CA2145767A1 - Cetp inhibitor polypeptide, antibodies against the synthetic polypeptide and prophylactic and therapeutic anti-atherosclerosis treatments - Google Patents

Abstract

A polypeptide and analogues thereof inhibit cholesteryl ester transfer protein (CETP). An anti-atherosclerosis composition comprises an anti-atherosclerosis effective amount of the polypeptide and a pharmaceutically acceptable carrier. An anti-atherosclerosis kit comprises in separate sterile containers at least one unit of the composition containing the polypeptide, one syringe and one needle. An antibody has speficity for the polypeptide of the invention, the baboon CETP 4kD polypeptide inhibitor, the 1-36 amino acid N-terminal fragment of apo C-I, modified apo A-I (MW:31kD) or modified apo E (MW:41kD). A method of preventing atherosclerosis in a mammal being predisposed to that condition comprises administering to the mammal a prophylactically effective amount of the polypeptide of the invention, and a method of treating a mammal afflicted with atherosclerosis the administration of a therapeutically effective amount of the polypeptide disclosed herein.

Description

WO 95/04755 214 5 ~ 6 7 PCT/US94/08624 CETP INHIBITOR POLYPEPTIDE, ANTIBODIES AGAINST
n THE SY~ ;l`lC POLYPEPTIDE AND PROPHYLACTIC AND
THER~APEUTIC ANTI-ATHEROSCLEROSIS TREATMENTS
BACKGROUND OF THE INVENTION
This applir~*on is a continuation-in-part of applir~ll;on, Serial No. 07/811,049, which was filed on December 18, 1991.
The work leading to the present invention was partially supported by N~tion~l Heart, Lung and Blood TnQtit~lte Grants Nos.
HL28972 and HL41256, and Contract No. HV53030. The governmQnt may hold rights in the present patent.
Field of the Invention This invention relates to an endogenous baboon plasma cholesteryl esters transfer protein (CETP) inhihitor polypeptide.
More sperifir~lly, this invention relates to the i~n*fir~*on and char~r.teri7.~tion of the polypeptide and to novel synthetic peptides possessing inhihitory activity of CETP. The endogenous inhihitory peptide has a molecular weight of 4000, is present in pl~.cm~ in the form of modified apo A-l and apo E having molecular weights of 31kD and 41kD, respectively, and has a commoT amino acid sequence with the N-termin~l fragment of apo C-1. This invention also relates to an anti-atherosclerosis compoQition, a kit, and to antibodies raised against the N-termin~l amino acid sequence of the inhihitory polypeptide. The inhih;tory peptide of the invention, fragments thereof and analogues thereof are useful for the 25 prophylactic and therapeutic tre~tment of atherosclerosis.
Description of the R~ck~round Atherosclerosis is one of the most widespread health problems in the United States today as are its attendant complir~*ons, r parfic~ rly coronary heart disease. A number of risk f~ctors have30 been associated with the development of premature atherosclerosis, primz~rily elevated pl~.cm~ cholesterol levels. Due to the crucial role cholesterol appears to play in the oc~:u- l~nce of heart disease, a great deal of atten*on has been devoted to studying its synthesis, transport and metabolism in the human body.

SlJBSTllUrE SHEET (RULE 26) , ~ 2 PCT/US94/08624 Of particular interest is the est~hli.qhment of r~1~*on.qhips between the levels of plt~-cm~ lipoproteins or serum lipids and the r risk of development of coronary heart disease. 13oth high density lipoproteins ~EIDL) and low density lipoproteins (LDL) carry 5cholesterol mainly in the form of cholesteryl esters (CE). There are some in-lic~*on.q, however, that while LDL cholesterol is a posi~ive risk factor, HDL cholesterol is an even more important negative ~isk factor. Although the exact filnc1;on.q of these lipoproteins have~ot been completely est~hli.~hed, HDL appears to serve for the removal of 10cholesterol from peripheral cells and its transport back to the liver, where a large proportion of the cholesterol excreted from the body is removed.
LDL and HDL are believed to play key roles in the development of cardiov~cll1~r disease by overloading the lysosomes 15of the walls of arterial cells with metabolites which are generally hydrolyzed slowly, such as CE and triglycerides. These products are ev~r,ll~te-l from the liver and intestine by pl~m~ LDL. When the amount of lipids to be transported exceeds the transporting capacity of HDL to the liver for excretion, CE become deposited in the cells in 20 cel~a~ rri*r~l areas, such as arterial walls. This overloading eventually results in impaired cell filnc1;on, and if continued may produce cell death. A continuous overloading results in the accumlll~*on of cellular debris and the form~*on of atherosclerotic plaque in the vessel wall. This, in turn, leads to the blockage of the 25 affected artery and/or muscular spasm, events which may m~nifest themselves as coronary heart disease or strokes. Thus, the level of HDL in pl~.qm~ has been negatively correlated with the probability of developing atherosclerosis in humans and experimental ~nim~
Although the level of HDL has been shown to vary 30 con.~iderably among indivi(lll~l.q, the means of regu1~ion of such pls~.qmz~ level rem~in.q to be eln-~id~qte~
CETP tr~n.~ferq CE from HDL to VLDL and LDL, and it has been suggested that it plays an important role in the regulation of pls~m~ HDL levels. Some hyperalphalipoproteinemic p~ ?ntq were 35 reported to have high levels of large HDL particles that were clearly separate from LDL. Plasma samples from these patients were shown SUBSTITUTE SHEET (RlJLE 2~) 214~767 to lack CETP activity ~Koizumi et al, Atherosclerosis 58:175-186 (1985))- A homozygous subject with f~milis~l hyperalphalipoproteinemia was found to have impaired transfer of CE from HDL to LDL (Yokoyama et al, Artery 14:43-51 (1986)). A
5 fraction of density d>l.21 g/ml from the subject's plasma evidenced substantial CETP activity with normal HDL. The HDL, how~v~r, proved to be a poor substrate for CETP.
Certain ~nim~l sires and their progeny possess unusual lipoproteins p~ttermc~ e.g., lipoproteins of a density intermediate to 10 that of LDL and HDL, or large high density lipoproteins. These lipoproteins have been ~le.cign~ted HDLl and the ~nim~l phenotype as "high HDLl". Baboon strains possessing, for instance, patterns of either high or low HDLl are known. In most cases, HDLl separates either as a distinct peak between LDL and HDL or as a shoulder to 15 the HDL peak, and is induced by a high cholesterol, high lard ~EICHF) diet. The proportion of HDLl ~limini.qh~.c when the baboons are fed a diet that is either enrirhed in polyunsaturated fat, with or without cholesterol. Oc ~.cion~lly, howevel, the amount of HDL
present in high HDLl baboons fed the chow diet is low.
In some baboon f~milies, the level of pl~.cm~ ~lOLl was shown to increase when the ~nim~l.c are rl~ nged with a HCHF diet.
VVhen fed a HCHF diet, the baboons also show higher pl~.cm~ HDL.
More generally, the accumul~tion of HDL in baboons as well as in hllm~n.c is associated with a slower transfer of CE from HDL as very low density lipoproteins (VLDL) and LDL. Thus, baboons with high HDLl pls-cm~ levels are exr~llent as ~nim~l models for the study of hyperalphalipoproteine_ia.
In a previous study, some of the present inventors reported that a slower transfer of CE from HDL to VLDL and LDL was 30 observed in high HDLl baboons. This was attributed to the presence of a CETP protein inhihitor associated with HDL and intermediate density lipoprotein (IDL) particles OE~ushwaha et al, J.P. Lipid Res.
31:965-974 (1990)). An accum~ tion of HDLl in the high HDLl baboons fed a HCHF diet was reported along with a slower tr~ncfer 35 of CE from HDL to LDL. A simil~r protein was found in human pls~.cm~ by Son and Zilversmit (Son and Zilv~r.cmit B.B.A. 795:473-SUBSTIME SHEET ~RUEE 26)

