JP2001520204A - Heterologous CETP for modulating cholesteryl ester-carrying protein (CETP) activity - Google Patents

Abstract

SUMMARY OF THE INVENTION Methods for modulating cholesteryl ester transfer protein (CETP) activity and plasma levels of lipoproteins involved in heart disease recognize in mammals the natural (endogenous) CETP of the mammal ( Administering a non-endogenous CETP or a plasmid-based vaccine for expressing such CETP to elicit the production of antibodies (which bind to it).

Description

DETAILED DESCRIPTION OF THE INVENTION

This application is a continuation-in-part of US patent application Ser. No. 08 / 954,643, filed Oct. 20, 1997.

TECHNICAL FIELD [0002] The present invention relates to the field of cardiovascular diseases, especially atherosclerosis. More specifically, the present invention provides compositions and methods for controlling, treating, or reducing the risk of atherogenic activity in the circulatory system of mammals, especially humans.

(Background Art) Cholesterol circulates predominantly in the body as a component of lipoprotein particles (lipoproteins). Lipoproteins consist of a protein portion called apolipoprotein (Apo) and various lipids, including phospholipids, triglycerides, cholesterol and cholesteryl esters. There are ten major classes of apolipoproteins. That is, Apo AI, Apo A-II, Apo-IV, Apo B-48, Apo B-100, Ap
o CI, Apo C-II, Apo C-III, Apo D and Apo E. Lipoproteins are classified by density and composition. For example, high density lipoproteins (HDLs), whose function is to mediate the transport of cholesterol from peripheral tissues to the liver, typically have a density in the range of about 1.063-1.21 g / ml. HDL contains various amounts of Apo AI, Apo A-II
Apo CI, Apo C-II, Apo C-III, Apo D and Apo E, as well as varying amounts of lipids such as cholesterol, cholesteryl esters, phospholipids and triglycerides (TG).

[0004] In contrast to HDL, low-density lipoprotein (LDL), whose density is typically about 1.019-1.063 g / ml, contains Apo B-100 along with various lipids. In particular, the amount of lipids, cholesterol and cholesteryl esters, is significantly higher in LDL than in HDL, measured as a percentage of dry mass. LDL is particularly important in transporting cholesterol to peripheral tissues.

[0005] Very low density lipoproteins (VLDL) have a density of about 0.95-1.006 g / ml and differ in composition from other classes of lipoproteins in both protein and lipid content. VLDL generally has much higher amounts of triglycerides than HDL or LDL, and is particularly important in transporting endogenously synthesized triglycerides from the liver to adipose and other tissues. The characteristics and functions of various lipoproteins have been discussed previously (eg, Mathews and van Holde, Biochemistry, pp. 574-576 and 626-630 (The Benjamin / Cummings Publishing Co., Redwood City, California).
a, 1990); The Metabolic Basis of Inherited Disease, 6th ed., pp. 1129-1138
Havel et al., “In,” described in (Scriver et al., Eds.) (McGraw-Hill, Inc., New York, 1989).
troduction: Structure and metabolism of plasma lipoproteins ”(“ Introduction: Structure and Metabolism of Plasma Lipoproteins ”); Advances in Human Genetics, Vol. 21, pp. 145-319, Zannis et al., Plenum Press, New York, 1993 , ”Geneti
c mutations affecting human lipoproteins, their receptors, and their enz
ymes "(" Gene mutations affecting human lipoproteins, their receptors and enzymes ")).

[0006] Decreased susceptibility to cardiovascular diseases such as atherosclerosis generally results in increased absolute levels of circulating HDL, decreased levels of LDL or VLDL, and also of HDL levels relative to circulating levels of VLDL and LDL. Coronation (eg, 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 J., 113: 589-597 (1987); T.
all, J. Clin. Invest., 89: 379-384 (1990); Tall, J. Internal Med., 237:
5-12 (1995)).

Cholesteryl ester transporter protein (CETP) mediates the transport of cholesteryl esters from HDL to TG-rich lipoproteins (such as VLDL and LDL) and the reciprocal exchange of TG from VLDL to HDL ( Tall, supra; Tall, J. Lipid Res., 34:12.
55-1274 (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 regulating the levels of cholesteryl esters and triglycerides associated with various classes of lipoproteins. The high cholesteryl ester transport activity of CETP correlated with elevated levels of LDL-related cholesterol and VLDL-related cholesterol. And it has also been correlated with an increased risk of cardiovascular disease (see, for example, Tato et al., Arterioscler. Thromb. Vascular Biol., 15: 112-120 (1995)).

[0008] CETP isolated from human plasma is a hydrophobic glycoprotein with a molecular weight of about 66,000 to 74,000 daltons on sodium dodecyl sulfate (SDS) -polyacrylamide gel with 476 amino acids (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)). The cDNA encoding human CETP has been cloned and sequenced (Drayna et al., Nature, 327: 632-634 (1987)). C
ETP includes cholesteryl esters, triglycerides, phospholipids (Barter et al., J. Lipid R
es., 21: 238-249 (1980)) and lipoproteins (eg, Swenson et al., J. Biol.
Chem., 264: 14318-14326 (1989)). More recently, the region of CETP defined by the carboxyl-terminal 26 amino acids, and especially amino acids 470-475, has been shown to be particularly important for neutral lipid binding involved in neutral lipid transport (Hesler et al. , J. Biol. Chem., 263: 5020-5023 (1988)).

Hereinafter, the term LDL-C refers to total cholesterol associated with low density lipoproteins, including cholesteryl esters and / or non-esterified cholesterol. The term VLDL-C is used to refer to total cholesterol associated with very low density lipoproteins, including cholesteryl esters and / or non-esterified cholesterol. HDL- refers to total cholesterol associated with high density lipoproteins, including cholesteryl esters and / or non-esterified cholesterol
Use the term C.

[0010] Numerous in vivo studies using animal models or humans have shown that CETP activity can affect the levels of cholesterol-containing circulating HDL. The increased cholesteryl ester transport activity of CETP is associated with HDL-dependent levels of LDL-C and / or VLDL-C.
-C, which correlates with reduced levels and subsequent predisposition to atherosclerosis. Injection of partially purified human CETP into rats, which usually lacks CETP activity, results in the transfer of cholesteryl esters from HDL to VLDL,
Consistent with cholesteryl ester transport from HDL to VLDL promoted by ETP
(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 (19)
87)). Transgenic mice expressing human CETP have been reported to show a significant decrease in cholesterol levels associated with HDL (eg, Hayek et al., J. Cl.
in. Invest., 90: 505-510 (1992); Breslow et al., Proc. Natl. Acad. Sci. USA, 9
0: 8314-8318 (1993)). Furthermore, while wild-type mice are usually highly resistant to atherosclerosis (Breslow et al., Same place), transgenic mice expressing monkey CETP have altered lipoprotein-associated cholesterol distribution, , LDL-C and VLDL-C levels were reported to increase and HDL-C levels were decreased (Marotti et al., Nature, 364: 73-75 (1993)). Transgenic mice that express monkey CETP also have higher
Large focal lesions susceptible to diet-induced severe atherosclerosis, and are significantly larger in area compared to those found in control mice, and more typical of those found in human atherosclerotic lesions Atherosclerotic lesions with arising in the aorta were developed (Marotti et al., Supra). Intravenous infusion of anti-human CETP monoclonal antibody (Mab) into hamsters and rabbits suppresses CETP activity in vivo, leading to significant increases in HDL-C levels, decreased HDL-triglyceride levels, and increased HDL size , In the distribution of cholesterol in circulating lipoproteins
We again suggested a significant role for CETP (Gaynor et al., Atherosclerosis, 110: 101-109 (1
994) (hamster); Whitlock et al., J. Clin. Invest., 84: 129-137 (1989) (rabbit).

