AU747709B2 - Xenogeneic cholesteryl ester transfer protein (CETP) for modulation of CETP activity - Google Patents

Description

XENOGENEIC CHOLESTERYL

ESTER

TRANSFER PROTEIN (CETP) FOR MODULATION OF CETP ACTIVITY Field of the Invention This invention relates to the field of cardiovascular disease, particularly atherosclerosis. More particularly, the present invention provides compositions and methods for the control, treatment, or reduction of the risk ofatherogenic activity in the circulatory system of mammals, particularly humans.

Background of the Invention Cholesterol circulates through the body predominantly as a component of lipoprotein particles (lipoproteins), which are composed of a protein portion, called apolipoproteins (Apo) and various lipids, including phospholipids, triglycerides, cholesterol and cholesteryl esters. There are ten major classes of apolipoproteins: Apo A-I, Apo A-II, Apo-IV, Apo B- 48, Apo B-100, Apo C-I, Apo C-II, Apo C-Im, Apo D, and Apo E. Lipoproteins are classified by density and composition. For example, high density lipoproteins (HDL), one function of which is to mediate transport of cholesterol from peripheral tissues to the liver, have a density usually in the range of approximately 1.063-1.21 g/ml. HDL contain various amounts of Apo A-I, Apo A-II, Apo C-I, Apo C-II, Apo C-III, Apo D, Apo E, as well as various amounts of lipids, such as cholesterol, cholesteryl esters, phospholipids, and triglycerides (TG).

*o In contrast to HDL, low density lipoproteins (LDL), which generally have a density of approximately 1.019-1.063 g/ml, contain Apo B-100 in association with various lipids. In particular, the amounts of the lipids, cholesterol, and cholesteryl esters are considerably higher in LDL than in HDL when measured as a percentage of dry mass. LDL are particularly important in delivering cholesterol to peripheral tissues.

Very low density lipoproteins (VLDL) have a density of approximately 0.95-1.006 g/ml and also differ in composition from other classes of lipoproteins both in their protein and lipid content. VLDL generally have a fluch higher amount of triglycerides than do HDL or WO 99/20302 PCTIUS98/22145 LDL and are particularly important in delivering endogenously synthesized triglycerides from liver to adipose and other tissues. The features and functions of various lipoproteins have been reviewed (see, for example, Mathews and van Holde, Biochemistry, pp. 574-576 and 626-630 (The Benjamin/Cummings Publishing Co., Redwood City, California, 1990); Havel et al., "Introduction: Structure and metabolism of plasma lipoproteins", in The Metabolic Basis of Inherited Disease. 6th ed., pp. 1129-1138 (Scriver et al., eds.) (McGraw-Hill, Inc., New York, 1989); Zannis et al., "Genetic mutations affecting human lipoproteins, their receptors, and their enzymes", in Advances in Human Genetics. Vol, 21, pp. 145-319 (Plenum Press, New York, 1993)).

Decreased susceptibility to cardiovascular disease, such as atherosclerosis, is generally correlated with increased absolute levels of circulating HDL, with lowered levels of LDL or VLDL, and also with increased levels of HDL relative to circulating levels of VLDL and LDL (see, Gordon et al., N. Engl. J. Med., 321: 1311-1316 (1989); Castelli et al., J.

Am. Med. Assoc., 256: 2835-2838 (1986); Miller, et al., Am. Heart 113: 589-597 (1987); Tall, J. Clin. Invest., 89: 379-384 (1990); Tall, J. Internal Med., 237: 5-12 (1995)).

Cholesteryl ester transport protein (CETP) mediates the transfer of cholesteryl esters from HDL to TG-rich lipoproteins such as VLDL and LDL, and also the reciprocal exchange of TG from VLDL to HDL (Tall, ibid.; Tall, J. LipidRes., 34: 1255-1274 (1993); Hesler et al., J. Biol. Chem., 262: 2275-2282 (1987); Quig et al., Ann. Rev. Nutr., 10: 169-193 (1990)).

CETP may play a role in modulating the levels of cholesteryl esters and triglyceride associated with various classes of lipoproteins. A high CETP cholesteryl ester transfer activity has been correlated with increased levels of LDL-associated cholesterol and VLDLassociated cholesterol, which in turn are correlated with increased risk of cardiovascular disease (see, Tato et al., Arterioscler. Thromb. Vascular Biol., 15: 112-120 (1995)).

CETP isolated from human plasma is a hydrophobic glycoprotein having 476 amino acids and a molecular weight of approximately 66,000 to 74,000 daltons on sodium dodecyl sulfate (SDS)-polyacrylamide gels (Albers et al., Arteriosclerosis, 4: 49-58 (1984); Hesler et al., J. Biol. Chem., 262: 2275-2282 (1987); Jarnagin et al., Proc. Natl. Acad. Sci. USA, 84: 1854-1857 (1987)). A cDNA encoding human CETP has been cloned and sequenced (see, Drayna et al., Nature, 327: 632-634 (1987)). CETP has been shown to bind choesteryl esters, trygicerides, phospholipids (Barter et al., J. Lipid Res., 21:238-249 (1980)), and lipoproteins (see, Swenson et al., J. Biol. Chem., 264: 14318-14326 (1989)). More recently, the -2- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 region of CETP defined by the carboxyl terminal 26 amino acids, and in particular amino acids 470 to 475, has been shown to be especially important for neutral lipid binding involved in neutral lipid transfer (Hesler et al., J. Biol. Chem., 263: 5020-5023 (1988)).

Hereinafter, LDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with low density lipoprotein. VLDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with very low density lipoprotein. HDL-C will be used to refer to total cholesterol, including cholesteryl esters and/or unesterified cholesterol, associated with high density lipoprotein.

A number of in vivo studies utilizing animal models or humans have indicated that CETP activity can affect the level of circulating cholesterol-containing HDL. Increased CETP cholesteryl ester transfer activity can produce a decrease in HDL-C levels relative to LDL-C and/or VLDL-C levels which in turn is correlated with an increased susceptibility to atherosclerosis. Injection of partially purified human CETP into rats (which normally lack CETP activity), resulted in a shift of cholesteryl ester from HDL to VLDL, consistent with CETP-promoted transfer of cholesteryl ester from HDL to VLDL (Ha et al., Biochim.

Biophys. Acta, 833: 203-211 (1985); Ha et al., Comp. Biochem. Physiol., 83B: 463-466 (1986); Gavish et al., J. Lipid Res., 28: 257-267 (1987)). Transgenic mice expressing human CETP were reported to exhibit a significant decrease in the level of cholesterol associated with HDL (see, Hayek et al., J. Clin. Invest., 90: 505-510 (1992); Breslow et al., Proc.

Natl. Acad. Sci. USA, 90: 8314-8318 (1993)). Furthermore, whereas wild-type mice are normally highly resistant to atherosclerosis (Breslow et al., ibid.), transgenic mice expressing a simian CETP were reported to have an altered distribution of cholesterol associated with lipoproteins, namely, elevated levels of LDL-C and VLDL-C and decreased levels of HDL-C (Marotti et al., Nature, 364: 73-75 (1993)). Transgenic mice expressing simian CETP also were more susceptible to dietary-induced severe atherosclerosis compared to non-expressing control mice and developed atherosclerotic lesions in their aortas significantly larger in area than those found in the control animals and having a large, focal appearance more typical of those found in atherosclerotic lesions in humans (Marotti et al., ibid.). Intravenous infusion of anti-human CETP monoclonal antibodies (Mab) into hamsters and rabbits inhibited CETP activity in vivo and resulted in significantly increased levels of HDL-C, decreased levels of HDL-triglyceride, and increased HDL size; again implicating a critical role for CETP in the -3- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 distribution of cholesterol in circulating lipoproteins (Gaynor et al., Atherosclerosis, 110: 101-109 (1994) (hamsters); Whitlock et al., J. Clin. Invest., 84: 129-137 (1989) (rabbits)).

CETP deficiency has also been studied in humans. For example, in certain familial studies in Japan, siblings that were homozygous for non-functional alleles of the CETP gene had no detectable CETP activity. Virtually no atherosclerotic plaques were exhibited by these individuals, who also showed a trend toward longevity in their families (see, e.g., Brown et al., Nature, 342: 448-451 (1989); Inazu et al., N. Engl. J. Med., 323: 1234-1238 (1990); Bisgaier et al., J. Lipid Res., 32: 21-23 (1991)). Such homozygous CETP-deficient individuals also were shown to have an anti-atherogenic lipoprotein profile as evidenced by elevated levels of circulating HDL rich in cholesteryl ester, as well as overall elevated levels of HDL, and exceptionally large HDL, up to four to six times the size of normal HDL (Brown et al., supra, p. 451). The frequency of this mutation in Japan is relatively high, and may account for an elevated level of HDL in a significant fraction of the Japanese population.

The above studies indicate that CETP plays a major role in transferring cholesteryl ester from HDL to VLDL and LDL, and thereby in altering the relative profile of circulating lipoproteins to one which is associated with an increased risk of cardiovascular disease decreased levels of HDL-C and increased levels of VLDL-C and LDL-C). Taken together, the current evidence suggests that increased levels of CETP activity may be predictive of increased risk of cardiovascular disease. Modulation or inhibition of endogenous CETP activity is thus an attractive therapeutic method for modulating the relative levels of lipoproteins to reduce or prevent the progression of, or to induce regression of, cardiovascular diseases, such as atherosclerosis.

