art and is further defined in detail herein, as a gene having a sequence ~Pntiztlly as set forth in SEQ ID NO:l, and that is associated with a cf ~.~ti~ ely-produced CCK-releasing factor in the LCRF family. Accordingly, sequences which have bet~,veen about 70% and about 8û%, or more preferably, between about 81% and 15about 90%; or even more preferably, bt;tw~t;ll about 91% and about 99%; of amino acids which are ~ ntic~1 or functionally equivalent to the atmino acids of SEQ ID NO: 1 will be sequences which are "ç~Pnti~lly as set forth in S~Q ID NO:l"

- In certain other embo~liment~, the invention concerns isolated DNA segmPnt.c 20 and recomhin~nt vectors that include within their sequence a nucleic acid sequence çss~nti~lly as set forth in SEQ ID NO:2. The term "PssPnti~lly as set forth in SEQ ID
NO:2," is used in the sarne sense as described above and means that the nucleic acid sequence s~lh.~t~nti~lly cc,l~ .,llds to a portion of SEQ ID NO:2, and has relatively few codons which are not itlPnti~l, or functionally equivalent, to the codons of SEQ ID
25 NO:2. The term "functionally equivalent codon" is used herein to refer to codons that encode the same arnino acid, such as the six codons for arginine or serine, as set forth in Table 1, and also refers to codons that encode biologically equivalent amino acids.

It will also be llntlç~tQod that amino acid and nucleic acid sequences may 30 include ~ lition~l rç~ , such as additional N- or C-t~min~l arnino acids or S' or 3' W O 97/15671 PCTAJS96/}7998 sequences, and yet still be çee~Mti~lly as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, in~ ing the m~ t~ ce of biological protein activity where protein t;x~les~ion is con~ ernlod The addition of t~rrnin~l se~luellces particularly applies to nucleic acid sequences which may, 5 for example, include various non-coding sequences fl~nkin~ either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

Excepting intronic or fl~nking regions, and allowing for the degeneracy of the 10 genetic code, sequences which have b~lwt;ell about 70% and about 80%; or morepreferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99%; of nucleotides which are i~lçntie~l to the nucleotides of SEQ ID
NO:2 will be sequences which are "ees~nti~lly as set forth in SEQ ID NO:2". Sequences which are ees~nti~lly the same as those set forth in SEQ ID NO:2 may also be 15 filnl~tiC)n~lly defined as sequences which are capable of hybridizing to a nucleic acid segm~nt cc-, ~ g the cQmplement of SEQ ID NO:2 under relatively strin~ent conditions. Suitable relatively stnn~ent hybritli7~tion contiitiQne will be well known to those of skill in the art and are clearly set forth herein, for example conditions for use - with Southern and Northern blot analysis, and as described in Example herein set forth.
Naturally, the present invention also ~;,.co...~ ee DNA segmente which are complement~ry, or ~c.ePnti~lly complem~nt~ry, to the sequence set forth in SEQ ID
NO:2. Nucleic acid sequences which are "compltom~nt~ry" are those which are capable of base-pairing according to the standard Watson-Crick complement~rity rules. As used 25 herein, the term "complement~ry sequences" means nucleic acid sequences which are s~lbst~nti~lly complem~nt~ry, as may be ~eeçeee~l by the same nucleotide comparison set forth above, or as defined as being capable of hybridi_ing to the nucleic acid segment of SEQ ID NO:2 under relatively stringent con-liti- ne W O 97/15671 PCT~US96/17998 The nucleic acid se~nents of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, ~Mition~l restriction c~l~yl.le sites, multiple cloning sites, other coding segmente, and the like, such that their overall length may vary S consi-lerAhly. It is therefore co~ tecl that a nucleic acid fragment of almost any length may be employed, with the total leng~ preferably being limited by the ease of prep~r~tic~n and use in the int~nfierl recombinant DNA protocol. For example, nucleic acid fr~grn~nt.s may be ~r~d which include a sho-rt stretch comrle~ . y to SEQ ID
NO:2, such as about 10 to 15 or 20, 30, or 40 or so nucleotides, and which are up to 200 10or so base pairs in length. DNA se~nPnt~ with total leng~s of about 500, 200, 100 and about 50 base pairs in length are also cont~mrl~t~l to be useful.

A ~ f~ d embodiment of the present invention is a nucleic acid segment which comrri~es at least a 14-nucleotide long stretch which col-~onds to, or is 15complement~ty to, the nucleic acid sequence of SEQ ID NO:2. In a more ~lerel.c,d embodiment the nucleic acid is fur~er defined as comrn~ing at least a 20 nucleotide long stretch, a 30 nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide long stretch, or at least an 200 nucleotide long stretch which CO~l~ ~onds to, or is complem~nt~ry to, the nucleic acid sequence of SEQ ID NO:2. The nucleic acid 20se~ nt may be further defined as having the nucleic acid sequence of SEQ ID NO:2.

An related embodiment of the present invention is a nucleic acid segrnent which comprises at least a 14-nucleotide long stretch which corresponds to, or is complemen~ly to, the nucleic acid sequence of SEQ ID NO:2, further defined as 25cf~mpri~ing a nucleic acid ~grnent of up to 10,000 basepairs in length. A moreer~ d embodiment if a nucleic acid fi~gment comprising from 14 nucleotides of SEQ ID NO:2 up to 5,000 bzl~e~n;l ~; in length, 3,000 b~ep~irs in length, 1,000 basepairs in length, 500 b~eeE~ir.s in leng~, or l O0 base~ in leng~.

W O 97/15671 PCT~US96/17998 Naturally, it will also be llnrler~tQod that this invention is not limited to the particular nucleic acid and amino acid se-luences of SEQ ID NOS:2 and 1.
~ecombinant vectors and isolated DNA segmPntx may thelcr~ variously include the LCRF coding regions themselves, coding regions bearing selected alterations or S modifications in the basic coding region, or they may encode larger polypeptides which nevertheless include LCRF-coding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acids sequences.

The DNA segmPnt~ of the present invention ~ nromp~x biologically functional 10 equivalent LCRF proteins and peptides. Such sequences may arise as a consequence of codon redl~ntl~n~y and functional equivalency which are known to occur n~tllr~lly within nucleic acid sequences and the proteins thus encoded. ~ltPrn~tively, functionally equivalent p~tGillS or peptides may be created via the application of recombinant DNA
technology, in which ~~h~ngPs in the protein ~I1U~;lU1G may be PnginPpred~ based on 15 considerations of the ~lVpGl ~ies of the amino acids being ç~h~nged. Ch~np~ec tie~i nPd by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the ~nti~enicity of the LCRF protein or to test LCRF ~ x in order to P~minP activity or detrrminP the presence of LCRF
- peptide in various cells and tissues at the molecular level.
A ~l~rG~Gd embodiment of the present invention is a purified (;;olll~o~ilion c~-mpri~in~ a polypeptide having an amino acid sequence in accordance with SEQ ID
NO:l. The term "purified" as used herein, is intPn~le~l to refer to a LCRF protein composition, wherein the LCRF protein is purified to any degree relative to its naturally-25 obtainable state, i.e., in this case, relative to its purity within a eukaryotic cell extract. A~lGr~lled cell for the isolation of LCRF protein is a pancreas or ;.~le~ l villi cell, however, LCRF protein may also be isolated from patient specimens, recombinant cells, tissues, isolated subpopulations of tissues, and the like, as will be known to those of skill in the art, in light of the present disclosure. A purified LCRF protein composition CA 02238940 l998-04-22 WO 97/15671 PCTrUS96/17998 therefore also refers to a polypeptide having the amino acid sequence of SEQ ID NO:l, free from the e~ ilolllllent in which it may naturally occur.

If desired, one may also prepare fusion proteins and peptides, e.g, where the 5 LCRF coding regions are aligned within the same c A~,. s.,ion unit with other proteins or peptides having desired functions, such as for pllrifi~tinn or immllnndetection pu-~oses (e.g., proteins which may be purified by affinity chromatography and enzyme label coding regions, respectively).

Turning to the GA~lGs~ion ofthe Icr gene whether from cDNA based or genornic DNA, one may proceed to prepare an CA~lG ,~ion system for the recombinant ~lc~dldlion of LCRF protein. The Pn~ ef ~ g of DNA se~ r~,l(s~ for eA~Gssion in a prokaryotic or eukaryotic system may be pclrolllled by techniques generally known to those of skill in recombinant ~A~lcssion. For exarnple, one may prepare a LCRF-GST (gltlt~thic-n~-S-15 I Ldll 7r~ ,dse) fusion protein that is a convenient means of bacterial GA~lession. However, it is believed that virtually any GA~l~ssion system may be employed in the GA~ iion of LCRF.

LCRF may be s~lcceccfillly expressed in eukaryotic GA~ies~ion systems, 20 however, the illVc~ i contt-n~rl~te that b~-~tPri~l ~A~l~7ion systems may be used for the plG~ n of LCRF for all ~ oSGs. The cDNA co~ g Icr gene may be s~d~Gly ~_A~,~ssed in bRrtPri~l systems, with the encoded ~lvlGills being ~AL,lessed as fusionc with ~3-galactosidase, avidin, ubiquitin, Schistosoma japonicum glutathione S-LL~dse, multiple hi~ti~lin~s, epitope-tags and the like. It is believed that b~ctPriz~l 25 G~les~ion will l-ltim~tely have advantages over eukaryotic GA~lG~ion in terms of ease of use and 4U~l~i~y of m~t-on~l ~ obtained thereby.

It is proposed that transformation of host cells with DNA segm~nt~ encoding LCRF will provide a convenient means for obtair~ing an LCRF protein. It is also 30 proposed that cDNA, genomic sequences, and combinations thereof, are suitable for =
CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 eukaryotic t;A~res~ion, as the host cell will, of course, process the genomic tr~n~cnI~ts to yield functional mRNA for translation into protein.

~ Another embodiment is a method of pl~ ll~g a protein composition c~-r ~ i . .g 5 growing recombinant host cell comprising a vector that encodes a protein whichinchl(les an amino acid sequence in accordance with SEQ ID NO:l, under conditions p~ ;llg nucleic acid ~;A~ ion and protein production followed by recovering the protein so produ~,ed. The host cell, con~1itiQns p ~ g nucleic acid tA~res~ion, protein production and recovery, will be known to those of skill in the art, in light of the 10 present disclosure of the Icr gene.

2.8.2 Gene Constructs and DNA Se~ t~

As used herein, the terms "gene" and "DNA segment" are both used to refer to a 15 DNA molecule that has been isolated free of total genomic DNA of a particular species.
Therefore, a gene or DNA segment encoding a LCRF polypeptide refers to a DNA
segment that contains sequences encoding a LCRF protein, but is isolated away from, or punfied free from, total genomic DNA of the species from which the DNA is obtained.
- Included within the term "DNA segment", are DNA segm~nt~ and smaller fr:~gment~ of 20 such segrnPnt~, and also recombinant vectors, inclllding, for example, pl~emi~
cosmids, phage, retroviruses, adenoviruses, and the like.

The term "gene" is used for simplicity to refer to a functional protein or peptide encoding unit. As will be understood by those in the art, this functional term includes 25 both genomic sequences and cDNA sequences. "Isolated sllhst~nti~lly away from other coding sequences" means that the gene of interest, in this case, a CCK-rele~cing factor gene, forms the siPnific~nt part of the coding region of the DNA segment and that the DNA scgment does not contain large portions of naturally-occ~lrring coding DNA, such as large chromosom~ nPnt~ or other functional genes or cDNA coding regions. Of 30 course, this refers to the DNA se~nent as originally isolated, and does not exclude W O 97/15671 PCTrUS96/17998 genes or coding regions, such as sequences encoding leader peptides or targetingsequences, later added to the segmPllt by the hand of man.

2.8.3 Recombfnant Ve~tors E~ , LCRF

A particular aspect of this invention provides novel ways in which to utilize LCRF-encoding DNA se~, . .~. .1.~; and recombinant vectors compri~ing Icr DNA
segment~ As is well known to those of skill in the art, many such vectors are readily available, one particular ~l~t~ (l exarnple of a suitable vector for cx~l~s~ion in 10m~mm~ n cells is that described in U. S. Patent 5,168,050, incolpol~tcd herein by reference. However, there is no ~ lent that a highly purified vector be used, so long as the coding segment employed encodes a LCRF protein and does not include any coding or reg~ tory sequences that would have an adverse effect on cells. TllerGrv.c, it will also be understood that useful nucleic acid sequences may include additional 15 residues, such as additional non-coding sequences fl~nking either of the S' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

- After identifying an ~plvpl;ate LCRF-encoding gene or DNA molecule, it may 20 be inserted into any one of the many vectors ~;ull~ltly known in the art, so that it will direct the e,~ ion and production of the LCRF protein when incv,~ cd into a hostcell. In a recombinant c~ ion vector, the coding portion of the DNA segment is positioned under the control of a promoter. The promoter may be in the form of the promoter which is naturally ~oçi~tP-l with a LCRF-encoding gene, as may be obtained 25 by isolating the 5' non-coding sequences located U~LIC~U11 of the coding segment or exon, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions ~ losecl herein.

In certain embo-limPnt~, it is co~ tp-t1 that particular advantages will be 30 gained by positioning the LCRF-encoding DNA se~"~ l under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is int~n-le~l to refer to a promoter that is not nf~rm~lly associated with a Icr gene in its natural environrnent. Such promoters may include those n~ lly associated - with other CCK-releasing polypeptide genes, and/or promoters i~ol~te~1 from any other S ~cter~ viral, eukaryotic, or m~mm~ n cell. Naturally, it will be i~ ulL~lL to employ a promoter that effectively directs the ~2~ules~ion of the DNA segm~nt in the particular cell co.~ .g the vector comr~i~ing the LCRF gene.

T~e use of recombinant promoters to achieve protein ~,u~ ion is generally known to those of skill in the art of molecular biology, for ~mp!e, see Sambrook et al., (1989). The promoters employed may be col~Li~ulive, or inducible, and can be used under the a~plupl~ate conditions to direct high level or regulated ~ ion of the introduced DNA segment The ~ ly ~.er~l.,d promoters are those such as CMV, RSV Ll~, the SV40 promoter alone, and the SV40 promoter in combination with the SV40 ~nh~nr~r 2.9 Methods of DNA Transfection Technology for introduction of DNA into cells is well-known to those of skill inthe art. Four general methods for delivering a gene into cells have been described: (1) chemical methods (C~raham and VanDerEb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Nel-m~nn, 1982; Frommet al., 1985) and the gene gun (Yang et al., 1990); (3) viral vectors (Clapp, 1993; Danos and Heard, 1992; Eglitis and Anderson, 1988); and (4) ~~;c~ol-m~ l mech~ni~m~
(Wu et al., 1991; Curiel et al., 1991; Wagner et al., 1992).

2.9.1 Liposomes and Nanor~ps~ s The formation and use of liposomes is generally known to those of skill in ~e 30 art (see for example, Couvreur et al., 1991 which ~iesrribçs the use of liposomes and WO.97/15671 PCT~US96/17998 nanoe~7rsllles in the targeted antibiotic therapy of intracellular ba(t~77Al infections and t1i~e~es). Recently, liposomes ~,vere developed with i~ lov~d serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).
The following is a brief description of these DNA delivery modes.

N~7nncApsl71es can generally entrap compounds in a stable and reproducible way (Henry-Mit~he11Anr7. etal., 1987). To avoid side effects due to intracellular polymeric overloading, such ~ e particles (sized around 0.1 mm) should be decigne~7. using polymers able to be degraded in vivo. Bio~legrA(lAl~le polyalkyl-cyanoacrylate 10 nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur ef al., 1984, 1988).

Liposomes are formed from phospholipids that are rlispersef7 in an aqueous 15 medium and :,l,onL~eously form mllltilAmell~r concentric bilayer vesicles (also termed mllltilAmellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4mm. Sonication of MLVs results in the ft rmAtion of small llnilAmt-llAr vesicles (SWs) withrliAmr-,tt-,rsintherangeof200to500A,c."llAill;llganaqueoussolutioninthecore.
In addition to the teAt~hin~s of Couvreur et al. (1991), the following information may be utilized in generating liposomal ft rmlllAtions. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the pl~rt;.l~d structure. The physical l~h~r~tf~ri~tics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated L~ ldlwes undergo a phase transition which mArk~rlly alters their pçrrn~Ahility. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered ~ ;Lule, known as the fluid state. This occurs at a c1~<1~-t~ ;c phase-trAn~itit)n L~l~c~alule and results in an increase in permeability to ions, sugars and drugs.

W O 97/15671 PCT~US96/17998 Liposomes interact with cells via four dirr~ L mf~.~h~ni~mc Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils;
adsorption to the cell s~ rç either by nonspecific weak hydrophobic or electrostatic S forces, or by specific int~r~ctions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with cimlllt~neous release of liposomal contents into the cytoplasm; and by L.d.~r~l of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome c~ L~illL~. It often is difficult to tlet~rmine which 10 me-~h~ni~m is operative and more than one may operate at the same time.

2.10 Expression of LCRF

For ~e ~x~.es~ion of LCRF, once a suitable (full-length if desired) clone or 15 clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an ~ e;,~ion system for the recombinant ~r~Lion of LCRF. The engin~çrin~ of DNA segment(s) for t;~ ssion in a prokaryotic or ~ ~yolic system may be performed by techniques generally known to those of skill in recombinant - e2,~l~ssion. It is believed that virtually any ~re;,~ion system may be employed in the 20 ~ res~ion of LCRF.

LCRF may be successfully t;~ ;ssed in ~uk~,yoLic t;x~ ion systems, however, it is also envisioned that bacterial ~lei,~ion systems may be ~l~;rell~;d for the a-dLion of LCRF for all pu~poses. The cDNA for LCRF may be sepa a~ly 25 ~ essed in b~t~ri~l systems, with the encoded proteins being c;~lc;ss~d as fusions with b-g~l~ctocidase, ubiquitin, Schistosoma japonicum glutathione S-lldll~L,ldse, green ffuorescent protein and the like. It is believed that b~cteri~ s~ion will llltim~t~ly have advantages over eukaryotic ~ ion in terms of ease of use and ~ liLy of m~tPri~1~ obtained thereby.

W O 97/15671 PCT~US96/17998 It is proposed that tran~r~ lion of host cells with DNA segmPnt~ encoding LCRF will provide a convenient means for obtaining LCRE~ peptide. Both cDNA and genomic se~llle~ces are suitable for euk~lL.yolic ex~ sion~ as the host cell will, of course, process the genomic tr~n~cripts to yield functional mRNA for translation into 5 protein.

It is similarly believed that almost any eukaryotic eA~lc:~iOn system may be utilized for the cA~Iession of LCRF, e.g, baculovirus-based, ~,l-.lii...;..~ synthase-based or dihydrofolate reductase-based systems could be employed. However, in plercL.~;d 10 embo-1imt~nt~, it is colllelll~lated that plasmid vectors illco~,ol~ g an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMVS, will be of most use.

For ex~l~ssiOn in this m~nn~r, one would position the coding sequences s~ rPnt 15 to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the S' end of the ll~s~ ion initiation site of the tr~n~rnrtional reading frame of the protein between about 1 and about 50 nucleotides "d~wl~ d~ of (i.e., 3' of) the chosen promoter.
-Where eukaryotic ex~lei~ion is cont~mplated, one will also typically desire to incorporate into the ~ scl;~Lional unit which in~lu~c LCRF, an a~ liate polyadenylation site (e.g, 5'-AATAAA-3') if one was not c.~ (1 within the original cloned segmPnt Typically, the poly A addition site is placed about 30 to 2000 nucleotides "dov~-lsL~ull" of the termin~tion site of the protein at a position prior to ll~ulscl;~lion ~ ?n.

Tr~n~l~ti(>n~l ~nh~nrf-rs may also be inc~ oldl~d as palt of the vector DNA.
Thus the DNA constructs of the present invention should also preferable contain one or more S' non-tr~n~l~tecl leader sequences which may serve to ~nh~nce ~A~les~ion of the 30 gene products from the rcsl-ltin~ rnRNA 1-,~ . Such sequences may be derived CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 from the promoter selected to express the gene or can be specifically modified to increase translation of the RNA. Such regions may also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence (~lriffith~ et al, 1993).
Such "enh~n-~Pr" sequences may be desirable to increase or alter the translational efficiency of the l~ull~ll mRNA. The present invention is not limited to constructs where the enh~n-~Pr is derived from the native 5'-nontr~nCl~te~ promoter sequence, but may also include non-tr~n~l~te~l leader scyuences derived from other non-related10 promoters such as other enhancer L-~lscliplional activators or genes.