[0011] CETP deficiency in humans has also been studied. For example, in a family study in Japan, siblings homozygous for a non-functional allele of the CETP gene had no detectable CETP activity. In effect, these humans did not show any atherosclerotic plaque, and they showed a tendency to live longer in families (see, eg, Brown et al., Nature, 342: 448-451 (1989): Inazu et al., N. et al. Engl. J. Med., 323
: 1234-1238 (1990); Bisgaier et al., J. Lipid Res., 32: 21-23 (1991)). Such homozygous CETP-deficient individuals also have cholesteryl ester-rich circulating
Elevated levels of HDL, and general elevated levels of HDL and exceptionally large HD
L (ie, 4-6 times the size of normal HDL) has been shown to have an anti-atherogenic lipoprotein profile (Br
own et al., supra, p. 451). The frequency of this mutation in Japan is relatively high, which can explain elevated levels of HDL in a fairly large part of the Japanese population.

The above studies show that cholesteryl esters are transported from HDL to VLDL and LDL, and thus the relative profile of circulating lipoproteins is associated with an increased risk of cardiovascular disease (eg, levels of HDL-C It has been shown that CETP plays a major role in lowering and lowering VLDL-C and LDL levels). Taken together, current evidence suggests that elevated levels of CETP activity may predict an increased risk of cardiovascular disease. Modulation or suppression of endogenous CETP activity is therefore attractive for modulating the relative levels of lipoproteins to reduce or prevent the progression of a cardiovascular disease such as atherosclerosis, or to induce regression of the disease. Is a typical treatment.

For example, co-pending patent application PCT / US96 / 06147 (WO
96/34888) and co-pending patent application PCT / US97 / 072 commonly assigned to the present application.
In our previous work, detailed in 94 (WO 97/41227), both incorporated herein by reference, we found that the peptide composition or endogenous CETP
An approach to modulate CETP activity in individuals by vaccination with a plasmid-based vaccine that results in the production of antibodies that recognize and neutralize E. coli was detailed. We have shown that administration of immunogenic peptides by direct inoculation or by in situ production following injection of a functional vaccine based on plasmids responds to the inoculated individual's own (endogenous) CETP. Has been shown to result in the production of sexual antibodies. Thus, a vaccine based on the vaccine peptide and plasmid disrupts tolerance of the individual receiving the vaccine and promotes the production of antibodies that recognize self proteins. In addition, administration of these vaccines to test animals resulted in reduced relative levels of total cholesterol and HDL-C, and reduced the incidence of atherosclerotic lesions. The elicited endogenous anti-CETP antibody promotes a physiological condition that is thus correlated with a reduced risk of cardiovascular disease. These antibodies then appear to have a direct effect on preventing or reducing the formation of atherosclerotic plaque.

The present inventors have found another approach to elicit the production of anti-CETP antibodies in mammals. We believe that all mammalian CETP molecules (ie, non-endogenous CETP
TP), which is reactive with its own endogenous CETP in mammals, modulates the activity of CETP, and reduces circulating CETP activity, lowers total cholesterol, lowers circulating LDL levels, LDL It was determined that antibodies could be elicited that would help provide an increased ratio of HDL-C to -C. The use of non-endogenous CETP to enhance the production of anti-endogenous CETP antibodies also results in a reduced incidence of atherosclerotic lesions compared to non-vaccinated controls.

SUMMARY OF THE INVENTION Accordingly, the present invention provides compositions and methods useful for modulating or suppressing cholesteryl ester transfer protein (CETP) activity. In particular, the use of a non-endogenous CETP, including a heterologous CETP, may increase the endogenous CETP of the mammal when administered to the mammal.
As a means for raising an antibody response against, and thereby promoting a prophylactic or therapeutic effect on, a cardiovascular disease such as atherosclerosis. Such non-endogenous CETP can be another mammalian CETP (heterologous CETP), such as rabbit CETP, mouse CETP or monkey CETP when administered to a human. The non-endogenous CETP can be a non-endogenous allelic variant or polymorph of mammalian CETP that is administered to a mammal of the same species (eg, administered to another human). A human CETP polymorph). Alternatively, the non-endogenous CETP can be a CETP from one species that has been modified to have a more similar amino acid sequence to a native CETP of another species (e.g., "humanized" for administration to humans). "Rabbit
CETP).

Described are vaccine compositions and plasmid-based vaccines that, when properly administered to a mammal, result in the production of antibodies reactive with the mammal's endogenous CETP and other benefits described herein. Is done.

The present invention provides a method for eliciting antibodies reactive with its own endogenous CETP in a mammal, a method for modulating mammalian CETP activity, and reducing circulating CETP activity in a mammal.
Lowers total cholesterol, lowers circulating LDL levels, and / or against LDL-C
Methods are provided that provide for an increase in the ratio of HDL-C. The present invention provides methods for reducing or preventing the development of atherosclerotic lesions in a mammal.

DISCLOSURE OF THE INVENTION As noted above, the reduced risk of atherosclerosis has been correlated with relatively low circulating levels of LDL and VLDL and relatively high levels of HDL. The level of such circulating lipoproteins is directly affected, at least in part, by the endogenous levels of CETP activity. In particular, high CETP activity promotes the transport of neutral lipids such as cholesteryl esters from HDL to VLDL and LDL. Thus, CETP is a relatively accurate target in humans and other animals to alter the relative levels of LDL, VLDL and HDL in the circulatory system (see, eg, Tato et al., Arteriosclero. Thromb.
Vascular Biol., 15: 112-120 (1995); Tall, J. Internal Med., 237-5-12 (1
995). The present invention provides a non-endogenous CETP molecule to an individual to promote an immune response to the endogenous CETP in the individual in the individual, thereby correlating the physiological condition (eg, HDL) with a reduced risk of atherosclerosis. (Increased levels or reduced LDL levels) by controlling endogenous CETP activity. Furthermore, promoting an immune response to endogenous CETP using the vaccine composition of the present invention can prevent or suppress the progression of lesions in tissues susceptible to atherosclerosis.

Compositions for Modulating CETP Activity The CETP vaccine compositions used in accordance with the present invention comprise, as an essential component, CETP or a portion thereof that is non-endogenous to the mammal to be vaccinated. For the purposes of the present invention, the term "non-endogenous CETP" means a cholesteryl ester-carrying protein that is not native CETP and is produced by a mammal to be vaccinated. For example, in the case of a human subject, non-endogenous CETP is CETP produced by another mammalian species, such as rabbit, mouse or monkey CETP, i.e., a heterologous CETP.
Including TP. Alternatively, the non-endogenous CETP for a particular human subject can be an allelic variant or polymorph of human CETP, such as CETP produced by another human individual.