In our previous work, detailed, in commonly assigned, copending patent application PCT/US96/06147 (WO 96/34888) and commonly assigned copending patent application PCT/US97/07294 (WO 97/41227), both incorporated herein by reference, we detailed an approach for modulating the CETP activity in an individual via vaccination with a peptide composition or with a plasmid-based vaccine that would lead to the production of antibodies recognizing and neutralizing endogenous CETP. We demonstrated that administration of immunogenic peptides either by direct inoculation or by in situ production following injection of a functional plasmid-based vaccine resulted in production of antibodies reactive with the inoculated individual's own (endogenous) CETP. Thus the vaccine peptides and the plasmid-based vaccines break tolerance in the vaccinated individuals and to promote -4- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 production of antibodies recognizing a self protein. Furthermore, administration of these vaccines to test animals resulted in a decline in the relative levels of total cholesterol and HDL-C and resulted in a decrease in the development of atherosclerotic lesions. The elicited endogenous anti-CETP antibodies thus promote a physiological condition correlated with decreased risk of cardiovascular disease, and they appear to have a direct effect on preventing or decreasing the formation of atherosclerotic plaques.

We have now discovered another approach to eliciting the production of anti-CETP antibodies in a mammal. We have now determined that whole CETP molecules of another mammal, that is, non-endogenous CETP, can be used to elicit antibodies in a mammal that will be reactive with its own, endogenous CETP and serve to modulate the activity of CETP and to provide lowered circulating CETP activity, lowered total cholesterol, lowered circulating LDL levels, elevated ratios of HDL-C to LDL-C. The use of non-endogenous CETP to promote production of anti-endogenous CETP antibodies also leads to a reduction in development of atherosclerotic lesions in comparison to unvaccinated controls.

Summary of the Invention Accordingly, the present invention provides compositions and methods useful for the modulation or inhibition of cholesteryl ester transfer protein (CETP) activity. In particular, the use of non-endogenous CETPs, including xenogeneic CETPs, is described as a means, when administered to a mammal, to raise an antibody response against the mammal's own endogenous CETP and thereby to promote a prophylactic or therapeutic effect against cardiovascular disease, such as atherosclerosis. Such non-endogenous CETP can be CETP of another mammalian species (xenogeneic CETP), such as rabbit CETP, mouse CETP or simian CETP for administration to a human; the non-endogenous CETP can be a nonendogenous allelic variation or polymorph of a mammalian CETP administered to the same species of mammal a human CETP polymorph administered to another human); or the non-endogenous CETP can be a CETP from one species modified to have an amino acid sequence more similar to the native CETP of another species a "humanized" rabbit CETP for administration to a human).

Vaccine compositions and plasmid-based vaccines are described which, when suitably administered to a mammal result in the production of antibodies reactive with the mammal's endogenous CETP and the other benefits described herein.

SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 The invention provides methods for eliciting antibodies in a mammal that will be reactive with its own, endogenous CETP, for modulating the activity of CETP in a mammal, and for providing in a mammal lowered circulating CETP activity, lowered total cholesterol, lowered circulating LDL levels, and/or elevated ratios of HDL-C to LDL-C. The invention provides a method for reducing or preventing in a mammal the development of atherosclerotic lesions.

Brief Description of the Drawings Figure 1A-C. An alignment of the amino acid sequences of mature rabbit CETP (SEQ ID NO: 3) with mature human CETP (SEQ ID NO: The rabbit CETP is shown over the aligned human CETP sequence. The rabbit sequence includes 20 more amino acid residues than the human sequence, and the human sequence shows a 1-amino acid and a 19amino acid gap (indicated with dashes, in the human sequence) in order to show the residue matches (indicated with a vertical line, I) most clearly.

Figure 2. Shows the end point titers of antibodies from rabbit plasma recognizing the C-terminal peptide of rabbit CETP from rabbits vaccinated with human chorionic gonadotropin ("HCG Vaccine"), a synthetic vaccine peptide having segments of tetanus toxoid and the C-terminal sequence of human CETP ("Peptide Vaccine", see SEQ ID NO: 7), and full-length recombinant human CETP ("rhuCETP"). The figure shows maximum anti- CETP antibody titers achieved for each rabbit in an ELISA detecting plasma antibodies specific for rabbit CETP C-terminal peptide (amino acids 477-496).

Figure 3. Shows inhibition of CETP activity in a commercial fluorescence-based assay (Roar Biomedical, Yonkers, New York) by protein A-isolated antibodies from the plasma of vaccinated rabbits from Groups I-III (see Examples, infra). The graph shows percent inhibition achieved in each of the vaccinated rabbits.

Figure 4. Shows the CETP activity in the vaccinated rabbits from week 1 to week 32.

Figure 5. Shows the percentage change in total cholesterol levels in the vaccinated rabbits from week 1 to week 12.

Figure 6. Shows the HDL-associated cholesterol levels in the vaccinated rabbits from week 1 to week 32.

Figure 7. Shows the percentage change in LDL-associated cholesterol levels in the vaccinated rabbits from week 1 to week 12.

-6- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 Figure 8. Shows the plasma lipoprotein levels for a rabbit vaccinated with nonendogenous CETP(recombinant human CETP, or rhuCETP). indicates the periodic vaccination boosts. HDL-associated cholesterol, total cholesterol level, and triglyceride level were assayed.

Figure 9. Shows a correlation between CETP activity and HDL as a percent of total lipoproteins and total CETP mass, in a rabbit vaccinated with non-endogenous CETP (rhuCETP) Figure 10. Shows the levels of cholesterol deposits in the irises of 48 rabbits vaccinated with human chorionic gonadotropin rabbits a synthetic vaccine peptide having segments of tetanus toxoid and the C-terminal sequence of human CETP ("Peptide", see SEQ ID NO: 7, rabbits full-length recombinant human CETP ("rhuCETP", rabbits and a CETP-tetanus toxoid conjugate composition.("Conjugate", rabbits The irises of each rabbit have been scored based on the percentage of deposits observed from 0-5, 0 representing no deposits and representing 100% deposits observed in the iris of the animal.

Figure 11. Shows percentage of the aorta covered by lesions observed in vaccinated rabbits fed an atherogenic diet. Values of individual animals are represented by open symbols.

Figure 12. Shows anti-CETP antibody titers for mice vaccinated with various plasmid-based vaccines.

Figure 13A-K. Shows antibody titers of 11 rabbits vaccinated with between weeks 1-32. The open diamond and open square symbols refer to the antibody titers of the rabbits on weeks 30 and 34 respectively.- Figure 14. Shows the effect of plasmid vaccination on the development of aortic lesions. The figure compares the mean percentages collected for control rabbits plasmid without the CETP coding sequence) and rabbits vaccinated with Detailed Description of the Invention As noted above, a decreased risk of atherosclerosis has been correlated with relatively low circulating levels of LDL and VLDL and relatively high levels of HDL. The levels of such circulating lipoproteins are directly influenced, at least in part, by the endogenous levels of CETP activity. In particular, high CETP activity promotes transfer of neutral lipids, such -7- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 as cholesteryl esters from HDL to VLDL and LDL. Accordingly, CETP is a relatively precise target in humans and other animals for altering the relative levels of LDL, VLDL and HDL in the circulatory system (see, Tato et al., Arteriosclero. Thromb. Vascular Biol., 112-120 (1995); Tall, J. Internal Med., 237: 5-12 (1995)). This invention is directed to the control of endogenous CETP activity by providing non-endogenous CETP molecules to an individual, for promoting an immune response in such individuals against their endogenous CETP, thereby promoting a physiological condition increased level of HDL or decreased level of LDL) correlated with a decreased risk of atherosclerosis. In addition, promoting an immune response against endogenous CETP using the vaccine compositions of this invention can provide, prevent, or inhibit the progression of lesions in tissue susceptible to atherosclerosis.

Compositions for Modulation of CETP Activity As used herein, a CETP vaccine composition for use according to the invention contains as an essential ingredient a CETP or a portion thereof, that is non-endogenous with respect to the mammal to be vaccinated. For the purposes of this invention, "non-endogenous CETP" means a cholesteryl ester transfer protein that is not the native CETP produced by the mammal to be vaccinated. For example, with respect to a human subject, non-endogenous CETP will include CETP produced by another mammalian species, xenogeneic CETP, such as rabbit, mouse or simian CETP; or non-endogenous CETP with respect to a particular human subject can be an allelic variant or polymorphism of human CETP, such as CETP produced by another human individual.

The non-endogenous CETP can also be a xenogeneic CETP that has been modified in order to make the amino acid sequence of the modified CETP more similar to that of the endogenous CETP of the mammal to be vaccinated. The term used herein to describe such modified non-endogenous CETP is "mammalianized CETP". This term is used with reference to the mammal to be vaccinated, and it means a non-endogenous CETP that has been modified to have an amino acid content more similar to the native CETP of said mammal. An example of a mammalianized CETP, where the subject to be vaccinated is a human, would be a rabbit CETP modified (or "humanized") to have an amino acid sequence more similar to the native human sequence. As a further example, reference is made to Figures 1A, 1B and 1C, which show the respective amino acid sequences of rabbit CETP (SEQ ID NO: 3) and human CETP (SEQ ID NO: 1) in alignment.

-8- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 Referring to Figures 1A-1C, it is seen that the structure of these two mammalian CETPs is similar, having the same amino acids at 80% of the positions of human CETP.

Rabbit CETP (SEQ ID NO: 3) is 20 amino acids longer than human CETP (SEQ ID NO: 1), and the alignment of the two proteins in Figures 1A-1C shows two segments, denoted with dashes where the proteins do not correspond structurally. With respect to a human subject to be administered a non-endogenous CETP in accordance with this invention, an example of a "mammalianized" non-endogenous CETP would be a rabbit CETP in which the 19-amino acid segment from amino acid Ala, 93 through Ala 4 1 1 of rabbit CETP has been deleted, making the modified CETP (see SEQ ID NO: 5) 477 amino acids in length and thus more similar to the human CETP (SEQ ID NO: Since the mammal of this example is a human, another term for such a modified CETP or mammalianized non-endogenous CETP would be a "humanized rabbit CETP".

