KR20030048138A - Apolipoprotein analogues - Google Patents

Abstract

The present invention provides an apolipoprotein structure, an apolipoprotein structure for use as a medicament, a nucleic acid sequence encoding the apolipoprotein structure, a vector comprising the nucleic acid sequence, a method for producing an apolipoprotein structure, and a pharmaceutical composition It relates to the use of the apolipoprotein structure for. The data presented show that the constructs according to the invention can bind to lipids, bind to a strong Apo AI receptor, cublin, are more potent than native Apo AI, and that the constructs of the present invention have a plasma half-life compared to native Apo AI. Document that it has been extended at least three times. Together these data demonstrate that the constructs according to the invention are potent candidates useful for the treatment of cardiovascular disease.

Description

Apolipoprotein analogs {APOLIPOPROTEIN ANALOGUES}

In the following, the term Apo A or Apolipoprotein A refers to Apolipoprotein, Apolipoprotein A I, Apolipoprotein A II, or Apolipoprotein A IV.

Cardiovascular diseases caused by atherosclerosis in blood vessels are the most common cause of death in industrialized countries worldwide. One of the causes of atherosclerosis is the deposition of cholesterol in the walls of blood vessels, which leads to plaque formation, which causes atherosclerosis to increase the risk of seizures.

Apolipoprotein A-1 (apo-A-1) is a major component of plasma high density lipoprotein (HDL), which is inversely proportional to the development of atherosclerosis. There is strong experimental evidence that this effect is caused by the so-called reverse cholesterol transport from peripheral tissues to the liver. There is also experimental evidence that this cholesterol reverse transfer could be promoted by apo-A-1 in mammals.

Apolipoprotein A-1 is rapidly removed from plasma. It is believed that Apo-A-1 is mostly removed from plasma by filtration in the kidney without degradation (Braschi et al., 1999, J Lipid Res, 40: 522-532; Braschi et al., 2000, Biochemistry, 39: 5441-5449; Glass et al., 1983, J Biol Chem 258: 7161-7167). The short half-life of Apolipoprotein A in plasma is limiting the availability of this protein in the treatment of atherosclerosis.

US 5,876,968 (SIRTORI et al.) Relates to a substantially pure dimer of the apo-A-1 variant called apolipoprotein A-1-Milano. Medicines containing this dimer can be used to prevent thrombosis or they can be used as prodrugs of monomers. One unique feature of this particular variant of apo-A-1 is that it can form a covalent dimer itself. The inventors of this patent assume that the plasma half-life seems to be prolonged due to the presence of Apo-A-I-M, but no definitive data have been presented.

US 5,643,757 (SHA-IL et al.) Discloses a high yield of pure, stable, mature and biologically active human apolipoprotein A-I.

US 5,990,081 (AGELAND et al.) Describes a method for treating atherosclerosis or cardiovascular disease by administering a therapeutically effective amount of Apolipoprotein A or Apolipoprotein E.

WO 96/37608 (RHONE-POULENC ROHRER et al.) Describes human homologous dimers of apolipoprotein A-I variants with cysteine at position 151. The presence of cysteine residues in the amino acid sequence allows dimers to be formed via disulfide bonds between monomers. This document further describes corresponding nucleic acid sequences and vectors comprising them, as well as pharmaceutical compositions comprising such variants and their use in gene therapy.

WO 90/12879 (Sirtori et al.) And WO 94/13819 (Kabi Pharmacia) disclose methods for preparing ApoA-1 and ApoA-IM in yeast and E. coli, respectively. These documents also describe the use of ApoA-I and ApoA-IM as a medicament for the treatment of atherosclerosis and cardiovascular disease.

In conclusion, the prior art focuses primarily on the use of these proteins as medicaments for the treatment of vascular diseases, despite the known disadvantages (mainly rapidly eliminated) of natural ApoA-I or ApoA-IM monomers or ApoA-IM dimers. have. There is no technique in the prior art to modify ApoA-I to obtain a structure that has improved ability to perform cholesterol reverse transfer and / or extended plasma half-life. It is therefore an object of the present invention to provide ApoA constructs that can be used to treat and / or prevent cardiovascular diseases.

Summary of the Invention

A first aspect of the invention relates to apolipoprotein structures having the following general formula and which can be used as medicaments:

apo A-X

Wherein apo A is an apolipoprotein A component selected from apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, analogs or variants thereof,

X is a heterologous moiety comprising one or more compounds selected from amino acid, peptide, protein, carbohydrate and nucleic acid sequences,

Provided that the constructs consist of exactly two identical and naturally occurring apolipoproteins, they are linked in series.

According to the present invention, novel structures are provided that can be used as medicaments. The prior art does not describe apolipoprotein structures as defined in the present invention for medical use. Apolipoprotein constructs according to the invention are broadly considered HDL analogs because of their ability to form complexes with cholesterol and other lipids and help deliver these compounds to the liver.

Apolipoprotein components or portions of these constructs are referred to as apo A or apolipoproteins throughout the present invention. In the following detailed description and claims, the heterologous moiety will be referred to as component X of this structure. Apolipoproteins or analogs or variants thereof are covalently linked to heterologous moieties.

Component X of this structure may be widely regarded as a heterogeneous part. Heterologous parts in the text refer to all parts of any kind that are not linked to apolipoproteins or their analogs or variants or functional equivalents under natural conditions. The heterologous moiety can therefore be a peptide or protein from a homologous or other species or a part of such a peptide or protein, even a single amino acid. It may also be a synthetic peptide. It may also be a type of carbohydrate or other polyol, nucleic acid sequence with other polymeric and biocompatible properties.

Functional equivalence with native apolipoproteins A-I, A-II, and A-IV can be readily determined using lipid binding assays. The ability of the construct to elicit substantially the same physiological response in mammals may be readily determined by measuring the ability to perform cholesterol reverse transfer in rodents such as mice or test animals such as rabbits.

Such constructs, including apolipoproteins and heterologous moieties, can perform cholesterol back-transfer equivalents, even better, than natural apolipoproteins, despite the modifications caused by the addition of the heterologous moieties. The plasma half-life of this construct is preferably increased compared to that of wild type apolipoprotein. The prolonged half-life is due to the increased size of the apolipoprotein structure that may reduce the filtration rate through the kidney, which may be due to increased binding to HDL or reduced structure of this structure compared to native Apo A. It may be due to decomposition.

Preferably the plasma half-life is at least 2 or 3 times, or at least 4 or at least 10 times. Likewise, the binding affinity of constructs such as lipid binding affinity and / or cholesterol binding affinity is also preferably increased compared to wild type apolipoproteins. Preferably, the lipid binding affinity is at least 5%, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least Preferably 75%, such as at least 100%, such as at least 150%, such as at least 200%, such as at least 300%. Even in cases where the lipid binding affinity of the constructs according to the invention is equal to or lower than the lipid binding affinity of native apolipoproteins, the constructs according to the invention may have better clinical effects due to their increased plasma half-life.

Increased plasma half-life and / or increased lipid binding affinity suggest a deep linkage that can utilize apolipoprotein structures in the treatment of atherosclerosis. Therefore, the clinical effect of the apolipoprotein construct according to the present invention is superior to that of the wild type apolipoprotein.

The invention also includes analogs or variants of wild-type apolipoproteins that can elicit substantially identical physiological responses in oil.

According to a second aspect of the invention, an apolipoprotein structure is provided having the general formula

apo A-X

Wherein apo A is an apolipoprotein component selected from apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, analogs or variants thereof,

X is a heterologous moiety selected from an oligomerization module and apolipoproteins linked at the ends.

According to another aspect, a nucleotide sequence is provided that encodes an apolipoprotein structure as defined above. Preferably the nucleotide sequence is operably linked to regulatory sequences for expression of the protein construct.

According to another aspect of the invention, a vector comprising a nucleotide sequence encoding an apolipoprotein structure as defined above and a transformed host cell comprising a nucleotide sequence are also provided.

Apolipoprotein structures according to the present invention can be prepared by various methods.

In a first method, transformed host cells are cultured and the protein constructs are recovered and optionally further processed, under conditions that promote expression of the protein constructs according to the invention encoded by the DNA inserted into the constructs.

This method is preferred when the whole structure is derived from a polypeptide and can therefore be encoded by one corresponding nucleic acid sequence.

In a second method, the apolipoprotein structure can be prepared by chemically synthesizing the heterologous moiety and then linking it to an apolipoprotein or analog to obtain the apolipoprotein structure, which is then separated and optionally further processed. This method is preferred when the heterologous moiety is from a non-peptide. However, even when the heterologous moiety is derived from a polypeptide, there may be conditions where it is desirable to chemically synthesize the heterologous moiety. This condition is where the heterologous moiety is rather short, such as less than 20 amino acids.

In a third method, transformed host cells are cultured under conditions that promote expression of apolipoprotein or apolipoprotein analogs encoded by nucleic acid fragments, and then the apolipoprotein or apolipoprotein analogs are shared covalently with the heterologous moiety. The apolipoprotein structure can be obtained by linking to the apolipoprotein structure, and the obtained apolipoprotein structure is separated, and then optionally further processed.

Finally, under conditions that promote expression of the protein encoded by the nucleic acid fragment encoding the oligomerising module, the transformed host cell is cultured, and then the module is linked to at least one apolipoprotein. Apolipoprotein structures can be prepared by obtaining apolipoprotein structures.

According to another aspect of the present invention there is provided a pharmaceutical composition comprising the apolipoprotein structure as described above. Preferably, the pharmaceutical composition may be administered parenterally, such as by injection.

The invention also encompasses the use for the preparation of pharmaceutical compositions of the apolipoprotein constructs as defined above. The pharmaceutical compositions may further contain pharmaceutically acceptable excipients, adjuvants, additives such as lipids, phospholipids, cholesterol or triglycerides.

The pharmaceutical composition of the present invention can be used intravenously, intraarterially, intramusculary, transdermally, pulmonary, subcutaneously, intratradermally, intactecally It may be administered orally or orally or through a cheek, anus, vagina, conjunctiva, or intranasal tissue, or into a tissue such as tumor tissue, or by implantation.

Apolipoprotein constructs as defined herein can also be used in gene therapy, where the DNA sequence encoding the apolipoprotein construct is used to transfect or infect at least one cell population.

The present invention relates to apolipoprotein constructs, the use of apolipoprotein constructs as a medicament, a nucleic acid sequence encoding an apolipoprotein construct, a vector comprising a nucleic acid sequence, a method for producing an apolipoprotein construct, an apolipoprotein construct And pharmaceutical compositions containing apolipoprotein structures for the preparation of pharmaceutical compositions.

Figure 1 shows the amino acid sequence (one letter code) of human apolipoprotein A-I.

2A shows CLUSTAL W (1.74) multiple sequence alignment of apolipoprotein A-I using BLOSUM. The following sequences are arranged in this figure:

Human sp│P02647│APA1_HUMAN Apolipoprotein A-I precursor (Apo-AI)-Homo sapiens (Human)

Sp│P15568│APA1_MACFA Apolipoprotein A-l precursor (Apo-AI) of Macaque-Macaca fascicularis (Crab eating macaque)

Bovine sp│P15497│APA1_BOVIN Apolipoprotein A-I precursor (Apo-AI) -Bostaurus (bovine).