It is cnntPmI l~ted that virtually any of the commonly employed host cells can be used in connection with the cA~.cs:jion of LCRFg in accordance herewith. F~mple~include cell lines typically employed for cuk~uyulic cx~ ion such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.

It is colll~;;lllplated that LCRF may be "o~ r~ ed", i.e., t;~lessed in increased levels re}a~ive to its natural ~x~les~ion in hurnan cells, or even relative to the ex~les~ion of other proteins in a recombinant host cell c~ g LCRF-encoding DNA
20 segn~nt~ Such u~- Ic~ ion may be ~q~sP~s~cA by a variety of methods, including radio-labeling and/or protein pl~rifi~tion. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein st~inin~ or Westernblotting, followed by ~ re analyses, such as dt;~ leLL;c sc~nning of the resultant gel or blot. A specific increase in the level of the recombinant protein or 25 peptide in c~,lll~ison to the level in natural LCRF-producing animal cells is indicative of o~ A~lession, as is a relative ablln~l~nre of the specific protein in relation to the other proteins produced by the host cell and, e.g, visible on a gel.

As used herein, the term "~nginPPred" or "recombinant" cell is intPntle~l to refer 30 to a cell into which a recombinant gene, such as a gene encoding a LCRF peptide has WO 97/15671 PCT~US96/17998 been introduced. T11e1G~1~G~ l ngine~red cells are distinguishable from n~t~ llyoCcllrrinf~ cells which do not contain a recomlfmcillLly introduced gene. Fngineered cells are thus cells having a gene or genes introduced through the hand of man.
Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned cf~nt to a promoter not naturally associated with the particular introduced gene.

It will be understood that recombinant LCRF may differ from naturally produced LCRF in certain ways. In particular, the degree of post-tr~n~l~tional 10 modifications, such as, for example, glycosylation and phosphoIylation may bedifferent between the recombinant LCRF and the LCRF polypeptide purified from a natural source, such as int~stin~l secretions Generally ~pe~kin~, it may be more convenient to employ as the recombinant 15 gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the si~e of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of m~nihl~le larger than the cDNA gene. However, the- inventors do not exclude the possibility of employing a genomic version of a particular 20 gene where desired.

After identifying an a~p~ iate DNA molecule by any or a combination of means as described above, the DNA may then be inserted into any one of the many vectors CU11G1I~1Y known in the art and transferred to a prokaryotic or eukaryotic host 25 cell where it will direct the expression and production of the so-called "recombinant"
version of the protein. The recombinant host cell may be selected from a group con~ictin~ of S. mutans, E. coli, S. cerevisae. BaciUus sp., Lactococci sp., Enterococci sp., or Salmonella sp. In certain preferred embo~lim~nt~, the recombinant host cell will have a recA phenotype.

W O 97/15671 PCT~US96/17998 Where the introduction of a recombinant version of one or more of the foregoing genes is required, it will be i~lly~3ll~ll to introduce the gene such that it is under the control of a promoter that effectively directs the Gxyres~ion of the gene in the cell type chosen for Pn~inpering. In general, one will desire to employ a promoter that allows 5 c~ ve (constant) t;xyl~s~ion of the gene of interest. Commonly used con~ ivG
promoters are generally viral in origin, and include the cytomegalovirus (CMV) promoter, the Rous sarcoma long-t~ n~l repeat (LTR) sequence, and the SV40 earlygene promoter. The use of these co~ ive promoters will ensure a high, consL~ll level of G~lGs~ion of the introduced genes. The level of G~ G~ion from the introduced 10 genes of interest can vary in diL~lellt clones, probably as a function of the site of insertion of the recombinant gene in the chromosomal DNA. Thus, the level of lG~ion of a particular recombinant gene can be chosen by evS~ ting di~ ll clonesderived from each llcu~re~;Lion ~ Pnt once that line is chosen, the CO~ I;VG
promoter ensures that the desired level of G~ ion is pk....~ ..Lly ...Z~ c(l It may 15 also be possible to use promoters that are specific for cell type used for en~ lhlg, such as the insulin promoter in insll~inom~ cell lines, or the prolactin or growth hormone promoters in anterior ~iLuiL~y cell lines.

2.10.1 Enhanced Production of LCRF
One of the problems with LCRF isolated from natural sources is low yields and extensive pllri~ir~tion processes. An aspect of the present invention is the Pnh~n(~e~i production of LCRF by recombinant methodologies in a b~t~ host, employing DNA
constructs to transforrn Gram-positive or Grarn-negative b~cteri~l cells. For example, 25 the use of Escherichia coli ~Lession systerns are well known to those of skill in the art, as is the use of other b~ct~ri~1 species such as Rn~i771~ subtilis or Streptococcus sanguis.

Fur~er aspects of the invention include high cA~,e~sion vectors incorporating DNA encoding the novel LCRF and its variants. It is co~ lllplated that vectors 30 providing ~rlh~n~ed c..~rei,~ion of LCRF in other ~y~ lls such as S. mutans will also be CA 02238940 l998-04-22 W O 97/lS671 PCT~US96/17998 obt~in~hle Where it is desirable, modifications of the physical properties of LCRF may be sought to increase its solubility or c;~ s~ion in liquid culture. The Icr locus may be placed under control of a high exples~ion promoter or the components of the c;~ ession system altered to ~nh~nt~e expression.

In further embor1im~nt~, the DNA encoding the LCRF of the present invention allows for the large scale production and isolation of the LCRF polypeptide. This can be accomplished by directing the ~ ion of the mutacin polhpeptide by cloning the DNA encoding the LCRF polypeptide into a suitable t;~ ;ssion vector. Such an10 e~ ssion vector may then be (~ rc l,--ed into a host cell that is able to produce the LCRF protein. The LCRF protein may then be purified, e.g., by means provided forin this disclosure and utilized in a biologically active form. Non-biologically active recombinant LCRF may also have utility, e.g, as an immlmogen to prepare anti-LCRF antibodies.
2.10.3 Cloning of LCRF Gene . .
In still another embodiment, the present disclosure provides methods for - cloning the DNA encoding the LCRF polypeptide. Using methods well known to 2() those of skill in the art, the DNA that encodes the purified LCRF of the present invention may be isolated and purified. For example, by ~eci~ning a degenerate oligonucleotide compri~ing nucleotides compl~ment~ry to the DNA encoding sequence of SEQ ID NO: 1, the LCRF-encoding DNA can be cloned from a pancreas cell library.
The DNA sequences disclosed by the invention allow for the ~ ud~ion of relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to a gene encoding the LCRF polypeptide. Such a gene, is here termed the Icr gene and is understood to mean the gene locus encoding the LCRF structural gene.
30 In these aspects, nucleic acid probes of an al~p~ ;ate length are pl'~.,d. Such W O 97/15671 PCT~US96/17998 probes are typically prespred based on the con~i~er~tion of the defined amino acid sequence of purified LCRF. The ability of such nucleic acid probes to specifically hybridize to Icr gene sequences lend them particular utility in a variety of embo-liment~ For example, the probes may be used in a variety of diagnostic assays S for detecting the presence of Icr genes in intestin~l mucosal samples; however, other uses are envisioned, inchl~1ing id~ ntific~tion of Icr gene sequences encoding similar or mutant polhpeptides related to the muLacill. Other uses include the use of mutant species primers, or primers to prepare other genetic constructs 10A first step in such cloning procedures is the screening of an ~plop.;ate DNA
library, such as, in the present case, genomic or cDNA prepared from an applopliate cell library; for example, pancreas cell. The screening procedure may be an ession screening protocol employing antibodies directed against the protein, or activity assays. ~ ivt;1y~ screening may be based on the hybrit1i7~ti~ n of 1~ oligonucleotide probes, designed from a con~i(ler~tion of portions of the amino acid sequence of the protein, or from the DNA sequences of genes encoding related proteins. Another cloning approach contemplated to be particularly suitable is the use of a probe or primer directed to a gene known to be generally associated with, e.g, - within the same operon as, the structural gene that one desires to clone. For ey~mp!e, 20 in the case of LC3~F, one may wish to use a primer directed to any conserved regions known to be associated with CCK releasing genes.

Another approach toward identifying the gene(s) responsible for the production of LCRF is tolocate genes known to be ~(1jacent to related CCK releasing 25 factor genes. From sequenced loci in genes that encode other CCK rele~ing peptides, it will be possible to ~letermine if several processing and export enzymes are highly conserved among the lantiblotic producers and share areas of common sequences. Aseries of oligonucleotide primers compl~ Pnt~ry to conserved sequences could be used in PCRTM reactons to amplify the intervening sequence, this amplicon could be 30 used as a probe to identify putative kansporter genes. PCRTM technology is described WO 97/15671 PCT~US96/17998 in U.S. Patent No. 4,603,102, incorporated herein by reference. Where such a transporter gene is found to be part of every known CCK releasing peptide gene, the structural gene for LCRF should be nearby and readily identified by a technique known as "chromosome walking".
3.0 Brief Des~ ,lion of the Dr.~w ~

FIG. 1. Effect of intr~ ln~l~n~l infusion of partially purified intçstin~l LCRF
on pancreatic protein and fluid secretion and on plasma CCK levels (insert). The10 bioactivity of LCRF is blocked by the CCK receptor antagonist, M K329.
*Significantly different from NaCl or M K-329 groups (n = 6, unpaired t-test).
**Significantly di~lcnL from NaCl group (insert, n = 6, u~ d t-test).

FIG. 2. Purification of LCRF by reverse phase high ~ies~ulc liquid 15 cl..ulll~tography (HPLC).

FIG. 3. High ~rullll~ce capillary electrophoresis (HPCE) of HPLC-purified LCRF.

FIG. 4. Effect of an intraduodenal infusion of pure intestin~l LCRF on pancreatic protein and fluid secretion. ~Significantly different from NaCl and 1 mg groups. tSignifir~ntly dirr~le~lL from NaCl group (unpaired t-test) FIG. 5. Effect of immuno~ iLy chromatography using a LCRFI 6 antiserum on LCRF bioactivity of partially purified LCRF.

FIG. 6. Changes in pancreatic protein and fluid secretion after an intraduodenal injection of purified LCRF or Monitor Peptide ~MP). * denotes significantly dirrc~cl~L from 9 dose for LCRF. ~ denotes ~i~nific~ntly different from 9 dose for MP.

W O 97/15671 PCT~US96/17998 FIG. 7. Dose-response relationship between intraduodenal LCRFl 35 and pancreatic secretion. Each point represents 6-8 ~pc;~ ent~ with the dose indicated, using the bioassay rat model (see text). *denotes significantly di~l~;nL from zero 5 dose for LCRF.

FIG. 8. Comparison between intraduodenal (i.d.) vs. hlLIdv~;llous (i.v.) infusion of LCRFI 3s. Results for upper panel are from the same ~ e~;lllcnt illuskated in FIG. 2. * denotes significant dir{~lence from zero dose.
FIG. 9. Changes in pancreatic protein and fluid secretion after an intraduodenal injection of various subfr~gment~ of LCRFl 3s. * denotes significantly different from zero dose. The only subfragment with significant with ~i~nific~ntbiological activity was LCRFll 25.
FIG. 10. Changes in pancreatic protein and fluid secretion after an 't intraduodenal injection of rat Diazepam Binding Inhibitor DBII 86 or ODN peptide DBI33 so. * denotes significantly diLrel.,~ll from zero dose.
-FIG. 11. Effect of CCK-receptor blockade with MK329 on LCRFl 3s-stim~ tecl pancreatic protein (upper panel) and fluid (lower panel) secretion during return of pancreatic juice to the intestine ("Physiological model"). At the arrow, LCRFl 3s was infused intraduodenally at 25 ~g/hour for 2 hours during the return of 10% of the secreted pancreatic juice to the duodenum. MK329 was infused at 0.5 mg/hour i.v. starting one hour before first basal collection. * denotes significantly different from basal.
-FIG. 12. IncrPment~l protein and fluid output in ~ ents described in legend of FIG. 6. Results demonstrate the stim~ tion of pancreatic protein and fluid W O 97/15671 PCT~US96/17998 secretion by LCRFI 3s is abolished by the CCK-receptor antagonist MK325. *
denotes significantly ~lirf~r~llL compared to NaCl and LCRFI 3s + MK329.

FIG. 13. Plasma CCK concentrations in blood samples taken 60 rnimltes after 5 start of infusion of test compounds in e~ ;lllent described in legend of FIG. 9, with the addition of studies with LCRFl~.

FIG. 14. Effect of trypsin digestion of LCRFI 35 on its CCK-releasing activity.
LCRFl 3s was inc~lb~t~l with purified bovine trypsin (1 mg/ml) at 37~ C for 24 hours.
10 Control LCRF was incubated under the same conditions but without trypsin. Trypsin control was 1 mg/ml trypsin inc~h~terl under the same conditions but without LCRFI
35. * denotes significantly dirr~rell- from control.

FIG. 15 LCRFl 35 stim~ tion of CCK release from dispersed rat inle~Lilla 15 cells. * denotes significantly dirr~ , from zero concentration of LCRFl 3s.

FIG. 16. Effect of anti-LCRF IgG on pancreatic secretory response to 5%
peptone infilsed intraduodenally in absence of pancreatic juice in the i-ltt'';~- Peptone was mixed with anti-LCRF IgG and infused together into the duodenum. *
20 denotes significantly different from peptone mixed with normal rabbit IgG. Results show that anti-LCRF IgG abolished the pancreatic secretory response to peptone.

FIG. 17. Effect of LCRF antiserum on the pancreatic secretory response to diversion of bile-pancreatic juice from the duodenlmn LCRF antiserurn or normal 25 rabbit serum (NRS) were infused intravenously as a bolus (0.1 ml) 1 hour prior to diversion of bile-pancreatic juice. Increment of pancreatic protein and fluid output is shown in insert. * denotes significantly dirr~,el" from NRS-infused group.

W O 97/15671 PCT~US96/17998 FIG. 18. Effect of LCRF antiserum on the plasma CCK response to diversion of bile-pancreatic iuice from the duodenum. * denotes significantly ~IirreLellt from NRS group and group receiving no serum.

FIG. 19. Lack of effect of LCRFl 3s on amylase-release from isolated pancreatic acini. CCK-8 stim~ tecl amylase in a dose-related fashion. At similarconcentrations LCRFl 3s was without effect. The results indicate that LCRFl 3s does not stim~ t~ the pancreas directly, but rather indirectly by stim~ ting CCK release.

FIG. 20. LCRF immunoreactivity (LCRF-IR) in small intPstin~l villi. FIG.
15A shows in~stin~l villi stained using LCRF antiserum 2243232 showing LCRF-IR
(dark structures and areas) at the tip and structures in the body of the villi. FIG. 1 SB:
intestinal villi following st~ining where antiserurn was preabsorbed with specific antigen (specific antigen control).
FIG. 21. LCRF-IR in enteric nerves ofthe small intestine. 21A: LCRF-IR
(antiserum 22322) in nerve fibers and nerve cell bodies in the myenteric plexus and submucosal neurons of the duodenum. 16B: Specific antigen control.
-FIG. 22. LCRF-IR in the nodose g~ngli~ 22A: Nerve fibers (dark streaks) and nerve cell bodies (dark patches) in the nodose ganglia stained using antiserum 22322. 17B: Specific antigen control.
.

FIG. 23 LCRF-IR in the adrenal gland. 23A: Nerve fibers (dark streaks) in the adrenal me~ stained using antiserum 22322. 23B. Specific antigen control.

FIG. 24. Western blot of rabbit antisera reactivity against pancreas, stomach muscle and stomach mucosa tissue. FIG. 24A Is a control ~,vith normal rabbit serum.
FIG. 24B. Is with rabbit polyclonal serum #QPDG.

W O 97/15671 PCT~US96/17998 FIG. 25. Western blot of rabbit antisera reactivity against pancreas, stromal mucosa, stroma mll~cle, ~ oA~n~l muscle, duodenal mucosa, abdominal muscle, ileum mucos~ ileum muscle. FIG. 20A is a control with normal rabbit serum. FIG.
20B is with rabbit polyclonal serum #1728.

4.0 Detailed Description of Prcr~. . ed Embodiments A novel CCK rele~in~ factor, luminal cholecystokinin releasing factor (LCRF) has been isolated and purified from int~tin~l secretions. LCRF is active in 10 ~tim~ ting CCK release and is found in enterocytes at the tips of small intestinal villi.
It has been identified as a putative n~ v~ ide found in the enteric, para~ylnpaLhetic and sympathetic nervous systems, but not in the brain. Tmmlln--~ffinity studies using antibodies raised against synthetic LCRFI~ and small intestin~l lumen infusion studies suggest that LCRF mPAi~tes negative fee~1h~cl~ regulation of pancreatic 15 enzyme secretion as well as CCK release.

For practical use, the LCRF peptide and active fr~gment~ or analogs thereof may be used to stimlll~t~ release of CCK in a marmer typical of ingested fats and proteins. Unlike these foods, LCRF effects CCK release at virtually zero caloric input 20 since the peptide is many orders of m~nit~lcle more potent in releasing CCK. LCRF
acts physiologically from within the lumen of the i. .le~ (i. e., not systçn i~lly, or blood-borne); thus it can be delivered to its site of action orally. This contrasts to other bioactive peptides used in medical tre~tm~nt~ e.g, insulin and growth hormone, which must be parenterally ~Amini~tered since they act on cells within int~rnz~l organs 25 or muscles.

Oral delivery of the LCRF peptide may encounter potential premature destruction by stomach acid and/or pepsin, and\or overly rapid destruction in the intestine by trypsin and other pancreatic proteolytic enzymes. Therefore one will 30 wish to consider embo~liment~ of the agent that include ancillary agents inhibiting W O 97/15671 PCTrUS96/17998 these digestive processes. Such agents are available and well-known to those skilled in the art. Potentially useful agents include medications ~u~les~illg stomach acid secretion or action (antacids and acid ~u~piess~l~ such as hi~t~nnine type II receptor antagonists ~Tagarnet, Zantac, Pepcid), or H~, K+ ATPase inhibitors (e.g Prolesec) as S well as agents ~u~pL~;s:jing trypsin activity (e.g., soybean trypsin inhibitor or potato trypsin/chymotrypsin inhibitor (POT II)). Such compounds have already been used in hnmzm.~.

Additionally, pepsin-resistant analogs of LCRF or smaller peptide fr~gm~nt~
10 po~eee~eing LCRF activity may be employed. The practical result of these embodiments would be to have a formulation mimicking the CCK release that food (particularly fat and protein) causes, but lacking the calories. An exemplary plel!~LLdlion might be synthetic LCR~ combined with agents to inhibit its digestive destruction, or chemical analogs (or small fragments) of LCRF that resist digestion.
4.1 ELISAs ELISAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides inc~ oldlillg LCRF smtip;enic sequences are immobilized onto a 20 selected surface, preferably a surface exhibiting a protein affinity such as the wells of a poly~y~ e microtiter plate. After washing to remove incompletely adsorbed m~t~rizll, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test ~ntic~r~ such as bovine serum albumin (13SA), casein or solutions of powdered rnilk. This allows for blocking of 25 n~ pecific adsorption sites on the irnmobilizing surface and thus reduces the background caused by non~ecirlc binding of ~nti~r~ onto the surface.