2! ~ ~ 5 7 6 ~ PCT/US94/08624 480 (1984)). The human protein has a molec~ r weight of 31,000and suppresses the transfer of triacyl~lycelol and CE.
Several other species including rat, pig, and dog have been reported to readily accumulate HDLI in plasma. Kurasawa et al.
5 (1985), supra, reported that a homozygous subject with f~rnili~l hyperalphalipoprot~in~?mi~ has impaired CE transfèr between HDL
and LDL (Kurasawa et al, J.B. Biochem. 98:1499-1408 (1985)).
Separately, Yokoyama, et al. reported that a pl~.cm~ fraction of d>1.21 g/ml of the same subject evidenced subst~nti~l CE transfer 10 activity when tested with norm~l HDL (Yokoyama et al, Artery 14(1):43-51 (1986)). The HDL particles accumulated by this subject were substantially larger in molec~ r size than ordinary HDL2.
HDL is generally divided into subfr~ction.q based on their particle sizes and ~len.qiti~s These fr~ction.q include HDLl, HDL2, 15 and HDL3. HDLl has the largest particles and is usually not present in the pl~qm~ of norm~l humans or non-human prim~tes. HDL2 and HDL3 are the norm~l components of human plasma. HDL2 is larger than HDL3 and differs between men and women.
Many ~tt~mpts have been made to interfere with the transport 20 and transfer of cholesterol in m~mm~ n.q in order to alter its pl:~qm~ levels. Among them are the following.
U.S. Patent No. 4,987,151 to Taboc discloses tl~iterpene derivatives that inl~ihit acyl coenzyme ~ rll~le.steral acyltransferase (ACAT) enzyme. The ACAT is a c~ r enzyme that is not present 25 in pl~qm~, and esterfies cellular cholesterol to form CE. This enzyme is dil`r~l~t from the CE transfer protein (CETP) present in plasma.
The CETP does not form CE as does the ACAT enzyme. Instead, the CETP tr~n.qfers CE slmonF~qt ~ le~lt pl~.cm~ lipoproteins.
U.S. Patent No. 4,643,988 to Segrest, et al discloses 30 amphipathic peptides which are capable of substituting for apo A-I in Rl~L. Apo A-I is kllown to stimulate the lerit~in cholesterol:acyl tr~n.cfer~qe (LCAT) enzyme, a plasma enzyme that forms CE in HDL. Plasma CETP, in contradis~;nction, tr~n.qf~rs CE from HDL to VLDL and LDL. The filnction of the CETP enzyme is, thelerole, 35 (li~ t from that of the LCAT enzyme, as well. The amino acid SUBSTIME SHEET (RULE 26) WO 9~/047~ ^ ~CT/US94/08624 sequence of the Segrest et al peptides are, in ~ *on, different from the sequences of the CETP inhibitor of this invention.
S~-mm~ ry of the Invention This invention relates to a substqnti~lly pure polypeptide 5 having activity inhibitory of CE transfer protein (CETP).
This invention also relates to an anti-atherosclerosis composit~on, comprising an anti-atherosclerosis effective amount of the polypeptide ll~scrihed above; and a ph~rm~ceutically-acceptable carrier.
In a-l-li*on, this invention relates to an anti-atherosclerosis kit, comprising in separate cont~iners at least one unit of the composition (les~rihed above;
at least one syringe; and at least one needle.
This invention also relates to an antibody having specificity for a polypeptide selected from the group consisting of the polypeptide described above;
baboon CETP polypeptide inhibitor;
1-36 amino acid N-termin~l fragment of apo C-I;
modified apo A-I ~NW:31kD);
modified apo E ~MW:41D).
In a .li~elellt aspect, this invention relates to a method of yl~vellting atherosclerosis in a m~mm~l being predisposed to that condition, c.~ lsing ~(lmini.qtering to the m~mm~l a prophyl~rtic~lly effective amount of the polypeptide described above.
This inven1ion also relates to a method of treating a m:~mm~l ted with atherosclerosis comprising ~imini~tering to the mzlmmz~l a therapeutically ~ecli~e amount of the polypeptide ~i~sl~rihed above.

SUB~I~UTE SHEET ~RULE 26) WO gs/047ss l ~l 5 7 6 ~ PCTIUS94/08624 Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following disc~ eion .
Dçscription of the Preferred Embodiment This invention arose from the desire by the inventors to provide a novel and unobvious approach to t~ie prevention and treatment of atherosclerosis in hllm:~n.c A schematic of what is known about the aSso~i~tion of cholesterol with the (li~elellt fr~ction.~ of lipoproteins in plasma and 10 in the liver is shown below:
The metabolic steps leading to the accumll1~tion (~) of HDLl, VLDL and LDL are shown above. HDL2+3 c-.lle~ct~ cholesterol from extrahepatic cells, which is then esterified by LCAT to form cholesteryl esters (CE) and stored in the core of the particles. The 15 HDL becomes larger in size (HDLl) and may pick up apo E to attain a particle which is removed from LDL receptors (LDL-R) on liver cells. The CE enriched HDLl may also donate CE to VLDL and LDL.
This is mediated by CETP. Due to the presence of the CETP
inhihitor, such as the one provided herein, CE transfer is slow (har) 20 and the reciprocal transfer of triglyceri(le.ci CTG) does not take place (X). The triglyceride-poor HDL is thus not a sllit~hle substrate for hepatic triglyceride lipase (HTGL). Due to the presence of the CETP
inl~ihjtor, in plzl.cm:~, VLDL and LDL are thus not av~ hle to the liver. As a consequence of this, the liver then increases the 25 expression of mçss~ges for the increased pro-lllctioI- of LDL receptor and 3 hy~y, methyl, ~lutal~l-coenzyme A (HMG-COA) synthase.
The increase in LDL receptor in the liver leads to an increase in uptake of LDL or HDL, with apo E, and consequently, to a greater delivery of cholesterol to the liver. An increase in synth~.c.i.~ of HMG-30 CoA synthase leads to an increase in synthesis of cholesterol in theliver to meet all cellular needs. Thus, the presence of a CETP
inhihitor in plzl~m~ will prevent the uptake of VLDL and/or LDL by tissues as weJl as the deposition of cholesteryl esters.
In general, high levels of HDL have an anti-atherosclerogenic 35 effect whereas high levels of LDL have an atherogenic effect. The SlJBSrITUTE S~IEET (RULE 26) 21~5767 WO 9~/047~;!; PCT/US94/08624 circu1~1~;on in blood of compounds, such as cholesterol, that are insoluble in water requires the form~tion of particles. The insoluble components, e.g., cholesteryl esters and triglycçri(les, are packed in the core of the particles and ~urlounded by polar components such as 5 proteins, phospholipids, and the like. These particles are called lipoproteins, and have, thus, an outer polar shell and a non-polar core. These associations of lipoproteins cont~ining CE in the core and, depending on their sizes and llen.~ es, have been named VLDL, IDL, LDL, and HDL.
VLDL is the largest lipoprotein secreted by the liver and is converted into IDL, and then to LDL, after the triglycerides contained by the VLDL are hydrolzyed by the lipoprotein lipase enzyme present on the surface of the arterial walls. LDL is the major lipoprotein that provides cholesteryl ester to extrapepatic and 15 hepatic tissues. HDL is also secreted by the liver and is divided in HDL2 and HDL3 on the basis of size and density. A function of HDL
is to pick up cholesterol from extrahepatic cells and deliver it to the liver, either through VLDL and LDL or through large HDL which is Qnrirhed with apo E. These large HDL particles are called HDLl and 20 they do not stay in the pl~.~m:~ for long periods of tilne. They are rapidly removed by the liver or converted back to HDL2 after rlon~ting their cholesteryl esters to VLDL and LDL.
HDLl is not present in either norm~1 humans or non-human primslteS. As inllic~te-l above, HDLl appears as a distinct bandin 25 the pl~.cm~ of baboons that have been fed a HCHF diet. From what is known, when cholesterol enters the blood stream it becomes s~csorizltQ~l with HDL in the form of a CE, with the help of the LCAT
enzyme. In HCHF-fed baboons, this appears as an HDLl-CE
fraction. The CE is then tr~n.~ferred from HDL to VLDL and LDL to 30 form VLDL-CE and LDL-CE with the aid of the CETP enzyme These particles then enter the liver cells through the LDL receptor (LDL-R). After being metabolized in the liver cells, the VLDL-CE is returned to the pl~.~m~ and thus to the periphery of the m~mm~ n body, where its deposition may occur leading to atherosclerosis.
An inhihitor of CETP, such as the one provided by this inven~ion, blocks the transfer of CE from HDL to VLDL and LDL

~lJBSTlTUrE SHEET (RULE ~6~

WO 9~;/0475!; PCT/US94/08624 2~5~ ~ 8 Instead, a shunt is favored that leads to the asso~i~tion of the CE
with apo E and to the form~hoI- of HDLl-CE-apo E particles that can enter the liver cells through the LDL receptor (LDL-R).
The apolipoprotein C-I of various species but not baboon, are 5 known. The Apo C-I is a single polypeptide of m~lec~ r weight 6600, consisting of 67 amino acids. It is a basic protein that is mainly present in VLDL and HDL, with HDL serving as a reservoir for this protein. LDL, on the other hand, contains little apo C-I. It has recently been shown that apo C-I displaces apo E from VLDL
10 and affects its binding to the LDL receptor.
The polypeptide inhihitor of CETP that is described herein has a common sequence with the N-t~rmin~l fr~gnlent of apo C-I. This fr~nent includes at least 36 amino acids as shown below.
The endogenous polypeptide (SEQ. ID. NO: 1) provided by this 15 iIlvention has a molec~ r weight of about 4,000 and becomes associated or binds to apo A-I and apo E in pl~qm~ Its N-termin~l 36 amino acids are shown below.
A polypeptide having the sequence corresponding to amino acids 1 to 36 of the following sequence (SEQ. ~. NO: 1) was synt~e.ci7.ed by the inventors and shown to be inhihitory of CETP in vitro. The peptide has the following sequence.