[0020] The non-endogenous CETP can also be a heterologous CETP whose amino acid sequence has been modified to be more similar to the amino acid sequence of the endogenous CETP of the mammal to be vaccinated. The terms used herein to describe non-endogenous CETPs so modified are:
"Mammalianized CETP". The term is used in reference to a mammal to be vaccinated, and refers to a non-endogenous CETP that has been modified to have an amino acid sequence more similar to the native CETP of the mammal. If the subject receiving the vaccine is a human, an example of a mammalianized CETP would be a rabbit CETP that has been modified (ie, “humanized”) to have an amino acid sequence more similar to the native human sequence. As a further example, reference is made to FIGS. 1A, 1B and 1C. These figures show the respective amino acid sequences of rabbit CETP (SEQ ID NO: 3) and human CETP (SEQ ID NO: 1) in the form of an alignment.

Referring to FIGS. 1A to 1C, the structures of these two mammalian CETPs are similar,
80% of the positions of human CETP have identical amino acids. Rabbit CETP (SEQ ID NO: 3) is 20 amino acids longer than human CETP (SEQ ID NO: 1), and the alignment of these two proteins shown in FIGS. 1A to 1C shows two structurally inconsistent segments (dashed line (- ---)). For human subjects to be administered non-endogenous CETP according to the present invention, examples of "mammalianized" non-endogenous CETP include rabbits
A segment consisting of 19 amino acids from amino acids Ala393 to Ala411 of CETP is deleted to give a modified CETP having a length of 477 amino acids (see SEQ ID NO: 5), which is more similar to human CETP (SEQ ID NO: 1). Would be a rabbit CETP. As the mammal in this example is a human, another term for such modified CETP or mammalianized non-endogenous CETP would be "humanized rabbit CETP".

Referring again to FIGS. 1A-1C, a further example of a humanized rabbit CETP would be the CETP set forth in SEQ ID NO: 6. FIG. 1C shows that there is only one difference in amino acid sequence in the C-terminal portions of human and rabbit CETP. That is, Lys485 of rabbit CETP
Corresponds to Glu465 of 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 known in the art, two or more administrations are required to obtain a high concentration of anti-endogenous CETP antibody sufficient to affect endogenous CETP activity in a mammal, or Would be desirable.

The immunogenicity of the vaccine peptides of the present invention can be further enhanced by binding the immunogenic non-endogenous CETP to itself or a related protein homologous to CETP. In this approach, dimers can be formed. The dimer is
It provides a multi-chain protein that is more immunogenic than non-endogenous CETP alone. Examples of CETP-related proteins that can be used in this approach include, for example, phospholipid-carrying proteins and neutrophil bactericidal proteins (see, eg, Day et al., J. Biol. Chem., 269: 9388-91 ( 1994); Gray et al., J. Biol. Chem., 264: 9505-9.
509 (1989)).

Other immunogenic carrier molecules such as keyhole limpet hemocyanin (KLH) can also be used in combination with non-endogenous CETP. For example, KLH has a number of lysine residues in its amino acid sequence. Each of these lysine residues binds a CETP molecule described herein (eg, maleimide-activated KLH; Cat. No. 77106, Pierce, Rockford, IL) to provide a multivalent non-endogenous CETP immunogen. Site). Another example of an immunogenic carrier molecule is M. tuberculosis (Mycob
acterium tuberculosis). This molecule has been shown to be a particularly potent antigen containing a number of B and T cell epitopes (see, for example, Suzue and Young, J. Immunol., 156: 873-879 (1996)). The hsp70 protein can be linked to non-endogenous CETP by standard cross-linking agents to enhance the immunogenicity of the vaccine composition.

Other peptides can be conjugated to non-endogenous CETP molecules to provide a source of helper T cell epitopes to increase the immunogenicity of the vaccine composition of the invention. Such peptides are "universal" antigenic peptides, such as tetanus toxoid and diphtheria toxoid, especially tetanus toxoid fragment: Gln Tyr Ile
Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu (amino acids 2 to 1 of SEQ ID NO: 7)
5) Including.

A pharmaceutically acceptable adjuvant, such as alum, can be mixed with a non-endogenous CETP described herein to make a vaccine composition of the invention. Alum is the only adjuvant currently approved for use in administering vaccines to humans (eg, Immunobiology of Proteins and Peptides V: Vacc
ines: Mechanisms, Design, and Applications (Atassi, MZ) (Plenum Pres
s, New York, 1989), p. 192). Alum in combination with a phthalyl sodium derivative of a lipopolysaccharide can also be used (Talwar et al., Proc. Natl. Acad. Sci. USA, 91: 8532-8536 (1994)). Other approved adjuvants can be used if approved for a particular use. For example, biodegradable microspheres composed of poly (DL-lactide-co-glycolide) (Eldridge et al.,
Supra, pp. 191-202); Freund's complete adjuvant (Sigma Chemical Co., St. Lou)
is, Missouri); Freund's incomplete adjuvant (Sigma Chemical Co., St. Louis)
, Missouri); and RIBI ^TM adjuvant system (RAS; RIBI ImmunoChem Research,
Inc., Hamilton, Montana); lipophilic N-palmitoyl-S- [2,3-bis (palmitoyloxy) -propyl)]-cysteine ("Pam3-Cys-OH"); Pam3-Cys-Ser-Lys4 and
Amphiphilic, water-soluble lipopeptides such as Pam3-Cys-Ser-Glu4; N-acetyl-glucosaminyl-N-acetylmuramyl-alanyl-D-isoglutamine ("GMDP"), muramyl dipeptide and alanyl-N- Glycopeptides such as adamantyl-D-glutamine; and polyamide gel-based adjuvants (Synthetic Vaccines (Nicholson), which can be easily attached to CETP peptides during in vitro chemical synthesis).
Blackwell Scientific Publications, Cambridge, Massachusetts, 1994, pp.
236-238).

When physically linking or conjugating a helper T cell epitope molecule or adjuvant species to a non-endogenous CETP, the CETP may be directly or via a crosslinker molecule.
Can be covalently bonded.

Suitable crosslinker molecules include, for example, amino acids using one or more glycine residues that form a “glycine bridge” between CETP and the carrier or adjuvant molecule. Formation of disulfide bonds between cysteine residues, or glutaraldehyde (Korn et al., J.
The use of crosslinker molecules such as Mol. Biol., 65: 525-529 (1972)) and EDC (Pierce, Rockford, IL) or other bifunctional crosslinker molecules is also contemplated. Bifunctional crosslinker molecules have two distinct reactive sites. One of them can be reacted with a functional group on the CETP and the other with a functional group on the carrier or adjuvant molecule, thereby integrating the CETP with the carrier or adjuvant molecule. General methods for cross-linking molecules are discussed by Means and Feeney (Bioconju
gate Chem., 1: 2-12 (1990)).

A homobifunctional crosslinker molecule has two chemically identical reactive sites. Examples of homobifunctional crosslinker molecules are glutaraldehyde; N, N'-bis (3-maleimido-propionyl) -2-hydroxy-1,3-propanediol (sulfhydryl-specific homobifunctional crosslinker) Certain N-succinimide esters such as disuccinimidyl suberate and dithio-bis- (succinimidyl propinate) and their soluble bis-sulfonic acids and salts (eg, Pierce Chemicals, Rockford, Illi);
nois; or available from Sigma Chemical Co., St. Louis, Missouri).

Preferably, the bifunctional crosslinker molecule is a heterobifunctional crosslinker molecule, which comprises at least two non-endogenous CETP and at least two covalently attachable carriers or adjuvant molecules. It has a reactive site. Heterobifunctional crosslinker molecules that can be used include m-maleimidobenzoyl-N-hydroxysuccinimide ester; m-maleimido-benzoylsulfosuccinimide ester; γ-maleimidobutyrate N-hydroxysuccinimide ester; and N-succinimidyl 3- (2-pyridyl-dithio) propionate.