After binding of ~nti~nic m~ttori~l to the well, coating with a non-reactive m~t~ri~l to reduce background, and washing to remove unbound m~t~ri~l, the 30 immobilizing surface is cont~-~te(1 with the ~ntisPr~ or clinical or biological extract to be tested in a manner conducive to ;,.n~ complex (antigen/antibody) formation. Suchconditions preferably include diluting the ~nti~Pr~ with diluents such as BSA, bovine gamma globulin (BGG) and phosphate burr~,d saline (PBS)/Tween~. These added agents also tend to assist in the reduction of n...,~ec;l;c background. The layered s ~nti~ra is then allowed to incubate for from about 2 to about 4 hr, at te~ dLulcs preferably on the order of about 25~ to about 27~C. Following im~llh~ti(~n, the antisera-contacted surface is washed so as to remove non-immlmocomr~ 1 m~t~ri~l A
~.er~.~ d washing procedure in~ d~ washing with a solution such as PBS/Tween~), or borate buffer.
Following formation of specific immlm~lcQmrl~ s b~lwwll the test sample and the bound antigen, and subsequent washing, the oc-;ul,ellce and even amount of immlmocomplex formation may be ~1e l. ",;..~o~l by subjecting same to a second antibody having specificity for the first. To provide a (lete cting means, the second antibody will 15 preferably have an associated enzyme that will generate a color development upon incllh~ting with an ~,pro~l;ate chromogenic :ju~:~Llale. Thus, for example, one will desire to contact and incubate the ~nti~Pr~-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under con-1iti~n~ which favor the - development of immlm~complex formation (e.g, incubation for 2 hr at room 20 t~m~dLule in a PBS-c~ g solution such as PBS/Tween(~)).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound m~tt~ l, the arnount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-25 ethyl-b~,.,ll.iz~,oline)-6-slllfonic acid (ABTS) and H2O2, in the case of peroxidase as the en~yme label. Q..~ n is then achieved by m.o~enring the degree of color generation, e.g., using a visible ~e~;LI.~ spectrophotometer.

W O-97/lS671 PCT~US96117998 4.2 Epitopic Core Sequences .

The present invention is also directed to protein or peptide compociti(m~ free from total cells and other peptides, which comprise a purified protein or peptide which incorporates an epitope that is immlln~logically cross-reactive with one or more anti-LCRF antibodies.

As used herein, the term "inco~ dLillg an epitope(s) that is immlmc-logically cross-reactive with one or more anti-LCRF antibodies" is int~n~led to refer to a peptide or protein antigen which in~ a ~lu~ , secondary or tertiary structure similar to an epitope located within a LCRF polypeptide. The level of ~imil~rity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the LCRFpolypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein ~nti~en Various immlmo~c~y methods may be employed in cl,nju,l~;Lion with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.

. .
The icientific~tion of LCRF ~iLopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Patent 4,554,101, incorporated herein by reference, which teaches the icl~ntific~tion and ~ al~Lion of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several otherpapers, and software programs based thereon, can also be used to identify epitopic core se-lut;llces (see, for example, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Patent Number 4,554,101). The amino acid sequence of these "epitopic core sequences" may then be readily incol~ol~Lt:d into peptides, either through the application of peptide synthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more CA 02238940 l998-04-22 preferably about 8 to about 20 amino acids in length. It is proposed that shorter antigenic LCRF-derived peptide sequences will provide advantages in certain ci~ " -~ ,rc~, for example, in the pl~didlion of vaccines or in immunologic detection assays. F~Pmplzry advantages include the ease of pl~.~dlion and purification, the 5 relatively low cost and improved reproducibility of profl~lctit~n~ and advantageous biodistribution.

It is proposed that particular advantages of the present invention may be reali~d through the pl~d~ion of :iy~ ic peptides which include modified and/or P~ctPn~lPd 10 epitopic/imml-nc-genic core sequences which result in a 'tuniversal" epitopic peptide directed to LCRF and LCRF-related sequences. It is proposed that tbese regions represent those which are most likely to promote T-cell or B-cell stimlllzlti~ln in an animal, and, hence, elicit specific antibody production in such an animal.
.

An epitopic core seq~enre, as used herein, is a relatively short stretch of amino acids that is "complernPntzlry" to, and therefore will bind, antigen binding sites on transferring-binding protein antibodies. Additionally or ztl~ "~lively~ an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide colll~o~i~ions of the present invention. It will be understood that in 20 the context of the present disclosure, the terrn ~complPTnentz~ refers to amino acids or peptides that exhibit an attractive force luw~ls each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the i~lentified core sequence or sequences. The ~mzlllest useful core seq~Pnre anticipated by the present disclosure would generally be on the order of about S amino acids in length, with sequences on the 30 order of 8 or 25 being more ~.~,l~d. Thus, this si~ will generally collc~.olld to ~e CA 02238940 l998-04-22 W 0 97/15671 PCT~US96/17998 .cm~llect peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it ccnt~in~ a basic epitopic core sequence.

The ~ ontific~tif)n of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Patent 4,554,101, inco ~o,~d herein by reference, which teaches the identification and p~ ualion of epitopes from amino acid sequences on the basis of hy~Lophilicity. Moreover, numerous coll~ lel programs are available for use in predicting antigenic portions of proteins (see e.g, J~rnescn and Wolf, 1988;
Wolfetal., 1988). C~ (e~ 1 peptide sequence analysis programs (e.g., DNAStar~
software, DNAStar, Inc., Madison, Wisc.) may also be useful in tl~igning synthetic LCRF peptides and peptide analogs in accordance with the present disclosure.

Syntheses of epitopic sequences, or peptides which include an ~nti~nic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synth~i7~r such as an Applied Biosystems Model 430A Peptide Synth~si7~r). Peptide ~ntig~n~ synth~ci7.od in this manner may then be aliquoted in prê~i~t~rrninerl amounts and stored in co,lvelllional manners, such as in aqueous solutions or, even morêpreferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g, up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of ~nti~nic activity. EIowever, where ~ten-le~l aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to m~int~in a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit rnicrobial growth, such as sodium azide or Merthiolate.
For ~xtton-lel1 storage in an aqueous state it will be desirable to store the solutions at 4~C, or more l~lcreldbly, frozen. Of course, where the peptides are stored in a lyophili7P-l or CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 powdered state, they may be stored virh~lly indefinitely, e.g., in metered aliquots that may be lchydldL~d with a pre~ rmin~d amount of water (preferably distilled3 or buffer prior to use.

5 4.3 Immunoprecipitation The antibodies of the present invention are particuldrly useful for the isolation of antigens by ;mm~moprecipitation. Immlin~precipitdtion involves the separation of the target antigen component from a complex nlix~ , and is used to ~ . ;,.,;"~1~ or isolate 10 minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into d~ ellL micelles. Nonionic salts are ~l~;rt;ll~ d, since other agents such as bile salts, plcci~iL~Le at acid pH or in the pl~sence of bivalent cations.

In an ~ltem~tive embodiment the antibodies of the present invention are useful 15 for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g, enzyme-:iul~sLI~L~ pairs.

4.4 Western Blots The compositions of the present invention will find great use in immllnoblot or western blot analysis. The anti-LCRF antibodies may be used as high-affinity primary reagents for the identification of ~lv~ehls immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immlmnprecipitation, followed by gel ele~ opholt;~is, these may be used as a single step reagent for use in cletccting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immlm~ globulins (preçhl-ting the use of immllnc~globulins binding bact~ri~l cell wall components), the antigens studied cross-react with the cletecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.

CA 02238940 l998-04-22 Tmmlln~logically-based detection methods for use in conjunction with Western blotting include e.~y..~ ic~lly-, radiolabel-, or fluorescentiy-tagged secondaryantibodies against the toxin moiety are considered to be of particular use in this regard.

4.5 Vaccines T_e present invention co~ tps vaccines for use in both active and passive ;.. ~.. i~lion embo-limt~nt~ Immunogenic compositions, p-~osed to be suitable for 10 use as a vaccine, may be prepared most readily directly from immllnfgenic LCRF
peptides prepared in a manner disclosed herein. Preferably the antigenic m~tPri~l is extensively dialyzed to remove undesired small molecular weight molecules andlorlyophili7PA for more ready f~rm~ tion into a desired vehicle.

15The ~l~aldLion of vaccines which contain LCRF peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorporated herein by reference. Typically, such vdccil,es are p~cp~ucd as injectables. Either as - liquid solutions or ~ cnsions: solid forms suitable for solution in, or slT~rPn~inn in, 20 liquid prior to injection may also be prepared. The prc~a~dLion may also be ~mlll.~ifie~l The active immlln~genic ingredient is often mixed with excipients which are ph~rrn~ceutically acceptable and colnr~tihle with the active ingredient. Suitable ~cipient~ are, for example, water, saline, dextrose, glycerol, eth~n~ or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of 25 auxiliary substances such as wetting or emulsifying agents, pH bllffering agents, or adjuvants which ~nh~nre the effe-;Li~ l-ess of the v~c~inPs Vaccines may be conventionally ~ cd parenterally, by injection, for example, either sub~ .,evu~ly or ;.,~ "~ rly. Additional fr~rmlll~tiQns which are 30 suitable for other modes of ~1mini~ tion include suppositories and, in some cases, oral W O 97/15671 PCT~US96/17998 fn7mnkltions For suppneitn7ies, traditional binders and carriers may include, for example, poly, Ik~lf n- glycols or triglycerides: such suppositories may be formed from mixtures co..l,l,.~ g the active ingredient in the range of about 0.5% to about 10%, preferably about 1 to about 2%. Oral form~ tif)n~ include such normally employed S excipients as, for e~mrl~, ph~rrn,~relltical grades of ...~....;I--l, lactose, starch, m,7gn~eium stearate, sodium s~crh,7rinP, cellulose, m,~lg~,fS~ c~l,oll~Le and the like.
These compositions take the form of solutions, .,u.,~e.~,ions, tablets, pills, ç,-lpslllee, sll~ts~in~d release fo7mll1~tions or powders and contain about 10 to about 95% of active ingredient, preferably about 25 to about 70%.
The LCRF-derived peptides of the present invention may be fnm7l71~tf f1 into thevaccine as neutral or salt forms. Ph,nm,7f~e~ltically-acceptable salts, include the acid ~flr7iti-)n salts (formed with the free amino groups of the peptide) and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or 15 such organic acids as acetic, oxalic, tartaric, m~nfi~lic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, pol~ ;..ll., ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are ~f7mini~tf-red in a manner col~ Lible with the dosage form~ ti~n~ and in such amount as will be therapeutically effective and immlmogenic.
The quantity to be ~ ;t~ ed depends on the subject to be treated, including, e.g, the capacity of the individual's immlme system to synth~si7P antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be ~rlmini~tered depend on the j~ rn~nt of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograrns active ingredient per vaccin~tinn Suitable regimes for initial ~1miniet~tion and booster shots are also variable, but are typified by an initial ,I~l"~ Lion followed by subsequent inoculations or other ~lmini~trations.

W O 97/15671 PCTrUS96/17998 The manner of application may be varied widely. Any of the convrntion~l methods for ~tlmini~tration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, pale~ dlly~ by injection or the like. The dosage of the vaccine S will depend on the route of ~lmini~tration and will vary according to the size of the host.

Various methods of achieving adjuvant effect for the vaccine includes use of agents such as al...";"l...- hydroxide or phosphate (alum), commonly used as about 0.05 10 to about 0.1% solution in I~hC sph~te buffered saline, ~tlmixhlre with synthetic polymers of sugars (Carbopol~)) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat l~ with lel,,~ Lul~s ranging between about 70~ to about 101~C
for a 30-second to 2-minute period, r~;~e~ rely. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mi~ c; with b~rteri~l cells such as C
parvum or endotoxins or lipopolysaccharide cwll~onents of Gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as m~nnirle mono-oleate(Aracel A) or rm~ n with a 20% solution of a perfluorocarbon (Fluosol-DA(~)) used as a block substitute may also be employed.
-In many ;~ r.ec~ it will be desirable to have mllltiple ;~-imini~tr~tions of thevaccine, usually not excee~lin~ six v~crin~tic)n~ more usually not .-~cee-ling four vaccin~tion~ and preferably one or more, usually at least about three v~rrin~tinn.C The vaccinations will normally be at from t~vo to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to . "~; " 1i1; " ~o~-;liv~; levels of the antibodies. The course of the i"""~ ;on may be followed by assays for antibodies for the supern~t~nt antigens.The assays may be performed by labeling with conventional labels, such as r~ nw.lides, enzymes, fluv,~escc~"L~i, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932;
4,174,384 and 3,949,064, as illustrative of these types of assays.

4.6 DNA Scv ~ b In other emborliment~ it is co~ le pl~tec~ that certain advantages will be gained S by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter As used herein, a recombinant or heterologous promoter isinten~led to refer to a promoter that is not normRlly associated ~,vith a DNA segment encoding a LCRF peptide in its natural e~lvir ~ Pnt Such promoters may include promoters n~rm~lly associated with other genes, and/or pr )mr~tPrs i~olRte~l from any 10 viral, prokaryotic (e.g, b~c.teriRl), eukaryotic (e.g, fungal, yeast, plant, or animal) cell, and particularly those of m~mm~liRn cells. Naturally, it will be illl~Ol~l~ to employ a promoter that ~Lre.iliv~ly directs the G~ie,~ion of the DNA se~ le~.l in the cell type, organism, or even animal, chosen for ~lei,:,ion. The use of promoter and cell type combin~tion~ for protein expression is generally known to those of skill in the art of 15 molecular biology, for example, see Sambrook et al., 1989 The promoters employed may be co~ re, or inducible, and can be used under the al.plo~.iate conditions to direct high level c~~ ion of the introduced DNA segrn~nt, such as is advantageous in the large-scale production of recombinant ~ ei ls or peptides Appropriate - promoter/~ res~ion systems contemplated for use in high-level c;x~cs~ion inclntle, but are not limited to, the Pichia ~ s~ion vector system (Pharmacia LK~
Biotechnology), a baculovirus system for ~lei,~ion in insect cells, or any suitable yeast or b~cteri~ e ~ion system.

In connection with expression embo-liment~ to prepare recombinant proteins and peptides, it is co ~ ,pl~tl d that longer DNA se~ i will most often be used, with DNA sçgment~ encoding the entire peptide se~uence being most plcr~ d However, itwill be appreciated that the use of shorter DNA segmentc to direct the e~lession of LCRF peptides or epitopic core regions, such ~ may be used to generate anti-LCRFantibodies, also falls within the scope of the invention DNA segments that encode LCRF peptide antigens ~om about 10 to about 100 atmino acids in length, or more W O 97/15671 PCT~US96/17998 preferably, from about 20 to about 80 amino acids in length, or even more preferably, from about 30 to about 70 amino acids in length are cont~mpl~tecl to be particularly useful.

S In ~ litic~n to their use in directing the exy~s~ion of LCRF peptides of the present invention, the nucleic acid sequences co~ t~cl herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hyhri-li7~tion emboc1iment~ As such, it is c~nt~mrl~t~l that nucleic acid segm~nt.c that comprise a sequence region that consists of at least an abQut 14-nucleotide longcontiguous sequence that has the same sequence as, or is compl~met~t~ry to, an about 14-nucleotide long contiguous DNA segm~nt of SEQ ID NO:2 will find particular utility. Longer contiguous i~1~nti~l or compl~m~nt~ry sequences, e.g., those of about 20, 30, 40, 50, 100, 200, ~inrlll-lin~ all int~orme~ te lengths) and even those up to and including about 220-bp (full-length) sequences will also be of use in certain embo~limt-nt~

The ability of such nucleic acid probes to specifically hybridize to LCRF-encoding sequences will enable them to be of use in cletecfing the presence of complem~nt~ry sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the ~lc~J~dlion of mutant species primers, or primers for use in prep~u~-lg other genetic constructions.

Nucleic acid molecules having sequence regions cnn~i~ting of contiguous nucleotide ~ clles of about 14, 15-20, 30, 40, 50, or even of about 100 to about 200 nucleotides or so, identical or compl~ment~ry to the DNA sequence of SEQ ID NO:2, are particularly cul.lelllplated as hyhri~li7~tinn probes for use in, e.g, Southern and Nor~ern blotting. Smaller fr~gm~nt~ will generally find use in hybricli7~ti~ n embod~ cl.l~, wl~ eil- the length of the contiguous complen-ont~ry region may bevaried, such as b~;;lw~ell about 10-14 and up to about 100 nucleotides, but larger W O 97/1~671 PCT~US96/17998 contiguous complempnt~rity ~llclclles may be used, accol.lillg to the length complem~nt~ry sequences one wishes to detect.

The use of a hybri-li7~tion probe of about 14 nucleotides in length allows the 5 formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 14 bases in length are generally pr~rc..c;d, though, in order to increase stability and selectivity of the hybrid, and thereby illl~ Ve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complPm~nts~ry 10 stretches of about 15 to about 20 contiguous nucleotides, or even longer where desired.

Of course, fr~gmPntc may also be obtained by other techniques such as, e.g, by merh~nir~l ehPs-rin~ or by restriction enzyme ~ oeti~)n Small nucleic acid segments or fr~gmPnte may be readily ple~hcd by, for example, directly synthPei7in~ the ~gmPnt 15 by chPmic~l means, as is c )mmonly practiced using an ~ d oligonucleotidesynthPei7~r. Also, fr~gmPnte may be obtained by application of nucleic acid reproduction technology, such as PCRTM, by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA
techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complen~Pnt~ry stretches of DNA
~mente Depending on the application envieionp~1~ one will desire to employ varying conditions of hybritli7~tion to achieve varying degrees of selectivity of probe towards 25 target sequence. For applications re~ui~ ulg high selectiv~ty, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g, one will select relatively low salt and/or high ~m~dlule c~-n-1iti- ne, such as provided by about 0.02 M to about 0.15 M NaCl at t~ C~d~ ;S of about 50~C to about 70~C. Such selective c~ n-liti-ne tolerate little, if any, miem~t ll b~lwt;en the probe and the t~mpl~t~ or target strand, and 30 would be particularly suitable for isolating LCRF-encoding DNA se~nPnte. Detecti~n W O 97/lS671 PCT~US96/17998 of DNA se~n~nt~ via hyhrifli7Ation is well-known to those of skill in the art, and the teaching~ of U.S. Patents 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridi7~tic-n analyses. Tt-~-hing.c such as those found in the texts of Maloy et al., 1994, Segal, 1976; Prokop, 1991, and Kuby, 5 1994, are particularly relevant.

Of course, for some applications, for example, where one desires to prepare z."~i employing a mutant primer strand hyhridi7~ocl to an underlying template orwhere one seeks to isolate LCRF -encoding sequences from related species, functional 10 equivalents, or the like, less stringent hyhri~1i7Ati-)n conditions will typically be needed in order to allow formation of the heteroduplex. In these cirCllm~t~nr~os~ one may desire to employ conditions such as about 0.15 M to about 0.9 M salt, at ten~cld~u-es ranging from about 20~C to about 55~C. Cross-hybricli7ing species can thereby be readilyitlçntifiç-l as positively hybridi7ing signals with respect to control hybri~li7Ations. In any 15 case, it is generally appreciated that conditions can be rendered more stringcnt by the addition of increasing amounts of fol",~l..itle which serves to destabili_e the hybrid duplex in the same manner as increased tclllpe~dlulc. Thus, hybridi_ation conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embo~limPnt~, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an a~pl~o~liate means, such as a label, for tlet~rmininp hyhricli7Ation~ A wide variety of a~pl~opliate in~lic~t(~r means are known in the art, inçhl-ling fluorescent, radioactive, enzymatic or other li~n~lc, such as 25 avidin/biotin, which are capable of giving a ~lçt~ct~ble signal. In ~lcr~ ,lcd embo~lim~-nt~, one will likely desire to employ a flu~lcscc~lt label or an enzyme tag, ~ such as urease, ~lk~lin~ pho~,halase or pero~ ç, instead of radioactive or other ellvilvl--- -elll~l undesirable lcagelll~. In the case of enzyme tags, colorimetric in~ t~r sub~ dLes are known that can be employed to provide a means visible to the human eye CA 02238940 l998-04-22 W 0 97/15671 PCT~US96/17998 or spectrophotometrically, to identify specific hyhricli7~tion with complem~.nt~ry nucleic acid-co,.li.i.~ s~mples In general, it is envisioned that the hyhr~ 7~tion probes described herein will be S useful both as reagents in solution hybridization as well as in embo-limente employing a solid phase. In embo~lim~nte involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-str~n-l~d nucleic acid is then subjected to specific hybri~1i7~tio~ with sel~cte~1 probes under desired con~litione The selected conditions will depend on the particular 10 cir~ ..eee based on the particular criteria required (clep~n~lin~;, for example, on the G~C content, type of target nucleic acid, source of nucleic acid, si_e of hyhricii7~ti~ n probe, etc.). Following washing of the hyhri~li7P~ surface so as to remove nons~e.;irlcally bound probe molecules, specific hyhritli7~tion is detected, or even q~ , by means of the label.
4.7 Biological Functional Equivalents ., Modification and changes may be made in the structure of the peptides of the present invention and DNA segmlont.c which encode them and still obtain a functional 20 molecule that en~o~es a protein or peptide with desirable ch~r~ccteri~irs The following is a ~ c~ ion based upon ~~h~n~in~ the amino acids of a protein to create an equivalent, or even an i~ r~ d, second-generation molecule. The amino acid changes may be achieved by f.h~nging the codons of the DNA sequence, according to the followingcodon table:

~=

W O 97/15671 PCTnUS96/17998 -5g-- Amino Acids Codons Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C UGC UGU
Aspartic acid Asp D GAC GAU
(~Tlllt~tmic acid Glu E GAA GAG
Phenyktl~ninP Phe F WC UUU
Glycine Gly G GGA GGC GGG GGU
~j~ti~line His H CAC CAU
Isoleucine Ile I AUA AUC AW
Lysine Lys K AAA AAG
T.ell-~ine Leu L WA WG CUA CUC CUG CW
Methionine Met M AUG
~A~p~tr~tgine Asn N AAC AAU
Proline Pro P CCA CCC CCG CCU
Glllt~tntinP. Gln Q CAA CAG
' Arginine Arg R AGA AGG CGA CGC CGG CGU
Serine Ser S AGC AGU UCA UCC UCG UCU
Threonine Thr T ACA ACC ACG ACU
Valine Val V GUA GUC GUG GW
Tryptophan Trp W UGG
Tyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other arnino acids in a 5 protein ~L~u;~ without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the inter~tive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and - 10 nevertheless obtain a protein with like ~up~llies. It is thus contcntrlated by the W O 97/15671 PCT~US96/17998 ~--v~nLul:, that various changes may be made in the peptide sequences of the disclosed compositions, or coll.,s~ol1ding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.

