AU683295B2 - CETP inhibitor polypeptide, antibodies against the synthetic polypeptide and prophylactic and therapeutic anti-atherosclerosis treatments - Google Patents

Description

WO 95/04755 PCT/US94/08624 CETP INHIBITOR POLYPEPTIDE, ANTIBODIES AGAINST THE SYNTHETIC POLYPEPTIDE AND PROPHYLACTIC AND THERAPEUTIC ANTI-ATHEROSCLEROSIS TREATMENTS BACKGROUND OF THE INVENTION This application is a continuation-in-part of application erit7 No. 07/811,049, which was filed on December 18, The work leading to th sent invention was partially supported by Natio art, Lung and Blood Institute Grants Nos.

HL2892 HL41256, and Contract No. HV53030. The government may hold rights in the present patent.

Field of the Invention This invention relates to an endogenous baboon plasma cholesteryl esters transfer protein (CETP) inhibitor polypeptide.

More specifically, this invention relates to the identification and characterization of the polypeptide and to novel synthetic peptides possessing inhibitory activity of CETP. The endogenous inhibitory peptide has a molecular weight of 4000, is present in plasma in the form of modified apo A-1 and apo E having molecular weights of 31kD and 41kD, respectively, and has a common amino acid sequence with the N-terminal fragment of apo C-1. This invention also relates to an anti-atherosclerosis composition, a kit, and to antibodies raised against the N-terminal amino acid sequence of the inhibitory polypeptide. The inhibitory peptide of the invention, fragments thereof and analogues thereof are useful for the prophylactic and therapeutic treatment of atherosclerosis.

Description of the Background Atherosclerosis is one of the most widespread health problems in the United States today as are its attendant complications, particularly coronary heart disease. A number of risk factors have been associated with the development of premature atherosclerosis, primarily elevated plasma cholesterol levels. Due to the crucial role cholesterol appears to play in the occurrence of heart disease, a great deal of attention has been devoted to studying its synthesis, transport and metabolism in the human body.

SSUBSTITUTE SHEET (RULE 26) \^kir Oc^ I I I 1~11 I L-La I I I I I I I WO 95104755 PCT/US94/08624 2 Of particular interest is the establishment of relationships between the levels of plasma lipoproteins or serum lipids and the risk of development of coronary heart disease. Both high density lipoproteins (HDL) and low density lipoproteins (LDL) carry cholesterol mainly in the form of cholesteryl esters There are some indications, however, that while LDL cholesterol is a positive risk factor, HDL cholesterol is an even more important negative risk factor. Although the exact functions of these lipoproteins have not been completely established, HDL appears to serve for the removal of cholesterol 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 cardiovascular disease by overloading the lysosomes of the walls of arterial cells with metabolites which are generally hydrolyzed slowly, such as CE and triglycerides. These products are evacuated from the liver and intestine by plasma 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 certain critical areas, such as arterial walls. This overloading eventually results in impaired cell function, and if continued may produce cell death. A continuous overloading results in the accumulation of cellular debris and the formation of atherosclerotic plaque in the vessel wall. This, in turn, leads to the blockage of the affected artery and/or muscular spasm, events which may manifest themselves as coronary heart disease or strokes. Thus, the level of HDL in plasma has been negatively correlated with the probability of developing atherosclerosis in humans and experimental animals.

Although the level of HDL has been shown to vary considerably among individuals, the means of regulation of such plasma level remains to be elucidated.

CETP transfers CE from HDL to VLDL and LDL, and it has been suggested that it plays an important role in the regulation of plasma HDL levels. Some hyperalphalipoproteinemic patients were reported to have high levels of large HDL particles that were clearly separate from LDL. Plasma samples from these patients were shown SUBSTITUTE SHEET (RULE 26) -II I L II' WO 95/04755 PCTUS94/08624 3 to lack CETP activity (Koizumi et al, Atherosclerosis 58:175-186 (1985)). A homozygous subject with familial hyperalphalipoproteinemia was found to have impaired transfer of CE from HDL to LDL (Yokoyama et al, Artery 14:43-51 (1986)). A fraction of density d>1.21 g/ml from the subject's plasma evidenced substantial CETP activity with normal HDL. The HDL, however, proved to be a poor substrate for CETP.

Certain animal sires and their progeny possess unusual lipoproteins patterns, lipoproteins of a density intermediate to that of LDL and HDL, or large high density lipoproteins. These lipoproteins have been designated HDL 1 and the animal phenotype as "high HDLi". Baboon strains possessing, for instance, patterns of either high or low HDL 1 are known. In most cases, HDL 1 separates either as a distinct peak between LDL and HDL or as a shoulder to the HDL peak, and is induced by a high cholesterol, high lard (HCHF) diet. The proportion of HDL 1 diminishes when the baboons are fed a diet that is either enriched in polyunsaturated fat, with or without cholesterol. Occasionally, however, the amount of HDL 1 present in high HDLI baboons fed the chow diet is low.

In some baboon families, the level of plasma HDLi was shown to increase when the animals are challenged with a HCHF diet.

When fed a HCHF diet, the baboons also show higher plasma HDL.

More generally, the accumulation of HDL in baboons as well as in humans is associated with a slower transfer of CE from HDL as very low density lipoproteins (VLDL) and LDL. Thus, baboons with high HDL, plasma levels are excellent as animal models for the study of hyperalphalipoproteinemia.

In a previous study, some of the present inventors reported that a slower transfer of CE from HDL to VLDL and LDL was observed in high HDL,,baboons. This was attributed to the presence of a CETP protein inhibitor associated with HDL and intermediate density lipoprotein (IDL) particles (Kushwaha et al, J.P. Lipid Res.

31:965-974 (1990)). An accumulation of HDL, in the high HDLI baboons fed a HCHF diet was reported along with a slower transfer of CE from HDL to LDL. A similar protein was found in human plasma by Son and Zilversmit (Son and Zilversmit, B.B.A. 795:473- SUBSTITUTE SHEET (RULE 26) _IC WO 95/04755 PCT/US94/08624 4 480 (1984)). The human protein has a molecular weight of 31,000 and suppresses the transfer of triacylglycerol and CE.

Several other species including rat, pig, and dog have been reported to readily accumulate HDLI in plasma. Kurasawa et al.

(1985), supra, reported that a homozygous subject with familial hyperalphalipoproteinemia has impaired CE transfer between HDL and LDL (Kurasawa et al, J.B. Biochem. 98:1499-1408 (1985)).

Separately, Yokoyama, et al. reported that a plasma fraction of d>1.21 g/ml of the same subject evidenced substantial CE transfer activity when tested with normal HDL (Yokoyama et al, Artery 14(1):43-51 (1986)). The HDL particles accumulated by this subject were substantially larger in molecular size than ordinary HDL.

HDL is generally divided into subfractions based on their particle sizes and densities. These fractions include HDL,, HDL 2 and HDL 3

HDL

1 has the largest particles and is usually not present in the plasma of normal humans or non-human primates. HDL 2 and

HDL

3 are the normal components of human plasma. HDL 2 is larger than HDL 3 and differs between men and women.

Many attempts have been made to interfere with the transport and transfer of cholesterol in mammalians in order to alter its plasma levels. Among them are the following.

U.S. Patent No. 4,987,151 to Taboc discloses triterpene derivatives that inhibit acyl coenzyme A:cholesteral acyltransferase (ACAT) enzyme. The ACAT is a cellular enzyme that is not present in plasma, and esterfies cellular cholesterol to form CE. This enzyme is different from the CE transfer protein (CETP) present in plasma.

The CETP does not form CE as does the ACAT enzyme. Instead, the CETP transfers CE amongst different plasma lipoproteins.