Asp -Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 1).
Fr~ ntq of that polypeptide (SEQ. ID. NO: 1) comp~ising 35 the C-~....i..~l fr~nentc of amino acids 28 to 36 and amino acids 16 to 36 showed limite-l inhihit~ry activity of CETP at 50 ~g. Howevel, the fr~gment comp~g the C-termin~l amino acids 28 to 36 showed at 200 ~g an activity inhihitory of CETP approxim~tely the same as SUBSlllUrE SHEET (R~JLE 26) 21g5767 that of the 36 amino acid peptide. The N-t~rmin ~1 fragments comprising amino acids 1 to 15, amino acids 1 to 20 and amino acids 1 to 10, as well as the intermediate fragments comprising amino acids 15 to 30 and the like, corresponding to the synthesized peptide 5 have been shown active as inhihitors of CETP.
Polypeptides having the sequences corresponding to the following two sequences (~ n~ted (SEQ. ID. NO:2) and (SEQ. ID.
NO:3) have also been synthesized by the inventors and shown to be inhihitors of CETP in vitro.
The first of the following two sequences (SEQ. ID. NO:2) is a baboon sequence li~e (SEQ. ID. NO: 1) except that it has two lition~l amino acids at the be~innin~ of the peptide. The second of the following two sequences (SEQ. ID. NO:3) is a human sequence and varies from (SEQ. ID. NO:2) in seven of the thirty-eight amino 15 acids in the sequence.
Ala-Pro-Asp -Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-val-Tle-Asn-Arg-Ile-Lys-Gln Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO:2) 20 Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp -Lys-Ala-Arg-Glu-Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO:3)--Analogues of the polypeptide of the invention and fr~gm~nts 25 thereof having inhihitory activity of CETP are also part of the invention The ~n~loEues may have one or more substitutions in their sequences while still preserving their inhihitory activity.
mples of s~n~logues sllit~hle as inhihitors of CETP are analogues of the peptide of the invention and fragments thereof, such as those 30 where one or more of the amino acids are substituted in accordance with the g lidelines provided below.
The substitute amino acids may be selected from the group consisting of Glu, Ca-methylAsp, and B-carboxyAsp for Asp;

SUBSTIT~E SHEET tRULE 26) WO 95/0475~ PCT/US94108624 ~1 21~S7 ~ lo isoVal, norVal, Leu, and Ca-methylVal for Val;
Gly, B-Ala, Ca-methylAla, and 2-amino butyric acid for Ala;
norLeu, isoLeu, and Ca-methylLeu for Leu;
ornit~ine, Arg, citr~llline and Ca-methylLys for Lys;
Ala and 2-amino isobutyric acid for Gly;
Gln, citruDine, and Ca-methylAsn for Asn;
P-Benzoyl Phe, Arg, and Ca-methylTrp for Trp;
2-amino adipic acid, Asp, and Ca-methylGlu for Glu;
Leu, norLeu, and Ca-methylIle for Ile;
Lys, homoArg, citrulline and Ca-methylArg for Arg;
Asn, citrulline, and Ca-methylGln for Gln;
2-amino-4-phenylbutyric acid, Leu and Ca-methylPhe for Phe;
Ser, Met and Ca-methylThr for Thr;
Thr and Ca-methylSer for Ser; and

3,4-DehydroPro, Ser and Ca-methylPro for Pro; and comhin~tion.~ thereo How~v~:r, other substitutions of a nature equivalent to that of the substituted amino acid as is known in the art may also be utilized, alone or in comhin~1;on with other substituents.
It is thelerole provided in accordance with this inven*on, a subst?nti~lly pure polypeptide having activity inhihitory of CETP.
In one embo~imçnt of the inv~ntion, the polypeptide is capable of inhihi*n~ the binding of an about 31kD modified apo A-I
polypeptide present in the pl~m~ of high HDLl baboons or a peptide 25 of the sequence.

Asp-Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. 1), to an antibody raised against the above peptide.

SUBSI ITUTE SHEET ~RULE 26) 21~5767 In another embodiment, the polypeptide of the invention is capable of inhihi*ng the binding of an about 4kD CETP inhibitor polypeptide present in the plasma of high HDLl baboons to an antibody raised against a peptide of the formula Asp -Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe-1 0 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO.1).
In still another embodiment, the polypeptide of this invention is capable of inhihi*ng the binding of an about 41kD modified apo E
polypeptide present in the plasma of high HDLl baboons with antibody raised against the 36 amino acid N-tDrmin~l fragment of apo C-I or a peptide of the formula Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. l).
In still another embodiment, the polypeptide is capable of inhihj*ng the binding of the 36 amino acid N-tD7~nin~1 fr~nent of apo C-I or a peptide of the formula Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-SUBSTIME SHEET ~RULE 2~

WO s5/0475~ PCT/US94/0862-1 ~
2~7 ~ 12 Gly-Asn-Thr-Leu-Glu-Asp -Lys-Ala-Trp -Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. 1), with an antibody raised against modified apo A-I.
Ple~elled polypeptides are the polypeptides of the sequence Asp -Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-ne-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. V, Ala-Pro-Asp -Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp -Lys-Ala-Trp -Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO:2), 25 and Thr-Pro-Asp -Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu-Leu-ne-Ser-Arg-Ile-Lys-Glln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO:3);
anti-4kD peptide antibody-binding inhibitory fr~ nts of (SEQ. ID NO.: 1) thereof; and anti-4kD peptide antibody-binding inhihitory analogues of (SEQ. ID NO:l) thereof having at least one substitute amino acid selected from the group con.~ ;ng of Glu, Ca-methylAsp, and ~-carboxy Asp for Asp;
SUBSTITUTE SHEET`(RlJLE 26) WO 95/04755 ~ 21 ~ $ 7 6 7 PCT/US94/08624 isoVal, norVal, Leu, and Ca-methylVal for Val;
Gly, ~-Ala, Ca-methylAla, and 2-amino butyric acid for Ala;
norLeu, isoLeu, and Ca-methylLeu for Leu;
ornit~ine~ Arg, citrulline and Ca-methylLys for Lys;
Ala and 2-amino isobutyric acid for Gly;
Gln, citrulline, and Ca-methylAsn for Asn;
P-BenzolyPhe, Arg, and Ca-methyl Trp for Trp;
2-amino adipic acid, Asp, and Ca-methylGlu for Glu;
2-amino adipic acid, Asp, and Ca-methylTrp for Trp;
Leu, norLeu, and Ca-methylIle for Ile;
Lys, homoArg, citrulline and Ca-methylArg for Arg;
Asn, citrulline, and Ca-methylGln for Gln;
2-amino-4-phenylbutyric acid, Leu and Ca-methylPhe for Phe;
Ser, Met and Ca-methylThr for Thr;
Thr and Ca-methylSer for Ser;
3,4-DehydroPro, Ser and Ca-methylPro for Pro;
and comhin~tion.q thereo In one particular lJlererled embo-liment, the polypeptide cont~in~q amino acids 1 through 36 of the above sequence (SEQ. ID.
20 NO. 1). In still another par~ir~ rly plererled embo(liment, the peptide is selected from the group consisting of peptide fragments (SEQ. ID. NO. 1) comprising amino acids 1 to 17, 1 to 20 and 1 to 25, and fragments thereof having anti-4kD peptide antibody/1-36 amino acid peptide binding inllihjtory activity.
In still another plere~led embodiment, the peptide fr~gmçnts are selected from the group consisting of peptides comprising amino acids 1 to 18, and 1 to 28 of (SEQ. ID. NO. 1), and fragments thereof having anti-4kD peptide antibody/1-36 amino acids peptide binding in~lihitory activity.