In making such changes, the hydlul dLl~.c index of amino acids may be considered. The illl~u-ku~ce of the hydLu~lic amino acid index in cc~.~..;.~g inter~ctive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, IllCUl~JUldLe herein by reference). It is accepted that the relative hydLo~dLhic ch~r~ t~r of the amino acid contributes to the secondary structure of the 10 res-llt~nt protein, which in turn defines the int~r~ctinn of the protein with other molecules, for ~x~mrle~e~yllles~ub~Lldles~lec~ DNA, antibodies, antigens, and t_e like.

Each amino acid h~ been ~cci~ntorl a hyLopdlllic index on the b~is of their 15 hydrophobicity and charge chd~ t~ tirs (Kyte and Doolittle, 1982), these are:isoleucine (+4.5); valine (+4.2); leucine (+3.8), phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); Ll..~ol~le (-0.7); serine (-0.8);
tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); hi.ctir~inP (-3.2); gliltz~mzlte (-3.5);
- ~h~ e (-3.5); a~ Ldl~; (-3.5); ~paragine (-3.5); lysine (-3.9); and arginine (-4.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hy~Lul~l~c index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. in making such changes, the substitution of amino acids whose hydropathic indices are 25 within +2 is pl~f~ d, those which are within ~tl are particularly preferred, and those within l 0.5 are even more particularly pl~er~ d.

It is also Im~ler~c)od in the art that the substitution of like arnino acids can be made erre~;Livt;ly on the b~is of hydrophilicity. U.S. Patent 4,554,101, incol~ol~L~d 30 herein by lcr~ ce, states that the ~Lle:~t local average llydlu~llilicity of a protein, ~

W O 97/15671 PCT~US96/17998 governed by the hy~ philicity of its ~clj~ nt amino acids, correlates with a biological ~rup~lly ofthe protein.

- As ~let~ in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); Iysine (+3.0); aspartate (+3.0 l); gll-t~m~te (+3.0 ~ 1); serine (+0.3); ~p~r~gin~? (+0.2); ~ (+0.2), glycine (0);
threonine (-0.4); proline (-0.5 ' 1), alanine (-0.5); hi~titlin~ (-0.5); cysteine (-1.0);
methi-~nine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenyl~l~nin~ (-2.5); LL~y~loplkLl~ (-3.4).
It is lm-i~rctQod ~at an arnino acid can be sl-hstib~t~ for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immlm~logically equivalent protein. In such changes, the ~ul~Li~uLion of amino acids whose llydro~hilicity values are within ~2 is pl~rt;ll~d, those which are within ~1 are particularly plc;f~ d, and those within ~0.5 are even more particularly ~ r~ ;d.
As outlined above, amino acid ~ub~LiLu~ions are generally therefore based on therelative ~imil~rity of the amino acid side-chain sul)~ ;, for example, their hydrophobicity, hydl~pl ilicity, charge, size, and the like. Exemplary ~ ;ons which take various of the foregoing characteristics into con~ er~tion are well known to those of skill in the art and incl~l~le: arginine and lysine; glut~m~t~ and a~dlLd1~; serine and threonine; ~,h~ ? and ~cp~r~gin~; and valine, leucine and isoleucine.

4.8 Site-SpecificMuta~. e~;~
Site-specific mutagenesis is a technique useful in the pl~dldlion of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incol~oldlil~g one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into W O 97/15671 PCT~US96/17998 the DNA. Site-specific mutagenesis allows the production of In~ ; through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of RAjRr~nt nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both 5 sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is ~ r.,~l~d, with about S to 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as 10 exemplified by various publicRt;on~ As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double strRnArA form.
Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double strRn~ iR~mi~l~ are also routinely employed 15 in site directed mutagenesis which eliminRtçs the step of hd.~ir~ g the gene of interest from a plasmid to a phage.

.
In general, site-directed mutagenesis in accu~ ce he~ is performed by first obtaining a single-stranded vector or m~l~ing apart of two strands of a double 2~ stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired ml1tRt~d sequence is p~ ;d, generally synthetically. This primer is then annealed with the single-strRn~lecl vector, and subjected to DNA polymeri7ing enzymes such as E. coli polymerase I
Klenow frRgrn-?nt, in order to complete the ~y~ le~is of the mutation-bearing strand.
2~ Thus, a heteroduplex is formed wherein one strand encodes the original non-mlltRt~l sequence and the second strand bears ~e desired mutation. This heteroduplex vector is then used to transform ~ ,iate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the m~ltRted seq~rnr-e RrrRnF~emrnt . . .

W O 97/15671 PCTrUS96/17998 The ~ udlion of sequence variants of the selected peptide-encoding DNA
segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be 5 obtained. For ~mple7 recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

4.9 Monoclonal Antibodies 10Means for ~ J~L;llg and l h~r~ct~ri7ing antibodies are well known in the art (See, e.g, Harlow and Lane, 1988; incorporated herein by reference).

The methods for g~ner~ting monoclonal antibodies (mAbs) generally begin along the same lines as those for plG~LlLlg polyclonal antibodies. Briefly, a polyclonal 15 antibody is ~rGl)~ed by ;~ IILI~ g an animal with an immlmogenic composition in accordance with the present invention and collecting antisera from that ;."".-...;~cl animal. A wide range of animal species can be used for the production of s~ntieçr~
Typically the animal ueed for production of anti-antisera is a rabbit, a mouse, a rat, a - h~metPr, a guinea pig or a goat. Because of the relatively large blood volume of rabbite, 20 a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immnnogenicity. It is often n~cçee~ry therefore to boost the host immlme system, as may be achieved by coupling a peptide or polypeptide immlmogen to a carrier.
25 Fxempl~ry and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum alburnin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumLn can also be used as c~rriprs- Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydn~xy~ cinimi~1t? ester, carbodiimide and bis-biazotized 30 bcn7i(1int?.

As is also well known in the art, the immnnogenicity of a particular imm~mogen composition can be ~nh~n(~eA. by the use of non-specific stim~ t~rs of the i..,...~
response, kno~-vn as adjuv~ll~. Exemplary and ~Ic;r~lr~d adjuvants include complete 5 Freund's adjuvant (a non-specific s*mnl~tQr of the i~ response c.~ ;..;r.g killed Mycobacterium tuberculosis), in~ompletc Freund's adjuv~ and ~IIl.llillll.l~ hydroxide adjuvant.

The amount of immlln(~gen composition used in the production of polyclonal 10 antibodies varies upon the nature of the immlm(~gen as well as the animal used for i~..",-"~ ion. A variety of routes can be used to ~mini~ r the immllnf~gen (subcutaneous, intramuscular, intr~Arrm~ dvellous and intraperitoneal). The production of polyclonal an*ibodies may be monitored by .c~mplin~ blood of the i..".~ A animal at various points following i..,..~ n. A second, booster, 15 in~ection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immlm-genicity is obtained, the i."""...i,~.l animal can be bled and the serurn isolated and stored, and/or the animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco,~ d herein by reference. Typically, this technique involves i~ a suitable animal with a s~lecte-l immlln~gen composition, e.g., a purified or partially purified LCRF protein, polypeptide or peptide.
The immllni7ing composition is ~mini~t~red in a manner effective to stim antibody producing cells. Rodents such as mice and rats are plcr~.~ed ~nim~
however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are plcr~ d, with the BALB/c mouse being most p,~re~l~,d as this is most routinely used and generaIly gives a higher p~ ~ge of stable fusions.

Following ;,.,...~",;,~;on, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb ~en~,dl,-lg protocol. These cells may be obtained from biopsied spleens, tonsils or ~ lymph nodes, or from a prriph.or~l blood sample. Spleen cells and peripheral blood cells 5 are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily c~csihle. Often, a panel of ~nim~l~ will have been il"",.,..i,~fl and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an i""""t,i,~-l mouse contains d~ xil~stt~ly 5 ' 107 to 2 108 lymphocytes.

The antibody-producing B lymphocytes from the il~ cl animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was ;I~ Myeloma cell lines suited for use in hybridoma-producing 15 fusion procedures plereL~bly are non-antibody-pro~ cing have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in cer~ain selective media which support the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the ill.. -lul.. ;G~d animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-ll, MPCll-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with 25 human cell fusions.

One plcr~;llc;d murine myeloma cell is the NS-l myeloma cell line (also termed P3-NS-l-Ag4-1), which is readily available from the NlGMS IIuman Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, ~ e~;LivGly~ in the presence of an agent or agents (ehemie~l or electrical) that promote the fusion of cell membranes. Fusion mtoth~ A~ using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use of electrically in~lur e~l fusion methods is also a~,v3~l;ate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10~ to 1 x 1 o-8. However, this does not pose a problem, as the viable, fused hybrids are di~ te~l from the parental, unfused cells (particularly the unfused myeloma cells that would norm~lly continue to divide indefinitely) by c~lltllring in a selective medium.
The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and ~erel,~,d agents are - ~lfillu~lGlill, methv~G~dlt;, and ~ Aminopterin and methuLLG~d~e block de novo synthesis of both purines and pyrimiAin~?~, whereas ~7~erine blocks only purine synthesis. Where aminopterin or methvLIG~dlG is used, the media is supplement~A with hypox~..ll-i"e and thymidine as a source of nucleotides aIAT meAilln ). Where ce~ e is used, the media is suppl~ment~A with hypox~. "h; ..e The preferred selection meAil-m is HAT. Only cells capable of O~ldLi,lg nucleotide salvage ~dl~lWdys are able to survive in HAT merlil-m ~he myeloma cells are defective in key enzymes of the salvage ~dlllWdy, e.g, hypû~n~hine phosphoribosyl r~ e (HPRT), and they caTmot survive. The ~-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks.

W 0 97/15671 PCTnUS96/17998 Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

~ This c ~ rin~ provides a population of hybridomas from which specific S hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in m-icrotiter plates, followed by testing the individual clonal sup~ c (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radio.,l,lllu.loassays, enzyme immllno~cs~ys, cytotoxicity assays, plaque assays, dot immlm-)binding assays, and the 1 0 like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be prop~ed in~ finitely to provide mAbs. The cell lines may be exploited for mAb production in 15 two basic ways. A sample of the hybridoma can be injected (often into the pGl;~oneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs 20 in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as ~L~ or affinity chromatography.
41 0 Pharm~eent;r~l Compositions The ~ c~u~;c~l compositions disclosed herein may be orally ~ . .C;d, for ~mplç, with an inert diluent or with an ~imil~hle edible carrier, or they may be 30 ~n~ sçd in hard or soft shell gelatin capsule, or they may be co~ e~ed into tablets, or W O 97/15671 PCT~US96/17998 they may be incc,,~u,~L~d directly with the food of the diet. For oral th,~ ulic~f1mini~tration~ the active compounds may be incol~uld~d with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and lJl~dldlions should contain at least 5 0.1% of active compound. The percentage of the compositions and ~L~ Llions may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit. The amount of active co,~ ds in such theLa~ Lically useful compositions is such that a suitable dosage will be obtained.

10The tablets, troches, pills, c~rs-llrc and the like may also contain the following:
a binder, as gum tr~ r~nth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate, a ~ te~ g agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as m~ ne~ium ~lc;~dle, and a ~w~ g agent, such as sucrose, lactose or saccl~ may be added or a flavoring agent, such as ~ lmilll, oil of 15 wi ll~ , .l, or cherry flavoring. When the dosage unit form is a c~rs-llP it may conl~in, in addition to m~teri~lc of the above type, a liquid carrier. Various other materials may be present as coatings or to oll,,.~ise modify the physical form of the dosage unit. For j,l!i(;,ll.~r tablets, pills, or c~rsl-lec may be coated with shellac, sugar or - both. A SylUp of elixir may contain the active compounds sucrose as a sweetening agent 20 methyl and p~ yl~dbens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any m:lterizll used in ~ mg any dosage unit form should be ph~rm~relltically pure and :~..b~ 11y non-toxic in the amounts employed. In addition, ~e active compounds may be incoll,u,,~Led into s -~t~ined-release L~r~ n and formnl~tions.
The active compounds may also be ~rlmini~trred l-~Glll~dlly or intraperitoneally. Solllti~n~ of the active compounds as free base or rh~ rologically acceptable salts can be ~ ,~, d in water suitably rnixed with a s~ rt~nt such ashydloxy~iu~ylcellulose. Di~r~rci~n~ can also be plep~d in glycerol, liquid 30 polyethylene glycols, and n.i2~ i, thereof and in oils. Under ordil~y conditions of t -W O 97/15671 PCT~US96/17998 storage and use, these p~r~aLions contain a preservative to prevent the growth of microorg~ni~m~

The ph~rrn~r,eutir~l forms suitable for injectable use include sterile aqueous S solutions or dispersions and sterile powders for the t;~Le~ vr~leous ~r~ Lion of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of m~mlf~r,tllre and storage and must be ~res~ ed against the c~ ting action of microor~ni~m~, such as bacteria and fungi. The carrier can be a solvent or 10 dispersion medium co..l~ ;..g, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable Lul~s thereof, and vegetable oils. The proper fluidity can be m~int~in~-tl for example, by the use of a co~tin~, such as lecithin~ by the m~inten~nce of the required particle size in the case of dispersion and by the use of sllrf~t~nt~ The prevention of 15 the action of microorg~ni~m~ can be brought about by various antib~t~ri~l ad antifungal agents, for ~Y~mple, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged al~sul~lion ofthe injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for exarnple, 20 al.l",;"l",~ monostearate and gelatin.

Sterile injectable solutions are prepared by illCul~Olalil~g the active compounds in the required amount in the ~ u~liate solvent with various of the other ingredients enumerated above, as required, followed by filtered st~rili7~tion. Generally, dispersions 25 are prepared by incol~o~ g the various sterili7~-l active ingredients into a sterile vehicle which contains the basic dispersion m~ m and the required other ingredients from those ~mlmPr~tecl above. In the case of sterile powders for the l,rep~L~lion of sterile injectable solutions, the ~ f~llGd methods of ~ ion are vacuurn-drying and freeze-drying techniques which yield a powder of the active ingredient plus ny 30 additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "ph~rm~relltically acceptable carrier" includes any and all solvents, ~licp~rcion media, co~ting~ antib~rteri~l and antifungal agents, isotonic and absorption delaying agents and the li~e. The use of such media and agents for 5 ph~rm~relltic~l active ~ b~ ces is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is co..l~ lated. Supplementary active ingredients can also be incol~ol~L~d into the compositions.

The phrase "rh~rm~relltic~lly acceptable" refers to molecular entities and compositions that do not produce an allergic or similar ullluw~ull reaction when~r1minictf-red to a human. The pr~aralion of an aqueous composition that collL~i ls a - protein as an active ingredient is well ~ml1er~tood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or ~u~ ; solid 15 forms suitable for solution in, or ~us~ ion in, liquid prior to injection can also be prepared. The ~lepal~.Lion can also be en~ if i~l ., The composition can be f(lrmlllAted in a neutral or salt forrn. Ph~rm~r,el~tically acceptable salts, include the acid addition salts (formed with the free amino groups of 20 the protein) and which are formed with inorganic acids such as, for example, hydrochloric or pho~horic acids, or such organic aGids as acetic, oxalic, tartaric, m~n-i~lic, and the like. Salts forrned with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodiurn, p~ mmrnillm, c~lcillm, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, hi~ti~in.o, 25 procaine and the like.

Upon formlll~tirn, solutions will be ~-1mini.ctt-red in a manner comr~fihle withthe dosage fnrm~ tion and in such arnount as is lL~a~;uLically effective. The f~rm~ tions are easily ~-lmini~tered in a variety of dosage forms such as injectable 30 solutions, drug release capsules and the l~e.

W O9711~671 PCTAUS96/17998 For p~ut;lll~.dl ~flmini~tration in an aqueous solution, for e~r~mple, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with s ]ffif~ient saline or glucose. These particular aqueous solutions are especially S suitable for illLldv~;llous, inLldlllus~;uldr, subcutaneous and il".,.l.. .;Lf l~f~l afllll.lli!iLldLion.
In this cf)nnf~c1ion~ sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infil~if)n, (see for ex~mple, I'Remin tonls Ph~rm~f~eutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).
Some variation in dosage will nf~Cç~rily occur depending on the condition of thesubject being treated. The person responsible for aflmini~tr~tion will, in any event, fletermine the appn,~llate dose for the individual subject. Moreover, for human a~lmini~tr~tion~ pl,;~dLions should meet sterility, pyrogenicity, general safety and 15 purity standards as required by FDA Office of Biologics standards.

Cholecystokinin secretion in rats and hllm~n~ is inhibited by pancreatic proteases and bile acids in the intestine. It has beerl hypoth~si7~d that the inhibition caused by pancreatic proteases is due to proteolytic inactivation of a cholecystokinin-20 releasing peptide present in intestin~l secretion. To purify this ~uldLive secretorypeptide,i,~ secretionswerecollectedby pP~ inF~ amodifiedThiry-Vella fistula of jejunum in awake rats and these secretions were used as starting m~teri~l A
peptide was concentrated from inle~Linal secretions by ultrafiltration and by low pressure reverse phase cll~ol.ldLography, and purified by reverse phase high pressure 25 liquid chromatography. Purity was confirmed by high ~l~,S:~Ul~ capil}ary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stiml-l~te pancreatic protein secretion when infused into the proximal small intPstinP.
of conscious rats.

CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 Partially-purified fractions strongly stimnl~ted pal~cle~ic secretion and cholecystokinin release, and cholecystokinin receptor blockade abolished the pancreatic response. Amino acid analysis and mass spectral analysis showed that the purified peptide has approximately 70 amino acid residues and a mass of about 8136 5 daltons. The amino acid composition of LCRF is as follows (amino acidlNo. of residues~: Ala~4; Arg/l; Asp/9; Cys/N.D.; Glu/l l; Gly/6; His/l; Ile/2; Leu/5; Lys/2;
Met/0; Phe/2; Pro/7; Ser/7; Thr/7; Trp/N.D.; Tyr/2; ValJ3 (N.D.= Not cletermined in analysis). Microsequence analysis of LCRF yielded an amino acid sequence for 41 amino acids, as follows:
1 0 STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV.