U.S. Patent No. 4,643,988 to Segrest, et al discloses amphipathic peptides which are capable of substituting for apo A-I in HDL. Apo A-I is known to stimulate the lecithin cholesterol:acyl transferase (LCAT) enzyme, a plasma enzyme that forms CE in HDL. Plasma CETP, in contradistinction, transfers CE from HDL to VLDL and LDL. The function of the CETP enzyme is, therefore, different from that of the LCAT enzyme, as well. The amino acid SUBSTITUTE SHEET (RULE 26) I laLI IILlrl~ sequence of the Segrest et al peptides are, in addition, different from the sequences of the CETP inhibitor of this invention.

Throughout the description and claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

Summary of the Invention This invention relates to a substantially pure polypeptide having activity inhibitory of cholesteryl ester transfer protein including 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-l le-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- S 15 Leu-Glu-Asp-Lys-AlaArgGlu-LeuIle-SerArgIle-Lys-Gln-Ser-Glu-Leu- Ser-Ala-Lys-Met (SEQ ID. NO:3); wherein said polypeptide is used to inhibit cholesteryl ester transfer protein activity.

This invention also relates to an anti-atherosclerosis composition, including an anti-atherosclerosis 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-l le-Asn-Arg-l le-Lys-Gn-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-l le-Ser-Arg-l le-Lys-Gln-Ser-Glu-Leu- Ser-Ala-Lys-Met (SEQ ID. NO:3), and a pharmaceutically-acceptable carrier; wherein said polypeptide is used to inhibit cholesteryl ester transfer protein activity.

C ,\WNWORDFIONA\STUARTSJSBSPEC75526 DOC II II In addition, this invention relates to a kit for the prevention of atherosclerosis when used in the method described above, including in separate sterile containers at least 10 to 400 mg of a composition described above; at least one syringe; and at least one needle; wherein a patient in need thereof is injected with said composition.

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-terminal fragment of apo C-I; modified apo A-I (NW:31kD); modified apo E (MW:41D).

In a different aspect, this invention relates to a method of preventing atherosclerosis in a mammal being predisposed to that condition, comprising administering to the mammal a prophylactically effective amount of the polypeptide described above.

This invention also relates to a method of treating a mammal afflicted with 20 atherosclerosis comprising administering to the mammal a therapeutically effective amount of the polypeptide described above.

I' CVWNWORDFIONALUARTIS SPECs55 DOC n' C~ ~III Replacement Page 6 Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion.

Description of the Preferred Embodiment This invention arose from the desire by the inventors to provide a novel and unobvious approach to the prevention and treatment of atherosclerosis in humans.

A schematic of what is known about the association of cholesterol with the different fractions of lipoproteins in plasma and in the liver is shown below: CE. ApoE S HDL t LDL-R mnRNA VLDL (CETP611 CE

LOL

The metabolic steps leading to the accumulation of HDL 1 VLDL and LDL are shown above. HDL 2 3 collects cholesterol from extrahepatic cells, which is then esterified by LCAT to form cholesteryl esters (CE) and stored in the core of the particles. The HDL becomes larger in size (HDL) and may pick up apo E to attain a particle which is removed from LDL receptors (LDL-R) on liver cells. The CE enriched HDL 1 may also donate CE to VLDL and LDL.

This is mediated by CETP. Due to the presence of the CETP inhibitor, such as the one provided herein, CE transfer is slow (bar) and the reciprocal transfer of triglycerides (TG) does not take place The triglyceride-poor HDL is thus not a suitable substrate for ~-~IPI -11 I IF1- s II Replacement Page 6A hepatic triglyceride lipase (HTGL). Due to the presence of the CETP inhibitor, in plasma, VLDL and LDL are thus not available to the liver. As a consequence of this, the liver then increases the expression of messages for the increased production of LDL receptor and 3 hydroxy, methyl, glutaryl-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 synthesis of HMG- CoA synthase leads to an increase in synthesis of cholesterol in the liver to meet all cellular needs. Thus, the presence of a CETP inhibitor in plasma will prevent the uptake of VLDL and/or LDL by tissues as well as the deposition of cholesteryl esters.

In general, high levels of HDL have an anti-atherosclerogenic effect whereas high levels of LDL have an atherogenic effect. The ~-~111 _1 ,-6 II~AB YTPII~-~ ~I I _I~ WO 95/04755 PCT/US94/08624 7 circulation in blood of compounds, such as cholesterol, that are insoluble in water requires the formation of particles. The insoluble components, cholesteryl esters and triglycerides, are packed in the core of the particles and surrounded by polar components such as proteins, phospholpids, and the like. These particles are called lipoproteins, and have, thus, an outer polar shell and a non-polar core. These associations of lipoproteins containing CE in the core and, depending on their sizes and densities, 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 hepatic tissues. HDL is also secreted by the liver and is divided in

HDL

2 and HDL 3 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 enriched with apo E. These large HDL particles are called HDLI and they do not stay in the plasma for long periods of time. They are rapidly removed by the liver or converted back to HDL 2 after donating their cholesteryl esters to VLDL and LDL.

HDL

1 is not present in either normal humans or non-human primates. As indicated above, HDL 1 appears as a distinct band in the plasma of baboons that have been fed a HCHF diet. From what is known, when cholesterol enters the blood stream it becomes associated with HDL in the form of a CE, with the help of the LCAT enzyme. In HCHF-fed baboons, this appears as an HDL 1

-CE

fraction. The CE is then transferred from HDL to VLDL and LDL to 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 plasma and thus to the periphery of the mammalian body, where its deposition may occur leading to atherosclerosis.

An inhibitor of CETP, such as the one provided by this invention, blocks the transfer of CE from HDL to VLDL and LDL.

SUBSTITITE SHEET (RULE 26) L I q I ~PP~I 1_11 1 5- WO 95/04755 PCT[US94/08624 8 Instead, a shunt is favored that leads to the association of the CE with apo E and to the formation of HDLi-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 known. The Apo C-I is a single polypeptide of molecular weight 6600, consisting of 57 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 and affects its binding to the LDL receptor.

The polypeptide inhibitor of CETP that is described herein has a common sequence with the N-terminal fragment of apo C-I. This fragment includes at least 36 amino acids as shown below.

The endogenous polypeptide (SEQ. ID. NO: 1) provided by this invention has a molecular weight of about 4,000 and becomes associated or binds to apo A-I and apo E in plasma. Its N-terminal 36 amino acids are shown below.

A polypeptide having the sequence corresponding to amino acids 1 to 36 of the following sequence (SEQ. ID. NO: 1) was synthesized by the inventors and shown to be inhibitory of CETP in vitro. The peptide has the following sequence.

1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Gl-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO: 1).

Fragments of that polypeptide (SEQ. ID. NO: 1) comprising the C-terminal fragments of amino acids 28 to 36 and amino acids 16 to 36 showed limited inhibitory activity of CETP at 50 pg. However, the fragment comprising the C-terminal amino acids 28 to 36 showed at 200 pg an activity inhibitory of CETP approximately the same as SUBSTTUTE SHEET (RULE 26) h _1L I Is I~O~P~ll I r ~i -rr WO 95/04755 PCTfUS94/08624 9 that of the 36 amino acid peptide. The N-terminal 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 have been shown active as inhibitors of CETP.

Polypeptides having the sequences corresponding to the following two sequences designated (SEQ. ID. NO:2) and (SEQ. ID.

NO:3) nfave also been synthesized by the inventors and shown to be inhibitors of CETP in vitro.

The first of the following two sequences (SEQ. ID. NO:2) is a baboon sequence like (SEQ. ID. NO:1) except that it has two additional amino acids at the beginning 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 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-Ile-Asn-Arg-Ile-Lys-Gln- Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO:2) 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 fragments thereof having inhibitory activity of CETP are also part of the invention. The analogues may have one or more substitutions in their sequences while still preserving their inhibitory activity.

Examples of analogues suitable as inhibitors of CETP are analogues of the peptide of the invention and fragments thereof, such as those where one or more of the amino acids are substituted in accordance with the guidelines provided below.