The non-endogenous CETP used in accordance with the present invention can be made by any of the methods known in the art for making proteins having a defined amino acid sequence. For example, one skilled in the art can utilize automated peptide synthesis using an automated peptide synthesizer (eg, Synerg from Applied Biosystems).
y ^TM Peptide Synthesizer; AMS 422 from Abimed, Langenfeld, Germany). Tailored synthesis of such proteins is available from a number of commercial peptide synthesis companies, such as Qu
quality Controlled Biochemicals, Inc. (Hopkinton, Massachusetts); Chiron M
imotopes Peptide Systems (San Diego, California); Bachem Bioscience, Inc
(Philadelphia, Pennsylvania); Seven Biotech Ltd. (Kiddeminster, Englan
It is implemented as a commercial service by d).

Alternatively, the proteins of the present invention can be made using synthetic and recombinant nucleic acid techniques. For example, those skilled in the art can design a nucleic acid sequence from the 5 'end to the 3' end that encodes the protein of the present invention from a known genetic code. Mature human CETP
Is known (see Drayna et al., Nature 327: 632-634 (1987)) and the corresponding DNA sequence (SEQ ID NO: 2). In addition, the amino acid sequence of mature rabbit CETP (SEQ ID NO: 3) and its corresponding DNA sequence (SEQ ID NO: 4) are known (see Nagashima et al., J. Lipid. Res., 29: 1643-1649 (1988)). A DNA molecule comprising the coding sequence for the desired CETP (or modified "mammalianized" CETP as described above) can be obtained from an automated DNA synthesizer (eg, Oligo 1000 DNA Synthesizer,
Beckman Corp.) or by contracting with a commercial DNA synthesis service company (eg, Genset Corp., La Jolla, California).

Next, standard methods available in the art (eg, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Volume 1-3, Cold Spring Harbor Laboratory, Col
d Spring Harbor, NY 1989) and / or specific commercially available gene expression systems (eg, pPROEX ^™ -1 bacterial cell expression system; SFV eukaryotic cell expression system; CHO (Chinese hamster ovary cell) expression System; BAC-TO-BAC ^TM baculovirus expression system
Life Technologies, Inc., Gaithersburg, Maryland) according to the manufacturer's instructions to convert synthetic or cloned DNA molecules into various available gene expression systems (eg, bacterial plasmids; bacteriophage expression vectors, retroviral expression vectors, Baculovirus expression vector).
Particularly preferred for CETP expression is the CHO expression system. The expressed CETP can then be isolated from the expression system using standard methods for purifying proteins.

Purification of the non-endogenous CETP of the present invention can be rapidly performed by employing affinity chromatography or immunoprecipitation based on the use of antibodies to a specific CETP domain. For example, Mab TP2 (Hesler et al., J. Biol. Chem., 263
: 5020-5023 (1988)) binds to the carboxyl-terminal 26 amino acids of human CETP and may be useful in one or more immunoaffinity steps in a purification scheme. Another method that can be used for protein purification is standard column chromatography (Weinberg et al., J. Biol. Chem., 269: 29588-29591 (1994)).

The non-endogenous CETP described herein, when administered to a mammal, specifically binds to the mammal's endogenous CETP and / or modulates the mammal's endogenous CETP activity (ie, , Reduce, or suppress) endogenous antibody production. The anti-CETP vaccine composition of the present invention comprises:
Or several different CETPs.

In addition, non-endogenous CETP can be linked to another molecule that can increase the immunogenicity of the peptide.

[0038] The vaccine composition of the present invention for administering non-endogenous CETP comprises adding non-endogenous CETP to the vaccine composition.
It is also advantageous to take the form of a plasmid-based vaccine for in situ production and for eliciting autoantibodies to endogenous CETP. Such plasmid-based vaccines are specifically DNA plasmids that are administered to an individual (eg, by intramuscular injection or intradermal ballistic administration). The administered DNA plasmid encodes an immunogenic non-endogenous CETP and elicits expression of the CETP. The present inventors have found that such immunogenic CETP elicits the production of autoantibodies that specifically react (ie, bind) to endogenous CETP in an individual.

Examples of recombinant plasmids that can be used to produce non-endogenous CETP for use in accordance with the present invention include the sequence encoding the vaccine peptide described in PCT / US96 / 06147, wherein the CMV promoter was mentioned above. PCMV-CET, a plasmid that directs the transcription of
P / TT. Escherichia coli carrying the plasmid pCMV-CETP / TT is American Type Cultur
Deposited with the e Collection (ATCC, Rockville, MD) and given accession number 98038. The DNA encoding the desired CETP is replaced with pCMV-CE in place of the vaccine peptide coding sequence.
It can be inserted into TP / TT and used for expression of full-length CETP molecules.

The production of anti-CETP antibodies promotes a physiological condition associated with a reduced risk of cardiovascular disease. The beneficial regulation of CETP activity provided by the DNA vaccines is due to significantly reduced or eliminated CETP activity; anti-atherogenic lipoprotein profiles (eg, LDL, LDL-C, VLDL or VLDL-C Increased levels of HDL or HDL-C compared); or by inhibiting (including inhibiting) or decreasing the development of atherosclerotic lesions in cardiovascular tissues such as the aorta.

General methods of administering and testing vaccines are well known to those of skill in the art (eg, Ta
Natl. Acad. Sci. USA 91: 8532-8536 (1994)). Endogenous CET
The immune response to P significantly suppresses CETP activity, alters the serum half-life of CETP, triggers clearance of CETP by forming an immune complex, alters the transport of HDL-cholesterol, Must alter the cholesterol content equilibrium, alter cholesterol catabolism, and / or reduce the incidence of atherosclerotic lesions. Control of LDL, VLDL and / or HDL levels is a generally accepted indicator or endpoint in the treatment of cardiovascular disease.
Because these levels are correlated with a reduced risk of cardiovascular disease or further progression of the disease (eg, Human Biology 4th ed., Pp. 83, 102).
(See Mader in Wm. C. Brown Publishers, Dubuque, Iowa, 1995). Thus, the desired prophylactic or therapeutic effect according to the present invention may be achieved by eliciting antibodies in an individual that bind to endogenous CETP and / or inhibit endogenous CETP activity, or against endogenous CETP. By a relative decrease in LDL and / or VLDL levels relative to HDL levels as the antibody titer increases, or by a reduction in absolute levels of VLDL due to the production of circulating LDL and / or anti-CETP antibodies, or Evidence is provided by the suppression or reduction of the development of atherosclerotic lesions in cardiovascular tissue.

As shown herein, non-endogenous CE using a rabbit model of atherosclerosis
Administration of TP resulted in a significant reduction in the development of atherosclerotic plaque. This evidence indicates that vaccination to elicit antibodies to endogenous CETP can be a useful way to treat or prevent atherosclerosis.