When infused intraduodenally, the purified peptide stim~ t~ pancreatic protein and fluid secretion in a dose-related manner in awake rats and significantly elevated plasma CCK levels. Immunoaffinity chromatography using antisera raised to 1~ synthetic LCR~I~ indicated that the CCK releasing activity of int~stin~l secretion was due to a peptide with the above arnino acid sequence. These studies demonstrate the first chemical char~cteri7~ti~ n of a luminally-secreted enteric peptide functioning as an intraluminal regulator of ;ntt?stin~l hormone release.
-The intraluminal mediator of protease-sensitive feeclb~ regulation of CCK
secretion was purified from intectin~l secretions collected by perfusing an isolated loop of jejunum in awake rats. Tntestin~l secretion appealcd to be a better source of this factor than intestin~l extracts. This may be because i.lle~ al extracts could contain other releasers of CCK that may not be released into the int~stin~l lumen.
To purify LCRF, int~stin~l secretions were collected by ~t;lru~illg a modified Thiry-Vella fistula of jejunurn in awake rats and these secretions were used as starting m~t~ri~l The peptide was conce~ Ied from i,~le~ l secretions by ultrafiltration and by low pressure reverse phase chromatography. It was purified by reverse phase high plei~:jUlC; liquid chromatography. Purity was co.~ I; " .~ç,l by high pressure capillary electrophoresis. Fractions were assayed for CCK-releasing activity by their ability to stim~ te pancreatic protein secretion when infused into the proximal small ;~te~ of conscious rats. Partially-purified fractions strongly stimulated pancreatic secretion and cholecystokinin release and cholecystokinin leCel)lOl blockade abolished S the pancreatic response.

Amino acid analysis and mass spectral analysis showed that the purified peptide has apprc~im~tely 70 amino acid residues and a mass of 8136 + 1% daltons.
Microsequence analysis of LCRF yielded an N-termin~l amino acid sequence for 41 10 ofthe amino acids, as follows:
STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV.

When infused intraduodenally, the purified peptide ~tim~ ted pancreatic protein and fluid secretion in a dose-related manner in awake rats and significantly 15 elevated plasma CCK levels. Immlm()~ffinity chromatography,'using antisera raised to synthetic LCRFI~, confirmed that the amino acid sequence described here was that of a CCK-releasing peptide present in intestin~l secretion. The present invention demonstrates the first chemical char~teri7~tion of a luminally-secreted enteric - peptide functioning as an intraluminal regulator of ;l-le~ l hormone release.
The dose-response studies with purified ;..~ l LCRF showed a biphasic curve, with the highest dose producing a subm~cim~l pa~ cdLic protein and fluid response. A similar biphasic dose-response curve for CCK release stimlll~ted by monitor peptide was reported by Cuber et al. (1990) in studies using isolated, 25 v~clll~rly-perfused rat intestine. These investigators suggested that the biphasic curve may reflect desen~iti7~tion of receptors on CCK secreting enteroendocrine cells at higher concentrations of the releasing peptide.

The parallel changes in fluid output and protein output in pancreatic juice 30 suggested that LCRF has secretin-releasing activity as well as CCK-releasing activity.

W O 97/1~671 PCTAUS96/17998 However, pancreatic fluid secretion in the rat during diversion of bile-pancreatic juice is highly dependent upon CCK, as demonstrated by Taguchi et al. (1992) who showed that the greatly elevated fluid output in bile-pancreatic juice-diverted rats was nearly abolished by CCK receptor blockade, in parallel with decreased protein output.
5 Because diversion of pancreatic juice in the rat stimulates sec~ release, the stim~ tion of fluid output by the int~stin~l LCRF may be intc.~lGLed as a reflection of increased levels of CCK augmenting fluid secretion stim~ tecl by a background ofelevated secretin secretion (Sun et al.,l982). This is also consistent with the virtual elimin~ti~ n of the pancreatic fluid response to partially purified LCRF, by the CCK
10 receptor antagonist, MK-329, in the studies presented here.

LCR~ is effective for releasing cholecystokinin in the rat at a dose of 3 micrograms (3 mg) delivered intraduodenally. This tr~n~l~tes to approxilllately lO
mg/kg rat. Conservatively, this suggests that an effective dose for CCK release in a l S 70 kg man would be approximately 1 mg. For effective tre~tment it is believed that this is the amount that would have to be available in the intestine (duodenum orjejunum).

Thus, approximately 1 mg of active LCRF should be present in the duodenum 20 to m~cim~lly elicit CCK release in a 70 kg human. Without protective measures other than a meal, it would be expected that only appro~im~tely 1-2% would survive digestive processes (DiMagno et al., 1986), meaning that 50-100 mg might be required as an err~ ivG oral dose. If accompanied by acid secretory :~UL)f~lCSSallt~i most (70-80%) of the peptide should survive stomach passage, and be delivered into 2~ the duodenum, i.e., a dose of 2-3 mg LCRF with Pepcid or Tagamet should be effective, especially if taken with a meal. If the peptide agent is fonn~ te-l with a pancreatic protease inhibitor and taken with acid ~UP~1GSS~1l medication, possibly 100% delivery could be expected, (a dose of 1 mg or less of LCRF then being effective). Likewise, if a chemic~lly-modified form of LCRF, resistant to digestion in 30 stom~h and int~Stine, iS made, it would be effective at doses of 1 mg or less.

W O 97/15671 PCT~US96/17998 As discussed, for a peptide given orally in an unprotected form, digestion of the peptide in the stomach and intestine could cause large losses of activitv. This is analogous to suppl~ment~tion with orally ~imini~tt~red digestive enzymes in S pancreatic ~ ç~ee~ in which most of the ~lmini~t~red enzymes are destroyed in the stom~ch by acid/pepsin. Neutralization of gastric contents with gastric acid seclt;lc.-y ~u~e;,sallL~ ~e.g, Tagamet, Zantac or Pepcid) prevents gastric inactivation of oral digestive enzyme supplement~ ~DiMagno et al.), and a similar protocol will protect orally-~-lmini~tçred LCRF form~ tions as well. Pepcid and Tagamet are now 10 available without pLescl;~Lion, and Zantac is expected to be so in the near future.
Additional ~ le~ e formulations could include enteric coating of microspheres that encapsulate the agent, such that the microspheres do not release their contents until they reach the duodenum. With these measures, it would be expected that 2-3 mg of LCRF taken orally would result in about 1 mg re~c~hing the duodenum. The oral 15 dosage form of LCRF, its active fr~gm~nt~, derivatives or analogs may be in any convenient ~iministrable form such as a solution, su~e~ ion, tablet, capsule or others known to those of skill in the art.

- 5.0 Examples The following examples are included to demonstrate ~.~;;r~l~;d embo-1imPnt.c of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow ~ ltse~lL techniques discovered by the inventors to function well in the practice of the invention, and thus can be c~n~iclered to constitute 25 pr~ rtlled modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embo~lim~nt.c which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

W O 97/15671 PCTrUS96/17998 SØ1 Materials Antisera #94 l l 3 and #22322 were raised in rabbits at the antibody core facility of CURE and by Quality Controlled Biochemic~lg, Inc. (HopkintQn, MA) to LCRFI 6 5 and LCRF7 23-Recombinant di~epam binding inhibitor (DBI~ 86) was provided by Jens~ml~ n (Odense University, Odense, Denm~rk)~ DBI 33-~0 (ODN) and Gastrin releasing peptide (GRP) were obtained from P~nin~ Laboratories Inc. (Belmont, 10 CA). Recombinant Monitor peptide (MP) was pl~,paled as described in Liddle (Liddle et al., l 984).

~;Ø2 Methods lS 5Ø2.1 Tissue preparation Wistar male rats weighing between 300 and 350 g were fasted ov~mi~ht Rats were anesthetized with pentobarbital (Nembutal, Abbott, Chicago, IL). The brain and hr~in~t~m were removed from rats perfused with 4% paraform~ hyde. The nodose 20 ganglia with sections of the vagus nerve, esophagus, stomach, duodenum, pancreas and adrenal glands were removed from non-perfused rats and fixed for 1-2 days in Zamboni~s solution. All tissue was subsequently cryoprotected in 2 changes of 30%
sucrose over 2 days. Some of each tissue sample except brain and br~in~tem was imbedded in egg yolk gel and sectioned into 30 um slices on a sliding micloL~"lle for 25 flo~ting section imm~ln~hi~toch~mi~try. Brain and b~ .l were sectioned into 30 um sections by sliding microtome without imbedding in egg yolk gel. Additional W O-97/15671 PCT~US96117998 tissue samples were frozen in Tissue-Tek OCT Compound (Miles Inc, Elkhart, rN), - sectioned on a cryostat, and thaw-mounted onto Superfrost slides (Fisher Scientific7 Pittsburgh, PA~ for antigen blocking studies using adjacent sectinne~

S 5Ø2.2 Egg gel eml~e(l~lin~

Following fixation and cryoprotection all tissues except brain and br~inet~m were embedded in "Egg gel" prior to floating section immnn~histo~h~n~ie~y. Gelatin was p~ cd at 6% and 12%, 2 hours prior to embedding and stored at 37~C to allow 10 bubbles to ~1ieeip~te A layer of 12% gelatin was poured into the mold which was to be used for the embedding and stored flat until hardened. The tissue was soaked in 6%
gelatin at 37~ C for 15 min then l~ rc~l~d and soaked in 12% gelatin just prior to embedding. Chicken eggs were brought to room lclll~cldlul~ before embedding. An egg was cracked and the white dec~nte-l A11 the white was removed by rolling the 1 5 yolk on filter paper and the yolk was mixed with 12% gelatin in a 1: 1 ratio. The tissue was removed from the 12% gelatin and put into the mold onto the gelatin base. The yolk gelatin ~ule was poured over the tissue being careful not to introduce bubbles and cooled in the refrigerator for 15 min. The molds were immersed in cold 4%
paraformaldehyde and refrigerated overnight then incubated at room temperature for 20 24 hours. The tissue block was removed from the mold and floated in 4%
paraform~ yde for several days then floated in 4% paldLo-...aldehyde with 2Q%
sucrose for 2 days.

W O 97/15671 PCT~US96/17998 5Ø2.3T~n~l~ohl~tochemistry Free-floating tissue sections underwent six 10 min washes in 0.05 M PBS, a 20 min inc~lbatiQn in 0.10% (v/v) phenylhydr~ine (F;sher, PilL~ , PA), followed by four additional 10 min washes in 0.05 M KPBS. Tissue sections were then incubate-l in primary antibody diluted 1:160,000 in 0.05M KPBS with 0.4% (v/v) Triton-X 100 for sixty min at 22~ C then for two days at 4~ C. Following incubation the tissues und~v~nL six 10 min washes in 0.05 M KPBS. Tissues were incubated ina solution of biotinylated-Goat anti-Rabbit IgG (Vector #BA1000) diluted to 1:600 in 0.05 M KPBS with Q.4% Triton-X 100 at room temperature for 1 hour then rinsed five times for 10 rnin with 0.05 M KPBS. Avidin and biotin with horseradish peroxidase (HRP, Vector, ABC Elite) was mixed at a ratio of 45 Al avidin with 45 Al biotin in 10 ml 0.05 M KPBS with 0.4% Triton-X 100 and then incllb~t~d for 30 min at room te~ )eldL lre. The tissue was incllb~t~d with the avidin-biotin complex for 1 hour at room t-;,ll~el~Lu.~. Following incubation the tissue was rinsed 3 times for 5 min with 0.05 M KPBS then 3 times for 5 min with 0.175 M sodium acetate. The chromogen used was 2 mg ~ min~ben7~fline (Fluka, Switzerland), 250 mg Nickel (II) sulfate, 8.3 Al of 3% hydrogen peroxide, and 10 ml of 0.175 M sodium acetate. Tissue sectionswere incubated in chromogen for eight to ten min under direct observation. When optimum st~ining was obtained the reactions were stopped with three 5 min rinses in 0.175 M sodium acetate followed by three S min rinses in 0.05 M KPBS. The floating sections were mounted on Superfrost plus slides, counter-stained with neutral red, and dehydrated through a series of alcohol rinses from 50% to 100%. The tissues werecleared with xylene and cover slips mounted with Histomount (Kimberly re3e~cl1, Atlanta, GA).

5Ø2.4 A~.lisel u~ characterization Optimal antiserum concentration for immnnnhi~tQchemical studies was cletermined across a 2-log concentration range. Specificity of st~ining was ~let~rmined CA 02238940 l998-04-22 W 0 97/15671 PCT~US96/17998 by pre-absorbing the santiserum solution with mtermin~l LCRFI 35 at 150 AM or - control solution for 1 hr before antiser 3m was added to the tissue sections. Optimal antiserum dilution for immllnohi~tochemical studies was ~et~rrnin~d by titration of the primary antibody through a series of dilutions ranging from 1:1,000 to 1:320,000.
5.02.4.~ Assays 5Ø2.4.5.1 Protein Assay Protein output in pancreatic juice was measured by ~et~. "~illil.~ optical density at 280 nm of samples diluted in 0.01 M Tris buffer (pH 7.8) and ex~lcssed as mg/30 min using bovine trypsinogen as standard. Fluid output was measured by Hamilton syringe and estim~te~l to the nearest 0.001 ml.

5Ø2.4.5.2 CCK bioass~.y Plasma CCK was ~let~rminPd by a validated bioassay based on amylase release from isolated pancreatic acini. ~he same plel,~dLion was used to test for direct effects of LCRFI 3s on pancreatic acini.

5.1 li'.Y:~mrle 1 LCRF Isolation and Characterization 5.1.1 Isolation Wistar male rats weighing belwt;ell 325-375 grams were fasted overnight Under methoxynuldile anesthesia (Metofane, Pitman-Moore), rats were p~ ,d with WO 97/15671 PCTrUS96/17998 a modified Thiry-Vella fistula of jejunum. The jejunurn was tr~n~ecte<l at two points, 5 cm and 30 cm from the lig~ment of Treitz. The proximal end of the jejunal fistula was closed and a Silastic infusion ç~nn~ was inserted. The distal cut end was brought to the exterior and secured to the peritoneurn and subcutaneous fascia. Gut 5 continuity was reestablished by an end-to-end anastomosis (duodenum to the rem~inin~ jejunum). Rats were allowed 3 days recovery from surgery before collection of int~stin~l secretions began. During recovery and between collections, the Thiry-Vella loop was continl-~lly perfused at 2 ml/hr for ~14 hr/day with anelement~l-type diet (Vital, 0.5 kcal/ml, Ross Laboratories, Columbus, OH). The 10 purpose of the diet infusion was to prevent mucosal atrophy of the isolated loop. The s~nim~l.c were allowed normal rodent chow and water ad libitum after surgery. The surgical procedures are standard techniques and are described in (Guan et al., 1990).

Saline (0.15 M NaCl) was infused at 0.5 mUmin for an hour to wash out any 15 diet remzlining in the lumen of the fistula, followed by saline at 1.0 mVrnin for 5 hours to flush out the i~te~ l secretions CO~ g the intestinal CCK releasing peptide (300 ml of diluted intestin~l secretion per rat per day). The diluted intestinalsecretions (intestin~l washout) were collected on ice and at the end of the collection period (5 hr), the washout was boiled for 10 min~lte~7 cooled, then filtered through 20 Whatman number 4 filter paper. The washout was stored at 5~ C before protein isolation was undertaken..

5.1.2 Purification In a cold room (5~ C), the intestinal washout was filtered through a YM-30 Amicon disc memhr~ne (MW cutoff of 30,000) using a high-output Arnicon stirred cell and then concentr.qt~l 1 00-fold using a YM- 1 Amicon disc membrane (MW
cutoff of 1000). Conce~ tes were stored at -70~C. The concentrated washout was further concentrated and purified by using a chain of Cl8 Sep-Paks (Millipore, Milford, MA). Five Cl8 Sep-Paks (classic model) were linked together using Silastic CA 02238940 l998-04-22 W o 97115671 PCTAUS96/17998 tubing (elution volume ~5 ml). The Sep-Pak chain was conditioned with 100%
ethanol, followed by 0.1% acetic acid. The concentrates (100 ml) were loaded onto the Sep-Pak chain. Subsequently, the chain was washed with 0.1% acetic acid. Theinfestin~l CCK releasing peptide was eluted from the Sep-Pak chain by washing the 5 chain with increasing concentrations of ethanol in 0.1% acetic acid. Ethanol extracts were stored at 5~ C prior to further purification by HPLC.

The conce--LldLed samples were diluted five fold with 0.1% trifluoroacetate and loaded by repeated 4 ml injections onto a Vydac C-18 reverse phase HPLC column 10 equilibrated in 0.1 % trifluoroacetate. After loading, the column was rinsed with 0.1 % trifluoro~çet~te, until the absorbance returned to the value before injection. The sample was then eluted with a gradient to 50% acetonitrile co..t~ g 0.1 %
trifluoroacetate. The absorbances at 220 and 280 nm were monitored, and peaks were collected.
5.1.3 Analysis . .
The HPLC protein-co~ ;,.il-g samples were analyzed by High Performance - Capillary Electrophoresis (HPCE) to assess sample purity. A S ml sarnple was diluted three-fold with 0.1 M sodium phosphate, pH 2, and placed onto a Beckman 9600 High Perform~nce Capillary Electrophoresis apparatus. The sample was run according to the m~nllf~rturers recommenl1c~1 conditions and data analyzed by System Gold Software.

HPCE revealed elution of a single major component (FIG. 3). A co--l;~ L
eluting at 20.7 min was less than 1% the area of the major peak. This cn..t~ ,.t was present in buffer controls and thlls did not represent a component isolated from the intestin~l w~hings The eluted m~tt-ri~l represented a single pure protein.

An aliquot (50 ml) of the HPCE sample was dried under a vacuum . The sample was hydrolyzed with gaseous HCl for 24 hours then dried by vacuum. The hydrolyzed sample was loaded onto an Applied Biosystems automated amino acid analyzer and analyzed in accordance with the m~nllf~ rers recomm~ntl~
5 procedures.

Analysis showed the amino acid composition of LCRF as shown in Table 4 Table 4 ~mino acid No. of residues~nnino acid No. of residues Ala 4 Lys 2 Arg 1 Met 0 Asp 9 Phe 2 Cys N.D. Pro 7 Glu 11 Ser 7 Gly 6 Thr 7 His 1 Trp N.D.
Ile 2 Tyr 2 - Leu 5 Val 3 About 7% ofthe purified LCRF (100 ml) was loaded onto an Applied Biosystems Peptide Sequencer with automatic PTH analysis. Three analyses were perfonneA on two separately purified samples. One sequence analysis gave conclusive residue ~ignment~ up to position 41. The other two sequence analyses 25 gave similar results except residue ~ nment was not conclusive after position 30.
The single letter ~ieCiFn~tion for the amino acid sequence fietermined is as follows:

STFWAYQPDGDNDPTDYQKYEHTSSPSQLI,APGDYPCVIEV (SEQ ID
NO: 1) Several small aliquots of LC~F (5-10 rnl) were injected by eleetrospray onto a Sciex quadrapole mass spectrometer operated in the positive mode. Analysis of LCRF detected one mass ion above background values. The mass of LCRF was measured as 8136.5 daltons, inclie~tin~ that approximately 2/3 ofthe sequenee ofS LCRF has been ~1etç~nined. LCRF has a moleeular size of 8136 daltons + 1%, as det~rrnined by mass speetral analysis. ~sllming an average molecular weight based on the composition analyses, the estim~t~-l number of amino aeid residues is somewhere around 69-73 amino aeid r~ci(l~l~s The amino aeid eomposition of LCRF indieates that it eontains three basic residues that can represent potential trypsin eleavage sites. Sueh sites are eon~i~tent with the observation that the rele~ing faetor is inaetivated by trypsin (Miyasaka et al.
1989).

The ~letermin~cl amino aeid sequenee for the first 2/3 of the LCRF moleeule was eomp~cd to sequenees in a search program that ineludes (l~t~b~es SWISS-PROT, PIR, GenPept, and GenPept. Closest homologies for sequences of 30 or so amino acids was no greater than about 35% while closest homology for shorter sequences of 5 amino acids or more was about 60%.
5.2 Example 2 Biological Activity of LCRF

5.2.1 Bioassays An in vivo bioassay for CCK-relç~cing activity was a modification of the methods described by Miyasaka et al. (1992). Male Wistar rats were ~lepaled withpancreatic, biliary, duodenal and jugular vein c~nm~ In these ~nim~l~, the pancreatie juice was diverted f~om the intestine to pl~ven~ proteolytic inactivation of 30 the infused peptides and taurocholate was infused i.d. to ~U~ iSS the high basal CCK

release caused by diversion of pancreatic juice. Two c~nm~ were inserted into the duodenum for return of bile-pancreatic juice and for infusion of bioactive peptides. A
jugular vein ~~nmll~ was inserted for blood samples for CCK bioassay. During recovery and between ex~ . ;,..ent~, pancreatic juice and bile were collected and S continuously returned to the intestine by a servomech~ni.~m consisting of a collecting tube in a liquid level detector coupled to a peristaltic pump. During experim~nt~, pancreatic juice was collected and 10% of the collected secretion was returned to the duodenum. This partial pancreatic juice return model has the advantage of ms~ p :iu~ ,s:~ion of basal pancreatic secretion, but reduces the threshold for 10 ctimlll~tion by trypsin inhibitors and dietary protein. The rationale for using this in the study of LCRF, 3s was to lower the threshold for stimulation of pancreatic secretion by the peptide, analogous to trypsin inhibitor infusion under the sameconditions.