Sp│P18648│APA1_PIG Apolipoprotein A-I precursor of pig (Apo-AI)-Sus scrofa (pig).

Sp│P02648│APA1_CANFA apolipoprotein A-l precursor (Apo-AI)-Canis familiars (dog).

Sp│P09809│APA1_RABIT Apolipoprotein A-I precursor (Apo-AI) -Oryctolagus cuniculus (rabbit) in rabbits.

Sp│O18759│APA1_TUPGB Apolipoprotein A-I precursor (Apo-AI) Tupaia glis belangeri (Common Wood Mole).

Sp│Q00623│APA1_MOUSE Apolipoprotein A-I precursor (Apo-AI) -Mus musculus (mouse) in mice.

SpSEQ ID NOPo4639SEQ ID NOAPA1_RAT Apolipoprotein A-I precursor (Apo-AI) -Rattus norvegicus (rat).

Tr│Q9TS49 apolipoprotein A-l, APOA-I = cholesterol transporter-Erinaceus europaeus (Western European hedgehog)

Sp│P08250│APA1_CHICK Apolipoprotein A-I precursor (Apo-AI) -Gallus gallus (chicken) of chickens.

Sp│P32918│APA1_COTJA Apolipoprotein A-I precursor (Apo-AI) -Coturnix coturnix japonica (Japanese quail) of Japanese quail.

Duck's sp│O42296│APA1_ANAPL Apolipoprotein A-I precursor (Apo-AI)-

Anas platyrhynchos (Duck duck).

Sp│O57523│AP11_ONCMY Apolipoprotein A-I-1 precursor of rainbow trout (APOA-1-1)-Oncorhynchus mykiss (rainbow trout) (Salmo gairdneri).

Sp│Q91488│APA1_SALTR Apo lipoprotein A-I precursor (Apo-AI) -Salmo trutta (Brown trout) of brown trout.

Sp│P27007│APA1_SALSA Apolipoprotein A-I precursor (Apo-AI) -Salmo salar (Atlantic salmon) of Atlantic salmon.

Sp│42363│APA1_BRARE Apolipoprotein A-I precursor of Zebrafish (Apo-AI) —Brachydanio rerio (zebrafish) (zebradani).

Sp | 042175│APAl-SPAAU Apolipoprotein A-1 precursor (Spo AI) -Sparus aurata (Sil bream).

FIG. 2B aligns the amino acid sequence (single character code) of apolipoprotein A-IV of human, macaque, mouse, baboon, pig and rat.

3 is the amino acid sequence of the amino terminal band of tetranectin (SEQ ID NO 12). Tetranectin amino acid sequence from E1 to L51 (single character code). Exon 1 contains residues from E1 to D16 and exon 2 comprises residues from V17 to V49, respectively. Alpha helix extends beyond L51 through K52, the C-terminal amino acid residues in alpha helix.

4 shows the alignment of amino acid sequences of trimerized structural elements of the tetranectin protein family. Amino acid sequence corresponding to V17 to K52 comprising the first three residues of exon 2 and exon 3 of human tetranectin (single-letter code) (Sorensen et al., Gene, 152: 243-245, 1995); Tetranectin homologous protein isolated from reef shark cartilage (Neame and Boynton, 1992, 1996); And tetranectin homologous proteins isolated from bovine cartilage (Neame and Boynton, database accession number PATCHX: u22298). The residues at positions a and d in the seven-suite repeats (referred to as heptad repeats) are indicated in bold letters. The listed consensus sequence of the tetranectin protein family that trimers structural elements, in addition to the other conserved residues in the band, includes residues located at positions a and d of the heptad repeats shown in the figure. "hy" refers to an aliphatic hydrophobic moiety.

Figure 5 shows the pT7 H6UbiFx Apo A-I plasmid and its corresponding amino acid sequence. This expressed and processed polypeptide consists of amino acids no 25-267 (SEQ ID NO 1) of human Apo A-I and gly-gly linked to the N-terminus thereof.

FIG. 6 shows the pT7 H6UbiFx Cys-Apo A-I plasmid and its corresponding amino acid sequence. FIG. This expressed and processed polypeptide consists of an N-terminal cysteine residue and amino acid no 25-267 from human Apo A-I and a gly-gly linked to the N-terminus thereof.

Figure 7 depicts the pT7H6 Trip-A-Apo AI-Amp ^R plasmid and its corresponding amino acid sequence. This expressed and processed polypeptide (SEQ ID NO 3) consists of TTSE, the linking sequence, and amino acids no 25-267 from human Apo AI.

Figure 8 depicts the pT7H6 Trip-A-Apo AI-del 43-Amp ^R plasmid and its corresponding amino acid sequence. This expressed and processed polypeptide (SEQ ID NO 4) consists of TTSE, the linking sequence, and amino acids no 68-267 from human Apo AI.

9 shows the pT7H6FXCysApoAI plasmid and its corresponding amino acid sequence. This expressed and processed polypeptide consists of N-terminal cysteine residues and amino acids no 25-267 from human Apo A-I (SEQ ID NO 2) and gly-gly linked thereto.

10A-10G illustrate amino acid sequences of plasmids and corresponding apolipoprotein structures according to the present invention.

Figure 10A: pT7H6-Trip-A-Apo AI K9A K15A: Corresponds to pTH6-Trip-A-Apo Ai, but two lysine residues in the trimerization zone face to remove heparin affinity. The mature protein product is named Tip-A-AI K9A, K15A (SEQ ID NO 5).

Figure 10B: pT7H6-Trip-A-FN-Apo AI: Corresponding to pT7H6-Trip-A-Apo AI, but the bases encoding the amino acid sequence SGH were inserted after the Trip A sequence and before the apo AI sequence. The mature protein product is named Trip-A-FN-AI (SEQ ID NO 6).

Figure 10C: pT7H6-Trip-A-FN-Apo AI-final: corresponds to pT7H6-Trip-A-FN-Apo AI, but removed the BamHI site of pT7H6 Trip-A-FN-Apo AI and inserted three altered amino acids Thus, the amino acid sequence between the tetranectin derived trimerization sequence and apo AI was changed from GSSGH to GTSGQ. This five amino acid sequence corresponds to the sequence of the linker band of fibronectin. The mature protein product is named Trip-A-FN-final (SEQ ID NO 7).

Figure 10D: pT7H6-Trip-A-FN-Apo AI-final K9AK15A: corresponds to pT7H6-Trip-A-FN-Apo AI-final, but two lysine residues in the trimerization zone were mutated to remove heparin affinity. The mature protein product is named Trip-A-FN-AI-final-K9A, K15A (SEQ ID NO 8).

FIG. 10E: pT7H6-Trip-A-TN-Apo AI: Corresponding to pT7H6-Trip-Apo AI, but the bases encoding amino acid sequence KVHMK were inserted after the Trip A sequence and before the apo AI sequence. The mature protein product is named Trip-A-TN-AI (SEQ ID NO 9).

10F: pT7H6-Trip-A-TN-Apo AI-final: corresponds to pT7H6-Trip-A-TN-Apo AI, but removes the BamHI site of pT7H6 Trip-A-TN-Apo AI, resulting in tetranectin-derived tri The amino acid sequence between the merization sequence and apo AI was changed from GSKVHMK to GTKVHMK. This seven amino acid sequence corresponds to the tetranectin sequence following the trimerization domain. The mature protein product is named Trip-A-TN-AI-final (SEQ ID NO 10).

Figure 10G: pT7H6-Trip-A-TN-Apo AI-final K9AK15A: corresponds to pT7H6-Trip-A-TN-Apo AI-final, but two lysine residues in the trimerization zone were mutated to remove heparin affinity. The mature protein product is named Trip-A-TN-AI-final-K9A, K15A (SEQ ID NO 11).

10H: pT7H6Fx-Hp (alpha) -ApoAI. This plasmid encodes a fusion protein between Hp (alpha) and ApoAI. The mature protein product is named Hp (alpha) -ApoAI (SEQ ID NO14).

FIG. 11 shows the binding test results of ApoA-I, TripA-ApoA-I, and TripA-FN-ApoA-I to DMPC in the assay described in Example 6. FIG.

FIG. 12 shows the results of binding test of ApoA-I and TripA-ApoA-I to immobilized cubilin, as described in Example 7. FIG.

Figure 13 is an analytical gel filtration diagram of Apo A-I, Trip-A-AI, Trip-A-TN-AI, Trip-A-FN-AI. BSA is also included as a control. See Example 5 for a detailed description.

Figure 14 shows the results of the plasma depletion experiment evaluation of apo lipoprotein A-I, TripA Apo-AI and TripA-fibronectin-linker Apo A-I in mice. See Example 8 for a detailed description of this experiment.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

The function of the construct according to the invention and the function of the apo-A component of the construct can be measured by a lipid binding assay, such as the DPMC assay described below. In vivo effects on cholesterol back-transfer have also been described in Miyazaki et al. (Arteriosclerosis, Thrombosis and Vascular Biology, 1995: 15: 1882-1888), as well as Apo E-deficient rabbits fed high cholesterol diets. Measurements can be made by administering to test animals such as mice (Sha PK et al., Circulation 2001, 103: 3047-3050).

Apolipoprotein or analog

In the following the term "apo-A" shall refer to apolipoprotein A-I, apolipoprotein A-II or apolipoprotein A-IV, variants or analogs thereof having the same lipid binding function.

Preferred apolipoprotein A-I analogs include those described in FIG. 2A. Preferred A-IV analogs include those described in FIG. 2B.

Known variants for the sequence of human Apo-AI in FIG. 1 include the following variants, followed by the location, modification, and, where appropriate, the name of the variant corresponding to the sequence of FIG. 1.

27P → H (IN MUSTER-3C).

27P → R.

28P → R (IN MUSTER-3B)

34R → L (IN BALTIMORE).

50G → R (IN IOWA).

84L → R (IN AUTOSOMAL DOMINANT AMYLOIDOSIS).

113D-E.

119A → D (IN HITA).

127D → N (IN MUNSTER-3A).

131 MISSING (IN MARBURG / MUNSTER-2).

131K → M.

132 W → R (IN TSUSHIMA).

133E → K (IN FUKUOKA).

151R → C (PARIS).

160E → K (IN NORWAY).

163E → G.

167 P → R (IN GIESSEN).

168 L → R (IN ZARAGOZA).

171E → V.

189P → R.

197R → C (IN MILANO).

222E → K (IN MUNSTER-4).

The term "apolipoprotein" according to the present invention includes both functional equivalents of at least one sequence shown in FIGS. 1, 2A and 2B or fragments of at least one sequence of FIGS. 1, 2A and 2B. Shall be. The term "fragment" is defined as follows:

i) a fragment comprising an amino acid sequence recognizable by an antibody that is also capable of recognizing the predetermined amino acid sequence shown in FIG. 1, 2A or 2B, and / or

ii) a fragment comprising an amino acid sequence capable of binding to a lipid such as dimyristoyl phosphatidylcholine or cholesterol, and / or capable of binding to any of the amino acid sequences shown in FIGS. 1, 2A or 2B.