The substitute amino acids may be selected from the group consisting of Glu, C(-methylAsp, and B-carboxyAsp for Asp; SUBSTITUTE SHEET (RULE 26) L~ I I _I I WO 95/04755 PCTIUS94/08624 isoVal, norVal, Leu, and Ca-methylVal for Val; Gly, B-Ala, Cm-methy1Ala, and 2-amino butyric acid for Ala; norLeu, isoLeu, and C(X-methylLeu for Leu; ornithine, Arg, citrulline and Ccc-methy]Lys for Lys; Ala and 2-amino isobutyric acid for Gly; Gin, citmufine, and Ca-methyIAsn for Asn; P-Benzoyl Phe, Arg, and Cot-methylTrp for Trp; 2-amino adipic acid, Asp, and Ca-methylGlu for Glu; Leu, norLeu, and Cotmethylle for Ile; Lys, homoArg, citrulline and Ca-methy1Mrg for Arg; Asn, citrulline, and Cot-methy1Gln for Gin; 2-amino-4-phenylbutyric acid, Leu and C.-methylPhe for Phe; Ser, Met and Ca-methy1Thr for Thr; Thr and CccmethylSer for Ser; and 3,4-DehydroPro, Ser and Ca-methylro for Pro; and combinations thereof.

However, 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 combination with other substituents.

It is therefore provided in accordance with this invention, a substantially pure polypeptide having activity inhibitory of CETP.

In one embodiment of the invention, the polyp eptide is capable of inhibiting the binding of an about 31k]) modified apo A-I polypeptide present in the plasma of high HDL 1 baboons or a peptide of the sequence.

1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-lle-Lys-Gln-Ser-Glu-Phe-Pro-Alda-Lys-Thr (SEQ. ID. NO. 1), to an antibody raised against the above peptide.

SUBSTITUTE SHEET (RULE 26)

I

WO 95/04755 PCT/US94108624 11 In another embodiment, the polypeptide of the invention is capable of inhibiting the binding of an about 4kD CETP inhibitor pnlypeptide present in the plasma of high HDLI baboons to an antibody raised against a peptide of the formula 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Le' i-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Aia-Lys-Thr (SEQ. ID. NO. 1).

In still another embodiment, the polypeptide of this invention is capable of inhibiting the binding of an about 41kD modified apo E polypeptide present in the plasma of high HDL, baboons with antibody raised against the 36 amino acid N-terminal fragment of apo C-I or a peptide of the formula 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. 1).

In still another embodiment, the polypeptide is capable of inhibiting the binding of the 36 amino acid N-terminal fragment of apo C-I or a peptide of the formula 1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- SUBSTITUTE SHEET (RULE 26) U I I II I WO 95/04755 PCTIUS94/08624 12 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Jle- 26 27 28 29 30 31 32 33 34 35 36 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.

Preferred poiypeptides are the polypeptides of the sequence 1 23 4 5 6 78 9 1011 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 26 27 28 29 3031 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr (SEQ. ID. NO. 1), 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-lle-Ser-Arg-lle-Lys-Gln- Ser-Glu-Leu-Ser-Ala-Lys-Met (SEQ. ID. NO:3); anti-4kD peptide antibody-binding inhibitory fragments of (SEQ. ID NO.: 1) thereof; and anti-4kD peptide antibody-binding inhibitory analogues of (SEQ. ID NO: 1) thereof having at least one substitute amino acid selected from the group consisting of Glu, Co-methy1Asp, and B-carboxy Asp for Asp; SUBSTITUTE SHEET,(RIJLE 26) WO 95/04755 PCT[US94/08624 isoVal, norVal, Leu, and Ca-methylVal for Val; Gly, B-Ala, Cc-methy1Ala, and 2-amino butyric acid for Ala; norLeu, isoLeu, and Coc-methylheu for Leu; ornithine, Arg, citrulline and C~x-methyILys for Lys; Ala and 2-amino isobutyric acid for Gly; Gln, citrulline, and CQI-methy1Asn for Asn; P-BenzolyPhe, Arg, and Cw~methyl Trp for Trp; 2-amino aclipic acid, Asp, and Ca-methylGlu for Glu; 2-amino aclipic acid, Asp, and CQ,-methylTrp for Trp; Leu, norLeu, and Coc-methylIle for le; Lys, homoArg, citrulline and C~x-methy1Arg for Arg; Asn, citrulline, and C~x-methylGhI for Gin; 2-amino-4-phenylbutyric acid, Leu and Cax-methyiPhe for Phe; Ser, Met and C.-methylThr for Thr; Thr and Ca-methylSer for Ser; 3,4-DehydroPro, Ser and Cot-methy1Pro for Pro; and combinations thereof.

In one particular preferred embodiment, the polypeptide contains amino acids 1 through 36 of the above sequence (SEQ. ID.

NO. In still another particularly preferred embodiment, 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 and fragments thereof having anti-4kD peptide antibody/1-36 amino acid peptide binding inhibitory activity.

In still another preferred embodiment, the peptide fragments are selected from the group consisting of peptides comprising amino acids 1 to 18, and 1 to 28 of (SEQ. ID. NO. and fragments thereof having anti-4kD peptide antibody/l-36 amino acids peptide binding inhibitory activity.

SUBSTITUTE SHEET (RULE 26) WO 95/04755 PCT/US94/08624 14 Also preferred are analogues (SEQ. ID. NO. 1) with the Lys, Asp and Asn amino acids substituting for the Arg, Glu, and Gin amino acids; the Ser, Leu, and Ala amino acids substituting from the Thr, Ile and Gly amino acids; the ornithine, citrulline and c aminoadipic acd amino acids substituting for the Lys and Glu amino acids.

Also preferred are the following analogues (SEQ. ID. NO. 1).

Peptides comprising amino acid sequences where amide bond(s) linking any pair, and up to all pairs, of amino acids comprising amino acds 1 to 17, 1 to 20, 1 to 25, 1 to 36, and fragments thereof having anti-4kD peptide antibody/1-36 amino acid peptide binding inhibitory activity are replaced by thioether bonds

-CH

2 alkyl such as ethyl (-CH 2

-CH

2 and/or amino CH2-NH 2 linkages. These analogues may be purchased commercially or prepared by methods know to those skilled in the art as long as the antibody 1-36 amino acid peptide binding and CETP inhibitory activities of the peptides (analogues) are retained to some degree.

However, other analogues (SEQ. ID. NO. 1) are also part of this invention as long as they preserve the inhibitory activity of the antibody/1-36 amino acid peptide binding.

The CETP inhibitory polypeptide of the invention may be provided as a powder, preferably in freeze-dried form, as a solution, preferably frozen at below -20°C, and the like, to prevent proteolysis.

This invention also provides an anti-atherosclerosis composition, comprising an anti-atherosclerosis effective amount of the polypeptide of the invention; and a pharmaceutically-acceptable carrier.

When the composition is used as preventative tool, it may contain 10 to 200 mg and more preferably 20 to 100 mg of the polypeptide. However, other amounts are also suitable. When the composition is intended for therapeutic use, the amount of polypeptide present is preferably about 10 to 400 mg, and more SUBSTITUTE SHEET (RULE 26) ill ~g sla WO 95/04755 PcTfUS94/08624 preferably about 20 to 300 mg. However, other amounts may also be utilized.

Any and all pharmaceutically-acceptable carriers known in the art for administration of peptides to mammals, and preferably to humans, are suitable for use herein. These are known in the art and need not be further described herein. Examples, however, are saline, human serum albumin and starch. However, others may also be utilized.