The successful use of non-endogenous CETP to elicit anti-endogenous CETP antibodies and modulate the activity of native CETP has been surprising in several respects. Earlier, full CE
The use of TP molecules had been avoided. This is because it was unknown whether the introduction of a complete non-endogenous CETP molecule would produce the desired immunogenic effect. For example, non-endogenous
CETP functions perfectly as CETP, and may already exacerbate undesirable cholesterol levels and metabolism. In addition, full-length CETP molecules may contain immunogenic segments that will induce antibodies that can react with or interfere with proteins or receptors that are outside the CETP metabolic pathway, The result may be dangerous cross-reactivity or side effects. Finally, the introduction of non-endogenous CETP disrupts tolerance in vaccinated subjects,
It was not known whether it could result in the production of antibodies that did not react with (or not only with the CETP) and react with native CETP. These uncertainties are now clear.

[0044] The non-endogenous CETP vaccine composition can be administered by any route used for vaccine administration. These routes include parenteral and oral routes, such as intraperitoneal, interperitoneal, intradermal, subcutaneous, intramuscular, intravenous, and the like. Preferably, the vaccine of the invention is administered parenterally, for example, intraperitoneally, interperitoneally, subcutaneously, intradermally, intramuscularly or intravenously. If oral administration of the vaccine peptide is desired, any pharmaceutically acceptable oral excipient can be mixed with the vaccine composition of the present invention. For example, solutions approved for use in oral polio vaccines. For other vaccines, such as against tetanus, it may be necessary to occasionally boost the CETP vaccine composition to effect or maintain the desired level of regulation or suppression of endogenous CETP. Biodegradable microspheres such as those composed of poly (DL-lactide-co-glycolide) have been shown to be useful for effective vaccine delivery and immunization by the oral or parenteral route.

Appropriate doses of non-endogenous CETP may be determined by common vaccine methods used in the art, including monitoring of possible contraindications such as hypersensitivity reactions, erythema, sclerosis, tenderness, etc. It can be established based on measurable parameters that the vaccine is proposed to affect (e.g., Physician's Desk Referen
ce 49th edition, Medical Economics Data Production Co., Mont Vale, New Jersey,
1995, pp. 1628, 2731 (referring to hepatitis B vaccine), pp. 1501, 1573,
1575 (referring to measles, mumps, and / or rubella vaccine),
pp. 904, 919, 1247, 1257, 1289, 1293, 2363 (referring to diphtheria, tetanus and / or pertussis vaccine); Talwar, GP et al., Proc. Natl. Acad. Sc.
i. USA 91: 8532-8536 (1994)]. A common and traditional approach to administering a vaccine to humans is to administer an initial dose of a particular vaccine to immunize the immune system and then administer one or more booster doses of the vaccine. And elicit a pre-existing response by the immune system sensitized by the first administration of the vaccine. Such "first and booster" administration methods are well known, e.g., measles, polio,
It is commonly used in the art in the development and use of tetanus, diphtheria and hepatitis B vaccines.

Initially, the amount of vaccine composition administered to an individual may be that amount necessary to neutralize the approximate level of endogenous CETP activity present in the individual prior to vaccine administration. The CETP activity can be determined by measuring the CETP activity in a serum or plasma sample derived from the individual, for example, using a commercially available CETP assay kit (eg, Roar Biomedical., Yonkers, New York).
York). Plasma or serum samples from vaccinated individuals are also monitored and administered following non-endogenous CETP administration using a commercially available CETP assay kit (eg, available from Sigma Diagnostics, Inc., Saint Louis, Missouri). One can determine whether there is a measurable increase in total HDL or HDL-C levels. An increase in the concentration (titer) of circulating anti-CETP antibody can be measured using a plasma or serum sample, for example, by an ELISA assay. Therefore, first of all, an increase in anti-CETP antibodies will result in an increase in HDL or LDL-C levels, LD
It is possible and recommended to determine if it can be correlated with a decrease in L or VLDL, or a decrease in CETP activity. Then, simply monitor the rise in anti-CETP antibodies to determine whether a sufficient dose of the vaccine peptide was administered, or whether the `` booster '' amount indicates an increase in anti-CETP antibody levels. Good. This is a common procedure for various established vaccinations, such as vaccination against hepatitis B virus.

[0047] Three-dimensional angiography is currently available that can be used to identify foci of arteries in an individual and monitor their development or regression (eg, McPher
son, Scientific American Science & Mecidine, pp. 22-31, March / April 1996). Thus, such imaging can be used to monitor the efficacy of vaccine administration according to the present invention. The following non-limiting examples will provide a more complete understanding of the present invention and the advantages thereof.

Example 1 Immunization of Rabbits to Endogenous CETP To test the ability of vaccine formulations to elicite an immune response to endogenous rabbit CETP, 12 New Zealand white rabbits (New Zeala) each were tested.
nd White Rabbit) were prepared for injection into four groups. Group I (negative control) included rabbits # 1- # 12, each rabbit was injected with a vaccine composition containing an irrelevant antigen, human chorionic gonadotropin (hCG). Group II (comparative embodiment of PCT / US96 / 06147) comprises rabbits # 13- # 24, each rabbit with human
A vaccine peptide containing part of the C-terminus of CETP and part of the tetanus toxoid was injected ("peptide"; see SEQ ID NO: 7). Group III (invention) is rabbit # 25- # 36
And each rabbit was administered a vaccine composition containing fully recombinant human CETP ("rhuCETP"). Group IV (the present invention) comprised rabbits # 37- # 48, each of which received a vaccine composition containing fully recombinant human CETP conjugated to complete tetanus toxoid using a chemical crosslinker ( "Conjugate").

The general protocol for administering and testing the vaccine in rabbits was as follows. That is, on the first day, complete Freund's adjuvant (Sigma Chemi
cal Co., St. Louis, Missouri) was injected once subcutaneously with 200 μg of the composition containing the immunogen. Each composition was prepared by suspending the respective immunogen in phosphate-buffered saline (PBS) and emulsifying with complete Freund's adjuvant (1: 1) to a final concentration of 100 μg / 100 μl. did. Each rabbit received a 200 μl dose of the vaccine mixture (200 μg of immunogen) at one subcutaneous site. Incomplete Freund's adjuvant (Sigma
A booster injection consisting of 200 μg of immunogen in Chemical Co.) was administered on days 28 and 49 as in day 1. Blood samples (about 1-5 ml) were collected from each rabbit's ears on days 1 ("pre-bleed"), 28, 49, 105, 147 and 217 (prior to injection). Plasma samples were prepared by standard centrifugation methods for separating cellular components from plasma. Plasma samples were stored at -70 ° C. Group I and II plasma samples were tested for the presence and increased titer of anti-CETP antibodies and for CETP activity,
CETP mass and plasma levels of various lipoprotein components (HDL, LDL, triglycerides) were analyzed.