Functional equivalents of apolipoproteins or fragments thereof in accordance with the present invention can be obtained by addition, substitution or deletion of at least one amino acid. If one amino acid sequence is substituted for another, such substitution will be a conservative amino acid substitution. Fragments of the sequences of FIGS. 1, 2A, and 2B may be used for two or more such substitutions, such as two conservative amino acid substitutions, such as three or four conservative amino acid substitutions, such as five or six conservative amino acid substitutions, such as Dog or eight conservative amino acid substitutions, such as 10 to 15 conservative amino acid substitutions, such as 15 to 25 conservative amino acid substitutions, such as 25 to 75 conservative amino acid substitutions, such as 75 to 125 conservative amino acid substitutions, such as And from 125 to 175 conservative amino acid substitutions. Substitutions may be made using one or more groups of certain amino acids.

Examples of fragments comprising one or more conservative amino acid substitutions include fragments comprising one or more conservative amino acid substitutions or a plurality of conservative amino acid substitutions within the same group of predetermined amino acids, wherein each conserved Proper substitution is one produced by substitution in a different group of a given amino acid.

Thus, variants or fragments of the sequences shown in FIG. 1, 2a or 2b according to the present invention are the same variant or fragments of the sequences of FIG. 1, 2a or 2b, or different variants of the sequences of FIG. 1, 2a or 2b or Among the fragments thereof, it may comprise at least one substitution, for example a plurality of substitutions introduced independently of one another. Variants of the sequences of FIG. 1, 2A or 2B or fragments thereof may thus comprise conservative substitutions independent of one another; That is, the variant wherein at least one glycine (Gly) of said variant of the sequence of FIG. 1, 2a or 2b or fragment thereof is substituted with an amino acid selected from the amino acid group consisting of Ala, Val, Leu and Ile, and independently 1, 2a or 2b wherein at least one of said alanines (Ala) of a variant of the sequence of FIG. 1, 2a or 2b or a fragment thereof is substituted with an amino acid selected from the amino acid group consisting of Gly, Val, Leu and Ile Or a fragment thereof, and, independently of it, at least one of said valine (Val) of said variant or fragment thereof of the sequence of FIG. 1, 2A or 2B is selected from the group of amino acids consisting of Gly, Ala, Leu and Ile Variants or fragments of the sequence of FIG. 1, 2a or 2b substituted with amino acids, and, independently of them, at least one of the aforementioned variants of the sequence of FIG. 1, 2a or 2b or fragments thereof A variant of the sequence of FIG. 1, 2a or 2b or a fragment thereof, wherein said leucine (Leu) of mine is substituted with an amino acid selected from the group of amino acids consisting of Gly, Ala, Val and Ile, and independently of FIG. 1, 2a or A variant of the sequence of FIG. 1, 2a or 2b wherein at least one of the above-mentioned variants of the sequence of 2b or a fragment thereof is substituted with an amino acid selected from an amino acid group consisting of Gly, Ala, Val, and Leu or Fragments thereof, and, independently of this, at least one such aspartic acid (Asp) of the aforementioned variant of the sequence of FIG. 1, 2A or 2B or a fragment thereof is substituted with an amino acid selected from the amino acid group consisting of Glu, Asn, and Gln A variant or fragment thereof of the sequence of FIG. 1, 2a or 2b, and at least one of the above variants or fragments thereof, independent of the sequence of FIG. 1, 2a or 2b A variant or fragment thereof of the sequence of FIG. 1, 2a or 2b wherein said phenylalanine (Phe) is substituted with an amino acid group consisting of Tyr, Trp, His, Pro and preferably an amino acid group consisting of Tyr and Trp; Independently, at least one of said tyrosine (Tyr) of said variant of the sequence of FIG. 1, 2a or 2b or the fragment of sequence of FIG. 1, 2a or 2b is an amino acid group consisting of Phe, Trip, His, Pro, A variant or fragment thereof of the sequence of FIG. 1, 2a or 2b, preferably substituted with an amino acid selected from the group of amino acids consisting of Phe and Trp, and independently of the above variant of the sequence of FIG. 1, 2a or 2b, or A variant of the sequence of FIG. 1, 2A or 2B wherein at least one of the arginine (Arg) of a fragment thereof is substituted with an amino acid selected from the group of amino acids consisting of Lys and His or 1, in which at least one said lysine (Lys) of said variant or fragment thereof of the sequence of FIGS. 1, 2A or 2B is substituted with an amino acid selected from the amino acid group consisting of Arg and His. Variants or fragments thereof of the sequence of 2a or 2b, and, independently of them, at least one of the asparagine (Asn) of said variant or fragment thereof of the sequence of FIG. 1, 2a or 2b is composed of Asp, Glue and Gln A variant or fragment thereof of the sequence of FIG. 1, 2a or 2b, which is substituted with an amino acid selected from the group, and, independently of it, at least one glutamine of said variant or fragment thereof of the sequence of FIG. Gln) is a variant of the sequence of FIG. 1, 2A or 2B, or a fragment thereof, which is substituted with an amino acid selected from the amino acid group consisting of Asp, Glu, and Asn, and independent thereof 1, 2a, wherein at least one of said proline (Pro) of said variant of the sequence of FIG. 1, 2a or 2b or its fragment is substituted with an amino acid selected from the amino acid group consisting of Phe, Tyr, Trp and His Or a variant or fragment thereof of the sequence of 2b, and, independently of it, at least one of said cysteines (Cys) of said variant or fragment thereof of the sequence of FIG. 1, 2a or 2b is Asp, Glu, Lys, Arg, His An example is a variant or fragment thereof of the sequence of FIG. 1, 2A or 2B which is substituted with an amino acid selected from the group of amino acids consisting of Asn, Gln, Ser, Thr, and Tyr.

It is apparent from the above schematic description that the same variant or fragment thereof may comprise two or more conservative amino acid substitutions from two or more groups of conservative amino acids as defined herein.

The addition or deletion of amino acids can be the addition or deletion of 2 to 10 amino acids, such as 10 to 20 amino acids, such as 20 to 30 amino acids, such as 40 to 50 amino acids. However, additions or deletions of more than 50 amino acids, such as 10 to 200 amino acids, are also included within the scope of the present invention. More specifically, the 43 N-terminal amino acid can be removed from the sequence of FIG. 1 without substantially altering the lipid binding effect of the protein. Such deletion variants are included in SEQ ID NO 4 as part of the apolipoprotein of the invention.

Accordingly, the present invention relates to an apolipoprotein comprising at least one fragment of the sequences of Figures 1, 2a or 2b capable of binding to a lipid such as DPMC, wherein the functional equivalents and any variants of these at least one fragment are also incorporated herein. Included.

Apolipoproteins according to the present invention may comprise less than 243 amino acid residues, such as less than 240 amino acid residues, such as less than 225 amino acid residues, such as 200, in one embodiment of the present invention, including functional equivalents thereof and fragments thereof. Less than amino acid residues, such as less than 180 amino acid residues, such as less than 160 amino acid residues, such as less than 150 amino acid residues, such as less than 140 amino acid residues, such as less than 130 amino acid residues, such as less than 120 Amino acid residues such as less than 110 amino acid residues such as less than 100 amino acid residues such as less than 90 amino acid residues such as less than 85 amino acid residues such as less than 80 amino acid residues such as less than 75 amino acid residues Eg, less than 70 amino acid residues, such as less than 65 amino acid residues, such as To less than 60 amino acid residues, such as less than 55 amino acid residues, such as less than 50 amino acid residues.

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Particular preference is given to fragments comprising the lipid binding zone of the native sequences of FIG. 1, 2a or 2b. However, the scope of the present invention is not limited to fragments comprising a lipid binding zone. The present invention also includes deletions of the aforementioned fragments, which do not include a lipid binding zone but give rise to functionally equivalent fragments of the sequences of FIGS. 1, 2A or 2B. Functional equivalent sequences of the FIG. 1, 2A or 2B peptides according to the invention and fragments thereof may comprise more or less amino acids than the lipid binding zone. Preferably, the fragment comprises amino acids 100-186 of Apo-A-I or a variant thereof or a functional equivalent thereof. This central domain and its alpha-helices have been shown to be directly involved in the interaction with phospholipids. Therefore, it is very likely that this band will play an important role in the functional properties of Apo-A-I.

The term "functional equivalency" in the present invention is in accordance with one preferred embodiment established with reference to the corresponding functionality of certain fragments of the sequences of FIG. 1, 2A or 2B.

Functional equivalents of the variants of the sequences of FIG. 1, 2A or 2B can be understood to represent amino acid sequences that gradually become different from the desired predetermined sequence as the number and range of substitutions, insertions, and deletions, including conservative substitutions, increases. . This difference is measured as a decrease in homology between the desired predetermined sequence and fragments or functional equivalents thereof.

All fragments or functional equivalents of apolipoproteins are included within the scope of the present invention regardless of the degree of homology they show for a given desired sequence of apolipoproteins. The reason is that certain bands of the sequence of FIG. 1, 2A or 2B may be extremely easily changed or deleted completely without any significant impact on the binding activity of the resulting fragments.

The functional variant obtained by substitution will exhibit the natural activity of the sequences of Figures 1, 2a or 2b in any form and to some extent, and its homology will be reduced if residues containing functionally similar amino acid side chains are substituted. Functionally similar here refers to important properties of the side chain, such as the presence of hydrophobic, basic, neutral or acidic, or three-dimensional bulk sizes. Thus, in one embodiment of the present invention, i) the degree of identity between a given sequence of FIG. 1, 2a or 2b fragments that can exert an effect and ii) the desired predetermined fragment is such that the fragment is in accordance with the invention of FIG. 1, 2a or It is not the primary measure of determination of whether a desired predetermined functional equivalent or variant of the sequences of 2b.

Homology between amino acid sequences is well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90 It can be measured using.

Fragments that share at least some degree of homology with the sequences of the Figures 1, 2a or 2b fragments have at least about 40% homology, such as at least about 50% homology with the apolipoprotein or fragments thereof, such as Figures 1, 2a Or at least about 60% homology with sequences of the 2b fragment, such as at least about 70% homology, such as at least about 75% homology, such as at least about 80% homology, such as at least about 85% homology For example, at least about 90% homology, such as at least about 92% homology, such as at least about 94% homology, such as at least about 95% homology, such as at least about 96% homology, such as at least about 97 It is considered to be within the scope of the present invention when it exhibits% homology, such as at least about 98% homology, such as at least about 99% homology. According to one embodiment of the present invention, percent homology refers to percent identity.

Additional factors to consider when measuring functional equivalence in accordance with the meaning used herein include: i) the fragments of FIG. 1, 2a or 2b capable of searching for fragments of the sequence of FIG. The ability of an antiserum to any of the sequences, or ii) the ability of a functional equivalent fragment to compete with the sequence of FIGS. 1, 2A or 2B in a lipid binding assay.