The composition of this invention may be provided in unit form, preferably in a sterile, closed container, 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 contain from about 1 to 20 units of the composition of the invention, but could contain 50 units or more. In addition, the kit may contain 1 to 20, but and sometimes 50 or more syringes if they are disposable, and 1 to 20, but sometimes up to or more needles if they are disposable. The components of the kit are provided in a sterile form, be it wrapped in a sealed, sterilized wrapping, or in some other way. If not disposable, the syringe and needle may be autoclaved between uses.

The composition of the invention is preferably administered intravenously, although it may also be administered intraperitoneally, subcutaneously or intramuscularly. The oral route is not permissible since the polypeptide would be degraded in the acidic pH of the stomach.

The composition may preferably have a pH of about 7 to 9, and more preferably about 8 to 9, which may be adjusted with the addition of a base, acid or buffer as is known in the art.

SUBSTITUTE SHEET (RULE 26) I I sp, PI--l sllrrrr WO 95/04755 PCT/S9/08624 16 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 inhibitor, fragments thereof and analogues thereof; the 1-36 amino acid N-terminal fragment of apo C-l; modified apo A-I (MW:31kD); and modified apo E (MW:41kD).

The antibodies of the invention may be raised in mammals as is known in the art. (Albers, J.J. Hazzard, 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 chicken. However, other mammals may also be utilized.

Preferred are rabbit antibodies. Also preferred are polychonal antibodies. However, monoclonal antibodies may also be prepared by methods known in the art (Kohler, and Milstein, Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity Nature (London) 256:495-497 (1975)).

In one preferred embodiment, the antibody of the invention is capable of specifically binding to modified apo A-I.

In another preferred embodiment, the antibody is capable of specifically binding to the baboon CETP inhibitor polypeptides of this invention.

In another preferred embodiment, the antibody of the invention is capable of specifically binding to the polypeptide of the sequence.

1 2 3 4 5 6 7 8 9 10 11 12 Asp-Val-Ser-Ser-Ala-Leu-Asp-Lys-Leu-Lys-Glu-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 Gly-Asn-Thr-Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-Ile- 26 27 28 29 30 31 32 33 34 35 36 Asn-Arg-Ile-Lys-Gln-Ser-Glu-Phe-Pro-Ala-Lys-Thr SUBSTITUTE SHEET (RULE 26) I I Is~ WO 95/04755 PCT/US94/08624 17 fragments thereof and analogues thereof as described above.

In still another preferred embodiment of the invention, the antibody is capable of specifically binding to modified apo E.

The antibody of the invention, in another preferred embodiment is also capable of specifically binding to apo C-1, and more preferably to the 1-36 N-terminal fragment thereof.

In another aspect of the invention, a method is provided for preventing atherosclerosis in a mammal being predisposed to that condition. The method comprises administering to the mammal a prophylactically effective amount of the polypeptide of the invention, or of the anti-atherosclerosis composition described above.

In a preferred embodiment, the polypeptide is administered in an amount of about 2 to 100 mg for preventative applications.

However, other amounts may also be administered. The polypeptide or composition thereof may be administered in a small volume of carrier, 0.2 to 1.5 ml of saline or other carriers, as is known in the art.

The polypeptide of the inventions may be administered intravenously, to a fragment of the population, particularly the human population, that is not afflicted by high blood cholesterol and hyperbetalipoproteinemia, but, as determined by other means, may be at risk of being afflicted by atherosclerosis. One such example may be a familial trait having been determined.

The polypeptide of this invention may be administered on a daily basis, or at longer intervals if provided as a slow release composition as is known in the art, such as depoestradiol provided by the UpJohn Co. (UpJohn Co., Kalamazoo, MI).

In another aspect, the present invention provides a method of treating a mammal afflicted with atherosclerosis. The method comprises administering to the mammal a therapeutically affective amount of the polypeptide of the invention. When administered for therapeutic purposes, the polypeptide may be injected in an amount SUBSITUTE SHEET (RULE 26) L -L1II WO 95/04755 PCT/US94/08624 18 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 administered.

In this case, as in the case of the prophylactic administration, the polypeptide may be administered intravenously, among other routes.

Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein for purposes of illustration only and are not intended to limiting of the invention or any embodiment thereof, unless so specified.

Examples Example 1: Animals and Diet Adult male and female baboons (papio sp.) held in a baboon colony at the Southwest Foundation for Biomedical Research in San Antonio, Texas, were used as blood donors for these studies. Among these, 24 baboons had a high HDLI phenotype and 32 had a low

HDL

1 phenotype (Williams, et al., Detection of Abnormal Lipoprotein In a Large Colony of Pedigreed Baboons Using High- Performance Gel Exclusion Chromatography, J. Chromat. 308:101- 109 (1984)).

Half of the high HDL 1 baboons (n=16) were maintained on a HCHF diet, the composition of which has been previously described (Kushwaha, et al., Metabolism of Apolipoprotein B. In Baboons with Low and High Levels of Low Density Lipoprotein. J. Lipid Res.

27:497-507 (1986)).

Most of the baboon donors of the low HDL, phenotype were maintained on a chow diet (Purina Monkey Chow, manufactured by Ralston Purina Co., St. Louis, Missouri). The monkey chow is low in fat (10% of total calories) and high in carbohydrate (62% of total calories). In addition, the chow has a very low cholesterol content (0.03 mg/Kcal).

The high HDL 1 baboons were progeny of two sires (X1672 and X102) who had a high HDL, phenotype. The low HDL, baboons were SUBSTITUTE SHEET (RULE 26) I WO 95/04755 PCT/US94/08624 19 progeny of a number of sires who did not have a high HDL 1 phenotype. The presence of HDL 1 was detected by high performance liquid chromatography (HPLC) as described previously (Williams, et al., Detection of Abnormal Lipoprotein in a Large Colony of Pedigreed Baboons Using High-Performance Gel Exclusion Chromatography, J.

Chromatography 308:101-109 (1984)).

Example 2: Preparation of 3H Cholesteryl Ester HDL High and low HDL, baboons were immobilized with 10 mg/kg of ketamine HC, and bled. The blood was collected in tubes containing 1 mg/ml EDTA, and plasma separated by low speed centrifugation at 6°C. The plasma was treated with sodium azide, choloramphenicol, gentamycin sulfate, phenylmethyl-sulfonyl fluoride and DTNB as described previously (Kushwaha, et al., Impaired Plasma CE Transfer with Accumulation of Larger High Density Lipoproteins in Some Families of Baboons (papio J.

Lipid Res. 31:965-973 (1990)).

to 60 pCi of tritiated cholesteryl linoleate were dissolved in ethanol and then added to the plasma. The plasma was flushed with nitrogen and incubated for 20 hours at 4 0

C.

After incubation, HDL 3 was isolated by density gradient ultracentrifugation (McGill, H.C. et al., Dietary Effects on Serum Lipoprotein of Dsylipoproteinemic Baboons with High HDLI, Atherioscerosis 6:651-663 (1986); Redgrave, et al., Separation of Plasma Lipoprotein by Density-Gradient Ultracentrifugation, Ana.Biochem. 65:42-49 (1975)), dialyzed against saline/EDTA, and used as a substrate for the CE transfer reaction.

The total and free cholesterol contents of HDL 3 were measured prior to use in the assay.

Example 3: Preparation of Acceptor Lipoproteins and CETP Source VLDL+LDL from low HDLi baboons was used as the acceptor of CE from HDL 3 A VLDL+LDL fraction of d<1.040 g/ml was isolated from 100-200 ml of blood by sequential ultracentrifugation as described previously (Kushwaha, et al. (1986), supra). Total and SUBSTITUTE SHEET (RULE 26) LII~'-LqCIP~ gr WO 95/04755 PCT/US94/08624 free cholesterol contents in acceptor lipoprotein were measured by enzymatic methods (Wako Pure Chemical Co.) (Allain, et al., Enzymatic Determination of Total Serum Cholesterol Clin. Chem.

20:470-475 (1974)).