Plasma titers containing anti-CETP antibodies were titrated using a direct ELISA sandwich enzyme-linked immunosorbent assay (ELISA) for titration of anti-CETP antibodies. The biotinylated C-terminal peptide of rabbit CETP (20 amino acids) is adsorbed to the wells of a microtiter plate coated with streptavidin, and various dilutions of rabbit plasma from rabbits of Groups I-III are added to each well. did. Non-specific binding was 1% solution of BSA in PBS and 0.05
% Tween can be added to each well and blocked by incubating for 2 hours at room temperature (20-22 ° C) on a shaker rotating at 150 rpm. Next, ELIS
The wells were washed four times using A washing buffer (PBS + 0.05% Tween). Next, the plasma samples were diluted with a dilution buffer (eg, 1% BSA in PBS) and then serially diluted with the same buffer. Diluted samples (100 μl) were added to the wells, incubated for 1.5-2 hours at room temperature on a shaker rotating at 150 rpm, and then washed four times with ELISA wash buffer (PBS + 0.05% Tween). To detect bound anti-CETP antibody, horseradish peroxidase (HRP) -labeled sheep anti-rabbit immunoglobulin (Southern Biotechnology A) diluted with dilution buffer.
ssociates, Inc .; Birmingham, Alabama; or Jackson Immunoresearch, Inc .;
100 μl of an optimized dilution of West Grove, Pennsylvania) was added and the plates were incubated for 2 hours at room temperature on a shaker rotating at 150 rpm. Next, ELIS
A Wash wells four times with wash buffer (see above) and add peroxidase substrate TMB (TMB peroxidase substrate, Kirkegaard & Perry Laboratories, Inc.,
Gaithersburg, Maryland) was added and the plates were incubated at room temperature for 30 minutes. The change in optical density is measured by an ELISA reader (eg, E-max, Molecular Device C).
orp., Menlo Park, California) at 450 nm.
In this assay, the OD was directly proportional to the amount of anti-CETP antibody present in the plasma sample.

FIG. 2 shows the assay results for each group of rabbits. Group I is rabbit C-terminal CETP
Although there was no plasma antibody detecting the peptide, most of the vaccinated individuals in the group vaccinated with the CETP immunogen (Groups II and III) showed high titers of anti-CETP antibodies. Analysis of group IV data is ongoing.

CETP Activity and Neutralization Assay To measure CETP activity in plasma, a commercially available fluorescence-based assay kit (Roar
Biomedical Inc .; Yonkers, New York) was used. CETP source (rhuCETP, huCETP
Or rabbit CETP) with the donor and acceptor particles included in the kit, results in CETP-mediated delivery of fluorescent neutral lipids. This fluorescent neutral lipid exists in a self-inactivating state when contained within the donor core. The above CETP-mediated transport is measured by the increase in fluorescence intensity as the fluorescent neutral lipid moves from the self-inactivating donor to the acceptor. To measure neutralization, anti-CETP antibodies are isolated using plasma A from plasma of vaccinated rabbits. Antibodies from different samples are added to the above reactions in equal amounts (measured by A280).

FIG. 3 shows the suppression of CETP activity by rabbit antibodies recovered from the plasma of vaccinated rabbits. The bar labeled "Positive control Mab" represents the anti-CETP monoclonal antibody TP2, which is known to inhibit CETP activity in vitro, and is included here for comparison. FIG. 4 shows the change in CETP activity from week 1 to week 32 in groups I and III. Analysis of group IV data is ongoing.

Cholesterol and HDL Levels in Vaccinated Rabbit Plasma Samples Plasma samples from rabbits in Groups I through IV were also obtained from standard commercial assay kits (Sigma Chemical Co., St. Louis, Missouri). Were assayed for total cholesterol (FIG. 5), HDL-C (FIG. 6) and triglyceride (FIG. 8) concentrations. LDL-C (Figure 7) is calculated as: Total cholesterol-HDL-C-0.2 x triglyceride levels. CETP levels were measured by slot blot analysis using the anti-CETP monoclonal antibody TB2 and chemiluminescence for detection. Band intensities obtained using various amounts of plasma samples were analyzed using Kodak ^™ DC40 camera and 1D Image Analysis.
Software (version 1.6) was quantified and then compared to band intensities obtained using known amounts of purified human CETP loaded on the same nitrocellulose filter.

FIG. 8 shows the plasma lipoprotein profile of Group III rabbit # 32, which showed the most significant reduction in LDL-C levels (FIG. 7). This profile shows a dramatic increase in HDL as a percentage of the total lipoprotein profile. FIG. 9 shows the correlation between reduced CETP activity, reduced cholesterol mass, and increased HDL as a percentage (%) of total lipoprotein for this rabbit.

Measurement of Cholesterol Deposits in the Iris of Vaccinated Rabbits The rabbits of Groups I through IV were also assayed for the amount of cholesterol deposits detected in the iris. The cholesterol deposition grade in the iris was determined as follows:
: 0 = no deposit, 1 = 20%, 2 = 40%, 3 = 60%, 4 = 80% and 5 = 100% deposited on the iris (ie the iris is completely covered by deposits) . One iris per rabbit was evaluated and the degree of cholesterol deposition was scored.
To avoid prejudice, the scorer was asked which group of rabbits to hide. FIG. 10 shows data collected from a total of 48 animals. Rabbits for which no data was obtained (# 3)
4, # 39 and # 42). These rabbits were euthanized due to unrelated complications, such as a furball in the stomach. These data indicate that cholesterol deposition was statistically lower in all groups vaccinated with CETP than in the control group.

Quantification of lesions in the aorta of vaccinated rabbits. Rabbit diet from the basic rabbit chow is known to produce rabbits with lesions similar to atherosclerosis, 0.25% cholesterol. (
w / w) (Daley et al., Arterioscler. Thromb., 14: 95-104 (
1994)). To determine whether vaccination affected the development of atherosclerosis, the aortas of these rabbits were examined for the development of atherosclerotic lesions. After a blood sample was collected on the last day, the rabbits were sacrificed. All aortas from each of Groups I through IV were removed and placed in fixative (3.7% v / v formaldehyde). Loose tissue, attached fat, and adventitia were dissected away from the aorta. Each artery was then cut longitudinally, pinned flat to expose the intimal surface, stained with Sudan IV, and then photographed. Sudan IV is a fat-soluble red dye that stains atherosclerotic plaque on the intimal surface of arteries. FIG. 11 summarizes the results of this experiment. The stained aorta of rabbits vaccinated with human chorionic gonadotropin ("HCG") showed the spread of atherosclerotic lesions throughout the length of the aorta, especially in the aortic segment from the thoracic region. In contrast, a synthetic vaccine peptide having a segment of the C-terminal sequence of tetanus toxoid and human CETP ("peptide", SEQ ID NO: 7)
Rabbit) vaccinated with full-length recombinant human CETP ("rhuCETP") and CETP-tetanus toxoid conjugate composition ("conjugate"), including the aortic portion from the thoracic region, The incidence of lesions was lower.

To quantify the significant difference in the presence or absence of atherosclerotic lesions in the rabbit aorta, the total pinned aortic and aortic lesion surface areas were measured using a digitized tablet with software (THET MORPHOMETER ^TM , Woods
Determined from photographs by two-dimensional morphometry (Daley et al., 1994) using Hole Educational Associated, Woods Hole, Massachusets. The percentage of the surface area of the aorta covered by the lesion was determined. The percentage is shown in FIG.

Examples 2 and 3 Plasmid-Based Vaccines in Mice and Rabbits Four groups of mice were vaccinated intramuscularly using one of the following: pCMV : A plasmid vector that has the cytomegalovirus immediate early promoter / enhancer, but has no operably linked structural gene. 2. pCIII-huCETP : Human CETP under the transcriptional control of the human Apo CIII promoter
A plasmid vector having the entire coding sequence (SEQ ID NO: 1). 3. pSV40-huCETP : A plasmid vector having the entire coding sequence of human CETP under the transcriptional control of the SV40 promoter. 4. pCMV-TT-rabCEPT: a plasmid vector having the coding sequence of the tetanus toxoid peptide (amino acids 2 to 15 of SEQ ID NO: 7) under the transcriptional control of the cytomegalovirus immediate early promoter / enhancer and the entire coding sequence of rabbit CETP.