Again referring to Figures 1A-1C, a further example of a humanized rabbit CETP would be a CETP as set forth in SEQ ID NO: 6. In Figure 1C it is noted that in the Cterminal portion of the human and rabbit CETPs there is only one difference in the respective amino acid sequences, Lys, 4 of the rabbit CETP corresponds to Glu 465 in the human

CETP.

In practicing the methods of the present invention, non-endogenous CETP is administered to a mammal in an amount effective to elicit an immune response. As is common in the field, more than one administration may be necessary or desirable to obtain a high enough concentration of anti-endogenous CETP antibodies in the mammal to affect endogenous CETP activity.

Immunogenicity of a vaccine peptide of this invention may be further enhanced by linking the immunogenic non-endogenous CETP to itself or to a related protein homologous to CETP. In this approach, a dimer could be formed, with the dimer providing a multi-chain protein that is even more immunogenic than the non-endogenous CETP alone. Examples of proteins related to CETP that might be used in this approach include, for example, phospholipid transfer protein and neutrophil bactericidal protein (see, Day et al., J. Biol.

Chem., 269: 9388-91 (1994); Gray et al., J. Biol. Chem., 264: 9505-9509 (1989)).

Other immunogenic carrier molecules such as keyhole limpet hemocyanin (KLH) may also be used in combination with the non-endogenous CETP. For example, KLH contains multiple lysine residues in its amino acid sequence. Each of these lysines is a potential site at -9- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 which a CETP molecule as described herein could be linked (for example, using maleimidc activated KLH, Catalog No. 77106, Pierce, Rockford, IL), to provide a multivalent nonendogenous CETP immunogen. Another example of an immunogenic carrier molecule is from Mycobacterium tuberculosis, which has been shown to be an especially potent antigen containing multiple B and T cell epitopes (see, Suzue and Young, J. Immunol., 156: 873 879 (1996)). The hsp70 protein can be linked by standard cross-linking agents to non-endogenous CETPs to enhance immunogenicity of the vaccine compositions.

Other peptides can be conjugated with the non-endogenous CETP molecules to provide a source of helper T cell epitopes and boost the immunogenicity of the vaccine compositions according to the invention. Such peptides include, for example, "universal" antigenic peptides, tetanus toxoid or diphtheria toxoid, especially the tetanus toxoid fragment: Gin Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu (amino acids 2 to 15 of SEQ ID NO: 7).

Pharmaceutically acceptable adjuvants, such as alum, may be mixed with the nonendogenous CETPs described herein to produce vaccine compositions of this invention.

Alum is the single adjuvant currently approved for use in administering vaccines to humans (see, Eldrige et al., in Immunobiology of Proteins and Peptides V: Vaccines: Mechanisms.

Design. and Applications (Atassi, ed.)(Plenum Press, New York, 1989), page 192).

Alum in combination with a sodium phthalyl derivative of lipopolysaccharide can also be used (see, Talwar et al., Proc. Natl. Acad. Sci. USA, 91: 8532-8536 (1994)). Other conventional adjuvants may be used as they are approved for a particular use. For example, biodegradable microspheres comprised of poly (DL-lactide-co-glycolide) (Eldridge et al., supra, pp. 191-202); Freund's Complete Adjuvant (Sigma Chemical Co., St. Louis, Missouri), Freund's Incomplete Adjuvant (Sigma Chemical Co., St. Louis, Missouri), and the

RIBI

T Adjuvant System (RAS; RIBI ImmunoChem Research, Inc., Hamilton, Montana); lipophilic N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl)]-cysteine ("Pam 3 -Cys-OH"); amphiphilic, water-soluble lipopeptides such as Pam 3 -Cys-Ser-Lys 4 and Pam 3 -Cys-Ser-Glu 4 glycopeptides such as N-acetyl-glucosaminyl-N-acetylmuramyl-alanyl-D-isoglutamine muramyl dipeptides, and alanyl-N-adamantyl-D-glutamine; and polyamide gelbased adjuvants which can easily be attached to CETP peptides during their in vitro chemical synthesis (see, Synthetic Vaccines (Nicholson, ed.) (Blackwell Scientific Publications, Cambridge, Massachusetts, 1994), pp. 236-238).

SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 Where helper T cell epitope molecules or adjuvant species are to be physically linke' or conjugated with the non-endogenous CETP, the CETP can be covalently linked directly or via a cross-linker molecule.

Suitable cross-linking molecules include amino acids, for example, using one or more glycine residues to form a "glycine bridge" between the CETP and the carrier or adjuvant molecule. Also contemplated are the formation of disulfide bonds between cysteine residues, or the use of cross-linking molecules such as glutaraldehyde (see, Korn et al., J. Mol. Biol., 525-529 (1972)) and EDC (Pierce, Rockford, IL) or other bifunctional cross-linker molecules. Bifunctional cross-linker molecules possess two distinct reactive sites; one of the reactive sites can be reacted with a functional group on the CETP and the other reactive site can be reacted with a functional group on the carrier or adjuvant molecule, uniting the two.

General methods for cross-linking molecules are reviewed by Means and Feeney (Bioconjugate Chem., 1: 2-12 (1990)).

Homobifunctional cross-linker molecules have two reactive sites which are chemically the same. Examples of homobifunctional cross-linker molecules include glutaraldehyde; N,N'-bis(3-maleimido-propionyl)-2-hydroxy-1,3-propanediol (a sulfhydrylspecific homobifunctional cross-linker); certain N-succinimide esters, such as disuccinimidyl suberate and dithio-bis-(succinimidyl propionate) and their soluble bis-sulfonic acids and salts as available from Pierce Chemicals, Rockford, Illinois; or Sigma Chemical Co., St.

Louis, Missouri).

Preferably, the bifunctional cross-linker molecule is a heterobifunctional linker molecule, meaning that the linker molecule has at least two reactive sites that can be separately covalently attached to the non-endogenous CETP and the carrier or adjuvant molecule. Heterobifunctional cross-linker molecules that may be used include mmaleimidobenzoyl-N-hydroxysuccinimide ester; m-maleimido-benzoylsulfosuccinimide ester; y-maleimidobutyric acid N-hydroxysuccinimide ester; and N-succinimidyl 3-(2pyridyl-dithio)propionate.

The non-endogenous CETP for use according to this invention can be produced by any of the available methods known in the art to produce proteins of defined amino acid sequence. For example, automated peptide synthesis is available to those skilled in the art by using automated peptide synthesizers Synergy Peptide Synthesizer by Applied Biosystems; AMS 422 by Abimed, Langenfeld, Germany). Synthesis of such proteins to -11- SUBSTITUTE SHEET (RULE 26)

I,

WO 99/20302 PCT/US98/22145 order is performed as a commercial service by many commercial peptide synthesizing servi.e companies, Quality Controlled Biochemicals, Inc., (Hopkinton, Massachusetts);-Chiron Mimotopes Peptide Systems (San Diego, California); Bachem Bioscience, Inc. (Philadelphia, Pennsylvania); Severn Biotech Ltd. (Kiddeminster, England).

Alternatively, the proteins of this invention may be produced using synthetic and recombinant nucleic acid technology. For example, one of ordinary skill in the art can design from the known genetic code a 5' to 3' nucleic acid sequence encoding a proteins of this invention. The amino acid sequence for a mature human CETP is known (SEQ ID NO: as well as its corresponding DNA sequence (SEQ ID NO: 2) (see, Drayna et al., Nature, 327: 632 634 (1987)). Furthermore, the amino acid sequence for a mature rabbit CETP is known (SEQ ID NO: as well as its corresponding DNA sequence (SEQ ID NO: 4) (see, Nagashima et al., J. Lipid. Res., 29: 1643 1649 (1988)). A DNA molecule containing the coding sequences of desired CETP (or a modified "mammalianized" CETP as described above) can readily be synthesized either using an automated DNA synthesizer Oligo 1000 DNA Synthesizer, Beckman Corp.) or by contracting with a commercial DNA synthesizing service Genset Corp., La Jolla, California).

The synthesized or cloned DNA molecule can then be inserted into any of a variety of available gene expression systems bacterial plasmids; bacteriophage expression vectors, retroviral expression vectors, baculoviral expression vectors), using standard methods available in the art Sambrook et al., Molecular Cloning: A Laboratory Manual. Vols. 1- 3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989)) and/or as directed by the manufacturer of a particular commercially available gene expression system pPROEXT-1 bacterial cell expression system; SFV eukaryotic cell expression system; CHO (Chinese Hamster Ovary cells) expression system; BAC-TO-BAC T baculovirus expression system; Life Technologies, Inc., Gaithersburg, Maryland). Especially preferred for the expression of CETP is the CHO expression system. The expressed CETP can then be isolated from the expression system using standard methods to purify proteins.

Purification of the non-endogenous CETPs of this invention may be expedited by employing affinity chromatography or immunoprecipitation based on using antibodies to the particular CETP domain. For example, the Mab TP2 (Hesler et al., J. Biol. Chem., 263: 5020-5023 (1988)) binds to the carboxyl terminal 26 amino acids of human CETP, and could be useful in one or more immunoaffinity steps in a purification scheme. Another method that -12- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 could be used in the purification of the proteins is standard column chromatography (Weinberg et al., J. Biol. Chem., 269: 29588-29591 (1994)).

The non-endogenous CETPs as described herein are used to make vaccines compositions that elicit, when administered to a mammal, production of endogenous antibodies which specifically bind to endogenous CETP of the mammal and/or modulate decrease or inhibit) endogenous CETP activity in the mammal. The anti-CETP vaccine compositions of this invention may contain one or several different non-endogenous CETPs.