SUBSI ITUIE SHEET (RULE 26) wo 95lo4755 rj ~ 6~ 14 PCT/US94/08624 Also plereLled are analogues (SEQ. ID. NO. 1) with the Lys, Asp and Asn amino acids substituting for the Arg, Glu, and Gln amino acids; the Ser, Leu, and Ala amino acids substituting from the Thr, ne and Gly amino acids; the ornit.hine, citrulline and o~
5 aminoadipic acid amino acids substituting for the Lys and Glu amino acids.
Also pl~Lelled are the following analogues (SEQ. ID. NO. 1).
Peptides comprising amino acid sequences where amide bond(s) (e.g., -C(O)-NH-) linkinE any pair, and up to all pairs, of amino acids 10 comprising amino acids 1 to 17, 1 to 20, 1 to 25, 1 to 36, and fr~gm~ntc thereof having anti-4kD peptide antibody/1-36 amino acid peptide binding inhihitory activity are replaced by thioether bonds (e.g., -CH2 -S-) alkyl such as ethyl (-CH2-CH2-), and/or amino (e.g., -CH2-NH2-) linkages. These analogues may be purchased 15 coTnmercially or prepared by methods know to those skilled in the art as long as the antibody 1-36 amino acid peptide hin(ling and CETP
inhihitory activities of the peptides (analogues) are retained to some degree.
How~ver, other analogues (SEQ. ID. NO. 1) are also part of 20 this invention as long as they preserve the inhihitory activity of the antibody/1-36 amino acid peptide hin(ling.
The CETP inhihitory polypeptide of the invention may be provided as a powder, ~ relably in freeze-dried form, as a solution, ~l~L~lably frozen at below -20C, and the like, to prevent proteolysis.
This invention also provides an anti-atheros~lerosis compo.Clhon, comprlsmg an anti-atherosclerosis effective amount of the polypeptide of the invent;on; and a ph ::lrm ~ceutically-acceptable carrier.
VVhen the composition is used as preventative tool, it may contain 10 to 200 mg and more preferably 20 to 100 mg of the polypeptide. Howevel, other amounts are also s~ hle. When the compositio~ is intended for therapeutic use, the amount of polypeptide present is preferably about 10 to 400 mg, and more SUBSTI~U~E SHEET tRULE 26) preferably about 20 to 300 mg. However, other amounts may also be utili7.ed.
Any and all ph~ eutically-acceptable carriers known in the art for a-lmini.qtration of peptides to m~mm~1s, and preferably to 5 hllm~n.q, are sllit~h1e for use herein. These are known in the art and need not be further lescrihed herein. ~,x~mI~le~q, however, are s~line, human serum albumin and starch. However, others may also be utilized.
The composition of this invention may be provided in unit 10 form, preferably in a sterile, closed cont~iner, and more preferably in a sealed container.
A kit, comprising in separate containers at least one unit of the anti-atherosclerosis composition of the invention;
at least one syringe; and at least one needle.
Typically, a kit may ccntain from about 1 to 20 units of the compo.qition of the invention, but could con~in 50 units or more. In lition, the kit may contain 1 to 20, but and sometimes 50 or more 20 syringes if they are disposable, and l to 20, but sometimes up to 50 or more needles if they are disposable. The components of the kit are provided in a sterile form, be it wrapped in a sealed, st~rili~.ed wla~l g, or in some other way. If not disposable, the syringe and needle may be autoclaved between uses.
The composition of the invention is preferably ~llmini~tered intravenously, although it may also be aflmini.~tered intraperitone~11y, subcutaneously or intramuscularly. The oral route is not p~rmi.~.~ihle since the polypeptide would be degraded in the acidic pH of the stomslrh.
30 The compo~ition may ~l~r~Iably have a pH of about 7 to 9, and more lJlefelably about 8 to 9, which may be adjusted with the on of a base, acid or buffer as is known in the art.

SUBSrl~E SJ IEET (RULE 26) WO 95/04755 2 l 4 S 7 ~ 7 16 PCT/US94/08624 This invention also provides an antibody having specificity for a polypeptide selected from the group consisting of the polypeptides of the invention;
the baboon CETP polypeptide inhihit~r, fragments thereof and 5 ~n~lo~ues thereof;
the 1-36 amino acid N-termin~1 fragment of-apo C-l;
modified apo A-I (MW:31k~)); and modified apo E (MW:4l~).
The antibodies of the invention may be raised in m~mm~1.c as 10 is _nown in the art. (Albers, J.J. ~7.7.~rd, W.R., Immunochemical Quantification of the Human Lp(a) Lipoprotein, Lipids 9:15-26 (1974)).
Typically, the antibodies may be raised in rabbit, goat, sheep, pig, and ~.hil~ken How~ver, other m~mm~ may also be utili7.ed.
15 Ple~Lled are rabbit antibodies. Also pr~felled are polychonal antibodies. Howevel, monoclonal antibodies may also be prepared by methods known in the art (~hl~r, G., and ~lstein, D., Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity Nature (London) 256:495-497 (1975)).
In one pleferled embodiment, the antibody of the invention is capable of sper.ifis~11y binding to modified apo A-I.
In another y~efelled embo~iment the antibody is capable of sper.ifir,~lly bin~ling to the baboon CETP inhihit~r polypeptides of this inv~nti~n In another pleLel-led embodiment, the antibody of the invention is capable of sperifis~11y binding to the polypeptide of the sequence.
2 3 4 5 6 7 8 9 lO 11 12 Asp -Val-Ser-Ser-Ala-Leu-Asp -Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-~e-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr SUBSTIlIITE ~HEET ~RULE 26) WO 95/04755 21 4 5 7 6 ~ PCT/USg4/08624 fr~gm~nts thereof and analogues thereof as (les~rihed above.
In still another plerelled embodiment of the invention, the 5 antibody is capable of spe~ific~lly binding to modified apo E.
The antibody of the invention, in another plererled embodiment is also capable of sperific~11y binding to apo C-l, and more preferably to the 1-36 N-termin~1 fragment thereo In another aspect of the invention, a method is provided for 10 preventing atherosclerosis in a m:3mm~1 being predisposed to that condition. The method comprises ~lmini~tering to the m~mm~1 a prophyl~c1;c~lly effective amount of the polypeptide of the invention, or of the anti-atherosclerosis composition lescrihed above.
In a plerel,ed embodiment, the polypeptide is ~llminiqtered in 15 an amount of about 2 to 100 mg for preventative applic~1;on.q.
However, other amounts may also be ~(lmini.qtered. The polypeptide or compo.cition thereof may be ~(lmini.st~red in a small volume of c~rrier, e.g., 0.2 to 1.5 ml of sP1ine or other c~rri~r.q, as is known in the art.
The polypeptide of the inv~ntion.q may be ~(lminiqtered intravenously, to a fr~gment of the pop~ on, particularly the human poplll~tion, that is not ~fflicted by high blood cholesterol and hyperbetalipoproteinemia, but, as de~e....i..ed by other me~n.q, may be at risk of being ~fflicte-l by atherosclerosis. One such ex~mple 25 may be a f~mili~1 trait having been determined.
The polypeptide of this invention may be ~(lminiqtered on a daily basis, or at longer intervals if provided as a slow release compo.qition as is known in the art, such as depoestradiol provided by - the UpJohn Co. (UpJohn Co., K~1~m~oo, MI).
In another aspect, the present invention provides a method of treating a m~mm~1 afflicted with atherosclerosis. The method comprises ~lmini.qtering to the m~mm~l a therapeutically affective amount of the polypeptide of the invention. When a-lmini.qtered for therapeutic purposes, the polypeptide may be injected in an amount S~JBSTlll~E SI~EE~ (RULE 26) 2~4~7~
WO 95/04755 ~ PCT/US94/08624 of about 10 to 400 mg, and more preferably 20 to 300 mg. However, other amounts as assessed by a practitioner in specific cases, may also be ~(lmini~tered.
In this case, as in the case of the prophylactic ~llmini.~tration~
5 the polypeptide may be ~(lmini.~tered intravenously, ~mong other routes.
Having now generally (les~rihed this invention, the same will be better understood by l~ereL,ce to certain specific examples, which are included herein for purposes of illustration only and are not 10 intended to limi~ing of the invention or any embodiment thereof, unless so specified.
~.Ynmrles Example 1: ~nim~l~ and Diet Adult male and female baboons (papio sp.) held in a baboon 15 colony at the Southwest Foundation for Biomedical Rese~rch in San ~ntonio~ Texas, were used as blood donors for these studies. Among these, 24 baboons had a high HDLl phenotype and 32 had a low HDLl phenotype (VVilli~m.~, M.C., et al., Detection of Abnormal Lipoprotein In a Large Colony of Pedigreed Baboons Using High-20 Performance Gel Exclusion Chromatography, J. Chromat. 308:101-109 (1984)).
Half of the high HDLl baboons (n=16) were maintained on a HCHF diet, the composition of which has been previously described (Kushwaha, R.S., et al., Metabol~sm of Apolipoprotein B. In Baboons 25 with Low and High Levels of Low Density Lipoprotein. J. Lipid Res.
27:497-507 (1986)).
Most of the baboon donors of the low HDLl phenotype were m~intqined on a chow diet ~urina Monkey Chow, manllf~ct~lred by R~ ton Purina Co., St. Louis, Missouri). The monkey chow is low in 30 fat (10% of total c~l-ries) and high in carbohydrate (62% of total c~loriç.s). In ~ on, the chow has a very low cholesterol content (0.03 mg/Kcal).
The high ~Ll baboons were progeny of two sires (X1672 and X102) who had a high HDLl phenotype. The low HDL~ baboons were SUBSIl~UTE SHEET (RULE 26) ~45767 WO 95/04755 ^ PCT/US94/08624 progeny of a number of sires who did not have a high HDLl phenotype. The presence of HDL1 was detected by high performance liquid chromatography (HPLC) as described previously (Willi~m.q, et al., Detection of Abnormal Lipoprotein in a Large Colony of Pedigreed 5 Baboons Using High-Performance Gel Exclusion Chromatography, J.
Chrom~tography 308:101-109 (1984)).
Example 2: Preparation of 3H Cholesteryl Ester HDL
High and low HDLl baboons were immobilized with 10 mg/kg of ketamine HCl and bled. The blood was collected in tubes 10 cont~inin~ 1 mgtml EDTA, and pl~.qm~ separated by low speed centrifugation at 6C. The plasma was treated with sodium azide, choloramphenicol, gentamycin sulfate, phenylmethy 1-sulfonyl fluoride and DTNB as (lesrrihed previously (Kushwaha, R.S., et al., Impaired Plasma CE Transfer with Accumulation of Larger High 15 Density Lipoproteins in Some Pamilies of Baboons (papio sp.), J.
LipidRes. 31:965-973 (1990)).
20 to 60 ~Ci of tritiated cholesteryl linole~te were dissolved in ethanol and then added to the pl~.qm~ The plasma was flushed with nitrogen and incubated for 20 hours at 4C.
After incubation, HDL3 was isolated by density gradient ultracentrifilg~tion (McGill, H.C. et al., Dietary Effects on Serum Lipoprotein of Dsylipoproteinemic Baboons with High HDL"
~e~osf~lerosis 6:651-663 (1986); Redgrave, T.G., et al., Separatwn of Plasma Lipoprotein by Density-Gradient Ultracentrifibgatwn, ~n~ P~iochem. 65:42-49 (1975)), dialyzed against saline/EDTA, and used as a substrate for the CE transfer reslction The total and free cholesterol cont~ntc of HDL3 were measured pl~ior to use in the assay.
E~ample 3: Preparation of Acceptor Lipoproteins and CETP
Source VLDL+LDL from low ~lDL~ baboons was used as the acceptor of CE from HDL3. A VLDL~LDL fraction of d<l.040 g/ml was isolated from 100-200 ml of blood by sequential ultracent~ffig~tion as (l~sf~rihed previously (Kushwaha, et al. (1986), supra). Total and SUBSr~ E SHEET ~RULE 26) WO 95/0475~ PCT/US94/08624 ~ ~57 ~ 20 free cholesterol contents in acceptor lipoprotein were measured by enzymatic methods (VVako Pure Chemical Co.) (Allain, C.C., et al., Enzymatic Determination of Total Serum Cholesterol Clin. Chem.
20:470-475 (1974)).
After separation of VLDL+LDL, ~he bottom fraction was adjusted to d=1.21 g/ml of ~ ing solid KBr, and total lipoprotein were isolated by ultracentrif~gqtion (Kushwaha, et al. (1986), supra).
The bottom fraction of d>1.21 g/ml was also collected.
All lipoprotein fractions and the lipoprotein--l~firient fraction of d>1.21 g/ml (LPDS) were dialyzed against sqline/EDTA. The LPDS was used as the source of CETP.
li Y~mrle 4: Cholesteryl Ester Transfer Assay The CE transfer activity of a sqmple was assayed by a mo(lifirq.1;on of a procedure described previously (Kushwaha, et al., (1986), surpa).
Briefly, 3H CE-labeled HDL3 cont-qining 50-100 llg of CE from low RT)Ll baboons was incubated with VLDL+LDL contqining 100-300 ~g CE in the presence of LPDS. The acceptor lipoprotein and the LPDS were obtained from low HDL, baboon pl~m~ (chow diet). In some cases, the HDL3 was obtained from high HDLl baboons mz~int~ined on the HCHF diet.
The incllh~tion.s were carried out at 4C (control) and 37C for