After separation of VLDL+LDL, the bottom fraction was adjusted to d=1.21 g/ml of adding solid KBr, and total lipoprotein were isolated by ultracentrifugation (Kushwaha, et al. (1986), supra).

The bottom fraction of d>1.21 g/ml was also collected.

All lipoprotein fractions and the lipoprotein-deficient fraction of d>1.21 g/ml (LPDS) were dialyzed against saline/EDTA. The LPDS was used as the source of CETP.

Example 4: Cholesteryl Ester Transfer Assay The CE transfer activity of a sample was assayed by a modification of a procedure described previously (Kushwaha, et al., (1986), surpa).

Briefly, 3 H CE-labeled HDL containing 50-100 pg of CE from low HDL, baboons was incubated with VLDL+LDL containing 100- 300 pg CE in the presence of LPDS. The acceptor lipoprotein and the LPDS were obtained from low HDL, baboon plasma (chow diet). In some cases, the HDL 3 was obtained from high HDL 1 baboons maintained on the HCHF diet.

The incubations were carried out at 4°C (control) and 37 0 C for 4-6 hrs. and terminated by placing the samples on ice. The assay mixture was then ultracentrifuged to separate VLDL+LDL having a d>1.040 g/ml, and the radioactivity in the lipoprotein was counted as described previously (Kushwaha, et al. (1986), supra). Any difference observed in the radioactivity transferred from HDL to VLDL+LDL at 4°C and 37°C was attributed to CETP activity in the

LPDS.

Time course experiments gave a linear response up to 7 hrs.

Similarly, the CETP activity was linear with increasing LDPS, up to 140 pl LPDS, which was derived from an equivalent volume of plasma.

SUBSTITUTE SHEET (RULE 26) -S~llll~ i ql l(r IIIl~r(l~ WO 95/04755 PCT/US94/08624 21 They polypeptide of the invention, and synthetic fragments thereof were added to the reaction mixture to determine their CETP inhibitory activities, along with an appropriate synthetic control peptide.

The percent difference between the control experiment and the assay with inhibitor peptide was expressed as the inhibitor activity.

Example 5: Identification of Inhibitor Polypeptide The bottom non-lipoprotein fraction obtained by ultracentrifugation for 72 hrs was analyzed for protein content by 10% SDS-polyacrylamide gel electrophoresis (Laemmli, U.K., Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4, Nature 227:680-685 (1970)), without fmercaptoethanol pretreatment.

To determine difference in small molecular weight apoliproteins, dilapidated lipoproteins of d<1.21 g/ml from high and low HDL 1 baboons were separated by 15-19% SDS-polyacrylamide gel electrophoresis with P-mercaptoethanol pretreatment prior to loading the samples onto the gels.

Example 6: Electroelution of Inhibitor Polypeptide The lipoprotein fraction of d<1.21 g/ml from high HDL 1 baboon plasma was dilapidated with ether-ethanol (Floren, et al., Estrogen-Induced Increase in Uptake of Cholesterol-Rich Very Low Density Lipoproteins in Perfused Rabbit Liver, Metabolism 30:367-374 (1981)), and separated by 15% SDS gel electrophoresis (Laemmli, (1970), supra) after addition of f-mercaptoethanol. A small molecular weight protein band was cut and transferred onto a tube gel A dialysis tube of molecular weight cut-off point 1000 was attached to the bottom of the gel tube to receive the electroeluted 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.

SUBSTITUTE SHEET (RULE 26) I I r Poll WO 95/04755 PCT/US94/08624 22 Example 7: Antibody Preparation The apolipoproteins were sei rated by 15% SDS-gel electrophoresis and stained (Laemmli, (1970), supra). The stained bands were transferred onto a nitrocellulose membrane. The bands corresponding to the inhibitor 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 mixture was injected intradermally into two rabbits. After 30 days, the rabbits were boosted with a similar amount of electroeluted protein band.

Antibody titer was measured by Western blotting. The rabbits were boosted again 3 times.

For the preparation of antibody against the synthetic polypeptide, 500 pg of polypeptide were dissolved in 400 p1 of titer max (CytRx Corporation, Atlanta, GA), and injected into rabbits intradermally. The rabbits were boosted with 500 pg of the synthetic peptide in 200 1p 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: Imnmuoaffinity Chromatography An immunaffinity column was prepared using CnBr-activated Sepharose beads (Pharmacia The bound ligand was IgG precipitated from the serum of rabbits having antibodies. The method used to precipitate IgG was similar to that described by McKinney and Parkinson (McKinney, M.M. and Parkinson, A Simple, Non-Chromatographic Procedure to Purify Immunoglobins From Serum and Ascites Fluid, J.Immunological Methods 96:271-278 (1987)).

Briefly, 5 ml of rabbit serum were diluted 4-fold with acetate buffer, pH 4.0. 625 pl of caprylic acid were added dropwise to precipitate albumin and non-IgG proteins. The insoluble materials were removed by centrifugation at 10,000. I for 30 min. The supernatant was mixed with phosphate buffered saline, and the pH adjusted to 7.4 with 1N sodium hydroxide. The solution was cooled to 4 0 C and ammonium sulfate was added to give a final concentration of 45% to precipitate the IgG.

SUBSTITUTE SHEET (RULE 26) L- II I~ I WO 95/04755 PCT/US94/08624 23 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 CnBr-activated Sepharose beads, and maintained in Tris-saline, pH 7.4, until ready to use.

8 ml of plasma was incubated overnight with IgG coupled beads in Tris-saline buffered with gentle rotation. The column was then washed with Tris-saline coupling buffer and sodium acetate buffer as described by Cheung and Albers (Cheung, and Albers, Distribution of High Density Lipoprotein Particles with Different Apolipoprotein Composition: Particles with A-I and A-II and Particles with A-IBut No A-HI, J. Lipid Res. 23:747-753 (1982)).

The bound proteins were eluted with 0.1 M acetic acid, pH and 1 ml aliquots were collected and read at 280 nm to visualize the peak. The protein fraction was dialyzed immediately against phosphate buffered saline and separated by electrophoresis in SDS-polyacrylamide gels.

Example 9: Immunoblotting The proteins separated with SDS-polyacrylamide gels were "-ausferred onto Immobilon-P sheets (Millipore, Beford, MA). The sneets were incubated with antibody against inhibitor peptides after blocking of nonspecific sites. The sheets were washed and incubated again with a secondary antibody containing horseradish peroxides.

The addition of boric acid buffer containing 3-amino-9ethylcarbozole, methanol and hydrogen peroxide produced a coloration.

Example 10: Amino Acid Analysis and Sequencing Stained bands of proteins were transferred onto Immobilon-P sheets. Selected bands were cut out and hydrolyzed with propionic acid, 50% 12 N HC1 for 2 hrs at 135 C. Amino acid analysis of each sample was performed using a model 6300 amino acid analyzer (Beckman Co., Palo Alto, CA) provided with System Gold software.

SUBSTITUTE SHEET (RULE 26) 1~IIIIC- I- i I I I L 4 WO 95/04755 PCT[US9408624 24 The same bands were sequenced using a model 477A protein sequencer (Applied Biosystems, Foster City, CA).

Example 11: Preparation of Synthetic Peptides The peptides were synthesized by solid-phase peptide synthesis as described by Barany and Merrifield (Barany, G. and Merrifield, The Peptides, Analysis, Synthesis, Biology; Gross, E.

and Meinehofer, eds. Vol. 2, Academic Press, New York, pp. 1-284 (1980)).

The 1-36 amino acid synthetic peptide was assembled from the C-terminus towards the N-terminus, with the a-carboxyl group of the amino acid attached to a solid support and was then characterized by HPLC.

Example 12: Data Analysis The values provided in the following examples are averages and are provided as mean standard error. These values were compared using variance analysis and, if significant differences were detected, the values were compared using Duncan's Multiple Range Test (Duncan, Multiple Range and Multiple F Tests, Biometrics 11:1-12 (1955)).