Conservative substitutions may be introduced at any position of the desired apolipoprotein or fragment thereof. However, non-conservative substitutions are particularly preferred, but not limited to, introduced into non-conservative substitutions at one or more positions.

Non-conservative substitutions that form the functional equivalent fragments of the sequences of FIG. 1, 2a or 2b, for example, i) actually change the polarity, ie, polar side chains such as Gly, Ser, Thr, Cys, Tyr, Asn or Gln Or a residue having a charged amino acid such as Asp, Glu, Arg, or Lys with a residue having a nonpolar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met), or Replace residues with polar or charged residues and / or ii) the orientation of the polypeptide backbone is actually different, e.g., a residue with a negative charge such as Glu or Asp, with a positively charged residue such as Lys, His or Arg. (Or vice versa), and / or iii) the electrical charge is actually different, and / or such that a positively charged residue such as Lys, His or Arg is replaced (or vice versa) by a negatively charged residue such as Glu or Asp, and / or iv) such as Ala, Gl For example, a residue having a small side chain such as y or Ser may be replaced with a bulky residue such as His, Trp, Phe or Tyr (or vice versa), and there is a difference in steric volume.

In one embodiment substitutions of amino acids may be made based on their relative similarity of amino acid side chain substituents, including their hydrophobicity and hydrophilicity values and charge, size, and the like. Examples of amino acid substitutions in view of the foregoing features are well known to those skilled in the art and include: arginine and lysine; Glutamate and aspartate; Serine and threonine; Glutamine and asparagine; And valine, leucine and isoleucine.

In addition to the variants described herein, three-dimensionally similar variants may be made to mimic the nucleus portion of the variant structure and such compounds may also be used in the same manner as the variants of the invention. This can be accomplished by chemical design and modeling techniques known to those skilled in the art. All such stereoscopically similar structures are within the scope of the present invention.

Component X

Preferably, component X of the protein construct according to the invention is essentially non-immunogenic. For example, component X can be an amino acid, a carbohydrate, a nucleic acid sequence, an inactive protein or a polypeptide, which does not substantially have a physiological effect and in particular an immunological effect on a mammal.

Preferably, component X is non-immunogenic and does not interfere negatively with respect to ligand binding; That is, the apolipoprotein component should not be directed to an undesirable position through the interaction of the X-component with the ligand.

In one embodiment, component X consists of only one amino acid, which preferably is a cysteine residue and may be located at the N-terminus, C-terminus or within the apolipoprotein component. Such structures may form dimers with other identical or similar structures. Preferably, a linker is introduced between the terminal cysteine residue and the apolipoprotein component to facilitate lipid interaction and accurate folding of the construct.

However, more preferably, component X has more than one amino acid, such as more than two amino acids, such as more than five amino acids, such as more than ten amino acids, such as more than 15 amino acids, such as 20 More than amino acids, such as more than 30 amino acids, such as more than 40 amino acids, such as more than 50 amino acids, such as more than 75 amino acids, such as more than 100 amino acids, such as more than 200 Many amino acids, such as more than 300 amino acids, such as more than 400 amino acids, such as more than 500 amino acids, such as more than 600 amino acids, such as more than 700 amino acids, such as more than 800 amino acids For example, more than 900 amino acids, such as 1000, more than 1250 amino acids, such as 1500, 2000 or 2500 baud It is good to include many amino acids.

When the X-component is a protein, preferably it is a mammalian protein, more preferably a human protein. Examples of suitable proteins include plasma proteins such as albumin, serum albumin or other non-immunogenic peptides or proteins such as serine protease fragments of plasminogen or another serine protease engineered to be inactive by destruction of the catalytic triad; Constant bands of heavy chains of immunoglobulins. More preferably, this protein comprises serum albumin. More preferably, the protein comprises an apolipoprotein containing an amphoteric helix containing an apolipoprotein.

According to a particularly preferred embodiment of the present invention, component X preferably comprises an apolipoprotein component selected from apolipoproteins A-I, A-II, A-IV, analogs, functional variants or fragments thereof. These two apolipoprotein components may be linked linearly or via additional non-natural terminal cysteine bridges.

Higher oligomers, as well as dimers of the apolipoprotein component comprising at least one non-natural cysteine residue, can be prepared and linked through cysteine bridges under appropriate conditions. Oligomers linked through disulfide bridges may be linked in series (apo-A-S-S-apo-A, or apo-A-S-S-apo-A-S-S-apo-A or higher oligomers).

The protein constructs according to the invention may also comprise two, three, or more apolipoproteins or analogs thereof which are continuously or covalently linked to one another. This can be accomplished by linking the C-terminus of the first apolipoprotein to the N-terminus of the next apolipoprotein. The protein may also be linked after transcription and translation has taken place or the nucleotide sequence may comprise only two, three or more sequences encoding the apolipoprotein construct in question as well as any linker peptide between the apolipoproteins. have.

Thus, there is no need for a heterogeneous part to perform the connection. In constructs with two, three or more apo-A units, substantially all apo-A units are expected to participate in lipid binding, contributing to the functionality of these constructs. Thus, these multi-apo-A constructs are expected to have increased lipid binding capacity comparable to native apo-A. An additional advantage of these structures over native apo-A is that they have a longer plasma half-life than native apo-A.

Such constructs comprising more than one apolipoprotein component may comprise a combination selected from:

Dimer:

A-I A-I; A-II All; A-IV A-IV; A-I A-il; A- A-IV; A-II A-IV.

Trimmer:

A-I A-II A-IV; A-I A- A-ll; A-I A-I A-I; A-I A-I A-IV; A-II A-II A-1; A-II A-il A-IV; A-II A-II A-II; A-IV A-IV A-IV; A-IV A-IV A-II; A-IV A-IV A-I.

Oligomerisation modules

According to a particularly preferred embodiment of the invention, the heterologous moiety is preferably an oligomerization module. Here, the oligomerization module is a peptide or protein or part of a protein that can interact with other, similar or identical oligomerization modules. An interaction is a type of reaction that produces a multimeric protein or polypeptide. This interaction may be due to covalent bonds between the multimer components, hydrogen bonding forces, hydrophobic forces, van der Waals forces, salt bridges. The invention also includes oligomerization modules derived from non-peptides such as nucleic acid sequences of DNA, RNA, LNA or PNA. Those skilled in the art are well known in the art for linking protein and nucleic acid sequences to each other.

The oligomerization module can be a dimerization module, trimerization module, tetramerization module or multimerization module.

When the apolipoprotein or analogue portion of this construct is coupled to an oligomerization module, the multimer of the construct can be made simply by mixing the construct solution (an oligomerization module linked to the apolipoprotein moiety) under appropriate conditions. . In this way, dimers, trimers, tetramers, pentamers, hexamers, or more higher -mers can be made depending on the type of oligomerization module that is linked to the apolipoprotein portion of the structure.

The multimers according to the present invention can be homomers or heteromers, since various apolipoproteins can be linked to oligomerization modules and incorporated into the multimers. In this way it is desirable to mix different types of apolipoproteins to obtain constructs with improved clinical effects. Preferred homomers include Apo-A-I trimers and Apo-A-IV trimers.

In a particularly preferred embodiment of the invention, the oligomerization module is derived from tetranectin and more specifically refers to trimerized structural elements (hereinafter referred to as TTSE, SEQ ID NO 12) as detailed in WO 98/56906. Tetranectin to trimerize. The amino acid sequence of TTSE is shown in SEQ ID NO 12. The trimerization effect of TTSE is due to the coiled coil structure interacting with the coiled coil structure of two other TTSEs to form trimers, which are extremely stable. Another advantage of TTSE is that it is a weak antigen (WO 98/56906).

Preferably, the heparin binding site located in the N-terminal band of exon 1 (FIG. 4) removes or mutates the N-terminal lysine residues (residues 9 and 14 of SEQ ID NO 12) without inhibiting trimerization. It is good to be avoided by. Preferably the lysine residues are mutated to alanine. Thus, TTSE comprising most or all of exon 1 confers affinity for sulfated polysaccharides to any designed protein comprising such TTSE as part of its structure. However, if desired, this affinity can be reduced or avoided by N-terminal truncation or mutation of lysine residues in the part of the TTSE corresponding to the N-terminal amino acid residues of tetranectin (Lorentsen et al. 2000, Biochem J 347). : 83-87).

The interaction domain of the trimerization module according to the invention is preferably of the same type as in TTSE, ie triple alpha helical coiled coils.

TTSE can be derived from human tetranectin, rabbit tetranectin, rat tetranectin or shark cartilage C-type lectin. Preferably the TTSE has at least 68%, such as at least 75%, such as at least 81%, such as at least 87%, such as at least 92% identity with the consensus sequence of SEQ ID NO 12. As such, analogs of TTSE having substantially the same trimerization effect are included in the scope of the present invention.

Preferably, the cysteine residue 50 (SEQ ID NO 12) of TTSE is a combination of serine, threonine, methionine or any other amino acid in order to avoid the formation of unwanted interchain disulfide bridges that may result in unwanted multimerization. May be mutated to a residue.

The presence of a trimer can be confirmed by known techniques such as gel-filtration, SDS-PAGE or native SDS gel electrophoresis, depending on the nature of the trimer. One preferred method for determining the presence of oligomers is to perform DMSI (dimethylsubiridate) and subsequently SDS-PAGE.

According to a preferred embodiment of the invention, this protein construct is obtained by linking two or more apolipoproteins into oligomerization modules. An advantage of this embodiment is that the linkages of the individual apolipoproteins to each other do not occur inside the apolipoprotein, but within the oligomerization module. This is advantageous in terms of its physiological function, since the properties of wild-type apolipoproteins are preserved and the apolipoproteins preserve secondary and tertiary structures. By introducing more peptide spacers between the apolipoprotein and the oligomerization module, both components of the construct ensure their own interactions with lipids and other oligomerization modules, respectively, without being affected by the interaction of the other components. can do. Preferably, the peptide spacer is non-immunogenic and basically has a linear three dimensional structure.

Oligomerization modules, such as dimerization modules, trimerization modules, tetramerization modules, pentamers, hexamerization modules or multimerization modules, can be used to oligomerize the same or different apo-A units. The oligomerization module may comprise coiled coils capable of interchain recognition and interaction.

Common methods for producing artificial trimers of proteins or peptides include identifying trimerization modules from proteins that form trimers in nature. With careful analysis, the domains responsible for protein-protein interactions can be linked to the proteins or peptides to be identified, isolated and trimmed. According to the present invention, such trimerization does not necessarily include trimer formation of apolipoproteins or analogs. It is also possible to connect only one apolipoprotein to a trimerization module to allow the peptide to be trimmered with two other trimerization modules. By doing so, the molecular weight of the apolipoprotein moiety can be increased and the plasma half-life can be increased compared to the native apolipoprotein.