4-6 hrs. and termin~ted by pl~ring the s~mples on ice. The assay mixt~lre was then ultr~rçntrifuged to separate VLDL+LDL having a d~1.040 g/ml, and the radioactivity in the lipoprotein was counted as ~esrrihed previously ~Kushwaha, et al. (1986), supra). Any ,3i~elellce observed in the r~-lio~ctivity tr~nsf~rred from HDL to VLDL+LDL at 4C and 37C was attributed to CETP activity in the LPDS.
Time course eXperim~nts gave a lillear response up to 7 hrs.
~imil~rly, the CETP activity was linear with increasing LDPS, up to 140 ~1 LPDS, which was derived from an equivalent volume of pls~.cm~

SUBSTITU~E SHEET fRUI E ~6 214576~

5 PCTIUS94/08624 They polypeptide of the invention, and synthet,ic fragments thereof were added to the re~ction mixture to determine their CETP
inllihjtory activities, along with an appropriate synthetic control peptide.
The percent ~ elellce between the control experiment and the assay with inhihitor peptide was expressed as the inhihitor activity.
Example 5: Identification of Tnhihitor Polypeptide The bottom non-lipoprotein fraction obtained by ultracentrifilg~tion for 72 hrs was analyzed for protein content by 10% SDS-polyacryl~mi(le gel electrophoresis (T.~mmli, U.K., Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature 227:680-685 (1970)), without ~-mercaptoet~n-~l pretre~tmçnt.
To determine ~ ellce in small moleclll~r weight apoliproteins, ~ pi~ ed lipoproteins of d<1.21 g/ml from high and low HDLl baboons were separated by 15-19% SDS-polyacryl~mille gel electrophoresis with ~-mercaptoet~l~nol pretre~tln~nt prior to loading the s~mpl~.q onto the gels.
Example 6: Electroelution of Tnhihitor Polypeptide The lipoprotein fraction of d< 1.21 g/ml from high HDLl baboon pls~:m~ was dilapidated with ether-ethanol (l~loren, C.H., et al., Estrogen-Induced Increase in Uptake of Cholesterol-Rich Very Low Density Lipoproteins in Perfused Rabbit Liuer, Metabolism 30:367-374 (1981)), and separated by 15% SDS gel electrophoresis (l~slçmmli, (1970), supra) after ~ on of ~-mercaptoethanol. A
small molecular weight ~lo~e~ band was cut and tr~n.~ferred onto a tube gel (15%).
A dialysis tube of molecular weight cut-off point 1000 was ~tt^^~e-l to the bottom of the gel tube to lec~ive the electroeluted 30 peptide. The thus electroeluted peptide was dialyzed and quantitated by comparing its absorbancy at 660 nm with a known amount of stained albumin electroeluted at the same time.

SUBSrIT~I SHEET (RULE 26) 2 i ~ 7 ~

Ex~qmple 7: Antibody Preparation The apolipoproteins were separated by 15% SDS-gel electrophoresis and stained (T ~emmli, (1970), supra). The stained bands were tr~n.sferred onto a nitrocellulose membrane. The bands 5 corresponding to the inhihitor polypeptide were cut out (0.05 mg) and dissolved in 0.5 ml of filtered DMSO. 0.5 ml Freund's adjuvant were then added and thoroughly mixed and the mixt~lre was injected intr?~lerm~lly into two rabbits. After 30 days, the rabbits were boosted with a .simil~r amount of electroeluted protein band.
10 Antibody titer was measured by Western blotting. The rabbits were boosted again 3 times.
For the preparation of antibody against the synthetic polypeptide, 500 ~g of polypeptide were dissolved in 400 ~l of titer max (CytRx Corporation, Atlanta, GA), and injected into rabbits 15 intra-l~rm~lly. The rabbits were boosted with 500 ~g of the synthetic peptide in 200 ~1 of Titer Max on the 28th day. The serum was tested on the 42nd day. The rabbits were bled and anti-serum was obtained as needed.
ExamPle 8: ImmunoaiEinity Chromatography An immlln~ff;nit.y column was prepared using CnBr-activated Sepharose beads (Ph~rm~ Co.). The bound ligand was IgG
precipitated from the serum of rabbits having antibodies. The method used to precipitate IgG was .simil~r to that described by M~Kinney and Parkinson (l\~l~Kinney, M.M. and Parkinson, A., A
Simple, Non-Chromatographic Procedure to Purify Immunoglobins From Serum and Ascites Fluid, J.Immunologic~l Methods 96:271-278 (1987)).
Briefly, 5 ml of rabbit serum were diluted 4-fold with acetate buffer, pH 4Ø 625 ~11 of caprylic acid were added dropwise to 30 precipitate albumin and non-IgG proteins. The insoluble m~t~ri~l.s were removed by centrif~lg~tion at lO,OOOxg for 30 min. The supern~t~nt was mixed with phosphate buffered saline, and the pH
adjusted to 7.4 with lN sodium hydroxide. The solution was cooled to 4C and ammonium sulfate was added to give a final 35 concentration of 45% to precipitate the IgG.