Example 13: Characterization of Proteins From Infranatant Fraction The CETP inhibitory activity was lost from the HDL when the lipoproteins were extensively ultracentrifuged, for 72 hrs, or by repeated ultracentrifugation. After ultracentrifugation, the inhibitory activity was found in the infranatant fraction.

To characterize the proteins, the infranatant fraction (d<1.21 g/ml) was separated by 10% SDS polyacrylamide gel electrophoresis in the absence of f-mercaptoethanol.

The infranatant fraction from high HDL, baboons contained albumin, a protein slightly larger than apo A-I and another protein larger than apo E. Lanes A and B show nonlipoprotein fractions from high and low HDL 1 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 SUBSTITUTE SHEET (RULE 26) WO 95/04755 PCT/US94/08624 and 5, respectively, correspond to proteins with molecular weights of 41,000 and 31,000, respectively. Protein samples from high HDLi baboons show only bands corresponding to albumin, a protein of molecular weight 41,000 and a protein of molecular weight 31,000.

The molecular weights were determined with standard proteins separated on similar gels (gel picture not shown).

The infranatant fraction from low HDL, baboons contains these proteins as well, but in addition, it also contains apo A-I and apo E. Both proteins in the apo A-I region were identified by immunoblotting with antibody to apo A-I.

Similarly, 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 difference of about 4kD (picture not shown). The estimated molecular weight of apo A-I is about 27,500 and that of the modified apo A-I is about 31,000. Similarly, the estimated 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.

Example 14: Detection of CETP Inhibitor Peptide in Plasma of High HDL 1 Baboons To determine if a common polypeptide of molecular weight 4,000 was modifying both apo A-I and apo E, plasma lipoproteins of d <1.21 g/ml were separated from high and low HDL, baboons by 18% SDS-polyacrylamide gel electrophoresis with -mercaptoethanol.

A higher amount of a 4kD protein was detected in lipoprotein from high HDL 1 baboons as compared to lipoprotein from low HDLi baboons (gel picture not shown).

To determine if the 4kD polypeptide inhibits CE transfer, the polypeptide and albumin were electroluted from the gels, and used in increasing concentrations in a CE transfer assay mixture with lipoproteins from low HDLI baboons as described in Example 4 above. Albumin had no effect on the transfer of CE from HDL to VLDL+LDL. The 4kD polypeptide, on the other hand, significantly inhibited CETP activity. (Results are not shown).

SUBSITUTE SHEET (RULE 26) I I I WO 95/04755 PCT/US94/08624 26 Example 15: Characterization of Polypeptide by Affinity Chromatography.

Rabbit antibody was prepared against the 4kD polypeptide isolated from lipoproteins obtained from high HDL 1 baboons. This antibody was used to prepare an immunoaffinity column. The lipoproteins of d<1.21 g/ml were passed over the immunoaffinity column. The bound lipoproteins eluted with 0.1 M acetic acid and separated in 15% SDS-polyacrylamide reducing gels. 4kD, and 31kD polypeptides, and a minor band corresponding to a 41kD polypeptide were detected.

The column bound peptides (100 pg) inhibited by 31.3 1.4% the transfer of CE (mean SE, n=3) in the CETP assay from low

HDL

1 baboons.

On the other hand, the addition of IgG to the assay from high

HDL

1 baboons increased CE transfer by 44.3 1.5% but had no effect on CE transfer from low HDL 1 baboons.

Example 16: Comparison of 4kD Inhibitor Polypeptide with Sequence Data Bank The sequence of this polypeptide was compared to sequence of known proteins using Sequence Data Bank (Reardon, W. R. and Lipman, PNAS (USA) 85:2444-2448 (1988)), and was found to have 100% homology 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% homologous (private communication from Dr.

Hixson of the Southwest Foundation for Biomedical Research).

Example 17: Characterization of Inhibitor Polypeptide with Synthetic Peptides Based on its molecular weight, it was determined that the 4kD polypeptide contained approximately 36 amino acids.

Three peptides were synthesized beginning from the Cterminal end of the apo C-I sequence. The first peptide contained 9 amino acids, the second peptide contained 21 amino acids and the third peptide contained 36 amino acids. The 36 amino acid peptide had an amino acid sequence similar to the 4,000 MVW polypeptide, SUBSTITUTE SHEET (RULE 26)

-I=-Y

r~ll~ lll~ a~ arman~ar~- WO 95/04755 PCT/US94/08624 27 the other two were fragments of the synthetic peptide starting from its C-terminus.

pg of these peptides were used in a CETP assay with lipoproteins of the low HDLi baboons as described in Example 4 above. A soluble helical peptide with a 1,900 molecular weight utilized as control. The 36 amino acid polypeptide significantly (p< 0.01) inhibited CE transfer from HDL to VLDL and LDL, while the others, including the control peptide, did not.

Example 18: Antibody Against 36 Amino Acid Inhibitor Peptide Antibody against the 36 amino acids inhibitor peptide was prepared in rabbits as described in Example 7, and used for immunoblotting. The thus prepared antibody recognized the 4kD peptide as well as a 31kD polypeptide from lipoproteins of high HDL 1 baboons.

To determine if both the apo C-I and the 4kD polypeptide were present in the plasma of high and low HDLi baboons, lipoproteins from both phenotypes were separated by 10% SDS gel electrophoresis and immuno-blotted. Two protein bands were detected with the antibody by immunoblotting of samples from high

HDL

1 baboons. Only a single band was detected in samples from low HDL, baboons.

In addition, isoelectric focusing patterns of the synthetic peptide suggest that the peptide is a slightly basic protein.

Example 19: CETP Inhibition by Various Peptide Fragments A CE transfer assay as described in Example 4 above was conducted using HDL 1 baboon plasma. 3 H HDL and the VLDL+LDL carriers, in the presence of CETP enzyme to mediate the exchange.

The reactions were conducted at 37 0 C and 4°C (control) in duplicate. The results are shown in Table 1 below.

SUBSTITUTE SHEET (RULE 26) Pilll_---l- Il WO 95/04755 WO 9504755PCT/US94/08624 TABLE 1: CETP INHIBITION BY VARIOUS PEPTIDE FRAGMENTS CETP [H 3 HDL SYNTHETIC VLDL+LDL TEMP, INHIBITION QA1) 125 125 125 125 125 125 125 125 125 125 125 125 125 125

PEPTIDE

(pg) (Jig) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 50 amino acict (Jig) 300 300 300 300 300 300 300 300 300 300 300 300 300 300 37 32 21.ain ciT 36 ainoaciT SUBST=UT SHEEr~ WO 95/04755 PCT/US94/08624 29 Example 20: Inhibition of CETP From Humans by Synthetic CETP Inhibitor Peptide.

Cholesteryl ester transfer activity from human plasma was assayed by the procedure described by the inventors (Kushwaha Rainwater Williams Getz and McGill Jr., Impaired Plasma Cholesteryl Ester Transfer with Accumulation of Large High Density Lipoproteins in Some Families of Baboons (Papio J. Lipid Res. 31:965-973, 1990). In short, [3H] cholesteryl esterlabeled HDL (10-pg of cholesteryl esters with a specific activity of 3-4 x 10 6 dpm/mg cholesteryl ester) from low HDLI baboons was incubated with 50-100 jig of VLDL+LDL cholesteryl ester from baboons. The incubations were carried out in the presence of 100 pl of lipoprotein deficient serum (LPDS) obtained from humans and 2mM DTNB. The total volume of the assay was 1 ml. The incubations were carried out for 1-2 h at 40 and 37°. At the end of the incubation, 40 pl of heparin (5000 units 0.5 ml of plasma, and 60 pl of 1 M MnC 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 scintillation spectrometry. The difference between 40 and 370 was considered to reflect the CETP-mediated transfer. At the same time each set of human CETP incubations were run in the presence of synthetic CETP inhibitor peptide (baboon apo C-I terminal peptide with 38 amino acids, 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 the transfer of cholesteryl ester from HDL to VLDL+LDL was determined in the presence of CETP inhibitor. In some cases synthetic CETP inhibitor was similar to human apo C-I terminal peptide with 38 amino acids (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).