One example of an oligomerization module is illustrated in WO 95/31540 (HOPPE et al.), Which describes a polypeptide comprising a collectin neck region. The amino acid sequence making up the collectin neck zone can be linked to any polypeptide selected. Subsequently, under appropriate conditions, trimerization can be performed between the three polypeptides comprising the collector neck neck amino acid sequence.

Another example of an oligomerization module is the α1-chain from Haptoglobin. The α1-chain has a cysteine residue that can bond with other α1-chains to form a dimer. The original variant is an α2-chain, with an α1-chain portion in the replicated disulfide bridges. α2-chains can form cymer bonds with cysteine residues in other α2 or α1-chains to form trimers, tetramers, pentamers, hexamers and higher -mers. In its natural form, the α-chain is linked to the β-chain. It is possible to make apo-A-α chain (haptoglobin) structures by substituting the β-chain with apolipoproteins.

Spacer peptide

It may also be advantageous that the protein construct comprises a spacer moiety and a heterologous moiety covalently linked between the apolipoprotein or apolipoprotein analog. The effect of the spacer is to provide a space, ie a space, between the heterologous portion of the structure and the apolipoprotein portion. By doing so, even if the apolipoprotein moiety is present, since the secondary structure of the apolipoprotein moiety is not affected, the physiological effect of the apolipoprotein moiety can be maintained. Preferably, the spacer is derived from a polypeptide. In this way, the nucleic acid sequence encoding the spacer is linked to the sequence encoding the apolipoprotein portion of the construct and optionally to the heterologous portion, so that the entire construct can be constructed simultaneously.

Design and preparation of suitable spacer moieties are well known in the art and can be conveniently performed by preparing fusion polypeptides having the format of apo-A-spacer-X (wherein the spacer moiety is a polypeptide fragment (often relatively Inert), which can avoid undesirable reactions between the spacer and the periphery of the structure).

The spacer moiety may also be inserted between two TTSEs, allowing both to interact with a third isolated TTSE to form a trimer complex, which is associated with two separate peptides, TTSEs. TTSE-spacer-TTSE. This embodiment provides apo-A-TTSE-spacer-TTSE, in which the main part of the trimer, which soon appears strictly as a dimer, designates a fusion partner (apo-A that designates any polypeptide sequence that forms the apolipoprotein part of the structure). Because it can be synthesized as one single polypeptide contained in -apo-A, it facilitates the production of apolipoprotein constructs.

In this embodiment where two TTSEs are present in the same monomer, the spacer portion preferably has a length and an arrangement such that it forms a complex associated with both TTSEs covalently linked by the spacer portion. In this way, the undesirable trimer formats (2 + 1 + 1), (2 + 2 + 2) and (2 + 2 + 1), where only one TTSE of each monomer participates in complex formation ) Can be reduced.

The spacer peptide may comprise at least two amino acids, such as at least three amino acids, such as at least five amino acids, such as at least 10 amino acids, such as at least 15 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, such as at least 40 amino acids, For example at least 50 amino acids, such as at least 60 amino acids, such as at least 70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as about 100 amino acids.

The spacer may be linked to the apo-A component and X via a covalent bond, preferably the spacer is essentially non-immunogenic and / or prone to proteolytic cleavage, and / or any cysteine residues It is preferable not to include.

Likewise, the three-dimensional structure of the spacer is preferably linear or substantially linear.

Next is illustrated a spacer sequence that is believed to be particularly suitable for linking apolipoprotein analogs to component X. Preferred examples of spacer or linker peptides include those used to link proteins without substantially inhibiting the function of the linked protein linked protein, or without at least substantially inhibiting the function of any of the linked proteins. More preferably, a linker or spacer is used to connect the protein comprising the coiled coil structure.

Tetranectin based linker:

The linker may comprise tetranectin residues 53-56 forming β-strand in tetranectin and residues 57-59 forming a turn in tetranectin (Nielsen BB, Kastrup JS, Rasmussen H, Holtet TL, Graversen JH, Etzerodt M, Thoegersen HC, Larsen IK, FEBS-Letter 412, 388-396, 1997). The sequence of this segment is GTKVHMK. This linker has the advantage that in natural tetranectin it is generally considered to be suitable for linking the trimerized domain with the CRD-domain to link the trimerized domain with other domains. Moreover, the resulting structure is not expected to be more immunogenic than the structure without the linker. Linkers based on tetranectin are particularly preferred when component X comprises TTSE.

Fibronectin based linker:

This linker may be selected as a sub-sequence from the linking strand 3 from human fibronectin, corresponding to amino acid residues 1992-2102 (SWISS-PROT numbering, entry P02751). Preferably, amino acid residues 2037-2049 covering the sequence: PGTSGQQPSVGQQ are preferred, with the GTSGQ segment corresponding to amino acid residues 2038-2042 within this sequence being particularly preferred. This structure is not known to have a high tendency to be cleaved by proteolysis, so the immunogenicity is not expected to be very high given the high concentration of fibronectin in the plasma.

Human IgG ₃ Linker based on upper hinge (Human IgG ₃ upper hinge basedliker)

PKPSTPPGSS, a ten amino acid residue derived from the upper hinge zone of mouse IgG ₃ , has been used in the production of dimerized antibodies via coiled coils (Pack P. and Pluckthun, A. Biochemistry 31, pp 1579-1584 (1992)), which may be useful as a spacer peptide according to the present invention. The corresponding sequence from the human IgG ₃ upper hinge zone would be more desirable. Sequences from human IgG ₃ are not expected to cause immunogenicity in humans.

Flexible linkers

Possible examples of flexible linker / spacer sequences include SGGTSGSTSGTGST, AGSSTGSSTGPGSTT or GGSGGAP. These sequences have been used to link the designed coiled coils to other protein domains (Muller, K.M., Arndt, K. M. and Alber, T., Meth. Enzymology, 328, pp 261-281).

Combination

Two components of the structure may be connected to each other by covalent bonds. Such a bond may be formed between the C or N terminal amino acid of the apo-A component and component X. These components may also be linked through two or more covalent bonds. Covalent linkages between components may also include bonds between S-S bridges, preferably cysteine residues. Such cysteine residues are located at the C terminus or N terminus of the apo-A component and at the terminus or inside of the component X.

carbohydrate

Regardless of the other components of the structure, the structure according to the invention may comprise a carbohydrate moiety.

Tetranectin trimerized structural element (TTSE)

One particularly preferred embodiment of the present invention is to trimerize or partially trimer apolipoprotein or an analog thereof with a trimerized module of tetranectin.

This technique is described in WO 98/56906 (THOGERSEN et al.), Incorporated herein by reference. Trimer polypeptides are constructed as monomeric polypeptide constructs comprising at least one tetranectin trimerising structural element (TTSE) covalently attached to at least one heterologous moiety. This tetranectin trimerized structural element can form a stable complex with two other tetranectin trimerized structural elements.

The term “trimerized structural element (TTSE)” herein refers to the polypeptide molecular portion of the tetranectin family responsible for trimerization between tetranectin polypeptides (SEQ ID NO 12) monomers. The term also encompasses variants of the TTSE of naturally occurring tetranectin family members, where the variants have substantially no effect on the trimerization properties compared to the trimerization properties of the native tetranectin family member molecule. , Means that the amino acid is modified.

Specific examples of such variants will be described below, but it is preferred that the TTSE is derived from human tetranectin, rat tetranectin, C-type lectin of human or bovine cartilage or C-type cartilage of shark cartilage. Particular preference is given to monomeric polypeptide constructs comprising at least one TTSE derived from human tetranectin.

The polypeptide sequence of 51 residues encoded by exons 1 and 2 of tetranectin (FIG. 3, SEQ ID NO 12) has not been established with significant sequence homology to other known polypeptide sequences. It appears to be specific for the tetranectin group (FIG. 4). For experimental investigation of the structure of tetranectin, the complete tetranectin, CRD domain (which generally corresponds to the polypeptide encoded by exon 3), the product corresponding to the polypeptide encoded by exon 2 + 3, exon 1 + 2 Recombinant protein collections were prepared, including the products corresponding to (Holtet et al., 1996). While tetranectin is actually a trimer, exon 2 coding polypeptides can actually perform trimerization on their own, as evidenced by the fact that the recombinant protein corresponding to exon 2 + 3 was actually observed as a trimer in solution. have.

Three-dimensional structural analysis of full length recombinant tetranectin crystals (Nielsen et al., 1996; Nielsen, 1996; Larsen et al., 1996; Kastrup, 1996) revealed that the polypeptide encoded by exon 2 and the three residues encoded by exon 3 It has been shown to form a triple alpha helical coiled coil structure.

From the combination of sequence and structural data, trimerization in tetranectin results in an unusual heptad repeat sequence by a structural element (FIG. 4) comprising the amino acid residues encoded by exon 2 and the first 3 residues of exon 3. Is evident and specific for meptanthectin and other members of this group. This amino acid sequence (FIG. 4) is characterized by two copies of heptad repeats (abcdefg) with hydrophobic residues at the a and d positions as do other alpha helical coiled coils. Following these two peptide repeats, a unique third copy of the heptad repeat in sequence (where glutamine 44 and glutamine 47 not only replaces hydrophobic residues at both a and d positions, but also has a triple alpha helical coiled coil structure). Directly involved in the formation). These heptad repeats are additionally flanked by two half-repeats with hydrophobic residues at the a and d positions, respectively.

It is known that the presence of beta-branched hydrophobic residues in the a or d positions of alpha helical coiled coils affects the oligomerization state. There is only one conserved valine (No. 37) in the tetranectin structural element. No specific aliphatic residues appear to be preferred at sequence position 29 of tetranectin.

In summary, it is clear that the triple strand coiled coil structure of tetranectin is highly dependent on unexpected interactions due to the characteristics of known coiled coil protein groups.

TTSE forms surprisingly stable trimer molecules. (1) the observation that a substantial portion of the recombinant protein is in oligomer state and can crosslink as a trimer molecule even at 70 ° C. and (2) the exchange of monomers between different trimers can only be detected after exposure to elevated temperatures. The observation that there is an increase in that the tetranectin trimerized structural element is extremely stable. These properties must be reflected in the amino acid sequence of the structural element. In particular, the presence and position of the glutamine-containing repeats in the sequential array of heptad repeats, together with the relative position and presence of other conservative residues in the consensus sequence (FIG. 4), contribute to the formation of these stable trimer molecules. Is considered important. In most practical applications, cysteine residue 50 is a serine, threo, to avoid the formation of unwanted interchain disulfide bonds, which may result in uncontrolled multimerization, aggregation and precipitation of the polypeptide products producing this sequence. Must be mutated to nin, methionine or other amino acid residues.

In particular with respect to the trimer-stabilized exon 1 encoded polypeptide, the tetranectin trimerized structural element produces a highly stable homotrimer complex, with or without peptide bonds to other proteins at one or both ends. It is a truly autonomous polypeptide module that retains its structural integrity and propensity.