Mice were injected once with 25 μl of PBS containing 100 μg of the above plasmid, blood samples were collected periodically and analyzed using ELISA to detect anti-human CETP antibody by the following method. .

A solution containing 5 μg of protein A / G per 1 ml of PBS was added to 100 μl per well.
A 96 well plastic microtiter plate was coated with protein A / G by incubating at 4 ° C. Empty plate, 200 μl
The wells were blocked for 2-8 hours at room temperature using a blocking buffer (PBS containing 4% BSA, 1% sucrose, 0.5% NP-40, 0.01% gentamicin). Antibodies from plasma samples were captured on protein A / G by incubating various dilutions (100 μl) of the sample for 1 hour at room temperature. After washing the wells, 100
Biotinylated CETP was captured by the plate-bound antibody by incubating 1 μl of the biotinylated CETP solution at room temperature for 1 hour. Incubate 100 μl of streptavidin-HRP solution at room temperature for 30 minutes, then add 100 μl of substrate, stop the reaction with 0.18 M sulfuric acid, and determine bound CETP by reading the optical density at 450 nm. Detected. FIG. 12 shows that the plasmids introduced in groups 2, 3 and 4 produce immunogenic heterologous proteins.

Next, a vector (pSV40-huCETP) carrying the human CETP coding sequence under the transcriptional control of the SV40 promoter enhancer or 300 μg of the same vector (SV40) containing no CETP coding sequence (6 quadriceps muscle) Rabbits were vaccinated using the same intramuscular site. The first injection was performed on day 1 and the same booster injections were performed at weeks 5, 8, 26 and 30. Blood samples were collected periodically throughout the experiment and rabbits were sacrificed at week 34.

Plasma samples from vaccinated rabbits were subjected to ELISA as described above for detection of antibodies to fully recombinant human CETP. Figures 13A-K summarize antibody titrations measured from weeks 1 to 34 in rabbits vaccinated with the SV40 promoter enhancer (pSV40-huCETP). Significant antibody production was detected in most rabbits at weeks 30 and 34 (shown by open diamonds and open squares in the graph). Two of the 11 rabbits (FIGS. 13A and 13E) were non-responders.

The rabbit diet was changed from basic rabbit diet to a diet supplemented with 0.25% cholesterol (w / w), which is known to cause rabbits to develop lesions similar to atherosclerosis. Lesions in rabbits vaccinated with pSV40-huCETP and control rabbits vaccinated with SV40 were visualized and quantified as described in Example 1. FIG. 14 shows the average percentage of lesion covered aorta in both groups. The results of this experiment show that the directly vaccinated rabbits (groups I to IV, FIG. 11)
), Indicating a reduction in aortic lesions following vaccination.

While a number of embodiments have been described, those skilled in the art will recognize that modifications and variations of the described compositions and methods can be made without departing from the spirit of the invention or the scope of the appended claims. Will be. The articles and publications cited herein are hereby incorporated by reference.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a diagram (A-C) showing the alignment of the amino acid sequences of mature rabbit CETP (SEQ ID NO: 3) and mature human CETP (SEQ ID NO: 1). Rabbit on aligned human CETP sequence
Indicates CETP. This rabbit sequence contains 20 more amino acids than the human sequence. Therefore, the human sequence is shown with a gap of 1 amino acid and 19 amino acids (indicated by a dashed line in the human sequence) to most clearly show the matching residues (indicated by vertical bar |).

FIG. 2. Human chorionic gonadotropin (“HCG vaccine”), a synthetic vaccine peptide having a segment of the C-terminal sequence of tetanus toxoid and human CETP (“peptide vaccine”, see SEQ ID NO: 7), and full length recombinant human Recognizes the C-terminal peptide of rabbit CETP from rabbits vaccinated with CETP (“rhuCETP”)
FIG. 3 shows the end-point titers of antibodies derived from rabbit plasma. This figure shows the maximum anti-CETP antibody titer achieved for each rabbit in an ELISA that detects plasma antibodies specific for the C-terminal peptide of rabbit CETP (amino acids 477-496).

FIG. 3: Fluorescence-based assay using a commercially available kit (Roar Biomedical, Yonkers, New York)
FIG. 7 shows suppression of CETP activity by antibodies isolated by plasma A from plasma of rabbits vaccinated in Groups I-III (see Examples) in York).
The graph shows the percent inhibition achieved in each vaccinated rabbit.

FIG. 4 is a view showing CETP activity in a rabbit to which a vaccine was administered from the first week to the 32nd week.

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

FIG. 6 shows LDL-related cholesterol levels from week 1 to week 32 in vaccinated rabbits.

FIG. 7 shows the percentage change in LDL-related cholesterol levels from week 1 to week 12 in vaccinated rabbits.

FIG. 8 shows plasma lipoprotein levels in rabbits. "V" indicates intermittent booster doses. HDL-related cholesterol, total cholesterol levels, and triglyceride levels were assayed.

FIG. 9. CETP activity in rabbits vaccinated with non-endogenous CETP (rhuCETP),
FIG. 3 shows the correlation between HDL as a percentage of total lipoproteins and total CETP mass.

FIG. 10: Synthetic vaccine peptide having a segment of the C-terminal sequence of human chorionic gonadotropin (“HCG”, rabbit # 1- # 12), tetanus toxoid and human CETP (“peptide”, see SEQ ID NO: 7, rabbit # 13- # 24), full-length recombinant human CETP ("rhuCETP", rabbit # 25- # 36), and CETP-tetanus toxoid conjugate composition ("conjugate", rabbit # 37- # 48). FIG. 2 shows the levels of cholesterol deposits in the irises of 48 vaccinated rabbits. The iris of each rabbit was scored from 0 to 5 based on the percentage of deposits observed. 0 indicates no deposits and 5 indicates that the rabbit iris is covered with 100% deposits.

FIG. 11 shows the percentage of lesion-covered aorta observed in vaccinated rabbits fed an atherogenic diet. The values of individual rabbits are represented by open marks.

FIG. 12 shows anti-CETP antibody titers of mice vaccinated with various plasmid-based vaccines.

FIG. 13 shows antibody titers (A to K) of 11 rabbits vaccinated with pSV40-huCETP during the first to 32 weeks. Open diamonds and open squares represent antibody titers in rabbits at weeks 30 and 34, respectively.

FIG. 14 shows the effect of administration of a plasmid vaccine on the development of aortic lesions. This figure shows control rabbits (pSV40 plasmid without CETP coding sequence) and pSV40
-compares the average percentage of rabbits vaccinated with huCETP.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/47 C07K 14/47 C12N 15/09 ZNA C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ , DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT , UA, UG, US, UZ, VN, YU, ZW (72) Inventor Thomas, Lawrence, J. United States 02375 Fox Ridge Road, Easton, MA 02375 1F Term (Reference) 4B024 AA01 BA31 CA04 DA02 EA04 FA02 FA06 GA11 HA15 4C084 AA13 NA14 ZA452 ZB092 ZC332 4C085 AA06 BB11 CC03 CC04 CC21 EE01 EE06 FF01 FF03 4H045 AA11 AA30 CA40 DA86 EA31 FA74

Claims (37)