At 0800 hr on postoperative days 4- 7, rats were fasted and their pancreatic juice was diverted from the duodenum. Three hours later bile was also diverted and 40 mM sodium taurocholate co~ ;r~ g 100 mM sodium bicarbonate was infused intraduodenally at 1 mVhr for 3 hours to establish a stable pancreatic secl~ Loly rate.
Samples were then injected intraduodenally and the L~dllcl~dLic protein and fluid 20 response was calculated by subtracting the output in the last 1 5-min basal collection period by the output in the first 1 5-min collection following the injection of the test solution.

In vitro bioassays based on the ability of LCRF to stim~ te CCK secretion 25 from isolated intestinztl mucosal cells (Bouras et al., Liddle 1995) or FACS-purified cholecystokinin cells (Liddle et al. 1992) were established. The in vitro pL~.dLions responded to CCK releasing factors such as monitor peptide, KCI and LCRF. One orthe other of these in vitro assays were used along with the described in vivo rat bioassay to follow the purification of LCRF from concentrated i ~ l washes. The 30 in vitro assays were used to CO~ the in vivo assays.

CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 To verify that CCK was the hormone stimnl~ting the pancreas in the bioassay, the effect of CCK-lecc~tor blockade (MK-329) on the pancreatic secl-,L~ responses to intr~ denal infusion of partially purified LCRF was determined. Partially 5 purified LCRF was infused intraduodenally as described above and pancreatic protein and fluid secretion ~leterminf~l following i.v. injection of MK-329 or vehicle. Plasma CCK levels were also measured during vehicle injection e~L.;...ent~ to insure that the bioassay was actually measuring the CCK-re1e~ing activity of the plcpdld~ions.

10 5.2.2 Bioactivity of LCRF

Fractions (100-200 rnl) collected from the HPLC of intestin~1 washings as described in Fxample 5. l, were subjected to Speed-Vac evaporation for ~30 mim-tes to remove the acetonitrile. l ml of 0. l % acetic acid was added and the sarnples were l5 then loaded onto a single C18 Sep-Pak. Sep-Paks were washed with 100% ethanolfollowed by 0.1% acetic acid. After loading, l.5 ml of 70% ethanol in 0.1% acetic acid was used for elution. Sample volume was reduced by Speed-Vac to xhllately 100 rnl; l ml of saline was added and pH was adjusted to ~6 to 7 with 0. l N NaOH.
Bioactivity was found in eluents from the Cl8 Sep-Pak chain in both the 40%
and 60% ethanol fractions. Reverse phase HPLC of the 60% ethanol fraction yielded a peak with weak bioactivity, but this peak also contained some hllpuL;Lies. Reverse phase HPLC of the 40% ethanol fraction yielded a single peak with absorbance at 220 25 and 280 nm that was associated with LCRF bioactivity (FIG. 2). Control tubes before and after this peak had no bioactivity. Once the ~let,i., ~ c n and chromatography conditions were ~t~nninecl, every ~ lcpaldlion of LCRF chlolllalographed (n=6) had bioactivity in the same position as shown in FIG. 2. Differences in ~lc~dLions included the amount of LCRF purified and the level of c~ x observed in other 30 regions of the chromatogram.

W O 97/1~671 PCT~US96/17998 The purified ;~ l LCRF was injected inkraduodenally at different doses and the pancreatic protein and fluid secretory response was monitored. 1 mg (n = 5), 2 mg (n = 5), 3 mg (n = 2), and 7 mg (n = 2) of pure LCRF or O. l S M NaCl (n = 5) 5 was slowly injected into the duodenum of the bioassay rats and the changes in pancreatic protein and fluid secretion were monitored. Responses seen with 3 mg and 7 mg were not evaluated statistically due to the small number of injections. Theinjection of 2 mg of the polypeptide significantly increased pancreatic protein and fluid secretion by 3.5-fold and 3. l-fold, respectively, compared to saline. The results, 10 illuskated in FIG. 4, show that the pancreatic secretory response to the purified intestin~l LCRF is dose-related and biphasic, with the highest dose (7 mg) ç~llcing a sllhst~nti~lly lower re:j~u~ se than the m~im~lly-effective dose (3 mg).

~ltr. ,-~lively, concellLd~d samples cl---ls-;--i--~ partially purified LCRF were 15 subjected to Sephadex gel filkration chromatography. The gel filkation increased the specific bioactivity 1 0û-fold compared to samples obtained after chain Sep-Pak separation. In vivo and in vitro bioassays of this partially purified ~.~p~ud~ion were cont1nete-1 as described above. One ml of blood was withdrawn 15 minllt~c after the injections of LCRF for plasma CCK tlt;l ~ tions. Plasma CCK was measured by 20 bioassay as described by Liddle et a~. (1984). LCRF injections were repeated in the presence of MK-329 (0.5 mg/kg i.v. bolus), a specific CCK-A receptor antagonist ~provided by Dr. Victor J. Lotti, Merck Sharp & Dohme, West Point, PA). MK-329 was dissolved in DMSO:Tween 80:saline (1:1:3) and injected i.v. 1 hr before the injection of the partially purified LCRF.
The effect of an intraduodenal infusion of partially purified LCRF on plasma CCK levels and on ~lCl~,dlic protein secretion was det~rmin~l Two hundred mg of LCRF in 1 ml of 0.15 M NaCl or the NaCl alone was slowly injected (~ one minute)into the duodenum ofthe bioassay rats. One ml of blood was withdrawn 15 mimltes 30 after the injections. LCRF injections were repeated the following day during CCK-A

CA 02238940 l998-04-22 W 0 97/15671 PCTÇUS96/17998 receptor blockade with MK-329. As shown in FIG. 1, LCRF had an effect that significantly increased plasma CCK levels 4.8-fold col,lp~ued to saline (0.15 M NaCl).
The increment~l pancreatic protein and fluid responses to LCRF were 4.2-fold and2.6-fold higher, respectively, than those seen with the infusion of saline. MK-329 S completely abolished the pancreatic secretory response to partially purified LCRF.
These results provided strong evidence that the factor being purified is a cholecystokinin-releasing peptide, and that the pancreatic secretory responses observed with the bioassay are due to the release of CCK.

10 5.3 Example 3 Immunoaffinity Experiments To confirm that the amino acid sequence reported was in fact that of a CCK-rele~eing peptide, immnn~ffinity chromatography studies were done to selectively15 remove LCRF bioactivity from i~lr~ l washes. These studies dete-min~l that the sequence attributed to LCRF was not that of protein cont~min~nt Polyclonal antibodies raised against several synthetic LCRF fr~gm~nt.e was found to specifically bind to LCRF and to block LCRF activity, thus confit min~ that the sequence - cletetmine~l was that of a CCK-releasing peptide.
Antisera were raised by standard methods in rabbits to synthetic LCRF (N-t~rrnin~l hex~Lide at positions 1-6 of SEQ ID NO:l) conjugated to KLH. This antisera (LCRF-Ab) or normal rabbit serum (NRS, control), was coupled to Bio-RadAf~l-Gel 10 gel. A LCRF sample obtained from ultrafiltration of rat intPstin~l washes 25 was applied to the NRS-coupled gel and to the LCRF-Ab-coupled gel and incubated overnight at 4~ C. After 16 hr each gel was tr~n~ferred to a column support and the unbound material was eluted from the column with l M NaCl (Elution Step 1).
Subsequently, 20 mM HCl was applied to each column with the objective of elutingthe material bound to the antibody by disrupting the antibody-antigen interaction (Elution Step 2). Eluents from Step 1 and Step 2 were concentrated using C-18 Sep-W O 97/15671 PCT~US96/17998 Paks and speed-vac. Eluents were assayed for CCK-releasing activity by stim~ tion of pancreatic protein secretion in conscious rats. The antisera was also found to selectively bind to some cells and tissues such as the small in~stin~, stomach, pancreas, nodose ganglion and brain.

Incubation of partially purified LCRF with the antiserum-coupled gel (Effluent from LCRF-Ab Column) significantly decreased the bioactivity of the m~tPri~l recovered offthe gel. LCRF was incubated overnight with an immlln~-~ffinity gel (Bio-Rad Affi-gel 10) to which either(LCRFl 6 antiserum (LCRFAb) or normal rabbit 10 serum (NRS) was coupled. On the following day, unbound mslt~ l was eluted from the column supports and assayed for LCRF bioactivity (pancreatic protein secretion).
The gel coupled to LCRF~ 6 antibody a~pdle~,Lly bound LCRF as indicated by significantly reduced bioactivity eluting from the column, cc,l~ d to NRS-coupled gel. The control was an equivalent arnount of partially purified LCRF ~ lion 15 which was not applied to affinity gels. In contrast, incubation with the normal rabbit serum-coupled gel (Fmn~nt from NRS Column) did not significantly affect the bioactivity of the material recovered off that gel. The results are illustrated in FIG. 5.
When the antibody-antigen interactions on the gels were disrupted and the gels were - eluted, significant amounts of LCRF bioactivity eluted from the antiserum-coupled 20 gel, but no LCRF bioactivity eluted from the NRS-coupled gel (results not shown).

Antisera to two different portions of the LCRF molecule were raised in rabbits. These antibodies were shown to neutralize the CCK-releasing effect of LCRF
in vivo. Rat Brain, nodose g~ngli~t, stomach, pancreas, duodenutn and adrenal were 25 prepared and sliced for immlm~histochemi~try Optimal antiserum concentration for immunohistochemical studies was detPrrnine-1 across a 2-log concenkation range.
Specificity of st~ining w~ ~letermined by pre-absorbing the antiserum solution with the specific LCRF antigen or nothing for 1 hr before antiserum was added to the tissue sections. Binding was localized using an avidin-biotin complex-horse radish W O 97/lS671 PCT~US96/17998 peroxidase secondary antibody system with nicke~ min~ben7~line chromogen.
Sections were counter-stained and analyzed by light microscopy.

~ Concentration-dependent and antigen-specific staining was identified in both 5 the duodenum and pancreas. Staining was observed in the lllyt;~ fic and submucosal plexus of t_e duodenum and stomach. St~ining was also identified in nerve fibersthroughout the pancreas, sensory fibers and cell bodies of the nodose ~npli~, and sympathetic nerve fibers in the adrenal medulla. The: immnno-histochemic~l evidence suggested that LCRF is a n~ulop~,~lide that may have several functions in 10 the gastrointestinal system and other systems.

The specificity of the binding was demonstrated by progle.,siv~ loss of binding with serial dilution, by the absence of staining with n~ llspe~iirlc rabbit ~Ihll~
antibody, and by blocking of the binding with the specific antigen used to; ~ P
the rabbits (FIGS. 20B, 21B, 22B and 23B)). Tmm-lnohistoch~mic~l st~ininp. of adjacent section with antiserum to LCRF-; 6 and LCRF7 23 in each of the tissue types demonstrated identical staining patterns, although the antiserum to LCRF7 23 wassuperior to the amin~ 1 antiserum for immlln-~hi.ctochemi~try. These data suggested that the immllnohistochemiç~l staining used for loç~l i7~tic n accurately 20 reflects LCRF distribution in vivo.

5.3.1 LCRF loc~li7.~t;~n in the upper i,ulcsLi~e and pancreas LCRF immunoreactivity was identified in nerve fibers within the proximal 25 two-thirds of the small int~ostin~l villi and in enterocytes at the tips of the villi (FIG.
20A and FIG. 20B). Lon~ in~l and cross-sectional views of the enterocytes demonstrate LCRF immunoreativity (LCRF-IR~ within discrete circular ~ cLul~s in the cytoplasm and fibers. Luminal mucus strands contain LCRF-IR but were incompletely blocked with preabsorbed antiserum. Although LCRF-IR mucus strands W O 97/lS671 PCTrUS96/17998 appeared to extend from the distal villi, goblet cells were LCRF-IR negative.
Enteroendocrine cells were also LCRF-IR negative.

Nerve fibers and nerve cell bodies in the llly~llL~;lic plexus and submucosal S neurons ofthe duodenum contain LCRF-IR (FIG. 21A and FIG. 21B). Nerve fibers e~rt.Qn-ling into the villi were traced to the subm~c oe~ neurons in some in~t~nc~e~
although the origin of most fibers could not be clet~rrnin~-1 LCRF-IR in the stomach was i~lPntified in nerve fibers and nerve cell bodies in 10 the myenteric and subm-l~os~l plexus. Enterocytes within the gastroesophagealjunction also displayed LCRF-IR. In addition, a nurnber of large LCRF-IR nerves coursed along the serosal surface of the stomach antrum. Large LCRF-IR nerve fibers appear to run through the pancreas, and are especially prominent in the interlobular connective tissue. Small immunoreactive nerves were occasionally seen around the15 periphery of the islets of Langerhans but these were not always observed.

5.3.2 LCRF immunoreactivity in the autonomic nervous system and brain.

- The para~y~ dLhentic nervous system was investig~tçd through evaluation of 20 the nodose ganglia with the adjacent vagus, and blci~n~LellL sections cont~ining the dorsal motor nucleus of the vagus and the nucleus ambiguous. Nerve cells bodies in the nodose ganglia and vagal fibers are LCRF-IR positive ~FIG. 22A and FIG. 22B), whereas the motor neurons in the brain stem are LCRF-IR negative. Thus, only thesensory arm of the vagus contains LCRF-IR.
The adrenal gland was used to screen nerves of the ~y~ tic nervous system. Cells of the adrenal medulla showed weak I,CRF-IR st~ining as well as distinct staining of ~yll~lhetic nerve fibers (FIG. 23A and FIG. 23B). However, no LCRF-IR perivascular ~yll~ lhetic fibers were observed in the adrenal gland, 30 int~stine or other tissues.

W O 97/15671 PCTrUS96/17998 The central nervous system was evaluated using regularly spaced sagittal sections covering the entire brain. No LCRF-IR was identified in the central nervous system. Thus, LCRF-IR localizes to nerves of the enteric nervous system, the sensory S arm of the vagus, and symp~thetic fibers of the adrenal gland.

5.4 F,Y~r1~1e4 Molecular Cloning of LCRF

The ~ ;on of the major portion of the LCRF amino acid sequence allows the relatively straightforward cloning of the encoding DNA~ using degenerate primers to probe an a~ru~liate DNA library. The length of the primer is generally a matter of cho;ce but will conveniently be on the order of 15-25 base pairs and could be up to the full length of the determined 41 amino acid sequence. Degenerate 15 primers synth~ei7~,cl from the sequenced N-~r~ l amino acids of the peptide will be used to produce, by RT-PCRTM~ a cDNA encoding that segment of LCRF. Once the cDNA is sequenced, primers generated from 3'-end of the cDNA sequence will be used as S'-primer, along with oligo(dT)l6 as 3'-primer, to RACE both ends of the- transcript in order to produce an intact full-length cDNA of LCRF.
Rapid amplification of cDNA end (RACE) The 3'-end of LCRF cDNA will be amplified in a 100 ml reaction Inixl~ue cont~ining 10 mM Tris-HCl (pH 8.4; at 23~C), 1.5 mM MgCl2, 40 mM KCl, 200 rnM
25 of each dNTP, 1 rnM each of a primer from the middle of the peptide already sequenced, 2 ml oligo(dT)I6, and 2 U Taq DNA polymerase. Thirty cycles of amplification will be carried out with denaturation at 94~C for 1 min, ~nn~-~ling at 40~C for 1 min., and extension at 72~C for 1 min, followed by an additional extension at 72~C for 20 min.

W O 97/15671 PCT~US96/17998 To ensure that the 5'-end of the LCRF transcript is fully sequenced, the latter will be reverse transcribed using the P3-primer. The ~ n~le~l primer will be tailed with poly A in a 20 ml reaction llfi~lule CO,.~ .g 50 rnM potassium cacodylate, 2 rnM CoCl2, 200 mM DTT, 200 mM dATP, and 10 U t~rrnin~l deoxynucleotidetidyl 5 transferase. The ext~n(le~l primer will be used as template and amplified as for the 3'-end described above, except that primers and first cDNA will be substituted by 0.2 mM oligo(dT)I6 primer, 0.5 mM of a specific primer obtained from the sequenced 123-bp cDNA, and 2ml of the tailed first strand cDNA. Finally, the overlapping 3'-and 4'-end RACE products will be combined to produce an intact full-length cDNA of 10 LCRF.

Cloning and Sequencillg PCRTM product will be purified and cloned into pVZI plasmid vector via the 15 TA cloning method from Invitrogen. The nucleotide sequences will be clet~nined by the dideoxynucleotide chain t~rrnin~tion method, using [a-3sS]dATP and the sequenase kit. An ~lt~rn~tive to PCRTM cloning would be a traditional plaque hybridization using a probe based on the known amino acid sequence of LCRF and acDNA library such as obtained from pancreas or brain cells. Once having the full-20 length cDNA encoding LCRF, the LCRF cDNA will be used to obtain the humanversion of this peptide. A h unan version of LCRF expected to be homologous to the rat LCRF would also be obtainable by analogous procedures.

The DNA sequences disclosed in the invention allow for the pr~a~ion of 2~ relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to Icr gene sequences by ~l~cuing nucleic acid probes of an ~ropliate length. Such probes are typically prepared based on the consideràtion of the defined gene sequence of the LCRF gene or derived from fl~nking regions of this gene.

W 0 97tlS671 PCTrUS96/17998 In order to clone the gene that encodes LCRF, two complement~ry strategies are contemplated. One approach has been to use the peptide sequence of SEQ ID
NO: 1 to design oligonucleotide primers for use in direct cloning by PCRTM
~polymerase chain reaction). In a second approach, serological reagents will be used 5 to screen a cDNA library to identify the sequence with immlmc reactivity. These two approaches are complPment~y, but are expected to identify the same DNA or RNA
sequence.

Oligonucleotide Approach From the 41 arnino acid sequence determined for the arnino te~ lllC of LCRF, the rnRNA sequence was predicted and the least degenerate regions were chosen. Six diLrci~elll oligonucleotide prirners (from 4 regions) were generated; their sequences and positions as shown.
STFWAYQPDGDNDPTDYQKYEHTSSPSQLLAPGDYPCVIEV (SEQ ID
NO:l) <
lcrf-5 lcr~-p lcrf-p2 lcrf-3' The sequences of the lcrf oligonucleotides are:

lcrf-5 (inosine) S'-TT(T/C) TGG GCI TA(T/C) CA(A/G) CCI GA(T/C) GG (SEQ ID NO: 4) lcrf-5 (degenerate) 5~-TT(T/C) TGG GC(A/C/T) CA(A/G) CC(A/C/T) GA(T/C) GG (SEQ ID NO: 5) W O 97/1~671 PCT~US96/17998 lcrf-p 5'-GA(T/C) AA(C/T) GA(T/C) CCI ACI GA(C/T) TA(T/C) CA (SEQ ID NO: 6) lcrf-p2 ~'-GT(A/G) TG(T/C) TC(A/G) TA(C/T) l~CI/C) TG
SEQ ID NO: 7 lcrf-3' (inosine) 5'-TCI ATI AC(A/G) CAI GG(A/G) TA(A/G) TCI CC
SEQ ID NO: 8 lcrf-3' (degenerate) 5'-TC(T/G/A) AT(C/G) AC(A/G) CA(T/A/G) GG(A/G) GG(A/G) TA(A/G) TCN CC
SEQ ID NO. 9 ~ - For each of the outermost oligonucleotides, two dirr~.e.l~ versions were - gen~,dLed, one in which the dege~ dl~ positions were filled with inosine and the other in which they contained the d~ iate mixture of nucleotides. In general, the LCRF-~' and LCRF-3' oligonucleotides were tiesigned to serve as primers in PCRTM, while the internal oligonucleotides were to be used rrim~rily as probes or if necessary, nested rrim~r~:.