SlJBSrlTUTE SHEET (I~JLE 261 The precipitate was recovered as a pellet after centrifugation, and resuspended in phosphate buffered saline. The IgG was dialyzed overnight in 100 volumes of phosphate buffered saline, and then dissolved in sodium acetate buffer, pH 8.3, coupled to 3 g of 5 CnBr-activated Sepharose beads, and maintained in Tris-saline, pH
7.4, until ready to use.
8 ml of pl~.cm~ was incubated overnight with IgG coupled beads in Tris-s~line bu~eled with gentle rotation. The column was then washed with Tris-saline coupling buffer and sodium acetate 10 buffer as described by Cheung and Albers (Cheung, M.C., and Albers, Distribution of High Density Lipoprotein Particles with Different Apolipoprotein Composition: Particles with A-l and A-II
and Particles with A-I But No A-II, J. Lipid Res. 23:747-753 (1982)).
The bound proteins were eluted with 0.1 M acetic acid, pH 3.0 15 and 1 ml aliquots were collected and read at 280 nm to visu~li7.e the peak. The protein fraction was dialyzed immediately against phosphate bu~eled saline and separated by electrophoresis in 15%
SDS-polyacrylamide gels.
Ti'.Y~ml-le 9: Tmmunoblotting The proteins separated with SDS-polyacrylamide gels were transferred onto Immobilon-P sheets ~llipore, Beford, MA). The sheets were incubated with antibody against inl~ihjtor peptides after hlofkin~ of nonspecific sites. The sheets were washed and incubated again with a secondary antibody cont~ining horseradish pero~ s The ~ on of boric acid buffer cont~ining 3-amino-9-ethylcarbozole, methanol and hydrogen peroxide produced a coloration.
mrle 10: Amino Acid Analysis and Sequencing - Stained bands of proteins were tr~n.~ferred onto Immobilon-Psheets. Selected bands were cut out and hydrolyzed with 50%
propionic acid, 50% 12 N HCl for 2 hrs at 135C. Amino acid analysis of each sample was performed using a model 6300 amino acid analyzer (Berkm~n Co., Palo Alto, CA) provided with System Gold software.

SUBSlIME SllEET (RULE 26) i WO 95/047~ A ~ 7 ~ ~ PCT/US94/08624 The same bands were sequenced using a model 477A protein sequencer (Applied Biosystems, Foster City, CA).
.Y;~mr~le 11: Preparation of Synthetic Peptides The peptides were synt~-e.qi7.ed by solid-phase peptide 5 synthesis as ~l~s~rihed by Barany and Merrifiel(l p3arany, G. and Marrifi~l(l, R.B., 17~e Peptides, Analysis, Synth,esis; Biology; Gross, E.
and ~Pin~hofer, J., eds. Vol. 2, ~ mic Press, ~ew York, pp. 1-284 (1980)).
The 1-36 amino acid synthetic peptide was assembled from the 10 C-t~rminus towards the N-terminus, with the o~-carboxyl group of the an~ino acid attached to a solid support and was then char~ctari7.ed by HPLC.
Example 12: Data Analysis The values provided in the following examples are averages 15 and are provided as mean + standard error. These values were compared using variance analysis and, if .signific~nt di~elellces were detected, the values were compared using Duncan's Multiple Range Test (Dllnc~n, D.B., Multiple Range and Mult~ple F Tests, Biometrics 12 (1955.
20 F~ynmr~le 13: Characterization of Proteins From Infr~n~t~nt Fraction The CETP inhihitc!ry activity was lost from the HDL when the lipoproteins were ext~nqively ultr~centrifuged, e.g., for 72 hrs, or by repeated ultr~o~ntrifilgf~tion After ultracentrifugation, the 25 inhihitory ac~vity was found in the infr~n?~t~nt fraction.
To char~cte~7.e the proteins, the infr~n~t~nt fraction (d<1.21 g/ml) was separated by 10% SDS polyacryl~mi(l~ gel electrophoresis in the absence of ,B-mercaptoethanol.
The in~n~t~nt fraction from high HDLl baboons contained 30 albumin, a protein slightly larger than apo A-I and another protein larger than apo E. Lanes A and B show nonlipoprotein fr~ctio~
from high and low HDLl baboons, respectively. The protein bands, 1, 3, and 5 correspond to albumin, apo E and apo A-I, respectively. The protein bands 2 and 4 of molecular weights between 1 and 3, and 3 SU~STITUTE SHEET (I~JLE 26) 21~767 t WO 95/04755 PCT/US94/08624 and 5, respectively, correspond to proteins with molec~ r weights of 41,000 and 31,000, respectively. Protein samples from high HDLl baboons show only bands corresponding to albumin, a protein of molecular weight 41,000 and a protein of molecular weight 31,000.
5 The molecular weights were determined with standard proteins separated on .cimil~r gels (gel picture not shown).
The infr~n~t~nt fraction from low HDLl baboons contains these proteins as well, but in a(l~ on, it also contains apo A-I and apo E. Both proteins in the apo A-I region were identified by 10 immunoblotting with antibody to apo A-I.
~ C~imil~rly, both proteins in the apo E region were identified by immunoblotting with antibody to apo E. The molecular weights of the proteins detected by immunoblotting with apo A-I and apo E
show a ~li~e~ ce of about 4kD (picture not shown). The estimated 15 moleclll~r weight of apo A-I is about 27,500 and that of the modified apo A-I is about 31,000. ~imil~rly, the estim~te(l molecular weight of apo E is about 37,000 and that of modified apo E is about 41,000.
Both apo A-I and apo E are modified by a protein of about 4,000 molecular weight.
20 F~y3~mrle 14: Detection of CETP Inhil~itor Peptide in Pl~:m~
of High HDLl Baboons To (letermine if a common polypeptide of molecular weight 4,000 was modi~ing both apo A-I and apo E, plz~ms~ lipoproteins of d < 1.21 g/ml were separated from high and low ~il)Ll baboons by 18%
25 SDS-polyacryl ~mi(le gel electrophoresis with ~-mercaptoet~ntll A higher amount of a 4kD protein was detected in lipoprotein from high HDLl baboons as compared to lipoprotein from low HDL
baboons (gel picture not shown).
To deL~ e if the 4kD polypeptide inl~ihit~ CE transfer, the 30 polypeptide and albumin were electroluted from the gels, and used in increasing conc~ntrations in a CE transfer assay mi~t~lre with lipoproteins from low HDLl baboons as described in ~.~r~m~le 4 above. Albumin had no effect on the transfer of CE from HDL to VLDL+LDL. The 4kD polypeptide, on the other hand, .~ignific~ntly 35 in~ihite~l CETP activity. (Results are not shown).
SUBS~I~lllE SHEET (RULE 26) WO 95/04755 ~ ~ ~5~ ~7 PCT/US94/08624 Example 15: Characterization of Polypeptide by Affinity Chromatography.
Rabbit antibody was prepared against the 4kD polypeptide i.cols~ted from ]ipoproteins obtained from high HDLl baboons. This 5 antibody was used to prepare an immuno ffinity column. The lipoproteins of d<l.21 g/ml were passed o~per the immunoaffinity column. The bound lipoproteins eluted with 0.1 M acetic acid and separatedin 15%SDS-polyacrylamidere~ ringgels. 4kD, and31kD
polypeptides, and a minor band corresponding to a 41kD polypeptide 10 were detected.
The column bound peptides (100 ,ug) inhihited by 31.3 + 1.4%
the transfer of CE (mean + SE, n=3) in the CETP assay from low Ll baboons.
On the other hand, the ~ on of IgG to the assay from high 15 ~iL)Ll baboons increased CE tr~ncfer by 44.3 + 1.5% (n=3), but had no effect on CE transfer from low HDLl baboons.
Example 16: Comp~rison of 4kD Tnhihitor Polypeptide with Sequence Data Bank The sequence of this polypeptide was compared to sequence of 20 known proteins using Sequence Data Bank (Reardon, W. R. and T.ipm~n, D.J., PNAS (USA) 85:2444-2448 (1988)), and was found to have 100% h- nnolo~y with human and crab-eating macaque apo C-I.
The sequence was then compared with apo C-I from baboons and found to be 100% hom~logous (private communic~tion from Dr.
25 Hixson of the Southwest Foundation for Biomedical Research).
h~.YnmI~le 17: Characterization of ~nhihitor Polypeptide with Synthetic Peptides Based on its molecular weight, it was (letermined that the 4kD
polypeptide contained approxim~tely 36 amino acids.
Three peptides were synt~e.ci7.ed beginning from the C-t~rmin~l end of the apo C-I sequence. The filst peptide cont~ined 9 amino acids, the second peptide cont~ined 21 amino acids a~d the third peptide cont~ined 36 amino acids. The 36 amino acid peptide had an amino acid sequence ~;imil~r to the 4,000 MVV polypeptide, SlJBSTl~UrE SHEET (RULE-26) ~ ~ 2145767 WO 95/0475~ PCT/US94/08624 the other two were fragments of the synthetic peptide starting from its C-t~rminus.
50 llg of these peptides were used in a CETP assay with lipoproteins of the low HDLl baboons as l~.s~rihed in F',x~mple 4 5 above. A soluble helical peptide with a 1,900 molecular weight utilized as control. The 36 amino acid polypeptide .ci~nific~ntly (p<
0.01) inhihited CE transfer from HDL to VLDL and LDL, while the others, including the control peptide, did not.
h'.Y~mrle 18: Antibody ~ nct 36 Amino Acid Tnhihitor 1 0 Peptide Antibody against the 36 amino acids inhihitor peptide was prepared in rabbits as ll~cf~rihed in F~x~mple 7, and used for immunoblotting. The thus prepared antibody recognized the 4kD
peptide as well as a 3 lkD polypeptide from lipoproteins of high HDL
1 5 baboons.
To det~rmine if both the apo C-I and the 4kD polypeptide were present in the pl~.cm~ of high and low HDLl baboons, lipoproteins from both phenotypes were separated by 10% SDS gel electrophoresis and immuno-blotted. Two protein bands were 20 detected with the antibody by immunoblotting of samples from high HDLl baboons. Only a single band was detected in samples from low HDLl baboons.
In ~(l(lition, isoPlect~ic focusing p~tt~rn.c of the synthetic peptide suggest that the peptide is a slightly basic protein.
25 ~y~mrle 19: CETP ~nhihition by Various Peptide Fragments A CE transfer assay as described in ~.x~mple 4 above was con(ll.cted using HDLl baboon plasma. 3H HDL and the VLDL+LDL
cS~rri~rs, in the presence of CETP enzyme to mediate the ~xrh~nge.
The re~C~ionc were conducted at 37C and 4C (control) in 30 duplicate. The results are shown in Table 1 below.