This unique property can be easily trimmered when polypeptide sequences derived from heterologous proteins are combined with tetranectin trimerized structural elements as fusion proteins. This is up to six mutually contiguous, in which the heterologous polypeptide sequence contributes to the formation of one molecular assembly containing six copies of one particular polypeptide sequence or functional entities, or each of which contributes to its individual functional properties. It is valuable regardless of whether it is located amino-terminally or carboxy-terminally in the trimmer element, which allows for one molecular assembly containing another polypeptide sequence.

Since the three TTSEs of naturally occurring human tetranectin form triple alpha helical coiled coils, it is preferred that the stable complexes formed by the TTSE of the present invention also form triple alpha helical coiled coils.

The term “tetranectin family” refers to the consensus sequence shown in FIG. 4, or polypeptides that share homology at the sequence level with this consensus sequence.

Accordingly, monomer polypeptide constructs of the invention comprising a polypeptide sequence having at least 68% sequence identity with the consensus sequence shown in FIG. 4 are preferred, with higher sequence identity, such as at least 75%, at least 81%, And at least 92% identity.

Tip A Module

In the expression plasmid according to the present invention, the TTSE module (SEQ ID NO 12) was modified by replacing Cys 50 with Ser and including it in the C-terminal lysine residue as described. The SPGT sequence was added at the N-terminus. This is the linking sequence for the trimmerization module. This inserted this sequence because the DNA strand gives the opportunity to be cleaved by Bgl II and Kpn K. The linking GS sequence is added at the C-terminus to provide an opportunity to cleave with Bam HI. This modified TTSE was named TipA and disclosed in SEQ ID NO 13. The trimerization module of the Apo A construct thus comprises a sequence having at least 68% sequence identity with this sequence or with the sequence of SEQ ID NO 13, preferably higher sequence identity, such as at least 75%, at least 81%, at least It is preferred to include sequences having 87%, at least 92% sequence identity.

Specific examples of structures that include a Trip A module are illustrated below.

Example of a structure according to the invention

The present invention includes the specific sequences SEQ ID NOs 2 to 11 and SEQ ID NO 14 disclosed in the accompanying examples. Preferably the present invention encompasses SEQ ID NOs 3 to 11 and SEQ ID NO 14. Sequences having at least 60% sequence identity, such as at least 70% sequence identity, with these sequences are also within the scope of the present invention, preferably at least 80% sequence identity, more preferably at least 90%, more preferably Is a sequence that shares at least 95%, more preferably at least 98% sequence identity.

Preparation of Protein Structures

To prepare the peptide component of the protein construct, the cDNA encoding this portion is inserted into an expression vector and transformed into a host cell.

The host cell (which is also part of the present invention) inserts a nucleic acid fragment (usually a DNA fragment) encoding the polypeptide portion of the monomer polypeptide construct of the present invention into an appropriate expression vector and transduces the appropriate host cell with the vector. After conversion, these host cells can be prepared by using traditional genetic engineering techniques, including culturing under conditions such that the polypeptide portion of the monomer polypeptide construct is expressed. Nucleic acid fragments encoding polypeptides may be located under the control of an appropriate promoter, which may be an inducible or structural promoter.

Depending on the expression system, the polypeptide can be recovered from the cytoplasm or extracellular state or periplasm of the host cell.

Suitable vector systems and host cells are well known in the art, as evidenced by the vast amount of relevant literature and data available to those skilled in the art. Since the present invention also relates to the use of the nucleic acid fragments of the present invention in the production of host cells and vectors, the following presents general considerations in particular for carrying out this aspect of the present invention and general points relating to such uses.

Of course, in general, prokaryotes are preferred for the initial cloning of nucleic acid sequences of the invention and for the construction of vectors useful in the invention. For example, in addition to the specific strains described in more detail below, strains such as, for example, E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. coli X 1776 (ATCC No. 31537). Can be. These examples are for illustrative purposes only and are not intended to limit the present invention thereto.

Prokaryotes are also preferred for expression because they can establish effective purification and protein refolding strategies for them. As well as the aforementioned strains, E. coli W3110 (F-λ, Prototropic, ATCC No. 273325), Bacillus genus such as Bacillus subtilis, or Salmonella typhimurim or Serratia marse Other enterobacteria, such as Serratia marcesand, and various Pseudomonas species are also available.

Generally, plasmid vectors containing control sequences and repolycons derived from species compatible with the host cell are used in connection with such hosts. Vectors generally have a replication site and a marking sequence that allows for selection of the phenotype in the transformed cell. For example, E. coli is often transformed using pBR322, a plasmid derived from E. coli species (see, eg, Boliver et al., 1977). This pBR322 plasmid contains ampicillin and tetracycline resistance genes, thus providing a convenient means of identifying transformed cells.

pBR plasmids or plasmids or phages of other microbial origin must also contain promoters available to these microorganisms for their expression.

The most widely used promoters in the field of recombinant DNA production include B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; EPO Publication No. 0036776). Although they are the most widely used, other microbial promoters have also been discovered and used, and details regarding their nucleotide sequences have been published so that those skilled in the art can functionally conjugate them to plasmid vectors (Siebwenlist et al., 1980). . Certain genes from prokaryotes can be effectively expressed in E. coli from their own promoter sequence, without the need to add another promoter by artificial means.

In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures are also available. There are many other strains, among which Saccharomyces cerevisiae and common baker's yeast are eukaryotic microorganisms in particular use. For expression in Saccharomyces, for example, plasmid YRp < " is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980).

This plasmid is for example ATCC No. It already contains a trpl gene that provides a selection marker for mutant strains of yeast lacking the ability to grow on tryptophan, such as 44076 or PEP4-1 (Jones, 1977). The presence of characteristic trpl lesions in enzyme host cells provides an effective environment to search for transformation by growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980), such as enolase, glyceralaldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate dekar Other such as carboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triocell phosphate isomerase, phosphoglucose isomerase and glucokinase Promoters for glycoproteinases (Hess et al., 1968; Holland et al., 1978). In constructing appropriate expression plasmids, the termination sequences associated with these genes can also be conjugated to the expression vector 3 'of the sequence to be expressed to provide polyadenylation and termination of the mRNA.

Other promoters with additional advantages of transcription controlled by culture conditions include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradable enzymes involved in nitrogen metabolism, and glyceraldehyde-3-phosphate dehydration as described above. It is the promoter zone for logenase and the enzymes responsible for maltose and galactose availability. Any plasmid vector containing a promoter, replication origin and termination sequence compatible with the yeast is suitable.

In addition to microorganisms, cell cultures derived from multicellular organisms can also be used as hosts. In theory, any of these cell cultures is feasible, whether cultures of vertebrate origin or invertebrate origin. However, the cells of vertebrates have been the most noted, and the diffusion of vertebrate cultures (tissue cultures) has become a common procedure (Tissue Culture, 1973). Examples of such useful host cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293 and MDCK cell lines.

Expression vectors for such cells include the origin of replication (if necessary), the promoter located in front of the gene to be expressed, any ribosomal binding site, RNA splice site, polyadenylation site, and transcription termination sequence.

For use in mammalian cells, control functions on expression vectors are often provided by viral agents. For example, commonly used promoters are derived from polyoma, adenovirus 2, and most commonly Simian Virus 40 (SV40). The early and late promoters of the SV40 virus are particularly useful because both are fragments that also contain the SV40 virus replication origin and are readily available from this virus (Fiers et al., 1978). Smaller or larger SV 40 fragments are also available as long as they contain approximately 250 bp sequences extending from the HindIII site located in the viral replication origin to the Bgl I site. In addition, if such control sequences are compatible with the host cell system, it is also possible and sometimes more desirable to employ promoter or control sequences that are generally associated with the desired gene sequence.

The origin of replication can be provided by constructing a vector comprising a foreign origin, such as may be derived from SV 40 or other viruses (eg polyoma, adeno, VSV, BPV), or by the chromosomal replication mechanism of the host cell. . If the vector is to be integrated into a host cell chromosome, the latter is often sufficient.

Once the polypeptide monomer construct is produced, by introducing a non-proteinaceous function into the polypeptide, subjecting the material to appropriate refolding conditions (e.g., using generally applicable strategies as set forth in WO 94/18227) Or it may be necessary to further process the polypeptide by cleaving the undesirable peptide portion of the monomer (eg, expression that enhances the unwanted peptide fragment in the final product).

As a vector capable of carrying and / or replicating a nucleic acid according to the invention in a host cell or cell line is part of the invention, in view of the above, a method for producing a monomer polypeptide construct of the invention in a recombinant manner is also disclosed. It is part of the invention. According to the invention, the expression vector may be a plasmid, cosmid, minichromosome or phage. Of particular note are vectors that are introduced into the host and then integrated into the host cell / cell line genome.

Another part of the invention are transformed cells capable of carrying and replicating a nucleic acid fragment of the invention (useful for the method above); The host cell may be from Bacillus, yeast or protozoa, or may be a cell derived from a multicellular organism such as a fungus, insect cell, plant cell, or mammalian cell. Of particular interest are the bacterial species Escherichia, Bacillus, and Salmonella, with the preferred bacteria being E. coli.

Another aspect of the invention relates to a stable cell line producing the polypeptide portion of the construct according to the invention, preferably which cell line carries and expresses the nucleic acid of the invention.

Receptor Coupling

The ability of a construct according to the invention can be analyzed by measuring the ability of the construct to bind to a receptor or HDL protein capable of binding to native apolipoprotein A-I, A-II or A-IV. These receptors and proteins include cublin, megalin, scavenger recepter class B type 1 (SR-B1), ATP-binding cassette 1 (ABC1: ATP-binding cassette 1), lecithin: cholesterol Acyltransferase (LCAT: lecithin: cholestero acyltransferase), cholesteryl-ester transfer protein (CETP), and phospholipid transfer protein (PLTP). The dissociation constant Kd of the complex between cubililine and natural apolipoprotein AI is 20 nM. It has been experimentally determined that the apolipoprotein A I trimer according to the present invention binds more strongly to cubilin (FIG. 12).

Affinity tags

The protein construct according to the invention may also comprise an affinity tag for use in the purification of the construct. Such tag preferably contains a polyhistidine sequence. This sequence can be advantageously used for purification of the product on a Ni ²⁺ column, which binds to the entire protein by binding to the polyhistidine sequence. After eluting from the column, the polyhistidine sequence can be cleaved off by a proteinase such as thrombin that recognizes a particular sequence made within the structure between the protein construct and the polyhistidine sequence.

Other examples of affinity tags include, but are not limited to, well known tags such as antigenic tags or GST tags. A proteolytic cleavage site can be inserted between the tag and the structure for cutting the tag.

Signal peptide

When the construct according to the present invention is expressed in E. coli or yeast, instead of the cumbersome process of separating the expressed protein from the cell in the cell, the expressed construct is secreted so that it can be harvested by secreting the expressed protein from the medium around the cell. It may be desirable to include signal peptides. Specific examples of signal peptides that can be used in the present invention for expression in yeast and E. coli are described in WO 90/12879 (Sirtori et al., Which describes signal peptides for expressing Apo-AI and Apo-AIM in yeast). ) And WO 94/13819 (Kabi Pharmacia), which describes signal peptides for expressing Apo-AI and Apo-AIM in E. coli.