The results of these experiments are provided in the following tables: SUBSTITUTE SHEET (RULE 26) 1- -31 WO 95/04755 PCT[US94/08624 TABLE 2. Ihibition of Hurm CETP bY CETP Inldbitor peptide (Blaboon sune).

S.N. Source of r 3i1 HDL Inhiitor VLDL+LD)L Term. CETP Ihbto h.maCETP (Jig) paptidle (jig) (0 jl (jig) 1L SH 10 00 50 4 200 00 60 37 200 00 10 100 60 4 200 100 50 37 200 2. RE 10 00 50 4 t=3 00 50 37 200 00 10 100 50 4 200 100 50 37 200 72 3. F.1 10 00 50 4 200 00 50 37 200 00 10 100 ISO 4 200 100 50 37 200 41. GL 10 00 50 4 200 00 50 37 200 CETP 10 100 50 4 200 NOT 100 50 37 200 ACTIVE ER 10 00 50 4 200 00 50 37 200 00 10 100 50 4 200 100 50 37 200 82 6. AB 10 00 50 4 200 00 50 37 200 00 10 100 so 4 200 100 50 37 200 7. GI, 10 00 50 4 200 00 50 37 200 00 10 100 50 4 200 100 50 37 200 a. 1(0 10 00 50 4 200 00 ISO 37 200 00 10 100 50 4 200 100 50 37 200 83 9. TH 10 00 50 4 200 00 50 37 200 00 10 100 50 4 200 100 50 37 200 86 um 10 00 50 4 200 00 50 37 200 00 10 100 50 4 200 SUBSTITUTE SHEET PCTIUS94/08624 WO 95/04755 100 11 EW 10 00 00 100 100 12. ML 10 00 00 100 100 50 37 50 4 50 37 50 4 IS0 37 50 4 150 37 50 4 50 37 TABLE 3. Comparisons of CETP inhibitor peptide from humyan and baboons on CETP transfer in baboons.

[3q HDL Inhibitor Inhibitor VLDL+LDL Temp. CETP Inhibition (i)peptide peptidle Ci)(C) (W1) (baboon) (human) (119) 019) 00 00 50 4 100 00 00 50 37 100 00 10 50 00 50 4 100 50 00 50 37 100 52 00 00 50 4 100 00 00 50 37 1010 00 10 00 50 50 4 100 00 50 50 37 100 SUBSTITUTE SHEET WO 95/04755 WO 9504755PCTIVS94/08624 100 60 37 200 11. EW 12. ML TABLE 8. Comparisons of CETP inhibitor peptide from human and baboons on CETP transfer in baboons.

[31j, IIDL Inhibitor Inhibitor VLDL+LDL Temp. Ch TP Inhibition (Jig) peptide peptide (49) (OC) (Wi) OA) (baboon) (hum-) (jig) (ni) 00 00 50 4 100 00 00 50 37 100 00 10 50 00 50 4 100 60 00 60 37 100 52 00 00 50 4 100 00 00 50 37 100 00 10 00 50 50 4 100 00 50 50 37 100 Example 21: Method of preparing [3H] HUL Cholesteryl esterlabeled HDL, VLDL+LDL LPDS (human).

3 cholesteryl ester-labeled HDL and acceptor lipoproteins (d 1.045 g/nil) were prepared as described by the inventors (Kushwaha, et al. J. Lipid Res. 3 1:965-973, 1990). Low HDL, baboons were bled after immobilization with ketamine HCl (10 mg/kg). Blood was collected in tubes containing EDTA (1 mg/mi) and plasma separated by low speed centrifugation at 6'C. Plasma was immediately treated with sodium azide (0.2 gentamycin sulfate 1 g/l), cbhloramphenicol (0.05 and phenylinethylsulfonyl fluoride mM). Tritiated cholesteryl oleate (20 pCilmil) dissolved in ethanol was added to the plasma. The plasma was flushed with nitrogen and incubated for 20 h at 4*C. After incubation, HDL was isolated by density gradient ultracentrifugation (Kushwaha, et al., 1990), SUBSTITUTE SHEET

I

WO 95/04755 WO 9504755PCTIUS94/08624 32A dialyzed against normal salinefEDTA (0.001iM) and used as a substrate for cholesteryl ester transfer reaction. VLDL+LDL (d 1.045 g/ml) and LPDS were isolated by sequential ultracentrifugation from 100-200 ml blood obtained from two to four SUBSTITUTE SHEET WO 95/04755 PCT/US94/08624 33 baboons as described by the inventors (Kushwaha, et al., 1990). Total and free cholesterol and HDL and VLDL+LDL were measured by enzymatic assay (Wako Pure Chemical Co.).

Experiment 22: Method of isolating human CETP To isolate human CETP, blood (5-10 ml) from human subjects was obtained in tubes containing EDTA (1 mg/ml). Plasma was obtained by low speed centrifugation. Plasma was kept on ice and the LDL was precipitated by adding 40 0l heparin/ml (5000 units /ml) of plasma along with 60 pl/ml of plasma of 1M MnC12. The plasma was vortexed and incubated on ice for 15 min. Following incubation, the supernatant was recovered by centrifugation. The procedure was repeated twice to completely precipitate VLDL+LDL in the plasma.

Afterwards, 80 pl/ml of 10% dextran sulfate per ml was added to the supernatant and incubated for 15 min. The mixture was centrifuged and the supernatant was collected and used as the source of CETP.

SUBSTITUTE SHEET I I IN WO 95/04755 PCTIUS94/08624 34 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: KUSHWAHA, RAMPRATAP McGILL JR., HENRY C.

KANDA, PATRICK (ii) TITLE OF INVENTION: CETP INHIBITOR POLYPEPTIDE, ANTIBODIES AGAINST THE SYNTHETIC POLYPEPTIDE, AND PROPHYLACTIC AND THERAPEUTIC ANTI-ATHEROSCLEROSIS

TREATMENTS.

(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: COX SMITH INCORPORATED STREET: 112 EAST PECAN STREET, SUITE 1800 CITY: SAN ANTONIO STATE: TEXAS COUNTRY: US ZIP: 78205 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: US FILING DATE:

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: HAYMOND, W. B.

REGISTRATION NUMBER: 35,186 REFERENCE/DOCKET NUMBER: S-0072.187 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 210-554-5260 TELEFAX: 210-226-8395 TELEX: 767609 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 36 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear SUBSTI1JTE SHEET (RULE 26) I- II I WO 95/04755 PCT/US94/08624 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: 6EQ ID NO:1: Asp Val Ser Ser Ala Asp Lys Leu Lys Glu Phe Gly Asn Thr Leu 1 5 10 Glu Asp Lys Ala Trp Glu Val Ile Asn Arg Ile Lys Gin Ser Glu Phe 25 Pro Ala Lys Thr INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Ala Pro Asp Val Ser Ser Ala Leu Asp Lys Leu Lys Glu Phe Gly Asn 1 5 10 Thr Leu Glu Asp Lys Ala Trp Glu Val Ile Asn Arg Ile Lys Gin Ser 25 Glu Phe Pro Ala Lys Thr INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 38 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Thr Pro Asp Val Ser Ser Ala Leu Asp Lys Leu Lys Glu Phe Gly Asn SUBSTITUTE SHEET (RULE 26) 'I L~ WO 95/04755 PCT/US94/08624 36 1 5 10 Thr Leu Giu Asp Lys Ala Arg Giu Leu Ile Ser Arg le Lys Gin Ser 25 Giu Leu Ser Ala Lye Met SUBSTITUJTE SHEET (RULE 26)