[Claims] 1. A vaccine composition effective in promoting the production of an antibody that binds to endogenous cholesteryl ester transport protein (CETP) in a mammal, comprising:
Such a composition, consisting essentially of non-endogenous CETP, optionally in combination with an adjuvant effective to non-specifically stimulate the immune response of the mammal. 2. The mammal is a human, and the non-endogenous CETP is rabbit CETP.
The vaccine composition of claim 1, wherein the vaccine composition is selected from the group consisting of: mouse CETP, monkey CETP, humanized rabbit, mouse or monkey CETP, and an allelic variant or polymorph of CETP of the human. 3. A vaccine composition effective in promoting the production of an antibody that binds to endogenous cholesteryl ester transport protein (CETP) in a mammal, comprising:
Such a composition, consisting essentially of mammalianized non-endogenous CETP, optionally in combination with an adjuvant effective to non-specifically stimulate the mammal's immune response. 4. The vaccine composition according to claim 3, wherein said mammalianized CETP is humanized rabbit CETP. 5. The vaccine composition according to claim 4, wherein said humanized rabbit CETP has the amino acid sequence shown in SEQ ID NO: 5. 6. The mammal is a rabbit, and the non-endogenous CETP is human CETP.
The vaccine composition according to claim 1, which is: 7. The vaccine composition according to claim 1, comprising an adjuvant selected from the group consisting of alum, complete Freund's adjuvant, incomplete Freund's adjuvant, and a RIBI adjuvant system. 8. A vaccine based on a plasmid, comprising a promoter sequence suitable for directing transcription of a nucleotide sequence in mammalian cells,
The vaccine, wherein the promoter sequence is operably linked to a nucleotide sequence encoding a cholesteryl ester transfer protein (CETP) that is non-endogenous to the mammal. 9. The method according to claim 9, wherein the mammal is a human and the non-endogenous CETP is rabbit CETP.
9. The plasmid according to claim 8, which is selected from the group consisting of, mouse CETP, monkey CETP, humanized rabbit, mouse or monkey CETP, and an allelic variant or polymorphism of said human CETP. vaccine. 10. The mammal is a human, and the non-endogenous CETP is a rabbit CETP.
The vaccine comprising the plasmid of claim 8 as a main component, which is TP. 11. The plasmid-based vaccine of claim 8, wherein said mammal is human and said non-endogenous CETP is humanized rabbit CETP. 12. The plasmid-based vaccine according to claim 11, wherein the non-endogenous CETP has the amino acid sequence shown in SEQ ID NO: 5. 13. The method of claim 13, wherein said mammal is a rabbit and said non-endogenous CETP is human CE.
The vaccine comprising the plasmid of claim 8 as a main component, which is TP. 14. The plasmid-based vaccine according to claim 8, wherein said promoter is a cytomegalovirus immediate early promoter / enhancer. 15. A method for promoting the production of an antibody in a mammal that binds to the endogenous CETP of the mammal, the method comprising optionally adding non-endogenous CETP or mammalianized non-endogenous CETP to the mammal. Such a method, comprising administering in combination with an adjuvant effective to nonspecifically stimulate an immune response in the animal. 16. A method for increasing the ratio of circulating low-density lipoprotein-related cholesterol to total cholesterol or circulating low-density lipoprotein-related cholesterol in a mammal, wherein the mammal is non-endogenous CETP or non-mammalian non-endogenous. Said method comprising administering sex CETP, optionally in combination with an adjuvant effective to non-specifically stimulate said mammalian immune response. 17. A method for reducing the level of endogenous CETP activity in a human or other animal, wherein the mammal comprises non-endogenous CETP or mammalianized non-endogenous CETP.
Such a method, comprising administering a TP, optionally in combination with an adjuvant effective to non-specifically stimulate an immune response in said mammal. 18. A method for altering the metabolism of low-density lipoprotein-associated cholesterol in order to reduce the incidence of atherosclerotic lesions in a mammal, wherein said mammal comprises non-endogenous CETP or mammalianized non-endogenous CETP. Optionally in combination with an adjuvant effective to non-specifically stimulate the immune response of the mammal. 19. A method for reducing the level of circulating low-density lipoprotein in a mammal, wherein said mammal comprises non-endogenous CETP or mammalianized non-endogenous CE.
Such a method, comprising administering a TP, optionally in combination with an adjuvant effective to non-specifically stimulate the immune response of said mammal. 20. A method of reducing the level of total circulating cholesterol in a mammal, comprising providing the mammal with a non-endogenous CETP or a mammalianized non-endogenous CETP, wherein the immune response of the mammal is non-specifically. Administering the composition in combination with an adjuvant effective for stimulating the disease. 21. A method of treating atherosclerosis therapeutically or prophylactically in a mammal, comprising administering to the mammal non-endogenous CETP or mammalianized non-endogenous CETP.
Such a method, comprising administering CETP, optionally in combination with an adjuvant effective to non-specifically stimulate the immune response of said mammal. 22. The mammal, wherein the mammal is a human, and the non-endogenous CETP is a rabbit CETP.
22. The method according to any of claims 15 to 21, which is a TP. 23. The mammalianized CETP is a humanized rabbit CETP.
22. The method according to any of 21. 24. The method of claim 23, wherein said humanized rabbit CETP has the amino acid sequence shown in SEQ ID NO: 5. 25. The method according to any of claims 15 to 21, wherein said CETP is administered in combination with an adjuvant selected from the group consisting of alum, complete Freund's adjuvant, incomplete Freund's adjuvant, and a RIBI adjuvant system. . 26. A method for promoting the production of an antibody reactive with endogenous CETP of a mammal in a mammal, comprising the steps of: Such a method, comprising transfecting at least one cell type. 27. A method for increasing the ratio of circulating low-density lipoprotein-related cholesterol or total cholesterol-related high-density lipoprotein-related cholesterol in a mammal, comprising using the plasmid-based vaccine according to claim 8 as a component. Said method comprising transfecting at least one cell of said mammal. 28. A method for reducing the level of endogenous CETP activity in a human or other animal, comprising using the plasmid-based vaccine of claim 8 to at least one cell of said mammal. Said method comprising transfecting 29. A method for altering the metabolism of low-density lipoprotein-associated cholesterol in order to reduce the occurrence of atherosclerotic lesions in a mammal, comprising using the plasmid-based vaccine according to claim 8 The above method, comprising transfecting at least one cell of the mammal. 30. A method for reducing the level of circulating low-density lipoprotein in a mammal, comprising transfecting at least one cell of the mammal with the plasmid-based vaccine according to claim 8. The above method. 31. A method for reducing the level of total circulating cholesterol in a mammal, comprising transfecting at least one cell of the mammal with the plasmid-based vaccine of claim 8. The above method, comprising: 32. A method for therapeutically or prophylactically treating atherosclerosis in a mammal, comprising using the plasmid-based vaccine according to claim 8 to at least one cell of the mammal. Said method comprising transfecting 33. The method of any one of claims 26 to 32, wherein said mammal is a human and said plasmid-based vaccine comprises a nucleic acid encoding rabbit CETP. 34. The method of any one of claims 26 to 32, wherein said mammal is human and said plasmid-based vaccine comprises a nucleic acid encoding humanized rabbit CETP. 35. The method of claim 34, wherein said humanized rabbit CETP has the amino acid sequence set forth in SEQ ID NO: 5. 36. The method of any of claims 26 to 32, further comprising administering to the mammal an adjuvant effective to non-specifically stimulate the immune response of the mammal. 37. The method of claim 36, wherein said adjuvant is selected from the group consisting of alum, complete Freund's adjuvant, incomplete Freund's adjuvant, and a RIBI adjuvant system.