In addition, the non-endogenous CETP may be linked to other molecules that may enhance the immunogenicity of the peptides.

The vaccine compositions for administration of non-endogenous CETP according to this invention can also advantageously take the form of plasmid-based vaccines for producing non-endogenous CETP in situ, eliciting autoantibodies directed to endogenous CETP. Such plasmid-based vaccines are, specifically, DNA plasmids which are administered (for example, by intramuscular injection or intradermal ballistic administration) to an individual.

The administered DNA plasmids encode and direct the production ofimmunogenic nonendogenous CETP. We have discovered that such immunogenic CETP elicits the production of autoantibodies that react specifically with bind to) endogenous CETP in the individual.

An example of a recombinant plasmid that can be used to produce a non-endogenous CETP for use according to this invention is plasmid pCMV-CETP/TT in which the CMV promoter directs transcription of a sequence encoding a vaccine peptide described in the previously mentioned PCT/US96/06147. E. coli bearing plasmid pCMV-CETP/TT has been deposited with the American Type Culture Collection (ATCC, Rockville, MD) and assigned Accession No. 98038. DNA coding for a desired CETP can be inserted in place of the vaccine peptide coding sequence in pCMV-CETP/TT and used for the expression of fulllength CETP molecules.

The production of anti-CETP antibodies promotes a physiological state associated with a decreased risk of cardiovascular disease. The beneficial modulation of CETP activity produced by the DNA vaccines is evidenced by a significantly decreased or eliminated CETP activity; by an anti-atherogenic lipoprotein profile (for example, an increase in the level of HDL or HDL-C compared to LDL, LDL-C, VLDL, or VLDL-C); or by an inhibition -13- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 (including prevention) or decrease in the development of atherosclerotic lesions in cardiovascular tissue, such as the aorta.

General methods of administering and testing vaccines are well known to those skilled in the art (see, Talwar et.al., Proc. Natl. Acad. Sci. USA, 91: 8532-8536 (1994)). The immune response to endogenous CETP should significantly inhibit CETP activity, alter the serum half-life of CETP, cause clearance CETP through formation of immune complexes,.

alter the trafficking of HDL-cholesterol, shift the equilibrium of cholesterol content of lipoproteins, alter cholesterol catabolism, and/or reduce development of atherosclerotic lesions. Control ofLDL, VLDL and/or HDL levels is an accepted indicator or endpoint in treatment of cardiovascular disease, as these levels are correlated with a decreased risk of cardiovascular disease or further progression of such disease (see, Mader, in Human Biology. 4th ed., pp. 83, 102 (Wm. C. Brown Publishers, Dubuque, Iowa, 1995)).

Accordingly, the desired prophylactic or therapeutic effect according to this invention is evidenced by eliciting antibodies in an individual that bind to endogenous CETP and/or inhibit endogenous CETP activity, or by a relative decrease in LDL and/or VLDL levels compared to HDL levels as the titer of antibody directed against the endogenous CETP rises, or by a decrease of absolute levels of circulating LDL and/or VLDL with the production of anti-CETP antibodies, or by an inhibition or decrease in development of atherosclerotic lesions in cardiovascular tissue.

As demonstrated herein, administration of non-endogenous CETP in a rabbit model of atherosclerosis led to a significant decrease in the development of atherosclerotic plaques.

This evidence indicates that vaccination to elicit antibodies to endogenous CETP may be a useful method of treating or preventing atherosclerosis.

The successful use of non-endogenous CETP to elicit anti-endogenous CETP antibodies and to modulate the activity of native CETP was surprising in a number of respects. Previously, the use of whole CETP molecules had been avoided, since it was not known whether introduction of a whole, non-endogenous CETP molecule would provide the desired immunogenic effects. For example, non-endogenous CETP might function perfectly well as a CETP and exacerbate already undesirable cholesterol levels and metabolism. In addition, it was contemplated that full-length CETP molecules might contain immunogenic segments that would elicit antibodies capable of reacting or interfering with proteins or receptors outside of the CETP metabolic pathway, resulting in dangerous cross-reactions or -14- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 side-effects. Finally, it was not known whether introduction of a non-endogenous CETP would be able to break tolerance in the subject vaccinated, leading to production of antibodies reactive not with (or not only with) the non-endogenous CETP but with the native CETP. These uncertainties have now been resolved.

The non-endogenous CETP vaccine compositions may be administered by any route used for vaccination, including: parenterally such as intraperitoneally, interperitoneally, intradermally, subcutaneously, intramuscularly, intravenously or orally. Preferably, the vaccines of this invention are administered parenterally, intraperitoneally, interperitoneally, subcutaneously, intradermally, intramuscularly, or intravenously. If oral administration of a vaccine peptide is desired, any pharmaceutically acceptable oral excipient may be mixed with the vaccine peptides of this invention, for example, such as solutions approved for use in the oral polio vaccine. As with certain other vaccines, such as for tetanus, an occasional booster administration of the CETP vaccine compositions may be necessary to produce or maintain a desired level of modulation or inhibition of endogenous CETP.

Biodegradable microspheres, such as those comprised of poly (DL-lactide-co-glycolide), have been shown to be useful for effective vaccine delivery and immunization via oral or parenteral routes.

Appropriate dosages of the non-endogenous CETP may be established by general vaccine methodologies used in the art based on measurable parameters for which the vaccine is proposed to affect, including monitoring for potential contraindications, such as hypersensitivity reaction, erythema, induration, tenderness (see, Physician's Desk Reference. 49th ed., (Medical Economics Data Production Co., Mont Vale, New Jersey, 1995), pp. 1628, 2371 (referring to hepatitis B vaccine), pp. 1501, 1573, 1575 (referring to measles, mumps, and/or rubella vaccines), pp. 904, 919, 1247, 1257, 1289, 1293, 2363 (referring to diphtheria, tetanus and/or pertussis vaccines)) Talwar, et al., Proc. Natl.

Acad. Sci. USA, 91: 8532-8536 (1994)). A common and traditional approach for vaccinating humans is to administer an initial dose of a particular vaccine to sensitize the immune system and then follow-up by one or more "booster" doses of the vaccine to elicit an anamnestic response by the immune system that was sensitized by the initial administration of the vaccine (vaccination). Such a "primary and booster" administration procedure has been well known and commonly used in the art, as for example, in developing and using measles, polio, tetanus, diphtheria, and hepatitis B vaccines.

SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 Initially, the amount of a vaccine composition administered to an individual may be that required to neutralize the approximate level of endogenous CETP activity present in the individual prior to vaccination, as can be determined by measuring CETP activity in serum or plasma samples from the individual, for example as determined using a commercially available CETP assay Roar Biomedical., Yonkers, New York). Plasma or serum samples from a vaccinated individual can also be monitored to determine whether a measurable increase in the levels of total HDL or HDL-C is seen after administration of the non-endogenous CETP using commercially available assays available from Sigma Diagnostics, Inc., Saint Louis, Missouri). A rise in the concentration (titer) of circulating anti-CETP antibodies can be measured in plasma or serum samples, for example using an ELISA assay. Thus, it is possible and recommended that initially it be established whether a rise in anti-CETP antibody can be correlated with an increase in the level of HDL or HDL-C, a decrease in LDL or VLDL, or with a decrease in CETP activity. Thereafter, one need only monitor a rise in titer of anti-CETP antibody to determine whether a sufficient dosage of vaccine peptide has been administered or whether a "booster" dose is indicated to elicit an elevated level of anti-CETP antibody. This is the common procedure with various established vaccinations, such as vaccination against hepatitis B virus.

Three-dimensional arterial imaging methods are currently available which can be used to identify arterial lesions and monitor their development or regression in an individual (see, for example, McPherson, Scientific American Science Medicine, pages 22-31, (March/April, 1996)). Thus such imaging methods can be used to monitor the effectiveness of vaccination according to the methods of this invention.

A more complete appreciation of this invention and the advantages thereof can be obtained from the following non-limiting examples.

EXAMPLE 1 Immunization of Rabbits Against Endogenous CETP Four vaccine preparations were made for injection into four groups of twelve New Zealand White Rabbits, to test the ability of the vaccine preparation to elicit an immune response against endogenous rabbit CETP. Group I (negative control) contained rabbits #1 #12, each of which was injected with a vaccine composition containing an irrelevant antigen, human chorionic gonadotropin (hCG). Group II (comparative embodiment of -16- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 PCT/US96/06147) contained rabbits #13 #24, each of which was injected with a vaccine peptide having a portion of the C-terminus of human CETP and a portion of tetanus toxoid ("Peptide"; see, SEQ ID NO: Group III (this invention) contained rabbits #25 #36, each of which received a vaccine composition containing whole recombinant human CETP ("rhuCETP"). Group IV (this invention) contained rabbits #37 #48, each of which received a vaccine composition containing whole recombinant human CETP conjugated with whole tetanus toxoid using a chemical crosslinker ("Conjugate").

The general protocol for vaccinating and testing the rabbits was as follows: On Day 1, each rabbit received one subcutaneous injection of a composition containing 200 gg of immunogen in Complete Freund's Adjuvant (Sigma Chemical Co., St. Louis, Missouri).

Each composition suspended the respective immunogen in phosphate buffered saline (PBS) and emulsified with complete Freund's adjuvant to yield a final concentration of 100 gg/100 gl. Each rabbit was administered the vaccine mixture in a 200 gl dose (200 gg immunogen) at one subcutaneous site. A boost of 200 gg of immunogen in Incomplete Freund's Adjuvant (Sigma Chemical Co.) was administered as on Day 1 at Day 28 and Day 49. Blood samples (approximately 1-5 ml) were withdrawn from the ear of each rabbit (prior to injections) on Days 1 ("prebleed"), 28 49, 105, 147 and 217. Blood plasma samples were prepared by standard centrifugation methods to separate cellular components from the plasma. Plasma samples were stored at -70* C. Plasma samples of both Groups I and II were analyzed for presence of and increase in titer of anti-CETP antibodies and for CETP activity, CETP mass, and plasma levels of various lipoprotein components (HDL, LDL, triglycerides).