In order to clone the LCRF coding sequence, RNA was ~i~alcd from several rat tissues, including int~stine, brain, pancreas, stomach, and nodose ~nglisl These RNAs were converted to cDNA for use in reverse tr~n~l.rirtase-coupled polymerasec_ain reaction (RT-PCR~); all were shown to be intact using an HPRT (hypox~nthine phosphoribosyl L~ r~.dse) control PCRTM. Standard PCRTM is employed. In addition, since the primers are highly deg~n~r~fe, step-down PCRTM is also lltili7.~or1 WO 97/15671 PCT~US96/17998 In addition, high molecular weight genomic DNA was isolated from rat liver for use in standard PCRTM arnplifications. Several PCRTM products have been obtained and cloned into a pUC for analysis. Next, step-down PCR~M will be used to 5 increase specificity with the DNA PCRTM reactions.

Serological Approach Prior to g~;nc.dLing an expression library, it was nece~y to identify a good 10 source of RNA which is likely to contain the LCRF mRNA sequence. In addition,one of more anti-LCR~ antibodies that could recognize delldLuled peptide were required. Thus, to address both issues, Western blots were pl~ed using protein extracts from several dir~ l sources. The protein blots were then inc~lb~te~l individually with 4 different antisera. In the pancreas extract; all 4 antisera ~let~cterl a 15 band of the same size ~20 kD. Thus, a cDNA ~,c;s~ion library will be constructed from p~l.;leas rnRNA and screened directly with the polyclonal anti-LCRF reagents.
The cDNAs detected will be sequenced to ensure that they contain the a~>~ru~liate coding information.

The identified LCRF cDNA will be used to clone the full-length cDNA from both rat and human cDNA libraries. The cDNAs will be cloned into G~?.e3~ion vectors in order to produce large arnounts of LCRF for physiological analysis. In addition, the LCRF gene will be cloned from human and mouse genomic libraries tofilrther define its regulatory actions. The inventors further contemplate using the murine gene to generate a knock-out mouse deficient for LCRF for use in assessing the biological role of this peptide.

CA 02238940 l998-04-22 W O97/15671 PCT~US96/17998 ~;.!; EYample ~;
Methods of utilizing the effect of LCRF on CCK Release LCRF ~imini~tration is superior to CCK or CCK agonists. This is because S LCRF releases endogenous cholecystc-kinin, which is predominately CCK-58 in blood of hnmzln~ and dogs. CCK-58 is too large a molecule to syntheci7~ economically for ph~ relltical purposes. However, CCK-58 released by LCRF would be preferable to the form of CCK a~pluvcd for medical use, i. e., injected CCK-8, because the former has a longer half-life and preferable receptor binding characteri~tics conlp~.cd 10 to CCK-8. Likewise, potential CCK agonists, peptide as well as non-peptide, would be less physiological than endogenous CCK.

The activity of LCRF inc1 j~ ~tec its utility in controlling CCK release and thus providing tre~t~nent methods for several conditions in which CCK is involved in a 15 regulatory capacity. LCRF and trlmc~te~ forms and active variants may be synthlosi7t-d by standard techniques and their ability to release CCK det~rmin~i in vitro and in vivo. In vitro methods are based on the ability of LCRF active peptides to release CCK from dispersed intestinal mucosal cells or from STC-l cells, a tumor cell line that secretes CCK in response to CCK-releasing peptides such as monitor peptide, 20 bombesin, as well as LCRF. ~n vivo methods include intraduodenal or intragastric or i~llldvcllous infusion of LCRFs.

S.S.l. Oral Pharmaceutical Composiffons Forms in which LCRF may be ~lmini~tered orally LCRF is a polypeptide, like insulin, so it is subject to digestion in the stomach, by acid/pepsin, and in the small int~tine by pancreatic proteases. But, unlike insulin (and CCK itseli9, LCRF ~c~u~ably acts on .ecc~lol~ on the luminal side of mucosal cells (CCK-releasing cells) so doesn't have to be absorbed. Insulin would have to be 30 absorbed intact to reach cellular receptors, and this is improbable. This makes LCRF

W O 97/lS671 PCT~US96/17998 unique as a regulatory peptide, and makes oral delivery practical whereas for other regulatory peptides (growth hormone, insulin, etc. oral ~imini~tration is impractical.

A~lmini~tration of LCRF orally would be practical in a mllltitucle of forms.
S The compound is heat stable (~UlViVC;~ boiling for 10 min, and survives incubation at 37~ for 24 hours, with loss of about 20% activity). It is water soluble, and effective at very low concentrations, such as 0.08 mg/kg body weight in the adult rat, given intraduodenally to stimlll~te CCK release, or 0.15 mg/kg to :iu~p~,s~ food intake in neonatal rats, ~lrnini~ered intragast$ically. Thus as little as 10 mg may effective be 10 orally in a 70 kg human.

l~orms in which LCRF can be ~ ~lministered orally:

Powder: As the pure peptide, mixed in a powder vehicle such as dry milk, dry 15 cocoa, sugar, which ~ e could then be dissolved in water or other suitable liquid vehicle. In this form, the peptide would be unprotected from gastric or intPctin~l digestion, as in neonatal rats, and therefore the dose would be expected to be in the range of 10 mg/kg. Although ~-imini~tration of LCRF orally without additional - efforts to prevent losses due to inactivation in stomach and inte~stine may seem 20 inefficient, it is not an illlpOl l~l~ barrier to successful treatment since it can be overcome by simply increasing the dose. This is not dangerous because the excess(wasted) peptide is simply digested like any other protein in the diet.

Such powdered forms would be taken in advance of a meal, to take advantage 25 of the "pre-load" phenomenon, in which giving a small meal 10 or 20 min before a regular meal can markedly reduce the amount of the meal co~ .~, .. "~

Capsule: LCRF can be ~mini~tered in a capsule such that it can be taken with a meal or before a meal. This would be convenient, whether or not the capsule is30 coated to resist digestion in the stomach and intestine.

W 0.97/15671 PCTAUS96/17998 Enteric coated pl~dLions: To reduce the dose of LCRF nPe~le~ a.dLions of LCRF can be in enteric coated capsules, or enteric coated. This technology has been in widespread use in the oral ?~riminietration of pancreatic enzyme supplements.
5 The pr~ions permit the en~rs~ ted plcpd,dLion to survive gastric digestive processes, releasing their conlel.L~ in the non-acid pH envi~ llent of the intestine.

Protease inhibitor ~.~a dLions: Oral protease inhibitors stim~ te CCK
release by protecting endogenous LCRF or other endogenous luminal CCK-releasing 10 peptides, according to the hypothesis of Miyasaka et al (1992). Thus, it is logical to con~i-ler mixing protease inhibitors, such as POT II, ie., potato pl.,~ase inhibitor II, with LCRF to make a plcl)dldLion that enhances the efficacy of LCRF by protecting it from digestion in the small i..l~ e. POT II (U.S. Pat. No. 5,468,727, the entire disclosure of which is incorporated by reference), stimlll~tes CCK release and inhibits 15 gastric ~ Lyillg in hllm~n~.

In hllm~n~ these effects presumably occur by plo~ Lillg an endogenous human versions of LCRF. ~hus, POT II could be made into a formulation which included - synthetic LCRF and incorporated into a capsule of microencapsulated for protection 20 from gastric acid/pepsin, and this formulation would be expected to survive both gastric and intestin~l protease digestive barriers and deliver nearly 100% of the ingested dose of LCRF to the a~ oL,.iate receptors on the intestin~l mucosa. With such a pL~LdLion, we predict that as little as 1 mg/70 kg of LCRF would be highly effective in .stimlll~tin~ CCK release in hllm~n.~, to effect in~ g satiety values for 25 foods taken prior to or with the LCRF p-c;~dLion~ to slow gastric elll~Lyillg and thereby slow glucose absorption and uptake, ameliorating postprandial hyper- andhypo-glycemia and hy~ lin~mi~ more complete elll~Lyillg of the gallbladder to reduce likelihood of stone forrnation, improved functioning of the gastro-colic reflex which promotes reflexive bowel movement and defecation after a meal.

W O 97/15671 PCTnUS96/17998 _99_ 5.5.1.1. I~ .v~ous Pharmaceutical Compositions -LCRF1-35 infused intravenously was as effective and potent as when given intr~chlo-lenzlly (FIG. 8B). This indicates that i.v. LCRF stimlllzlt~s CCK release, 5 because LCRF does not stimulate the pancreas directly as inflic~t~-l by its lack of effect on amylase release from isolated pancreatic acini. Because i.v. z~lminiet~red LCRF can stimulate CCK release, the i.v. route of ~rlministration may be useful in some situations and be superior to i.v. infusion of CCK itse}f, for the reasons described above, because LCE~F stimulates the release of endogenous, natural cholecystokinin The situations in which i.v. rather than oral z~1ministration might be w~ d are in patients in which the oral route is impractical or difficult, such as in patients (adults and children) l'eCc;ivillg i~ avellous feedings because of bowel surgery or 15 bowel dysfunction. They frequently develop gallstones because of lack of ~timulztfion of the gallbladder, and this can be prevented by intravenous z~lminietration of CCK-8.
.
For intravenous z~-iminisfration, LCRF could be supplied in sterile vials for injection or for drip infusion. Based on animal studies, the dose rate for human inlldVellOUS infusion 20 would be expected to be in the range of 0.1-l.0 ~g/kg body weight/hr. This is less than for oral route because there is no digestive enzyme inactivation of the peptide infused intravenously.

5.5.2 Control of Tn~--lin Secretion LCRF compositions are contemplated to be useful for the stimulz~tiQn of insulin secretion. CCK has been ~1~monctrated to potentiate amino acid-infl~ e~
insulin secretion in humz nc. Therefore, in conditions in which insulin secretion is deficient, such as type I or II diabetes mellihlc, CCK may be useful, and therefore a WO 97/1~671 PCTAUS96/17998 CCK-releasing peptide that is orally active, such as LCRF, will be valuable. In this case, LCRF may be a~lmini~tered orally in compositions as described above.

In early stages of type II diabetes, insulin secretion is in excess due to insulin 5 insensitivity. It is considered desirable to reduce hy~c~ linernia in type II diabetes, and it has been shown that endogenous and exogenous CCK in hlm~n~ can reduce hyperineulenim~ by slowing the emptying of carbohydrate from the stom~ h 5.5.3 Regulation of Gastric Emptying Gastric emptying in h~lm~n~ is regulated by CCK, and that both CCK and trypsin inhibitors slow gastric cunpLyillg in diabetic patients who have abnormally rapid gastric e~ yillg. This is important because rapid gastric clll~lyillg is now recognized as a symptom of early ~ betes~ and it exacerbates postprandial 15 hyperglycemia and hyperin~--tinf mi~

Diabetic subjects, both type I (insulin-dependent) and type II (adult onset, non-insulin dependent), would benefit from LCRF by taking it prior to and with high - carbohydrate meals, as this type of meal empties the fastest in such subjects. For 20 example, a diabetic subject may take LCRF as a pre-load in a liquid vehicle 10-20 minutes prior to a meal to slow the gastric c~ lyillg of the subsequent meal. This would also be expected to reduce food intake, as gastric rlist~nti~n is an important factor in satiety. If a high carbohydrate, high calorie beverage is being consumed, it would be rec~-mmen-led that LCRF, as a powder, be mixed in with the beverage to 25 slow its cnl~yillg from the stomach and enhance its satiety value.

5.5.4 Reduction in Gallbladder Stasis ~increased gallbladder emptying) Gallbladder stasis is a completion of ~iimini~h~d food, especially fat, in the 30 intestine, as in people on weight reduction diets, and absence of food in the intestine, dS in patients on total p~c~,hldl nutrition. This leads to gallstones in many cases. In the former case, subjects on low fat, low calorie weight reduction regimens would be advised to take LCRF prior to each meal, to enhance the ability of that meal to release CCK and thereby more fully contract the gallbladder. More frequent contraction of the gallbladder by exogenous CCK is known to prevent g~ tQnPs in susceptible S subjects, and it would therefore be expected that LCRF taken orally would do likewise.

5.5.5 Appetite Suppression and Control of Food Intake.

To test the ability of LCRF to induce satiety and reduce food consumption, a recognized ~ hllental design for testing the effect of endogenous CCK on food intake was employed. In this procedure, young rats approxim~t~ly 12 days old were removed from their nest and weighed. They were then rapidly infused intragastrically with 1 ml of isotonic saline (control) or LCRFI 3s in saline. They were then re 15 weighed and housed in a groups at 33~ C. Ten ~ es later they were transferred to individual cont~iner~s at room ltlllpC~dLu~e and allowed access to 4 ml of milk diet ~colllnleLcial half and hal~ for 30 min. After the test, rats were dried and weighed, and the mil intake was expressed as the percent of body weight gained during the test (%BWG) Two separate studies were carried out with separate sets of rats, but using 20 the same pl~udLion of LCRFl 35.

W O 97/15671 PCT~US96/17998 TABL~: 5 N ~n SD ~in 0.0 (saline) 11 1.5136 0.81426 0.39 3.17 1.438 0.88547 0.10 2.80 3.0 ~lg LCRF 11 1.18818 0.76448 0.46 2.39 0.0 (saline) 7 1.181857 0.53909 1.2 2.79 1.5 ~g LCRF 8 1.63375 0.73455 0.54 2.50 3.0 ,~Lg LCRF 8 1.39500 0.58756 0.84 2.32 Linear re~lession analysis using the SAS statistical analysis system was used S to evaluate the dose effect of LCRFI 3s on food intake. The data showed: (1) lack of fit: the lack of fit from the liner trend was not signifirs7nt ~p= >0.30); (2) the rate of decrease for each ~Lg of dose was 0.11% BWG, and 0.14 % BWG. The linear trend for decreasing food intake is found to be highly significant, in both c;~c.; ...en~, with p<O.OOl. These ~ nt~ establish in a ms7mms71is7n model that LCRF acts as a 10 satiety agent at very low doses to reduce food intake.

Use of LCRF for reduction of food intake in hllms7n.~. LCRF is expected to reduce food intake in the above c;~ hllc~ because previous studies in hl-msln~
showed that soybean trypsin inhibitor :ju~ ssed food intake. It has been proposed 15 t_at LCRF mediates the s1iml7ls7tion of CCK release by trypsin inhibitor. Because oral trypsin inhibitors also increase CCK release in hnms7n~ and reduce food intake in hllmsln~, it is expected that LCRF will stimlllSlt~ CCK release and reduce food intake in hllmsln~

LCRF, incorporated into the compositions described previously for oral delivery, would be taken prior to a meal to induce and Slu~m~nt the "pre-load"
phenomenon ~at helps reduce food intake normally. It would be expected that the LCRF p~ ion would be taken prior to each large meal, and prior to or with highlycalorie-rich liquid beverages, e.g, cola beverages. ~imllm induction of the satiety actions of LCRF would be achieved by taking LCRF 10-20 min~ltes prior to a meal,- and once again just prior to or with the meal. The dosage of LCR~ would depend on the form taken, e.g., enteric coated or as a powder. LCRF would not be taken in-between meals, as it acts to ~llgrnent the satiety value of foods, but may not have less satiety actions if given alone.

5.6 Example 6 LCRF Variants and fr~gments have been previously described. Several of the variants and truncated species have been R~sesse~l and found to have biological activity. Examples include, but are not limited to LCRFI~, LCR~I 35, LCRF7 23, LCR~I 37 and LCRFI 3s, Lys~ala at position 19~.
5.6.1 LCRFl 35 Bioactivity TheN-te....i..l~c sequence of LCRF including amino acids 1-35 was synthPsi7~l The peptide significantly stimulated pancreatic protein and fluid 20 secretion in conscious rats when infused either intravenously or intraduodenally.
Tntr~ lc denal infusion significantly s1im~ tec~ increased plasma CCK concentration but had no effect on amylase release from pancreatic acini. The CCKA-receptor antagonist MK329 abolished the pancreatic stimlll~tory activity. Under similar conditions, DBI 1-86 and DBI 33-50 did not significantly stim~ t~ pancreatic 25 secretion. Trypsin-digestion abolished the CCK-re~easing activity of LCRFl 3s.

W O 97/15671 PCT~US96/17998 5.6.1.2 Pancreatic secretoly response to intraduodenal infusion of Monitor Peptide and native purified LCRF

The dose/response relationships between increment~l protein and fluid output in rats infused with recombinant moni$or peptide and native LCRF are illustrated in FIG. 6A and 6B. Monitor peptide and native LCRF ~ignific:~ntly stim~ t~A
pancreatic protein and fiuid secretion at doses of 1-2 ~lg, respectively, with fluid output closely paralleling protein output. Both peptides exhibited supr~mz~imz~linhibition at higher doses in this mode.
5.6.1.3 Pancreatic secretory response to intraduodenal infusion of LCRFl 35 The dose/relationships between incr~ment~l pa~lcre~Lic protein and fluid output with LCRFI 35 and LCRFI 6 (as control) are illu~trzltçd in FIG. 7A and 7B. LCRFl 3s significantly stimlll~tecl protein secretion at doses from O.l to 0.5 llg/rat, with peak response at O. l ,ug. Fluid output followed a similar dose response curve. LCRF 1-6 did not stimlll~te pancreatic protein or fluid secretion.

5.6.1.4 Comparison between intravenous vs. intraduodenao routes for stimulation of pancreatic secretion by LCRFl 35 FIG. 8A and 8B illustrates the co~ ~ison between i.v. vs. i.d. routes of sl~mini~tration of LCRFl 3s. The dose-response curve was quite similar via both routes, with peak response occllrring at the same dose, 0.1 llg, via either route. These 25 results in~lic~te LCRFl 3s infused i~llldv~ ously may have access to CCK secreting cells of the small intestin~, since other results, described below, show that LCRFI 35 does not stiml~l~te pancreatic secretion directly.

CA 02238940 l998-04-22 W O97/15671 PCTrUS96/17998 5.6.1.~; Pancreatic secretory response to various subfragments of LCRFI 3s To ~lct~rmine the minim~l fragment of LCRF possç~cing CCK-releasing - activity, several fr~ments within the sequence of LCRFI 35 were synth~si7~1 and tested, using the "bioassay" model. As illustrated in FIG. 9, only fragment LCRFll 25 significantly stim~ ted pancreatic protein secretion, with increased potency butdecreased efficacy compared to LCRFl 3s.

~.6.1.6 Pancreatic secretory response to intraduodenal infusion of diazepam binding inhibitor ~DBI) and DBI fragment and GRP

These studies were carried out in the "bioassay model", described in Example 2. Across a wide dose range (FIG. 1 OA and 1 OB), none of the peptides significantly stim~ tçd p~lclG~lic protein or fluid secretion, under con-lition~ in which LCRF1 3s 15 and native LCRF strongly stimulated pancreatic secretion. This result in~liç~tçs that the peptide, diazepam binding inhibitor, reported to be a CCK-rele~in~ peptide in fhe rat by Herzig, et al. (1995), does not stimlllate CCK release in conscious rats fully recovered from surgery. These results indicate that DBI does not me~ t~ feedbackregulation of CCK release in the rat, contrary to claims by Herzig, et al.
5.6.1.7 Effect of CCK I ~iC~ lor blockade on the pancreatic secretory response to intraduodenal LCRFI 35 and effect of inl~ ' ~denal LCRFI 3s on plasma CCK conc~ lion These studies were carried out in a physiological model, z.e., with bile and pancreatic juice returned to the intestine. FIG. 1 lA and 1 lB show the time course of pancreatic protein and fluid secretion during continuous intr~duodçn~l infusion of 25 ~lg of LCRFI 3s and saline control for 2 hours, and the effect of the CCK rec~k,r antagonist MK329 on the response to LCRF~ 3s. LCRFI 35 significantly stim~ t~l pancreatic fluid and protein secretion, compared to basal, and this response wasabolished by MK329. The incrçment~l pancreatic protein and fluid responses are W O 97/15671 PC~US96/17998 illustrated in FIG. 12A and 12B. FIG. 13 illu~ es the plasma CCK responses in the same experim~ntc, determined on blood samples withdrawn 60 ~ eS after the start of infusion of the test compounds. LCRFl 35 ~i nific~ntly increased plasma CCK
concentration compared to basal levels with NaCl or LCRFI.6. Basal levels of plasma S CCK were higher than previously reported in rates with 100% of pancreatic juice returned to the int~stine, possibly because partial return of pdn~lea~ic juice does not completely ~U~lCSs ~ol~ eous secretion of CCK under these conditions. The results illustrated in FIGS. NO. 11-13 strongly in~ te that the stim~ tion of pancreatic secretion by LCRFI 3s is mP~ tecl by release of CCK.
5.6.1.8 Effect of tryptic digestion of LCRFI 3s Oll CCK-rele~cin~ activib FIG. 14 illustrates the effect of incubation of LCRFI 3s with purified bovine trypsin (lmg/ml) at 37~ C for 24 hours. Control LCRF in(1ir~tt-s LCRFl 3s incubated under the same conditions but without trypsin. Trypsin Control consisted of a solution of trypsin inc lh~t~l under the sarne conditions but without LCRFI 3s.
Tryptic digestion completely abolished the pancreatic secretory response to LCRFI 3s.
Trypsin Control did not contain any residual trypsin activity, insuring that the lack of effect of LCRFI 3s incubated with trypsin was not due to a ~u~lc;ssive ef~ect oftrypsin on pancreatic secretion. This result shows that LCRFI 3s meets the requirement for a trypsin-sensitive CCK-rele~eing peptide secreted by the intestin~
and has activity similar to that of the native polypeptide.