SUBSllTUTE S~EET (RULE 26) 2l-4~7 ~

TABLE 1: CETP INHIBITION BY VARIOUS ~l ll~E FRAGMENTS
CETP[H3] HDLSYNTHETIC VLDL+LDL TEMP. INHIBITION
PEPTIDE
g) ~g) ~g)(C) (~) 125 100 ~ 3004 125 100 9 amino acid 30037 0 Zlammoacid 36 ammo acld 9 ammo acld 125 100 Zl amino acid 30037 26 36 amino acid SV~STITUIE SHEET

~ 2lq5767 Wo 95/0475~ PCT/US94/08624 zg Example 20: Tnhihition of CETP From Hllm~n~ by Synthetic CETP Tnhihitor Peptide.
Cholesteryl ester transfer activity from human plasma was - assayed by the procedure ~l~scrihed by the inventors (Kushwaha 5 R.S., Rainwater D.L., Willi:lm.c M.C., Getz G.S., and McGill H.C., Jr., Impaired Plasma Cholesteryl Ester Transfer with Accumulation of Large High Density Lipoproteins in Some Families of Baboons (Papio sp.). J. Lipid Res. 31:965-973, 1990). In short, [3Hl cholesteryl ester-labeled HDL (10-~1g of cholesteryl esters with a specific activity of 3-4 10 x 106dpm/mg r~olest~ryl ester) from low HDL, baboons was incubated with 50-100 ,ug of VLDL+LDL cholesteryl ester from baboons. The incubations were carried out in the presence of 100 ,ul of lipoprotein ~l~firient serum (LPDS) obtained from hllm~ns and 2mM DTNB. The total volume of the assay was 1 ml. The 15 incubations were carried out for 1-2 h at 4 and 37. At the end of the incubation, 40 ~11 of heparin (5000 units /ml), 0.5 ml of pls~.cm~, and 60 ~1 of 1 M MnCl2 were added in that order. The mixture was vortexed, incubated for 0.5 h on ice, and centrifuged for 10 minutes.
The radioactivity was measured in the supernatant fraction by 20 scin~ t;on spectrometry. The ~li~el~l.ce between 4 and 37 was con.cillered to reflect the CETP-mediated transfer. At the same time each set of human CETP incubations were run in the presence of synthetic CETP inhihitor peptide (baboon apo C-I termin~l peptide with 38 amino acids, Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-25 Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. 2) and the transfer of cholesteryl ester from HDL to VLDL+LDL was det~rmined in the presence of CETP inhibitor. In some cases synthetic CETP inhihitor was .~imil~r to human apo C-I t~rmin~l 30 peptide with 38 amino acids CThr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp -Lys-Ala-Arg-Glu-Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID.
NO. 3).
The results of these experiments are provided in the following 35 tables:

SUBSTITUTE SttEET (RULE 26) WO 95/04755 21 4~7 ~7 PCT/US94/08624 1~

ao TABLE Inl~lbition o~ Hu~ G15TP by C~:TP l~ib~ ptlde ahbool~ ~equen~e~.
8.N. &~ o~~Hl HDL Inhibitcr VLDL~LDL T mp C~TP Inhibib hwD~n CETP (Iy~ pcpbdo ~ ~?

1. 8H 1000 60 4 200 1000 60 tl7 200 ' ~ 00 100 60 ~ Z00 `~
10100 50 :~7 Z00 60 2. RK 1000 60 4 200 lo00 60 ~7 200 00 lo loo ~o 'I 200 10100 30 ~7 Z00 72 1000 60 ~ 200 1000 50 ~7 Z00 00 lo loo 60 ~ Z00 1000 30 :17 200 CETP
lo loo so 4 200 NOT

6. KR 1000 60 4 200 1000 60 ~7 200 00 lo loo 60 ~ Z00 1000 60 ô7 900 00 lo loo 60 4 200 10100 60 ~7 200 60

7. GL 1000 30 4 200 lo loo so ~ 200

8. RC 1000 50 4 200 lo loo 60 4 200

9 TH 1000 60 4 200 1000 50 37 200 oo lo loo 50 4 200

10 OM 1000 60 4 200 1000 50 ~7 200 00 ~i~ 10 100 50 4 Z00 SVBSTlTlJTE SHEET

lo loo ~o ~7 200 90

11 EW lo oo 50 ~ 200 oo bO ~7 200 oo lo loo60 ~ 200 lo loo50 ~7 200 71

12. ~L 1000 ~0 4 200 00 ~0 ~7 200 oo ~o 100~0 ~ 200 10060 ~7 200 77 TABLE ~. C----r - - of CETP ;nh;hitor peptide from hum~n and b-~o-- - on 15 CETP ~ rer inb-h~ -[3Hl HDL InhibitorInhibitor VLDL+LDL Temp.OETP T~
(~g) peptide peptide (~e) ~ 1) (%) (bllboon)(hum~n) ~e) ~g) 00 60 ~ 100 00 00 50 4 loo lo oo 60 50 4 100 SU~STI~UIE SHEET

WO 95/04755 45rt 6~ 8~Z PCT/US94/08624 100bO :17200 90 Il. EW 10 00 ~0 ~ 200 00 150 :S7200 00 lo loo~o 4 200 `
100~0 ~S7200 ~ 71 12. ML 10 00 bO 4 200 00 bO 37 200 00 1 0 10 100 ~ 00 ICO~0 ll7200 77 TABLE 3. Cc , - ~ of CETP ;~h;hit,^r peptide f~om hum n ~nd ~ - on 15 CETP ~. ' r in b ~~^~~
3HI HDLInhibitorIn}~ibitor VLDL+LDL Temp. CETP I ~ ~
(~g)peptide peptide (~) C) (~1) (%) (baboon~ lhum~) (l~e) (~) 25 10 50 00 50 4 lO0 30 10 00 50 50 ~ 100 F,Y~n~rle 21: Method of preparing I~EI HDL Cholesteryl ester-labeled HDL, VLDL+LDL LPDS (human).
[3Hl r.hole.steryl ester-labeled HDL and acceptor lipoproteins (d c 1.045 g/ml) were prepared as fl~.s~^rihed by the inventors ~Kushwaha, et al. J. Lipid Res. 31:965-973, 1990). Low HDL, baboons were bled after immobili7:~tion with kPt~mine HCl (10 mg~kg). Blood was 40 c-llected in tubes cont~ining EDTA (1 mg/ml) and pl~.cm~ separated by low speed cent~filg~tion at 6C. Plasma was immediately treated with sodiwn azide (0.2 g~ gentamycin sulfate (0.1 g/l), chloramph~nir,ol (0.05 g~). and phenylmethylsulfonyl fluoride (0.5 ml!~. Tritiated cholesteryl oleate (20 ~lCi/ml) dissolved in ethanol was 45 added to the plasma. The pl~cm~ was flushed with nitrogen and incllh~ted for 20 h at 4C. After incubation, HDL was ;sol~ted by density gradient ultr~cen~ifilg~1;on (Kushwaha, et al., 1990), SUBSTlllJTE SHEET