Direct ELISA for Titering Anti-CETP Antibodies A sandwich enzyme-linked immunosorbent assay (ELISA) was used to titer plasma samples containing anti-CETP antibody. A biotinylated C-terminal peptide (20 amino acids) of rabbit CETP was adsorbed to wells of a microtiter dish coated with streptavidin, and various dilutions of rabbit plasma from the rabbits of Groups I III were added to each well.

Non-specific binding can be blocked by adding a 1% solution ofBSA in PBS and 0.05% Tween to each well and incubating for 2 hours at room temperature (20 -220 C) on a rotating shaker at 150 rpm. The wells were then washed four times with ELISA wash buffer (PBS 0.05% Tween). Plasma samples were then diluted in dilution buffer BSA in PBS), followed by serial dilutions in the same buffer. Diluted samples (100 pl) were added to the wells, incubated for 1.5-2 hours at room temperature on a rotating shaker at 150 rpm, and -17- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 then washed 4 times with ELISA wash buffer (PBS 0.05% Tween). To detect bound anti CETP antibodies, 100 gl of an optimized dilution of horseradish peroxidase (HRP) labeled goat anti-rabbit immunoglobulin (Southern Biotechnology Associates, Inc.; Birmingham, Alabama; or Jackson Immunoresearch, Inc.; West Grove, Pennsylvania) in dilution buffer was added, and the plates were incubated for 2 hours at room temperature on a rotating shaker at 150 rpm. The wells were then washed four times with ELISA wash buffer (see above), peroxidase substrate TMB (TMB peroxidase substrate, Kirkegaard Perry Laboratories, Inc., Gaithersburg, Maryland) was added, and the plates were incubated 30 minutes at room temperature. Change in optical density was monitored spectrophotometrically at 450 nm using an ELISA reader E-max, Molecular Device Corp., Menlo Park, California). In this assay, the O.D. was directly proportional to the amount of anti-CETP antibodies present in the plasma samples.

The results of the assay for each of the rabbit groups is shown in Figure 2. Group I showed no plasma antibodies detecting the rabbit C-terminal CETP peptide, whereas the groups vaccinated with CETP immunogens (Groups II, III) showed significant titers of anti- CETP antibodies in almost all vaccinated rabbits. Analysis of the Group IV data is in progress.

CETP Activity and Neutralization Assays In order to measure the activity of CETP in plasma, a commercial fluorescence-based assay (Roar Biomedical Inc.; Yonkers, New York) was used. Incubation of a CETP source (rhuCETP, huCETP or rabbit CETP) with the donor and acceptor particles, included in the kit, results in the CETP-mediated transfer of a fluorescent neutral lipid. This fluorescent neutral lipid is present in a self-quenched state when contained within the core of the donor.

The CETP-mediated transfer is determined by the increase in fluorescence intensity as the fluorescent neutral lipid is removed from the self-quenched donor to the acceptor. To measure neutralization, anti-CETP antibodies are isolated from the plasma of vaccinated rabbits with protein A. Identical amounts (measured by A280) of the antibodies from various samples are added to the above reaction.

Figure 3 shows inhibition of CETP activity by rabbit antibodies collected from the plasma of the vaccinated rabbits. The bar designated "Positive Control Mab" refers to anti- CETP monoclonal antibody TP2, which is known to inhibit CETP activity in vitro and is -18- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 included for comparison. Figure 4 shows the change in CETP activity in Groups I and III from week 1 to week 32. Analysis of the Group IV data is in progress.

Cholesterol and HDL Levels in Plasma Samples of Vaccinated Rabbits The plasma samples taken from rabbits of Groups I IV were also assayed for the concentration of total cholesterol (Figure HDL-C (Figure and triglycerides (see Figure 8) using standard commercial assays (Sigma Diagnostics, Inc., Saint Louis, Missouri). LDL- C (Figure 7) is calculated as total cholesterol minus HDL-C minus 0.2 x tryglyceride level.

CETP levels were determined by a slot blot analysis using anti-CETP monoclonal antibody TP2 and chemiluminescences for detection. The band intensities obtained with various amounts of plasma samples were quantified with the aid of a Kodak® DC40 camera and 1D Image Analysis software (version then compared to that obtained with known amounts of purified human CETP loaded on the same nitrocellulose filter.

The plasma lipoprotein profile for the Group III rabbit #32, which showed the most pronounced reduction in LDL-C levels (Fig. 7) is shown in Figure 8. The profile shows a dramatic rise in HDL as a percent of the total lipoprotein profile. Figure 9 shows, for this rabbit, the correlation between decreasing CETP activity, decreasing cholesterol mass and increasing HDL as a percentage of total lipoportein.

Measurement of cholestrol deposits in the irises of vaccinated rabbits The rabbits from Groups I IV were also assayed for the amount of cholestrol deposits detected in the irises. A scale of cholestrol deposition in the iris was established, with 0 no deposit, 1 20%, 2 40%, 3 60%, 4 80%, and 5 100% deposits on this iris iris completely covered with deposits). One iris per rabbit was evaluated and scored for degree of cholestrol deposition. The groups of animals were blinded to the scorer to avoid bias. Figure 10 shows the data collected from all 48 animals. The Xs indicate animals for which no data were obtained (rabbit #34, #39, and these animals were euthanized due to unrelated complications such as furballs. The data indicate that all CETP vaccinated groups had statistically less cholestrol deposits than the control group.

Ouantitation of lesions in aorta of vaccinated rabbits The rabbits were switched from a diet of basic rabbit chow to diets supplemented with 0.25% cholesterol known to produce atherosclerotic-like lesions in rabbits (Daley et al., Arterioscler. Thromb., 14: 95 104 (1994)). To determine whether the vaccination may affect the development of atherosclerosis, the aortas of these rabbits were examined for the -19- SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 development of atherosclerotic lesions. After blood samples were taken on the last day, rabbits were sacrificed. The entire aortas from each of Groups I-IV were removed and placed into fixative solution (3.7 v/v formaldehyde). Loose tissue, adherent fat, and the adventitia were dissected free from the arteries. Each artery was then cut lengthwise, pinned flat to expose the intimal (luminal) surface, stained with Sudan IV, and then photographed. Sudan IV is a fat soluble red dye that stains atherosclerotic plaques on the intimal surface of arteries.

Figure 11 summarizes the results of this experiment. The stained aortas of rabbits vaccinated with human chorionic gonadotropin revealed a prevalence of atherosclerotic lesions along the length of the aortas and particularly in the portion of the aortas from the thoracic region. In contrast, the aortas of rabbits vaccinated with a synthetic vaccine peptide having segments of tetanus toxoid and the C-terminal sequence of human CETP ("Peptide", see SEQ ID NO: full-length recombinant human CETP ("rhuCETP"), and a CETP-tetanus toxoid conjugate composition ("Conjugate") had lower incidence of lesions, including the portion of the aorta from the thoracic region.

To quantitate the noticeable difference in the presence of atherosclerotic lesions in the aortas of rabbits or lack thereof, the total surface area of the pinned aortas and that of the aortic lesions was determined from photographs by planar morphometry (Daley et al., 1994) using a digitizing tablet with associated software (THE MORPHOMETER

T

Woods Hole Educational Associates, Woods Hole, Massachusetts). The percentage of the surface area of the aortas covered by lesions was determined and the percentages are represented in Figure 11.

EXAMPLES 2 and 3 Plasmid-based Vaccines in Mice and Rabbits Four groups of mice were vaccinated intramuscularly with one of the following: 1. pCMV: a plasmid vector having the cytomegalovirus immediate early promoter/enhancer but without any operably-linked structural gene 2. pCIII-huCETP: a plasmid vector having the full coding sequence for human CETP (SEQ ID NO: 1) under the transcriptional control of the human Apo CIII promoter 3. pSV40-huCETP: a plasmid vector having the full coding sequence for human CETP under the transcriptional control of the SV40 promoter SUBSTITUTE SHEET (RULE 26) WO 99/20302 PCT/US98/22145 4. pCMV-TT-rabCETP: a plasmid vector having a tetanus toxoid peptide (amino acid: 2 to 15 of SEQ ID NO: 7) coding sequence and the full coding sequence for rabbit CETP (SEQ ID NO: 3) under the transcriptional control of the cytomegalovirus immediate early promoter/enhancer The mice were injected once with 25 1l of PBS containing 100 p.g of the plasmid and blood samples were periodically collected and were analyzed with an ELISA detecting antihuman CETP antibodies with the method described below: Plastic 96-well microtiter plates were coated with Protein A/G, by incubating 100 p.l of a 5 pg/ml PBS solution per well overnight at 40C. The plates were emptied and the wells were blocked with 200 p.1 of blocking buffer (PBS with 4% BSA, 1% sucrose, 0.5% 0.01% Gentamycin) for 2 to 8 hours at room temperature. Antibodies from the plasma samples were captured on the Protein A/G by incubating 100 l.1 of various dilutions of the samples for 1 hour at room temperature. Following washing of the wells, biotinylated CETP was captured by the plate bound antibodies by incubating 100 pl of a biotinylated CETP solution at room temperature for 1 hour. The bound CETP was detected by incubating 100 p1 of a streptavidin-HRP solution for 30 minutes at room temperature, followed by adding 100 l.1 of substrate, stopping the reaction with 0.18M sulfuric acid and reading the optical density at 450 nm. Figure 12 shows that plasmids delivered to Groups 2, 3, and 4 produce immunogenic xenogeneic protein.