5.6.1.9 Effect OI I,CRFl 35 on CCK secretion by di~ cd rate intestinal mucosal cells in vitro F~G. 15 illustrates the dose-response relationship of CCK release to LCRFl 3s in dispersed rat ;i~le~ cells. LCRFI 3s significantly increased CCK release, compared to basal release, at 5 nM and 50 nM concentrations of LCRFI 3s. These 30 results show that LCRFI 3s directly stimulates CCK release from intestin~l mucosal W O 97/15671 PCT~US96/17998 cells, presurnably from CCK "I" cells, and may mediate the indirect stim~ tion caused by nutrients in the same system.

- 5.6.2 LCRFl 35 Imml~r oneutr~ ti Immunoneutralization of LCRF inhibits the pancreatic secretory and CCK
response to diversion of bile-pancreatic juice and peptone infusion Peptone stim~ tee pdl~Cl~d~iC secretion when infused intraduodenally in 10 absence of pancreatic juice in the intestine, and this response is me~ te~l by CCK and by endogenous LCRF. To determine whether endogenous LCRF truly me~ tee this response, the effect of ~ o~len~l peptone infusion on pa~ aLic secretion was tested in rats infused concoll,i~llly intr~cluodenally with purified IgG (antiserum #22322) obtained from rats ;~ ..i7ed with LCRF7 23 (Quality Controlled Biochemic~l~, Inc., 15 Hopkinton, MA). As illustrated in FIG. 1 6A and 1 6B, anti-LCRF IgG infused ~imnlt~neously with 5% peptone completely abolished the pancreatic secr~lol~
response to this nutrient solution. Control rabbit IgG from 1~ i7~l rabbit plasma had no inhibitory effect on the pancreatic secretory response to peptone under - the same conditions. These results strongly indicate that the pancreatic secl~o.
20 response to peptone is mefli~te-l by LCRF.

To (1etennine the role of LCRF in the pancreatic secretory and plasma CCK
rcspunses to diversion of bile-pancreatic juice in the rat, a dirrel~,.ll antisera was used.
Antisera were raised in rabbits to the fragment LCRF22 37. This antisera was used 25 without further purification. Antisera, 0.1 ml, were injected i.v. in rats ~ 1 hour prior to diversion of bile-pancreatic juice from the duodenum. The results were colllp~Led to results obtained in the same rats the day before who had received 0.1 ml. NRS in similar manner. The results are illustrated in FIGS. 1 7A, 1 7B and 18.

W O 97/1~671 PCTrUS96/17998 Diversion of bile-pancreatic iuice significantly ct;m~ ted p~ Llic protein and fluid secretion in both groups. To dete~rnine whether the LCRF antiserum inhibited this response, as would be predicted, the increment (output above basal) was calculated and the peak responses for each group co~ ed. These results S (inserts in FIG. 1 7A and 1 7B) show that the LCRF antisera (LCRF Ab) significantly inhibited the pancreatic fluid and protein responses to diversion of bile-pancreatic Julce.

FIG. 18 illustrates the plasma CCK responses in the same expr~riment 10 deterrnint-~l on blood samples withdrawn 30 min~ltes after diversion of bile-pancreatic juice. LCRF antiserum cignific~ntly suppressed plasma CCK collcellLlalions~
col~ d to rats receiving no antiserum and co..~ ed to rats receiving NRS. The results of this t;~ .;...ent strongly indicate that LCRF me~ tt-cJ in part, the pancreatic secretory and plasma CCK responses to bile-pancreatic juice diversion.
FIG. 19 illustrates the lack of direct effect of LCRFl 35 on pancreatic cells.
Isolated pancreatic acini were incubated with increasing concentrations of CCK-8 or LCRFI 35 and amylase release into the medium measured. LCRFI 3s had no effect - onarnylase release at concentrations at which CCK-8 dose-depPnd~ntly increased 20 amylase release. These results indicated that LCRF2 35 does not directly stimnl~te the pancreas. Therefore the stim~ tion of pancreatic secretion by i.d. and i.v. LCRF1 3s is probably indirect, via release of CCK.

~;.6.3 LCRF Fragments and Epitopes The smallest LCRF fragment with full LCRF agonist activity will be dett?rrninçtl This biological activity will be determin~d with the in vivo and/or in vifro test described above. Because LCRF activity is destroyed by the proteolytic activity of trypsin and because there are only three trypsin sen~ilive sites (two lysines 30 and one arginine) initial fragment s~ ree~ g will be con~ cted around these basic -W Q 97/15671 PCT~US96/17998 amino acid recicl~les Peptides having appro~imz~tely 30 amino acids with a centered lysine or arginine will be p~ al~ed, based upon the LCRF sequence already known or to be ~let~rmined. When the active fragment is identified, the link to peptide - surrounding the basic amino acids will be shortened systematically. After each 5 shortening, biological activity will be determin~(l until full biological activity with a minim~l size fragment is cletermint-cl Once this is done, then the central basic amino acid may be replaced by an amino acid such as, e.g, homoarginine that results in a peptide not sensitive to hydrolysis by trypsin but le~ biological activity.
~ltern~tively, arginine or lysine may be substituted by a nonbasic amino acid. The 10 final step will be to assure that the trypsin in~en~itive fragment also has the biological CCK-releasing activity desired.

It is understood, of course, that non-peptide LCRF analogs of the ~ lly sized active fragment may be pl.,~.~ed by methods well known to those of skill in the 15 art. Such non-peptide bonds may elimin~t~ the need to replace the basic amino acid si~n~linP trypsin sensitivity.

* * * * *
- The following references are incorporated in pertinent part by reference herein for the reasons cited above.

6.0 Ref~ ce~s Agerberth et al., FEBS Lett, 281: 227-30, 1991.
Agerberth et al., Proc Natl Acad Sci, 86:8590-8594, 1989.
Ayalon et al., Digestion, 24:118-125, 1982.
Berghorn K and Bonnett J, GE. H. "cFos Tmmlln~reactivity is F.nh~n~e-1 with Biotin Arnplification," JHistochem Cytochem; 42:1635-1642, 1994.
Blundell J.E., Hill A. J., Peikin S. R., Ryan C. A., Physiol Behav, 48:241-246, 1990.
Chan~ et al., JPhysiol (Lond), 320:393-401, 1981.
Chey et al., Am JPhysiol, 246:G248-G252, 1984.

WQ 97/15671 PCT~US96/17998 Cuber et al., Am JPhysiol, 259:G191 -G197, 1990.
DiMagno et al., "Chronic Pancreatitis," In: THE EXOCRlNE PANC~EAS, Go VLW, Brooks et al. ed., New York Raven Press, 1986:541-575.
Eysselein V. E., et al., Am JPhysiol; 258:G951-7, 1990. .
S Folsch U, Cantor P, Wilms H, Schafinayer A, Becker H, Creutzfeldt W., "Role of Cholecystokinin in the Negative Fee~1hs~ek Control of Pancreatic Enz~me Secretion in Conscious Rats," Gastroenterology; 92(2):449-458, 1987.
Franco-Saenz et al., Can. J: Biochem., 57:548-553, 1979.
Fried et al., Gastroenterology, 101 :~03-511, 1991.
Fushiki et al., FASEB J., 3:121-126, 1989.
Green G and Lyman R., "Fee~ ek Regulation of Pancreatic Enzyme Secretion as a Merh~ni~m for Trypsin Inhibitor-Tn~ ed Hy~ se~lclion in Rats," Proc Soc Exp Biol Med; 140:6-12, 1972.
Green G, Olds B, Matthews F, Syman R., "Protein, as a Regulator of Pancreatic Enzyme Secretion in the Rat," Proc Soc Exp Biol Med, 142: 1162- 1167, 1973.
Green et al., Am JPhysiol, 245:G394-8, 1983.
Green G. and Levan V., "Inhibition of Rat Pancreatic Secretion by Fl~t~e," IRCS
MedSci; 13:153-154, 1985.
- - Guan et al., Pancreas, 5:677-84, l 99û.
Herzig, et al. (1995) Gut 37 (Suppl. 2) A70.
Hof~nan G, Srnith M, Fi~ ns M., "Detecting Steroidal Effects on Immediate Early Gene Expression in the Hypoth~l~m-l~," Neuroprotocols. A Companion toMet*odsinNeurosciences; 1:52-66, 1992.
Iwai K., et al., JBiol Chem, 262:8956-9, 1987.
Iwai K., Fushiki T., Fukuoka S., Pancreas, 6:720-728, 1988.
Jordan etal.,Am JSurg, 128:336-339, 1974.
Lake-Bakaar et al., Horm. Metab. ~es., 13 :682-685, 1981.
Li etal., JClinIn~est, 86:1474-9, 1990.
Liddle et al., Gaslroenterology, 87:542-9, 1984.
Liddleetal.,ProcNatlAcadSci USA, 89:5147-51, 1992.

W O 97/1~671 PCT~US96/17998 Liddle R., "Integrated Aetions of Cholecystokinin on the Gastroint~stin~l Traet: Use of the Cholecystokinin Bioassay," Gastroenterol Clin North Am; 18:735-756, 1989.
- Liddle R., "Regulation of Cholecystokinin Secretion by Intr~ min~l Releasing Factors," Am JPhysiol; 269:G319-G327, 1995.
Louie D, May D, Miller P, Owyang C., "Cholecystokinin Mediates Fee<lb~c k Regulation of Pancreatic Enzyme Secretion in Rats," Am J Physiol; 250 (2 Pt l):G252-G259, 1986.
Lu L., Louie D., Owyang C., Am JPhysiol; 256:G430-5, 1989.
Marx et al., In: Cholecystokinin, eds. Thompson, J. C., Greeley, G. H., Jr., Rayford, P.
L. & Townsend, C. M., Jr. (McGraw-Hill, New York), pp. 213-222, 1989.
Miyasaka et al., Pancreas, 7:536-42, 1992.
Miyasaka K., Guan D. F., Liddle D. F., Green G. M., Am J Physiol, 257:G175-81, 1989.
Miyasaka K. and Green G., "Effect of Rapid Washout of Proximal Small Tntestin~ on Pancreatic Secretion in Conscious Rat," Gastroenterology, 84:1251 (abstr.), 1983.
Owyang et al., In: Pancreatic enzymes in feedback regulation of cholecystokinin release, ed. Thompson, J. C. (~c~ mic Press, Inc., New York), pp. 297-306, 1990.
Owyang C, Louie D, Tatum D., "Fee-lh~rlr Regulation of Pancreatic En~yme Secretion. Suy~ei.~ion of Cholecystc-kinin Release by Trypsin," J Clin Invest, 77(6):2042-2047, 1986.
Reeve J.R., et al., Am JPhysiol, 33:G860-G868, 1996.
Reeve J.R., Jr., et al., Ann N YAcad Sci, 713:11-21, 1994.
Ritter et al., Peptides, 9:601 -612, 1988.
~ Rushakoff et al., JClin Endocrinol Metab, 76:489-93, 1993.
Sarfati et al., Pancreas, 3:375-82, 1988.

W Q 97/1~671 PCTnUS96117998 Schneeman B and Lyman R., "Factors Involved in the Tnt~stin~l Feecl~ Regulation of Pancreatic Enzyme Secretion in the Rat," Proc Soc Exp Biol Med; 148:897-903, 1975.
Schuster M.M., Gastrointestinal Disease M. H. Sleisenger, J. S. Fordtran, Eds. (W. B.
S ~ m~1~r~ Co., phil~ lrhia, vol. 1, pp. 917-933, 1993.
Schwartz J.G., Green G.M., Guan D., Phillips W.T., Diabetes Care; 17: 255-262, 1994.
Sharara A, Bouras E, Misukonis M, Liddle R., "Evidence for Indirect Dietary Regulation of Cholecystokinin Release in R~ts," Am J Physiol; 265:G107-G112, 1993.
Sit7rn~nn J.V., Pitt H.A., Steinborn P.A., et al., Surg Gynecol Obstet, 170:25-31, 1990.
Slaff J, Jacobson D, Tillman C, Curington C, Toskes P., "Protease-Specific Su~ple~ion of Pancreatic Exocrine Secretion," Gastroenterology; 87(1):44-52, 1984.
Sp~nn~n~el A, Green G, Guan D, Liddle R, Faull K, Reeve-Jr J., "Purification andChar~;t~ l;on of a Luminal Cholecystokinin-l'cele~ing Factory from Rat T~ l Secretion," Proc Natl Acad Sci USA; 93:4415-4420, 1996.
- Sun et al., Gastroenterology, 96: 1173-9, 1989.
Taguchi etal.,IntJPancreatol, 11:67-73, 1992.
Uvnas-Wallensten K., Clin Gastroenf, 9:545-553, 1980.

W O 97/15671 PCT~US96/17998 SEQUENCE LISTING

(1) G~.NF:R ~L INFORMATION:

(i~ APPLICANT:
(A) NAME: BOARD OF REGENTS, THE UNIVERSITY OF
TEXAS SYSTEM
(B) STREET: 201 West 7th Street (C) CITY: Austin (D) STATE~ Texas (E) COUNTRY: USA
(F) POSTAh CODE (ZIP): 78701 (A) NAME: DUKE UNIVERSITY
(B) STREET: 011 Allen Building (C) CITY: Durham (D) STATE: North Carolina (E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 27708 , (ii) TITLE OF INVENTION: LUMINAL
- CHOLECYSTOKININ-RELEASING
FACTOR
(iii) NUMBER OF SEQUENCES: 9 (iv) COM~Ul~ READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPhICATION DATA:
(A) APPLICATION NUMBER: US 60/005,872 (B) FILING DATE: 26-OCT-1995 W O 97/15671 PCTrUS96/17998 ~ o m S~ ~
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(D) TOPOLOGY: l inear (ix) FEATURE:
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CA 02238940 l998-04-22 W O 97/15671 PCT~US96/17998 (B) LOCATION:12..15 (D) OTHER INFORMATION: /mod_base= OTHER
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W O 97/15671 PCT~US96/17998 (i) SEQUENCE CHARACTERISTICS:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
. 20

Claims (31)

CLAIMS:

1. An isolated choleeystokinin-releasing polypeptide which specifically binds with antibodies raised against a polypeptide having at least the arnino acid sequence of SEQ ID NO: 1.

2. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

3. The polypeptide of claim 2 further defined as having a mass as determined by mass spectrometry of about 8136 daltons.

4. The polypeptide of claim 1 that is isolated from luminal secretions of small intestine.

5. The polypeptide of claim 2 that stimulates cholecystokinin release.

6. The polypeptide of claim l that has the amino acid sequence of SEQ ID NO: 1.

7. The polypeptide of claim 1 further defined as having at least 85% homology tothe amino acid sequence of SEQ ID NO: 1.

8. An isolated cholecystokinin releasing polypeptide comprising:

a) the amino acid sequence of SEQ ID NO: l; or b) the amino acid sequence of SEQ ID NO: 1 from position 1 to position 35; or c) the amino acid sequence of SEQ ID NO: 1 from position 11 to position 25; or d) the amino acid sequence of SEQ ID NO:l from position 1 to position 6, or e) the amino acid sequence of SEQ ID NO: 1 from position 7 to position 23; or f) the amino acid sequence of SEQ ID NO: 1 from position 22 to position 37; or g) the amino acid sequence of SEQ ID NO:l from position 1-35 where Lysine is replaced with alanine at position 19; or h) functional or homologous variants thereof.

9. A composition comprising the polypeptide of claim 1 or claim 2.

10. The composition of claim 9 further defined as comprising a physiologically acceptable excipient.

11. A purified antibody that specifically binds to the polypeptide of claim 2.

12. The antibody of claim 11 wherein the antibody is linked to a detectable label.

13. A method of generating an immune response, comprising administering to a mammal a pharmaceutical composition comprising an immunologically effective amount of the composition of claim 9.

14. A method for detecting luminal cholecystokinin-releasing peptide of claim 8 in a biological sample, comprising the steps of:

a) obtaining a biological sample suspected of containing a luminal cholecystokinin releasing peptide;

b) contacting said sample with a first antibody that binds to the protein or peptide of claim 8 under conditions effective to allow formation of an immune complex; and c) detecting the immune complex so formed.

15. An immunodetection kit comprising, in suitable container means, one or more protein or polypeptides as defined by claim 8, or an antibody that binds to a protein or peptide as defined by claim 8, and an immunodetection reagent.

16. An isolated nucleic acid segment that encodes a cholecystokinin-releasing polypeptide which specifically binds with antibodies raised against a polypeptide having at least the partial amino acid sequence of SEQ ID NO: 1.

17. An isolated nucleic acid segment that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

18. The nucleic acid segment of claim 16 or claim 17 further defined as comprising the nucleic acid sequence of SEQ ID NO: 2 or the complement thereof or a sequence which hybridizes to SEQ ID NO: 2 under conditions of high stringency.

19. The nucleic acid segment of claim 16 or claim 17 wherein the encoded polypeptide has the amino acid sequence of SEQ ID NO: 1.

20. The nucleic acid segment of claim 16 or claim 17 further defined as an RNA
segment.

21. A recombinant vector comprising a DNA segment which comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1.

22. A recombinant vector comprising a DNA segment which comprises a cholecystokinin-releasing polypeptide that specifically binds with antibodies raised against a polypeptide having at least the partial amino acid sequence of SEQ ID NO: 1.

23. The recombinant vector of claim 21 or 22 wherein said DNA segment comprises a nucleotide sequence in accordance with SEQ ID NO: 2.

24. A recombinant host cell comprising a recombinant vector in accordance with claim 21 or claim 22.

25. The recombinant host cell of claim 24 wherein the host cell is S. mutans.

26. A method of suppressing appetite comprising:

providing a composition in accordance with claim 10; and administering said composition to a subject in need thereof in an amount effective to suppress appetite.

27. A method for stimulating gallbladder contraction or treating gallbladder disease related to gallstone formation, the method comprising:

providing a composition in accordance with claim 10; and administering said composition to a subject in need thereof in an amount effective to stimulate gallbladder emptying.

28. A method of inhibiting gastric emptying, the method comprising:

providing a composition in accordance with claim 10; and administering said composition to a subject in need thereof in an amount effective to.
delay gastric emptying.

29. A method of stimulating insulin secretion comprising:

providing a composition in accordance with claim 10; and administering said composition to a subject in need thereof in an amount effective to stimulate insulin secretion.

30. A method of preparing an orally administerable preparation useful to suppress appetite, stimulate gallbladder emptying, inhibit stomach emptying, or stimulateinsulin secretion, the method comprising formulating an orally acceptable preparation comprising a therapeutically effective amount of the polypeptide of claim .1 or claim 2.

31. A method of using a DNA segment that includes an isolated cholecystokinin-releasing gene encoding the polypeptide of claim 1 or claim 2, comprising the steps of:

a) preparing a recombinant vector in which a cholecystokinin-releasing gene encoding the polypeptide of claim 1 or claim 2 is positioned under the control of a promoter;

b) introducing said recombinant vector into a recombinant host cell;

c) culturing the recombinant host cell under conditions effective to allow expression of an encoded cholecystokinin-releasing protein or peptide; and d) collecting said said expressed cholecystokinin-releasing protein or peptide.