Preparation of apo-A-TTSE

To prepare a construct comprising an apolipoprotein moiety and a TTSE, a cDNA encoding the apolipoprotein moiety is conjugated to the 3 'and 5' ends of the cDNA encoding the TTSE. Additional TTSE units and more apolipoprotein units may be conjugated. The sequence encoding the enzyme cleavage site is conjugated to the 3 'end of the sequence encoding TTSE, and finally the sequence encoding polyhistidine is also conjugated. This can be done by conventional PCR techniques. Bound cDNAs are inserted into expression vectors and transformed into host cells.

After expression in E. coli, the polyhistidine sequence is used to capture heterologous proteins on a Ni 2+ column. After elution, the polyhistidine tail can be removed by proteinases such as Fx, which cleaves heterologous proteins at specific sites inserted between the TTSE and the polyhistidine sequence. The resulting apo-A-TTSE peptide can then be further modified by trimmering with another or the same apo-A-TTSE peptide. In order to enhance expression in E. coli, it may be advantageous to express the construct as a fusion protein that can be cleaved later, such as with ubiquitin.

Use of apo-A constructs for the manufacture of pharmaceutical compositions

It is also possible to use apo-A structures for the preparation of pharmaceutical compositions. The composition may include pharmaceutically acceptable excipients, adjuvants, additives such as phospholipids, cholesterol or triglycerides.

The pharmaceutical composition is intravenous, intraarterial, intramuscular, percutaneous, pulmonary, subcutaneous, intradermal, intradural, or through the cheeks, anus, vagina, conjunctiva, or intranasal tissue, or tumors It may be inoculated into a tissue, such as tissue, or administered by or transplanted orally.

Preparation of the pharmaceutical compositions according to the invention is preferably carried out using techniques well known to those skilled in the art. This technique includes the addition of pharmaceutically acceptable excipients, adjuvant or additives such as phospholipids, cholesterol or triglycerides.

Administration of the apo-A construct

The apo-A construct according to the present invention is administered to prevent and / or treat diseases associated with cholesterol, phospholipids and triglycerides, LDL and HDL disorders such as hypercholesterolemia, and atherosclerosis diseases such as atherosclerosis and myocardial infarction. Can be. Other symptoms include angina, plaque angina, unstable angina, such as artery stenosis, such as carotid artery stenosis, claudication, or cerebral artery stenosis. Apolipoprotein structures can also be used to remove endotoxins.

In one embodiment, such administration comprises administering at least 50 mg of the construct each week so that a plasma concentration of about 0.5 g / L can be achieved. Preferably, the construct is administered parenterally, such as via injection, suppositories, implants, and the like. Preferably the composition is in an amount comprising at least 50 mg of apolipoprotein structure per week, such as at least 100 mg / week, such as at least 250 mg / week, such as at least 500 mg / week, such as at least 750 mg / week, For example at least 1000 mg / week, such as at least 1250 mg / week, such as at least 1500 mg / week, such as at least 2000 mg / week, such as at least 2500 mg / week, such as at least 5000 mg / week. Administration can be daily, once every two or three days, once a week, once every two weeks, or once every three weeks, or once every four weeks.

In another embodiment, the construct is administered once, twice or three times in large amounts for rapid treatment of angina and plaque angina or unstable angina. Such administration may be up to 1 day, 2 days 3 days, 4 days, 5 days, 6 days, 7 days, 8 days or 10 days. Such amount may be at least 10 mg / kg body weight, such as at least 20 mg / kg body weight, such as at least 30 mg / kg body weight, such as at least 40 mg / kg body weight, such as at least 50 mg / kg body weight, such as at least 60 mg / kg body weight, such as At least 70 mg / kg body weight, such as at least 75 mg / kg body weight, such as at least 90 mg / kg body weight, such as at least 100 mg / kg body weight, such as at least 125 mg / kg body weight, such as at least 150 mg / kg body weight, such as at least 200 mg / kg body weight, such as at least 250 mg / kg body weight, such as at least 300 mg / kg body weight, such as at least 400 mg / kg body weight, such as at least 500 mg / kg body weight, such as at least 600 mg / kg body weight, such as at least 700 mg / kg body weight, such as at least 800 mg / kg body weight, such as at least 900 mg / kg body weight, such as at least 1000 mg / kg body weight.

The constructs of the present invention may be administered orally. For this route of administration, the techniques described in WO 99/46283, US 5,922,680, US 5,780,434 or US 5,591,433, US 5,609,871 or US 5,783,193 can be applied to the protein construct according to the invention. Such references are hereby incorporated by reference in their entirety.

Cell population

The invention also encompasses the use of the nucleotide sequences according to the invention for gene therapy.

Transfer the gene to a macrophage colony and then to a patient in need of treatment. In this way, macrophages have a limited lifespan in the blood vessels, resulting in transient expression of the gene.

Permanent transfection can be performed by transforming liver cells.

Hereinafter, the present invention will be described in more detail with specific embodiments of the present invention. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. Designing additional structures according to the present invention falls within the general skill level of those skilled in the art or practitioners.

Example 1: Cloning of Apo A-I

Standard PCR techniques were used to amplify the cDNA encoding Apo A-I from the human liver cDNA library (Clontech). The primers used for the production of Ubi-AI were as follows: 5'-CAC GGA TCC ATC GAG GGT AGG GGT GGA GAT GAA CCC CCC CAG AGC-3 'and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3 '. This product was cloned using Bam HI and Hind III cloning sites into Ellgaard L. et al., Eur. J. Biochem. 1997; 244 (2): 544-51) and cloned into the pT7H6Ubi vector. Primers used for the construction of Trip-A-A-I were 5'-AAG GGA TCC GAT GAA CCC CCC CAG AGC CCC-3 'and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3'. This PCR product was cloned into the pT7H6tripa vector described in WO 98/56906 using Bam HI and Hind III cloning sites. Primers used for the construction of Trip-AI-del43 were 5'-AGG GGA TCC CTA AAG CTC CTT GAC AAC TGG G-3 'and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3' . This PCR product was cloned into the pT7H6tripa vector described in WO 98/56906 using Bam HI and Hind III cloning sites. Primers used for the preparation of Ubi-Cys-AI were as follows: 5'-GGT GGA TCC ATC GAG GGT AGG GGT GGA TGT GAT GAA CCC CCC C-3 'and 5'-TCC AAG CTT ATT ACT GGG TGT TGA GCT TCT TAG TG-3 '. This product was cloned using Bam HI and Hind III cloning sites into Ellgaard L. et al., Eur. J. Biochem. 1997; 244 (2): 544-51) and cloned into the pT7H6Ubi vector. The prepared plasmids are shown in Figures 4, 5, 6 and 7.

Example 2: E. coli Expression of Apolipoprotein A-I (apo A-I) in

Ubi-A-I and Trip-A-I as well as other constructs shown in the figure are readily expressed in E. coli AV-1 cells (Stratagene Inc.). Other cell lines can also be used. Cell culture and expression induction were performed as described in WO 98/56906.

Example 3: Isolation and Modification of Proteins

Isolation by phenol extraction as described for tetranectin in WO 98/56906. Re-dissolved pellets from 6 liters of expression culture were centrifuged to separate undissolved material and then the batch was taken up in 50 ml Ni ²⁺ -NTA-Sepharose prepared as described in WO 98/56906. The column material is charged to the column followed by 500 ml 8M free radicals, 500 mM NaCl, 50 mM Tris-HCl pH 8.0 followed by 200 ml of 6M guanidinium-HCl, 50 mM Tris-HCl pH 8.0 and finally 300 ml 500 mM NaCl, Washed with 50 mM Tris-HCl pH 8.0. Protein was extracted with 500 mM NaCl, 50 mM Tris-HCl pH 8.0 and 10 mM EDTA. Protein was mixed with 0.5 mg Factor Xa and cleaved overnight at room temperature. Thrombin can also be used for this purpose. Proteins in 500 mM NaCl, 50 mM Tris-HCl pH 8.0 buffer were gel filtered on a G-25 Sephadex (Pharmacia) column. Proteins that were not cleaved by passing the protein solution through a Ni ²⁺ -NTA-Sepharose column pre-washed at 500 mM NaCl, 50 mM Tris-HCl pH 8.0 and then washed with 500 mM NaCl, 50 mM Tris-HCl pH 8.0 Was removed. Uncleaved protein was eluted with 500 mM NaCl, 50 mM Tris-HCl pH 8.0 and 10 mM EDTA. Further purification can be performed using Sp Sepharose Ion Exchange.

Example 4: Removal of Lipids from Proteins

The protein was gel filtered with a 10 mM (NH 4) 2 CO 3 pH 8.8 solution and then lyophilized. Lyophilized protein was resuspended in 25 ml of cold 1: 1 methanol / chloroform, incubated for 30 minutes on ice and then centrifuged at 3000 g for 20 minutes. The pellet was resuspended in 25 ml of cold 1: 2 methanol / chloroform, equilibrated on ice for 30 minutes and then centrifuged again. The supernatant was removed and the pellet briefly air-dried and redissolved overnight at 6M guanidine-HCl, 50 mM Tris-HCl pH 8.0.

Bridge

Multimerization can be measured by measuring crosslinking of the multimer followed by analytical SDS-PAGE.

60 μl of 0.2 mg / ml protein dissolved in 150 mM Na-borate pH 9.0 equilibrated to the desired temperature for 30 minutes is added to 5 μl of 20 mg / ml dimethylsuberimidate and incubated for 30 minutes at the desired temperature. Crosslinking was stopped by adding 5 μl 3M Tris-HCl pH 9.0.

Dimethylsuberimidate forms covalent bonds by placing lysine residues at short distances from each other. As a result, the multimer-forming proteins covalently bind to each other. The molecular weight of the multimer can be assessed by subsequent SDS-PAGE.

The crosslinked product was analyzed by SDS-PAGE on an 8-16% polyacrylamide gel. Adjuvants such as lipids may optionally be included in the crosslinking mixture, in which case the proteins were pre-incubated with the adjuvant.

Analytical Gel Filtration

Multimerization can also be measured by analytical gel filtration.

The protein was dissolved in 500 mM NaCl, 50 mM Tris-HCl pH 8.0 buffer and then gel filtered on a Superdex 200 HR 10/30 column with the desired buffer at room temperature, 0.25 ml / min. For the process in the table the buffer was 100 mM NaCl, 50 mM Tris-HCl pH 8.0.

It can be seen from FIG. 13 that Apo A-I elutes as the complex peak, which is the main peak centered at about 14.5 and 16.5 mL. BSA with a molecular weight of 68 kDa elutes at about 14.5 ml, indicating that the Apo A-I peak at 16.5 ml corresponds to the monomer Apo A-I, while the other main peak corresponds to the apo A-I self-binding complex. The structures fused to the trimerized domain all elute at about 10.7 ml main peak and 14 ml minor peak. The peak at 14 ml probably corresponds to the trimerized form of the structure, while the main peak at 10.7 ml corresponds to the high molecular weight product. This product is assumed to be formed by the binding of trimers. This is because the apo AI fusion to the trimerization domain not only results in a trimer, but also allows the Apo AI unit to interact with other apo AI units, just as native apo AI can interact with other Apo AI molecules. Refers to the formation of complex formation.

