Note: Descriptions are shown in the official language in which they were submitted.
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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).
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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
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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
.
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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.
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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
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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'-
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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~
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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
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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
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-10-
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
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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
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-12-
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).
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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
-
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-14-
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
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-15-
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
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-16-
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.
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-17-
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
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r-- ~ cr o c~ ~o o CJ~ U~ 00 00
o o
.o _. .__ _ _ _ ~ _
~V¢~ ~V¢V ~V¢V
.~ ~ VVVV VVVV VV~V
~: ~ ~V ~ ~ ~ :=V C~ V V ~ ¢ ¢ ¢
~
~C ~~ Cd Cr~ ~ ~ -- ~ ~ ~~
o ;~, _ o ~ t~
~ t-- O O ct~ ~ ~ ~ ~o ~ ~ ~
g ~V¢V ~V¢V ~V¢V
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 ¢ ¢ ¢ ¢
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_19_
X ~ ~ o ~
E-- ~ ~ o
O ~ t_ ~D
~ D ~
D ~ ~ ~ _I ~
~ ~ ~ V V :~
O
O O 00 00
~ O ~
V ~ V C~ ~ --
~ O ~ ~ ~
~ O ~ ~
00 ~ ~ ~_ V
~ C~ ~ V
~ V g V
V V V C~ g
~ ~: ~ O~ CJ~ O ~ O
O ~ ~D ~ ~~ V
~ V ~ ) V ~J D
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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
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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
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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
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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
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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'
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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
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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~.
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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
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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
=
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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
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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
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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
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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.
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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.
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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
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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
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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.
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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
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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
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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
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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.
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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
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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.
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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.
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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
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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
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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.
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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
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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
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.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
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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.
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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
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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.
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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.
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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
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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
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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
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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
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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:
~=
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TABLE 3
- 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
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~--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, ~
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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
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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
. . .
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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?.
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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.
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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
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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.
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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
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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
-
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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.
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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.
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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.
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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
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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.
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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.
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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
2Q
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.
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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
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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.
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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
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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
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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
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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.
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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)
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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
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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.
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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.
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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
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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-
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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
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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
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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.
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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.
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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.
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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)
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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
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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.
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~;.!; 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
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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.
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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.
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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
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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
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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.
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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
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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.
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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.
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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
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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
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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.
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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
-
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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.
CA 02238940 1998-04-22
WQ 97/15671 PCT~US96/17998
-1 10-
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.
CA 02238940 1998-04-22
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.
CA 02238940 1998-04-22
W Q 97/1~671 PCTnUS96117998
-112-
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.
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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
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-114-
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CA 02238940 1998-04-22
W O 97/15671 PCT~US96117998
-115-
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CA 02238940 1998-04-22
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-116-
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(D) TOPOLOGY: l inear
(ix) FEATURE:
(A) NAME/KEY: modi~ied_base
(B) LOCATION:3.. 21
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "Y = T or C"
(ix) FEATURE:
(A) NAME/KEY: modi~ied_base
(B) LOCATION:9..18
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "N = Inosine"
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:15..16
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "R = A or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
-
TTYTGGGCNT AYCARCCNGA YGG 23
(2) INFORMATION FOR SEQ ID NO: 5:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: single
(D) TOPO~OGY: l inear
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:3..18
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- 118 -
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "Y = T or C"
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:9..15
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "H = A, C or T"
10(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:12..13
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "R = A or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
TTYTGGGCHC ARCCHGAYGG 20
-. 20
~2) INFORMATION FOR SEQ ID NO: 6:
-
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: 1 inear
(ix) FEATURE:
(A) NAME/~ Y: modified_base
(B) LOCATION:3..21
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "Y = T or C~
(ix) FEAlu~E:
(A) NAME/KEY: modified_base
.
...
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-- 119 --
(B) LOCATION:12..15
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "N = Inosine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GAYAAYGAYC CNACNGAYTA YCA 23
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: l inear
tix~ FEATURE:
(A) NAME/KEY: modi~ied_base
. 20 (B) LOCATION:3.. 9
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "R = A or G"
(ix) FEATURE:
(A) NAME/KEY: modi~ied_base
(B) LOCATION:6..15
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "Y = C or T"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
GTRTGYTCRT AYTTYTG 17
(2) INFORMATION FOR SEQ ID NO: 8:
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- 120 -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D~ TOPOLOGY: l inear
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION: 3..21
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "N = Inosine"
(ix) FEATURE:
(A) NAME/ ~ Y: modified_base
(B) LOCATION:9.. 18
(D) OTHER INFORMATION: /mod_base= OTHER
/note= " R = A or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
TCNATNACRC ANGGRTARTC NCC 23
-
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: modified_base
(B) LOCATION:3..12
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "D = G, A or T"
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- 121 -
(ix) FEATURE:
(A) NAME/KEY: modi~ied_base
(B) LOCATION:6..7
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "S = G or C
(ix) FEATURE:
(A) NAME/KEY: modi~ied_base
(B) LOCATION:9.. 21
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "R = A or G"
(ix) FEATURE:
(A) NAME/KEY: modi~ied_base
( B) LOCATION:24.. 25
(D) OTHER INFORMATION: /mod_base= OTHER
/note= "N = T, A, C or G"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
. 20
TCDATSACRC ADGGRGGRTA RTCNCC 26