WO 95/04755 21~ 5 7 6 7 PCT/US91/08'~4 , 32A

dialyzed against nortns~l saline/EDTA (O.OOlM) and used as a substrate for cholesteryl ester transfer re~rtion VLDL+LDL (d <
1.045 g/ml) and LPDS were isolated by seqllen*~l ultr~-~nt~ifilg~tion from 100-200 ml blood obtained from two to four SIJBSTlllJTE SHEET

WO 95/047S5 2~ 6~ PCT/US94/08624 ~

baboons as lles~rihed by the inventors (Kushwaha, et al., 1990). Total and free cholesterol and HDL and VLDL+LDL were measured by enzymatic assay (Wako Pure Ch~mi-~ql Co.).
F'Yl~eriment 22: Method of isolating hu~an CETP
To i.c~lqte human CETP, blood (5-10 ml) from human subjects was obtained in tubes contqining EDTA (1 mg/ml). Plasma was obtained by low speed centrif~gqtion Plasma was kept on ice and the LDL was pre.;;l.ilqte-l by q~ltling 40 ~1 heparin/ml (5000 units /ml) of plqcmq. along with 60 ~l/ml of pl~qcmq of lM MnCl2. The plqc~n~q. was 10 vortexed and incubated on ice for 15 min. Following incllh~tion~ the sup~rn~qtqnt was laoovelad by cantrifilgqtion The procedure was repeated twice to complePly ~ qte VLDL+LDL in the plscmq.
v~ds, 80 ~l/ml of 10% tl~xtrqn sulfate per I was added to the sUpernstsnt and incubated for 15 min. The ...i~ . e was centrifuged 15 and the sup~rn~stqnt was c- llected and used as the source of CETP.

r S~STITUIE SHEET

WO 95/04755 2 1 ~ 5 7 6 7 PCT/US94/08624 ~Q~ ~ LISTING

(1) ~NT'RAT- lN~ORhATION:
(L) APPLICANT: KUSHWAHA, R~MPRATAP
McGILL JR., HENRY C.
KANDA, PATRICK

(iL) TITLE OF lwv~hlION: CETP INHIBITOR POLY~ll~E, ANTIBODIES AGAINST THE SYh~ lC POLY~ll~E, AND
PROPHYLACTIC AND THERAPEUTIC ANTI-~ROSCLEROSIS
TRTi~ MT~NTs .
(iii) NUMBER OF ~U~N~S: 3 (iv) CORR~.~PONDENCE ADDRESS:
(A) ADDRESSEE: COX & SMITH lNCORPORATED
(B) STREET: 112 EAST PECAN STREET, SUITE 1800 (C) CITY: SAN ANTONIO
(D) STATE: TEXAS
(E) ~Uh 1~Y: US
(F) ZIP: 78205 (v) COh~ul~K R~n~RT-Ti! FORM:
(A) MEDIUM TYPE: Floppy di~k (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn R~le~e #1.0, Ver~ion #1.25 (vi) ~U~h.~. APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
( C ) CLASSIFICATION:
(viii) AL-ORN~Y/AGENT lN~OR~ATION:
(A) NAME: ~AY~Nn, W. B.
(B) REGISTRATION NUMBER: 35,186 (C) ~K~N~/DOCRET NUMBER: S-0072.187 (iX) TT~T.~:C~ ~NlCATION INFORNATION:
(A) TELEPHONE: 210-554-5260 (B) TELEFAX: 210-226-8395 (C) TELEX: 767609 (2) lNrORMATION FOR SEQ ID NO:1:
( i ) ~QDL ._~ CHARACTERISTICS:
(A) LENGTH: 36 amino acid~
(B) TYPE: amino acid (C) s~R~NnT!nNT!-~S: ~ingle (D) TOPOLOGY: linear SUBS~IME SHEET ~RIJL~ 26~

WO 95/0475~i 2 1 ~ 5 7 ~ I PCTJUS94/08624 (ii) MOLECULE TYPE: peptide (xi) S~U~N~ DESCRIPTION: SEQ ID NO:1:
Asp Val Ser Ser Ala Leu ABP Lys Leu LYB Glu Phe Gly Asn Thr Leu Glu Asp LYB Ala Trp Glu Val Ile Asn Arg Ile Ly~ Gln Ser Glu Phe Pro Ala LYB Thr (3) INFORMATION FOR SEQ ID NO:2:
(i) SLYU~:N~L CH~RACTERISTICS:
(A) LENGTH: 38 amino acid~
(B) TYPE: amino acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ( xi ) S~Q~hL._~ DESCRIPTION: SEQ ID NO:2:
Ala Pro ABP Val Ser Ser Ala Leu A~p Lys Leu Lys Glu Phe Gly A~n 35 Thr Leu Glu A~p Ly~ Ala Trp Glu Val Ile Asn Arg Ile Lys Gln Ser Glu Phe Pro Ala Lys Thr (4) lN ~ORhATION FOR SEQ ID NO:3:
(i) S~:YUL.._~ CHARACTERISTICS:
(A) LENGTH: 38 amino acids ( B) TYPE: amino acid (C) STRANDEDh'ESS: single (D) TOPOLOGY: linear ( ii ) M~T~CUT~T~ TYPE: peptide (xi) ~yu~.._~ DESCRIPTION: SEQ ID NO:3:
55 Thr Pro ABP Val Ser Ser Ala Leu Asp LYB Leu Lys Glu Phe Gly A~n SUBSl IME SHEET ffHJLE 26~

~WO 95/04755 21~ 5 7 6 7 PCT/US94/08624 Thr Leu Glu ABP Ly~ A1a Arg Glu Leu Ile Ser Arg Ile Lys Gln Ser Glu Leu Ser A1a Lys Met SU~Sl ITUI~ SHEET (RULE 26)

Claims (18)

1. A substantially pure polypeptide having activity inhibitory of cholesteryl ester transfer protein comprising at least a part of peptides selected from the group consisting of:

Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO: 3),

2. The cholesteryl ester transfer protein inhibitory polypeptide of claim 1, in freeze dried form.

3. An anti-atherosclerosis composition, comprising an anti-athero-sclerosis effective amount of at least a part of a polypeptide selected from the group consisting of:

Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO: 3), and a pharmaceutically-acceptable carrier.

4. An anti-atherosclerosis kit, comprising in separate sterile containers at least 10 to 400 mg of the composition of claim 3;
at least one syringe; and at least one needle.

5. An antibody having specificity for a polypeptide comprising at least a part of a polypeptide selected from the group consisting of:

Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO: 3),

6. The antibody of claim 5, being a polyclonal antibody.

7. The antibody of claim 5, being capable of specifically binding to modified apo A-I.

8. The antibody of claim 5, being capable of specifically binding to the baboon CETP inhibitor polypeptide.

9. The antibody of claim 5, being capable of specifically binding to modified apo E.

10. The antibody of claim 5, being capable of specifically binding to apo C-I.

11. A method of preventing atherosclerosis in a mammal being predisposed to that condition, comprising administering to the mammal a prophylacically effective amount of a polypeptide comprising at least a part of a peptide selected from the group consisting of:

Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO: 3),

12. The method of claim 11, wherein the polypeptide is administered in an amount of about 10 to 200 mg.

13. The method of claim 22, wherein the polypeptide is administered intravenously.

14. The method of claim 22, wherein the mammal is a human.

15. A method of treating a mammal afflicted with atherosclerosis comprising administering to the mammal a therapeutically effective amount of a polypeptide comprising at least one part of a peptide selected from the group consisting of:

Ala-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu Val-Ile-Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 2) and Thr-Pro-Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys Glu-Phe-Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Arg-Glu Leu-Ile-Ser-Arg-Ile-Lys-Gln-Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO: 3)

16. The method of claim 15, wherein the polypeptide is administered in an amount of about 10 to 400 mg.

17. The method of claim 15, wherein the polypeptide is administered intravenously.

18. The method of claim 15,wherein the mammal is a human.