Subsequently, rabbits were vaccinated with 300 p.g (equally split in six intramuscular sites in the quadriceps) of a vector carrying the human CETP coding sequence under the transcriptional control of the SV40 promoter enhancer (SV40-huCETP) or the same vector without the CETP coding sequence (SV40). The primary injection occurred on Day 1 with an identical boost on weeks 5, 8, 26, and 30. Blood samples were taken periodically throughout the experiment and animals were terminated on week 34.

The plasma samples from the vaccinated rabbits were subjected to the ELISA for detection of antibodies to whole recombinant human CETP, as described above.

Figure 13A-K summarizes the titration of the antibody measured in the rabbits vaccinated with the SV40 promoter enhancer (SV40-huCETP) between weeks 1-34. Significant antibody production was detected on weeks 30 and 34, depicted by the open diamond and open square symbols of the graphs, in most animals. Two of the 11 rabbits (Figure 13A, and 13E) were non-responders.

-21- SUBSTITUTE SHEET (RULE 26) The rabbits were switched from a diet of basic rabbit chow to diets supplemented Aith 0.25% cholesterol known to produce atherosclerotic-like lesions in rabbits. The lesions in rabbits vaccinated with pSV 4O-huCETP and control rabbits vaccinated with pSV4O were visualized and quantitated as described in Example 1 above. Figure 14 shows the mean percentage of aorta covered with lesions in both groups of rabbits. The results of this experiment are in line with the trend observed with directly vaccinated rabbits (Groups I-IV, see Figure 11), showing a decrease in aortic lesions due to vaccination.

Although a number of embodiments have been described above, it will be understood by those skilled in the art that modifications and variations of the described compositions and methods may be made without departing from either the spirit of the invention or the scope of the appended claims. The articles and publications cited herein are incorporated by reference.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the *common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context ***requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

-22- EDITORIAL NOTE FOR 11048/99 THE FOLLOWING SEQUENCE LISTING IS PART OF THE DESCRIPTION THE CLAIMS FOLLOW ON PAGE 23 WO 99/20302 WO 9920302PCT/US98/22145 SEQUENCE LISTING <11:0> Rittershaus, Charles W.

Thomas, Lawrence J.

Avant Immunotherapeutics, Inc.

<120> Xenogeneic Cholesteryl Eater Transfer Protein (CETP) for Modulation of CETP Activity <130> TCS-420.1 PCT seqlit <140> PCT/US9B/22145 <141> 1998-10-20 <150> 08/954,643 <151> 1997-10-20 <160> 7 <170> Patentln Ver. <210> 1 <211> 476 <212> PRT <213> Homo sapiens <300> <301> Drayna, Dennis <302> Cloning and Sequencing of Human Cholesteryl Ester Transfer cDNA <303> Nature <304> 327 <306> 632-634 <307> 1987-06-18 <313> 1 TO 476 <400> 1 Cys Ser Lys Gly Thr 1 5 Lys Pro Ala Leu Leu Ser His Giu Ala Ile Val Cys Arg Ile Thr 1s Val Leu Asn His 25 Glu Thr Ala Lys Val Ile Gin Thr Ala Phe Gin Arg Ala Ser Tyr 40 Pro Asp Ile Thr Gly Giu Lys Ala Met Met Leu Leu Gly Gin Val 55 Lys Tyr Gly Leu His Asn Ile Gin Ile SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCTIUS98/22145 Ser His Leu Ser Ile Ala Ser Ser Gin Val Giu Leu Val Giu Ala Lys 70 75 Ser Ile Asp Val Ser Ile Gin Asn Val Ser Val Val Phe Lys Gly Thr 90 Leu Lys Tyr Gly Tyr Thr Thr Ala Trp Trp Leu Giy le Asp Gin Ser 100 105 110 Ile Asp Phe Glu Ile Asp Ser Ala Ile Asp Leu Gin Ile Asn Thr Gin 115 120 125 Leu Thr Cys Asp Ser Gly Arg Val Arg Thr Asp Ala Pro Asp Cys Tyr 130 135 140 Leu Ser Phe His Lys Leu Leu Leu His Leu Gin Gly Giu Arg Giu Pro 145 150 155 160 Gly Trp Ile Lys Gin Leu Phe Thr Asn Phe Ile Ser Phe Thr Leu Lys 165 170 175 Leu Val Leu Lys Gly Gin Ile Cys Lys Giu Ile Asn Val Ile Ser Asn 180 185 190 Ile Met Ala Asp Phe Val Gin Thr Arg Aia Ala Ser Ile Leu Ser Asp 195 200 205 Gly Asp Ile Gly Val Asp Ile Ser Leu Thr Gly Asp Pro Val Ile Thr 210 215 220 Ala Ser Tyr Leu Giu Ser His His Lys Giy His Phe Ile Tyr Lys Asn 225 230 235 240 Val Ser Giu Asp Leu Pro Leu Pro Thr Phe Ser Pro Thr Leu Leu Giy 245 250 255 Asp Ser Arg Met Leu Tyr Phe Trp Phe Ser Giu Arg Val Phe His Ser 260 265 270 Leu Ala Lys Val Ala Phe Gin Asp Gly Arg Leu Met Leu Ser Leu Met 275 280 285 Giy Asp Giu Phe Lys Ala Vai Leu Giu Thr Trp Gly Phe Asn Thr Asn 290 295 300 Gin Giu Ile Phe Gin Giu Vai Vai Gly Giy Phe Pro Ser Gin Ala Gin 305 310 315 320 2 SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCT/US98/221 Val Gly Pro Thr Leu 385 Ser Gly Met Ile Thr Val Asp Thr 370 Asp Ser Ile Asn Thr 450 Vai Val Gin 355 Val Phe Glu Pro Ser 435 Arg His Cys 325 Val Asn 340 Gin His Gin Ala Gin Ile Ser Ile 405 Giu Vai 420 Lys Giy Asp Gly Leu Lys Met Pro Lys Ile Ser Cys Gin Asn Lys Ser Ser Ser Thr 390 Gin Met Vai Phe Ser Val Tyr 375 Pro Ser Ser Ser Leu 455 Val Ala 360 Ser Lys Phe Arg Leu 440 Leu Met 345 Tyr Lys Thr Leu Leu 425 Phe Leu 330 Val Thr Lys Val Gin 410 Giu Asp Gin Phe Giu Leu 380 Asn Met Vai Ile Asp 460 Leu Giu 365 Phe Leu le Phe Asn 445 Phe Phe 350 Asp Leu Thr Thr Thr 430 Pro Giy Pro Ile Ser Giu Aia 415 Al a Giu Phe Arg Vai Lau Ser 400 Val Leu Ile Pro Giu His Leu Leu Vai Asp Phe Leu Gin Ser Leu Ser 465 <210> <211> <212> <213> <300> <301> <302> <303> <304> <306> <307> <313> <400> 2 470 1428

DNA

Homo sapiens Drayna, Dennis Cioning and Sequencing of Human Cholesteryl Ester Transfer cDNA Nature 327 632 -634 1987-06-18 1 TO 476 2 3 SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCT/US98/22 145 tgctccaaag ctggtgttga cc agata tc a aacatccaga tccattgatg tacaccactg attgacctcc cctgactgct gggtggatca ggacagatc t agggc tgcc a cccgtcatca gtctcagagg ctgtacttct ggccgcctca ttcaacacca gtcaccgtcc aattcttcag tacacatttg ttcttaagcc agctccgagt gtcatgtctc ttcgacatca tttggcttcc gcacctcgca accacgagac cgggcgagaa tcagccactt tctccattca cctggtggct agatcaacac acctgtcttt agcagctgtt gcaaagagat gcatcctttc cagcctccta acctccccct ggttctctga tgctcagcct accaggaaat actgcctcaa tgatggtgaa aagaggatat tcttggattt ccatccagag ggctcgaggt tcaaccctga cigagcacct cgaggcaggc tgccaaggtg ggccatgatg gtccatcgcc gaacgtgtct gggtattgat acagctgacc ccataagctg cacaaatttc caacgtcatc agatggagac cctggagtcc ccccaccttc gcgagtcttc gatgggagac cttccaagag gatgcccaag attcctcttt cgtgactacc ccagattaca cttcctgcag agtgtttaca gattatcact gc tggtggat atcgtgtgcc atccagaccg ctccttggcc agcagccagg gtggtcttca cagtccattg tgtgactctg ctcctgcatc atctccttca tctaacatca attggggtgg catcacaagg tcgcccacac cactcgctgg gagttcaagg gttgtcggcg atctcctgcc ccacgcccag gtccaggcct ccaaagactg tcaatgatca gccctcatga cgagatggct ttcctccaga gcatcaccaa ccttccagcg aagtcaagta tggagc tggt aggggaccct acttcgagat gtagagtgcg tccaagggga ccctgaagct tggccgattt acatttccct gtcatttcat tgctggggga ccaaggtagc cagtgctgga gcttccccag aaaacaaggg accagcaaca cctattctaa tttccaactt ccgctgtggg acagcaaagg tcctgctgct gcttgagc gcctgccctc agccagctac tgggttgcac ggaagccaag gaagtatggc cgactctgcc gaccgatgcc gcgagagcc t ggtcctgaag tgtccagaca gacaggtgat ctacaagaat ctcccgcatg tttccaggat gacc tggggc cc aggc ccaa agtcgtggtc ttctgtagct gaaaaagc tc gac tgagagc catccctgag cgtgagcctc gcagatggac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1428 <210> 3 <211> 496 <212> PRT <213> Oryctolagus cuniculus <300> <301> Nagashima, Mariko <302> Cloning and UiPNA tissue distribution of rabbit cholesteryl ester transfer protein <303> J. Lipid Res.