Claims (16)

1. 3. An anti-atherosclerosis composition, including an anti-atherosclerosis effective amount of at least a part of a polypeptide selected from the group consisting of: Al a- Pro-Asp-Val-Se r-Se r-Ala-Le u-Asp- Lys- Leu-Lys-G Iu-P he-G ly-As n-Th r- Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-lle-Asn-Arg-lle-Lys-Gln-Ser-Glu-Phe-Pro- Ala-Lys-Thr (SEQ ID. NO:2) and C NOOINSUR OPCV56O 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-l le-Ser-Arg-lle-Lys-Gln-Ser-Glu-Leu- Ser-Ala-Lys-Met (SEQ ID. NO:3), and a pharmaceutically-acceptable carrier; wherein said polypeptide is used to inhibit cholesteryl ester transfer protein activity.
2. 4. An antibody having specificity for a molecule having inhibitory activity of cholesterol ester transfer protein and specificity for a polypeptide including 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- 15 Leu-Glu-Asp-Lys-Ala-Trp-Glu-Val-lle-A,;n-Arg-le-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-l le-Ser-Arg-lle-Lys-Gln-Ser-Glu-Leu-Ser- Ala-Lys-Met (SEQ.ID.NO:3); wherein said polypeptide is used to inhibit cholesteryl ester transfer protein activity. An antibody according to claim 4, being a polyclonal antibody.
3. 6. An antibody according to either claim 4 or 5, being capable of specifically binding to modified apo A-I. tt CtN ATINTAVSS!EhS"O I ~II~PI*~ R*II~ I~ WC~-
4. 7. An antibody according to any one of claims 4 to 6, being capable of specifically binding to the baboon CETP inhibitor polypeptide.
5. 8. An antibody according to claim 4, being capable of specifically binding to modified apo E.
6. 9. An antibody according to claim 4, being capable of specifically binding to apo C-l.
7. 10. A method of preventing atherosclerosis in a mammal being predisposed to that condition, including administering to the mammal a prophylactically effective amount of a polypeptide including 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-l e-Asn-Arg-lle-Lys-GIn-Ser-Glu-Phe-Pro- Ala-Lys-Thr a (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-lle-Lys-GIn-Ser-Glu-Leu-Ser- Ala-Lys-Met (SEQ.ID.NO:3); wherein said polypeptide is used to inhibit cholesteryl ester transfer protein activity.
8. 11. A method according to claim 10, wherein said polypeptide is administered in an amount of 10 to 200 mg. PA C, C I 6MNVPOFIONA uAURnSJ8SPECn552 DOC 1 I
9. 12. A method of treating a mammal afflicted with atherosclerosis including administering to the mammal a therapeutically effective amount of a polypeptide including 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-lle-Lys-GIn-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-lle-Lys-GIn-Ser-Glu-Leu-Ser- Ala-Lys-Met 15 (SEQ.ID.NO:3); wherein said polypeptide is used to inhibit cholesteryl ester transfer protein activity.
10. 13. A method according to claim 12, wherein said polypeptide is administered in an amount of 10 to 400 mg.
11. 14. A method according to any one of claims 10 to 13 wherein the polypeptide is administered intravenously. A method according to any one of claims 10 to 14 wherein the mammal is a human. 4 4* 44 \isC T-
12. 16. A kit for the prevention of atherosclerosis when used in the method according to claim 10, including in separate sterile containers at least 10 to 400 mg of a composition according to claim 3; at least one syringe; and at least one needle; wherein a patient in need thereof is injected with said composition. C'\WINWORDFIONA<STUARTSJBSPECI75526 OCC t- I I II I CP IIIIRI- slRI~ICI TCI ~l~ 41
13. 17. A kit for the treatment of atherosclerosis when used in the method according to claim 12 including in separate sterile containers at least 10 to 400 mg of a composition according to claim 3; at least one syringe; and at least one needle; wherein a patient in need thereof is injected with said composition.
14. 18. A polypeptide according to claim 1 substantially as hereinbefore described with reference to any one of the examples.
15. 19. An antibody according to claim 5 substantially as hereinbefore described with reference to any one of the examples. *0 0 o 0* 0 0 ar~=+ A-~ V" C.WNWORDRO'lONASTUARTIOSPECI\75526 DOC lu JNMhRNATIONAL SJUARCII RBPOXV PCTIUS94/08624 A. CLASSIFICATION OF SUBJECT MATR IPC(6) :Please See Extra Sheet. US CL :Please See Extra Shed,. According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) U.S. :Please See Extra Shedt. Documentation searched otherthan minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) Please See Extra Sheet. C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X J. LIPID RESEARCH, VOL. 28, ISSUED 1987, E.S. KRUL ET 1-3, 6-10 AL., "EXPRESSION OF A MONOCLONAL ANTIBODY Y DEFINED AMINOTERMINAL EPITOPE OF HUMAN APOC-I ON NATIVE AND RECONSTITUTED LIPOPROTEINS", PAGES
16. 818-827, ENTIRE DOCUMENT. Y US, A, 4,904,481 (FATHMAN) 27 FEBRUARY 1990, 4 COLUMN 10, KITS. X J. LIPID RESEARCH, VOL. 34, ISSUED 1993, R.S. 1-3, 5-13 KUSHWAHA ET AL., "CHARACTERIZATION Y CHOLESTERYL ESTER TRANSFER PROTEIN INHIBITOR 4, 14-18 FROM PLASMA OF BABOONS (Pap/c PAGES 1285- 1297, ENTIRE DOCUMENT. Further documents are listed in the continuation of Box C. 11 See patent family annex. Spociti eategorics of citd docinnent -r Wer document publiahed after the interational filing date or priority A donidfinog te gnerl slte f teauiwhih date and not inconflict with the application but cited to undcratand the ocum~deinin 6cgeneal s oftlacRn hichis nt cnsidredprioeiple or theory unerlying the invention to be of particullar relevance X.document of particular relevance; the claimed invetion cannot be .F earlicr domamet published on or after the inantioeWaling date considered novel or cannot be considerut to involve an inventivoep *L -ytrw ohsonpirtycama)o hc when the document is taken alone citd o atalih hepublication daof nte ctto or other Y. documcut ofparticular relevance; the claimed invention canot be spciaj ern(specified) considered to involve an inventive step when the document in .0 documnent referring to an oral disclosure, wse, exhibition or other combined with one or tmome other such documents, such combination being obvious to a person skilled in the ant .P documnent published prior to the international filing date but later than document member of the same patent family the prior*t dae elairno Date of the actual completion of the international search Date of mailing of the international search report 17 NOVEMBER 1994 2 D0EC Name and mailing address of the ISA/US Authorized officer Commrissioner of Patents and Trademarb (s' BOX CTI CHRIS EISENSCHENK Washington, D.C. 20231 Facsimile No. (703) 305-3230 Telephone No._ (703) 308-0196 Form PCTIISA/210 (second sheet)(July 1992)* INTERNATIONAL SEARCH! REPOW International app1loation No, PCT/US94(08624 A. CLASSIFICATION OF SUBJECT MATTER: IPC C07K 14/435, 16/18; A61K 38/17 A. CLASSIFICATION OF SUBJECT MATTER: US CL: 530/30 1, 388.25, 389.3; 51412, 824 B. FIELDS SEARCHED Minirnur documentation searched Classification Systemn: U.S. 530/301, 388.25, 389.3; 514/2, 824 B. FIELDS SEARCHED Electronic data base consulted (Name of data base and where practicable terms used): APS, MEDLINE, BIOSYS, EMBASE, WPI, LIFESCI, SEQUENCE SEARCH Form PCTIISA/210 (extra sheet)(iuly 1992)*