<304> 29 <306> 1643-1649 <307> 1988 <313> 1 TO 496 <400> 3 Cys Pro Lys Gly Ala 1 5 Lys Pro Ala Leu Leu Ser Tyr Glu Ala Ile Val Cys Arg Ile Thr Val Leu Asn Gln 25 Glu Thr Ala Lys Val Val Gin Thr Ala Phe Gln Arg Ala Gly Tyr Pro Asp Val Ser Gly Glu Arg Ala 4 SUBSTITUTE SHEET (RULE 28) WO 99/20302 PCTIUS98/22145 Val Met Leu Leu Gly Arg Val Lys Tyr Gly Leu His Asn Leu Gin Ile r. 1 55 Ser Thr Leu Val Leu Leu 145 Gly Leu Ile b Gly 1 2 Ala T 225 Val S Asp S His lie Asn Asp Thr 130 Ala rrp Cle Lei Asi Ty3 Phe 115 Cys Phe Leu ~eu u Se Va Sei Gli Asp His Lys Lys 180 Asp Gly Leu Ala Met r 11 1 Ale 81 r Tyz SIle Ala Lys Gin 165 Arg Phe Val Glu Phe 245 Leu e Al 7 Thl Asi Glj Leu 150 Leu Gin Val Asp Ser 230 Pro Tyr a Se 0 a Gli r Sel i Sez Ser 135 Leu Phe Val Gin Ile 215 His Leu Phe Gin r Ser I Asn Ala Ala 120 Val Leu Thr Cys Thr 200 Ser His Arg I Trp I 2 Glu G 280 Gi: Va Trj 10! Ile Arc His Asn Asn 185 Arg VTal -ys ~la 'he 65 Ily n Val L Ser 90 i Gly Asp Thr Leu Phe 170 Glu Ala Thr Gly 1 Phe P 250 Ser A Arg L Glu 75 Val Leu Leu Asn Gin 155 Ile Ile kla [is I !35 Ero I Sp C eu V

L

V

G

G:

A:

Se As Se ki 22 ?h rr aa eu Val al Phe ly Ile Ln lie 125 La Pro I0 .y Glu !r Phe :n Thr r Ile 205 a Pro 0 e Thr I o Gly I n Val 2 L Leu S 285 As Lys Asn 110 Asn Asp Arg Thr Ile 190 Leu lal Lis jeu .eu er Al Gi' 9! Gl Th, Cyc Glu Leu 175 Ser Ser Ile Lys Leu 255 Asn a Lys y Thr Ser r Glu I Tyr Pro 160 Lys Asn Asp Thr Asn 240 Gly Ser Thr et sp 10 'hr er er Ala 195 Ile Tyr Glu Arg 260 Leu Ala Arg Ala Ala Phe 275 Leu

L

Gly Asp Giu Phe Lys Lys Val Leu Gu Thr Gin Gly Phe Asp Thr Asn SUBSTITUTE SHEET (RULE 26) WO 99/20302 290 Gli Gu Ile Phe Gin Giu 305 310 PCT/US98/22145 Gin Val Arg Giy Arg Pro Ile Thr 370 Leu Leu 385 Ala Asn Leu Thr Ile Ala Phe Thr 450 Asn Pro 465 Phe Gly Ala Val Asp 355 Thr Asp Leu Giu Thr 435 Ala Giu Phe Val Val 340 Gly Val Phe Ser Ser 420 Val Leu Ile Pro His 325 Vai Arg Gin Gin Vai 405 Arg Gly Met le Lys 485 Cys Ser Glu Ala Cys 390 Ala Ser Ile Asn Thr 470 His 295 Leu Leu Ser Aia Ser 375 Val Leu Giu Pro Ser 455 Leu Leu Ser Lys Ser Val 360 Tyr Pro Arg Ser Giu 440 Lys Asp Leu Arg Val Val 345 Aia Ser Ala Thr Leu 425 Vai Gly Gly Val Gly Pro 330 Ala Tyr Gin Ser Glu 410 Gin Met Leu Cys Asp 490 300 Pro le Thr Phe Lys 380 Arg Lys Ser Arg Leu 460 Leu Leu Thr Ser Phe Giu 365 Leu Ala Ala Leu Leu 445 Phe Leu Gin Gly Cys Arg 350 Glu Phe Gly Val Arg 430 Glu Giu Gin Ser Gin Gin 335 Phe Asp Leu Ser Ser 415 Ser Val le Met Leu 495 Ala 320 Asn Pro le His Ser 400 Asn Leu Ala le Asp 480 Ser <210> 4 <211> 1488 <212> DNA <213> Oryctolagus cuniculus <300> SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCT[US98/221 <301> Nagashima, Mariko <302> Cloning and mRNA tissue distribution of rabbit zcholesteryl ester transfer protein <303> J. Lipid Res.

<304> 29 <306> 1643-1649 <307> 1988 <313> 1 TO 496 <400> 4 tgtcccaaag ttggtgttga ccggacgtca aacctccaga accatcgacg tac acgagtg attgacctcc cccgactgct gggtggctca cgacaggtct agggccgcca cctgtcatca gtc tccgagg ctctacttct ggccgtctcg ttcgacacca caggtagccg gtgtcttctt gcctacaggt ctcttcctac gcaaatctct cgc tccgagt gtcatgtctc ttcgaaatca ttcggttttc gcgcctccta accaagagac gcggcgagag tcagccacct tcgccatcca cctgggggtt agatcaacac acctggcttt agcagctctt gcaatgagat gcatcctctc cagccaccta ccttccccct ggttctccga tgctcagcct accaggaaat tccactgcct ccgtcgccgt ttgaggagga acctcttgga ccgtggccct ccctgcagag ggctcgaggt tcaaccccga ccaagcacct cgaggc tggc ggccaaggtg ggccgtgatg gtccatcgcc gaacgtgtcc gggcatcaat agagctgacc ccataaactg cacaaacttc caacaccatc agatggagac cctggagtcc ccgcgccttc tcaagtgctc gacaggggat cttccaggag taaggtgccc gacgttccgc tatcatcacc tttccagtgc caggactgag ctctctccgc ggcgttcaca gattatcac t gctggtggat atcgtgtgtc gtccagacgg ctcctcggcc agcagccagg gtggtcttca cagtctgtcg tgcgacgc tg ctcctgcacc atctccttca tccaacatca atcggggtgg catcacaagg ccgcccggtc aactccctgg gagttcaaga ctttccagag aagatctcct ttcccccgcc accgtccagg gtgccggcca gctaaggctg tccctgatcg gccctcatga ctcgatggct ttcctgcaga gcatcaccaa ccttccagcg gggtcaagta tggagc tggt aggggaccct acttcgagat gcagtgtgcg tccaggggga ccctgaagct tggctgactt acatttccgt gtcacttcac ttctggggga c cagggccgc aagtgctgga gccttcccac gccagaaccg c agatggccg cctcctactc gcggaagggc tttccaacct ccacggtggg acagcaaagg gcctgctgct gcctgagc gcccgccctc cgccggctat cgggctgcac ggacgccaag gaactacagc cgactctgcc caccaatgcc gcgcgagccg gattctgaag tgtccagacg gacgggggc c gcacaagaac ctcccgcatg cttccaggag gacccagggt cggccaggcc gggtgtcgtg agaagctgtg ccagaaaaag aggcagc tca gac tgagagc catcccggag cctggacctc gcagatggac 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1488 <210> <211> 477 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial sequence: humanized rabbit CETP protein <400> Cys Pro Lys Gly Ala Ser Tyr Glu Ala Gly Ile Val Cys Arg Ile Thr 1 5 10 7 SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCT[US98/22145 Lys Thr Val Ser Thr Leu Val Leu Leu 145 Gly Leu Ile Gly Ala 225~ Val Asp Pro Al a Met His Ile Asn Asp Thr 130 Al a Trp Ile Met Asp 210 Thr Ser Ser Al a Phe Leu Leu Asp Tyr Phe 115 Cys Phe Leu Leu Ala 195 Ile Tyr Giu Arg Leu Gin Leu Ser Val Ser 100 Glu Asp His Lys Lys 180 Asp Gly Leu Ala Met 260 Leu Val Leu Asn Gin Giu Thr Ala Lys Val Val Gin 25 Arg Gly Ile Al a Tyr Ile Al a Lys Gin 165 Arg Phe Val Giu Phe 245 Leu Ala Arg Ala 70 Ile Thr Asp Gly Leu 150 Leu Gin Val Asp Ser 230 Pro Tyr Gly Tyr 40 Val Lys Ser Ser Gin Asn Ser Ala Ser Ala 120 Ser Val 135 Leu Leu Phe Thr Val Cys Gin Thr 200 Ile Ser 215 His His Leu Arg Phe Trp Pro Tyr Gin Val Trp 105 Ile Arg His Asn Asn 185 Arg Val Lys Al a Phe 265 Asp Gly Val Ser 90 Gly Asp Thr Leu Phe 170 Giu Ala Thr Gly Phe 250 Val Leu Giu 75 Val Leu Leu Asn Gin 155 Ile Ile Ala Gly His 235 Pro Ser His Leu Val Gly Gin Al a 140 Gly Ser Asn Ser Ala 220 Phe Pro Gly Asn Val Phe Ile Ile 125 Pro Giu Phe Thr le 205 Pro Thr Gly Giu Leu Asp Lys Asn 110 Asn Asp Arg Thr Ile 190 Leu Val His Leu Arg Gin Ala Gly Gin Thr Cys Giu Leu 175 Ser Ser Ile

LYS

Leu Ala Ile Lys Thr Ser Giu Tyr Pro 160 Lys Asn Asp Thr Asn 240 Gly Ser Asp Gin Val Leu Asn Ser 270 SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCT/US98/22 145 Leu Ala Arg 275 Gly Asp Giu 290 Gin Glu Ile 305 Gin Val Ala Arg Gly Val Arg Pro Asp 355 Ile Thr Thr 370 Lau Leu Asp 385 Ser Arg Ser Val Gly Ile Leu Met Asn 435 Ile Ile Thr 450 Pro Lys His 465 Ala Phe Phe Val Val 340 Gly Val Phe Glu Pro 420 Ser Leu Leu Ala Phe Gin Giu Gly Arg Leu Val Leu Ser Leu Thr 280 285 Lys Gin His 325 Vai Arg Gin Gin Ser 405 Giu L~ys Asp Lau Lys Glu 310 Cys Ser Giu Ala Cys 390 Leu Val Gly Gly Val 470 Val 295 Leu Leu Ser Ala Ser 375 Vai Gin Met Lau Cys 455 Asp Leu Ser Lys Ser Val 360 Tyr Pro Ser Ser Asp 440 Lau Phe Giu Thr Arg Gly_ Vai Pro 330 Val Ala 345 Ala Tyr Ser Gin Lys Ala Ser Leu 410 Arg Leu 425 Leu Phe Leu Leu Leu Gin Gin Lau 315 Lys Val Arg Lys Val 395 Arg Giu Giu Gin Ser 475 Gly 300 Pro Ile Thr Phe Lys 380 Ser Ser Val Ile Met 460 Leu Phe Thr Ser Phe Glu 365 Leu Asn Lau Al a Ile 445 Asp Ser Asp Gly Cys Arg 350 Glu Ph.

Leu Ile Ph.

430 Asn Ph.

Thr Gin Gin 335 Ph.

Asp Leu Thr Al a 415 Thr Pro Gly Asn Ala 320 Asn Pro Ile His Giu 400 Thr Ala Glu Ph.

<210> 6 <211> 496 <212 PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: humanized 9 SUBSTITUTE SHEET (RULE 26) WO 99/20302 rabbit CETP protein PCT/US98/22145 <t00> 6 Cys Lys Thr Val Ser Thr Leu Val Leu Leu 145 Gly Leu Ile Giy Ala 225 Pro Pro Ala Met His Ile Asn Asp Thr 130 Al a Trp Ile Met Asp 210 Thr Lys Ala Phe Leu Leu Asp Tyr Phe 115 Cys Phe Leu Leu Ala 195 Ile Tyr Gly Leu Gin Leu Ser Val Ser 100 Glu Asp His Lys Lys 180 Asp Gly Leu Ala Leu Arg Gly Ile Ala Tyr Ile Ala Lys Gin 165 Arg Phe Val Glu Ser Val Ala Arg Ala 70 Ile Thr Asp Gly Leu 150 Leu Gin Val Asp Ser 230 Tyr Leu Gly Val 55 Ser Gin Scr 5cr Ser 135 Leu Phe Val Gin Ile 215 His Glu Ala Gly Ile Val Cys Arg Ile Thr Asn Tyr 40 Lys Scr Asn Al a Al a 120 Val Leu Thr Cys Thr 200 Ser His 10 Gin Glu 25 Pro Asp Tyr Gly Gin Val Val Ser 90 Trp, Gly 105 Ile Asp Arg Thr His Leu Asn Phe 170 Asn Glu 185 Arg Ala Val Thr Lys Gly Thr Val Leu Glu 75 Val Leu Leu Asn Gin 155 Ile Ile Ala Gly His 235 Ala Ser His Leu Val Gly Gin Ala 140 Giy Ser Asn Ser Ala 220 Phe Lys Giy Asn Val Phe Ile Ile 125 Pro Glu Phe Thr Ile 205 Pro Val Giu Leu Asp Lys Asn 110 Asn Asp Arg Thr Ile 190 Leu Val Val Arg Gln Ala Gly Gin Thr Cys Giu Leu 175 Ser Ser Ile Gin Ala Ile Lys Thr Ser Glu Tyr Pro 160 Lys Asn Asp Thr Asn 240 Thr His Lys SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCT/US98/22 145 Val Asp Leu Gly Gin 305 Gin Arg Arg Ile Leu 385 Ala Leu Ile Phe Asn 465 Ser Ser Ala Asp 290 Glu Val Gly Pro Thr 370 Leu Asn Thr Ala Thr 450 Pro Giu Arg Arg 275 Glu Ile Ala Val Asp 355 Thr Asp Leu Glu Thr 435 Al a Glu *Ala Phe 245 *Met Leu 260 Ala Ala Phe Lys Phe Gin Val His 325 Val Val 340 Gly Arg Val Gin Phe Gin Ser Vai 405 Ser Arg 420 Val Gly Leu Met Ile Ile Pro Leu Arg Ala Phe Pro Pro Gly Leu Leu Gly 250 255 Tyr Phe Lys Giu 310 Cys Ser Glu Ala Cys 390 Ala Ser Ile Asn Thr 470 Phe Gin Val 295 Leu Leu Ser Al a Ser 375 Val Leu Giu Pro Ser 455 Leu Trp Glu 280 Leu Phe 265 Giy Glu Ser Arg Lys Val Ser Val 345 Val Ala 360 Tyr Ser Pro Ala Arg Thr Ser Leu 425 Giu Val 440 Lys Giy Asp Gly Leu Val Ser Asp Arg Leu Thr Gin Gly Leu 315 Pro Lys 330 Ala Val Tyr Arg Gin Lys Ser Gly 395 Giu Ala 410 Gin Ser Met Ser Leu Asp Cys Leu 475 Gin Val Gly 300 Pro Ile Thr Phe Lys 380 Arg Lys Ser Arg Leu 460 Leu Val Leu 285 Phe Thr Ser Phe Giu 365 Leu Ala Ala Leu Leu 445 Phe Leu Leu 270 Ser Asp Gly Cys Arg 350 Giu Phe Gly Val Arg 430 Glu Glu Gin Asn Leu Thr Gin Gin 335 Phe Asp Leu Ser Ser 415 Ser Val Ile Met Ser Thr Asn Ala 320 Asn Pro Ile His Ser 400 Asn Leu Ala Ile Asp 480 Phe Gly Phe Pro Giu His Leu 485 Asp 490 Phe Leu Gin Ser Leu Ser 495 SUBSTITUTE SHEET (RULE 26) WO 99/20302 WO 9920302PCTIUS98/22 145 ':210> 7 ':211> 31 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: fusion protein of a tetanus toxoid segment and human CETP C-terminus '400> 7 Cys Gin Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Phe 1 5 10 Gly Phe Pro Glu His Leu Leu Val Asp Phe Leu Gin Ser Leu Ser 25 312 SUBSTITUTE SHEET (RULE 26)

Claims (11)

1. A humanized rabbit cholesteryl ester transfer protein comprising the amino acid sequence of SEQ ID

2. A humanized rabbit cholesteryl ester transfer protein comprising the amino acid sequence of SEQ ID NO:6.

3. A method of modulating the level of endogenous, active cholesteryl ester transfer protein (CETP) in a mammal comprising immunizing the mammal one or more times with a whole, non-endogenous CETP in an amount or amounts effective to reduce CETP activity below 20% of that of the untreated mammal. S4. A method of modulating the level of endogenous cholesteryl ester transfer protein 15 (CETP) in a mammal comprising administering to the mammal a whole, non-endogenous CETP in an amount effective to achieve a level of essentially 0 ig of CETP per milliliter of blood of the mammal. The method according to any one of Claims 3 and 4, wherein the mammal is a 20 human.

6. The method according to any one of Claims 3 and 4, wherein the whole, non- endogenous cholesteryl ester transfer protein (CETP) is selected from the group consisting of a xenogeneic CETP, an allelic variant of the mammal's endogenous CETP, and a mammalianized non-endogenous CETP in which the amino acid sequence of a non- endogenous CETP has been altered by deletion or substitution of one or more amino acids so as to make the amino acid sequence of said non-endogenous CETP more similar to the mammal's endogenous CETP.

7. The method according to Claim 6, wherein the mammal is a human. P MOPERVEHMR. Clm\M.,dh\2285745 dh.d-Z6/0302 -24-

8. The method according to any one of Claims 3 and 4, wherein the whole, non- endogenous cholesteryl ester transfer protein (CETP) is administered to the mammal by administering a plasmid-based vaccine comprising a promoter sequence suitable for directing the transcription of a nucleotide sequence in a cell of the mammal operably linked to a nucleotide sequence coding for the whole, non-endogenous CETP, wherein the plasmid-based vaccine expresses the whole, non-endogenous CETP in an. amount effective to modulate the level of endogenous CETP, the level of LDL-cholesterol, or the level of HDL-cholesterol in the blood.

9. The method according to Claim 8, wherein the mammal is a human.

10. Use of a cholesteryl ester transfer protein (CETP) non-endogenous to a mammalian subject to achieve a level of essentially 0 gg of CETP per milliliter of blood in said S. mammalian subject.

11. Use of a humanized, rabbit cholesteryl ester transfer protein according to any one of Claims 1 and 2 in the manufacture of a medicament.

12. Use of a humanized, rabbit cholesteryl ester transfer protein according to any one of Claims 1 and 2 to treat atherosclerosis.

13. A protein according to any one of Claims 1 and 2, a method according to any one of Claims 3 to 9 or a use according to any one of Claims 10 to 12 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this 2 6 th Day of March, 2002 Avant Immunotherapeutics, Inc. by its Patent Attorneys ,N AVIES COLLISON CAVE