Example 6: Kinetics of the binding of protein constructs with dimyristoyl phosphatidylcholine (DMPC)

By using known assays, such as binding to dimyristoyl phosphatidylcholine (DPMC), the binding capacity to the lipids of the constructs according to the invention can be readily determined.

This assay is described in Bergeron J. et al. (1997), Biochem. Biophys. It was performed as described in Acta, 1344, 139-152. Dry DPMC was suspended in 100 mM NaCl, 50 mM Tris-HCl pH 8.0 and 0.25 mM EDTA at a concentration of 0.5 mg / ml above its transition temperature. Protein samples, buffers, and DMPC suspensions were incubated at 24 to 10 minutes, then mixed so that the final concentration of DMPC was 0.5 mg / ml and the concentration of protein was 5.2 M (monoconcentration). Determination of the absorbance of the mixture at 325 nm showed a decrease in turbidity of the mixture, reflecting increased lipid-protein binding. This analysis was performed four times for apo AI, Trip-A-AI and Trip-A-FN-AI, and once without protein addition.

From FIG. 11, it can be seen that all Apo A-I structures bind to DMPC. After 24 hours, all three constructs in the test were completely cleared of turbidity, indicating that the fusion protein's ability to bind to DMPC was present in the fusion protein. However, both Trip-A-Apo A-I and Trip-A-FN-Aop AI bound more slowly than native Apo A-I bound to DMPC at 24 ° C, the only temperature at which this assay functions.

Example 7 Surface Plasmon Resonance Analysis of Binding of Analogs to Cubilin

This analysis was performed as described in Kozyraki R, Fyfe J, Kristiansen M, Gerdes C, Jacobsen C, Cui S et al. Cubilin, an intrinsic factor-vitamin B12 receptor, is a high-affinity apolipoprotein A-I that facilitates endocytosis of high density lipoproteins. Nat Med 1999; 5 (6): 656-661. The concentration of apolipoprotein constructs used was 0.5 M. Results (FIG. 12) are shown for Trip A-AI and apo A-I only. Similar binding was observed for TripA-FN-AI and TripA-TN-AI as observed for Trip A-AI. The response was particularly increased when trimming the apo AI, in particular when a reduction in of-rate based on the obtained "avidity" of the interaction between the multimer and the immobilized target compared to the monomers occurred. (Reward bonus). The fact that apo AI is able to bind to cubilin in the trimmer state, and that two or more apo AIs are in an array that can interact with quillin indicates that the apo AI unit is correctly folding within the trimmer structure. .

Example 8 Plasma Clearance Evaluation of Apolipoprotein A-I, TripA Apo-A-I and TripA Fibronectin-Linker Apo-A-I in Mice

Three groups of five mice each were injected with 1 mg of apo A-I, Trip-A-AI or Trip-A-FN-AI, respectively. Protein was dissolved in the following buffer at a concentration of 0.33 mg / ml: 1 x PBS pH 7.4, and 8.9 mg / ml dipalmitoylphosphatidylcholine. Blood samples were drawn from each mouse at the following time points after injection: 10 minutes later, 4 hours later, 24 hours later and 48 hours later.

Plasma concentrations of Apolipoprotein A-I and its derivatives were determined using the ELISA assay as follows:

Nunc Immuno PolySorp plates were used and 100 μl of each vial was added for each addition. MB corresponds to the following buffer composition: 2 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM HEPES, 140 mM NaCl.

Plates were coated in cold room overnight with 4 μg / ml of polyclonal anti-human apo AI (DAKO A / S) from rabbits dissolved in 50 mM NaHCO ₃ pH 9.6. Plates were washed at MB + 0.05% Tween-20 pH 7.8 and blocked for 1 hour at MB + 0.05% Tween-20 + 1% BSA pH 7.8. Samples were dissolved in MB + 0.05% Tween-20 + 1% BSA pH 7.8 and then incubated for 1 hour and monoclonal anti-apo AI from mice for 1 hour at MB + 0.05% Tween-20 + 1% BSA pH 7.8. (PerImmune Inc, clones 10-A8) were incubated for 1 hour at a concentration of MB + 0.05% Tween-20 + 1% BSA pH 7.8 1 μg / ml. Plates were washed and incubated with secondary anti-mouse IgG antibodies linked to hose radish peroxidase. The plate was washed again and developed using OPD-tablet and H ₂ O ₂ and then the reaction was stopped using 1M H ₂ SO ₄ . The results were compared with standard results based on known concentrations of apo AI and derivatives, respectively. The dilution effect of Apo AI in mouse plasma was not observed by the standard method.

The results shown in FIG. 14 show that plasma removal of construct Trip A Apo A-I is increased by at least 3 hours relative to the removal time of native Apo AI. Preliminary data indicate that the removal time of Trip A FN Apo A-I is at least equal to Trip A Apo A-I. These experimental data, together with the cublin binding data and the DMPC binding data, document that the construct according to the invention can be a strong candidate to treat the diseases mentioned in this application.

Example 9 Plasmids

The structure according to the present invention can be prepared using the plasmid described below.

Linker Sequence Insertion into Trip-A-I

That is, by PCR amplification of Apo A-I (and linker sequence), constructs containing the basic linker with the mutations described above were inserted into the pT7FxH6-Trip-A plasmid, as with the linkerless construct. The reverse primer was the same as that used for the structure of pT7H6FxTrip-A-AI, while the forward primer used the following:

pT7H6FX-Trip-A-FN (-2) -AI:

5'-CGC GGATCC TCG GGT CA G GAT GAA CCC CCC CAG AGC CCC-3 '

Unfortunately all the clones isolated contained the highlighted G , which was mutated to T, indicating that the sequence of the primer was incorrect.

pT7H6FX-Trip-A-TN-AI-Bam-S

5'-cgc gga tcc aag gtg cac atg aag gat gaa ccc ccc cag agc ccc-3 '

The mutations described above were corrected by site-directed mutations using the Stratagene QuickChange kit and the following primer sets.

pT7H6FX-Trip-FN-AI:

5'-acg gtc tcc ctg aag gga acc tcg ggt cag gat g-3 '

5'-cat cct gac ccg agg ttc cct tca ggg aga ccg t-3 '

pT7H6FX-Trip-A-TN-AI:

5'-acg gtc tcc ctg aag gga acc aag gtg cac atg aag g-3 '

5'-cct tca tgt gca cct tgg ttc cct tca ggg aga ccg t-3 '

Removal of Heparin-binding Sites in Trip-A

As an additional induction of this construct, heparin binding sites of the Trip-A sequence (Lorentsen RH, Graversen JH et al., Biochemical Journal (2000), 347 pp 83-87), the site-directed mutation kit from Stratagene and the following primer sets Mutagenesis using:

Mutations of Lysine 9 from Trip-A:

5'-cca acc cag aag ccc aag gcg aat gta aat gcc-3 '

5'-gtg ttc aca aca tct gcc ttg gca ttt aca atc-3 '

Mutations of Lysine 15 from Trip-A:

5'-ggc att tac aat cgc ctt ggg ctt ctg ggt tgg-3 '

5'-cca acc cag aag ccc aag gcg att gta aat gcc-3 '

Such mutations are designed to be performed on all relevant Trip-A-apo-AI derivatives, possibly all of them. Double mutations K9A, K15A of the trip-A derivative were produced and named Trip-A-FN-AI-K9AK15A, TripA-TN-AI-K91K15A and TripA-AI-K91K15A.

N-terminal truncation also eliminated heparin affinity without removing trimerization. Holtet et al. Protein Science (1997), Lorentsen et al. Biochem. Journal (2000), Nielsen et al. See FEBS (1997) and Nielsen et al Acta Cryst D. (2000). The N-terminal residue is preferably located between Val16 and Met22.

Construction of Expression Plasmids for Hp-AI

First plasmid pT7H6Fx-Hp (alpha) (FIG. 10H) was prepared as follows:

Hp-alpha sequences from cDNA library (Clontech fetal liver) were PCR amplified using the following primer sets:

Nonsense Primer: 5'-cac aag ctt tcc get aga tct ctg cac tgg gtt age eggatt ctt ggg-3 '

Sense Primer: 5'-ggt gga tcc ate gag ggt agg ggt gtg gac tca ggc aat gat gtc acg g-3 '

PCR products have flow characteristics:

BamH I-site-Hp alpha sequence-BgI II site-Hind III site.

Cysteine disulfide bonds of HP with the beta-chain in the Hp-alpha sequence were mutated to alanine.

The product was digested with BamH I and HIn III and inserted into the pT7H6FX plasmid to determine the sequence. PCR products encoding human Apo AI were made using standard nonsensor primers, which are also used to make pT7H6-Ubi-Fx-ApoAI and pT7H6Fx-TripAI. The sense primer used was the sense primer used in the construction of pT7H6FX-TripA-AI. PCR products were endowed with the following characteristics:

BamH I site-Apo AI sequence-Hind III site, this product was inserted into the plasmid cut with BamH I and Hind III and cut with Bgl II and HindIII. The sequence of the resulting plasmid pT7H6FX-Hp (alpha) -Apo AI was analyzed and expression tests were performed in E. coli.

pT7H6 TripA-apoAI (FIG. 7):

This plasmid comprises the plasmid pT7H6FxtripA described in WO 98/56906 as in Example 1.

Expression is dictated by the T7 promoter. This plasmid also contains the H6 sequence, which is a hexa-His affinity tag to be used for purification. Subsequently, Factor Xa recognition sequence (IQGR) is inserted

SPGT is the linking sequence for subsequent trimerization modules. This sequence was incorporated because it provides an opportunity to cleave DNA strands with Bgl II and Kpan I.

Trip A is a trimerization module from tetranectin.

GS is another linking sequence that confers cleavage opportunities by Bam HI.

Finally the plasmid contains human apolipoprotein A-I cDNA encoding amino acids 25-267 from human apolipoprotein A-I. The expressed and purified protein corresponds to SEQ ID NO 3.

pT7H6TripA-apoA1-del43 (FIG. 8):

This plasmid contains the sequences as described above, but the apolipoprotein moiety has been replaced with a cDNA encoding amino acids 68-267 from human apolipoprotein A-I. The expressed and purified protein corresponds to SEQ ID NO 4.

pT7H6UbiFxApoA (FIG. 5):

This basic plasmid has been described in Ellgaard et al. (1997).

This plasmid contains the following sequences:

Expression is governed by the T7 promoter

H6: hexa-His affinity tag for purification of protein constructs

Ubi: cDNA encoding human ubiquitin inserted to stabilize protein in E. coli.

Fx: recognition sequence for Factor Xa

DNA encoding two Gly residues required for optimal cleavage by Factor Xa

Apo AI: cDNA encoding amino acid residues 25-267 from human apolipoprotein A-I

The expressed and purified protein corresponds to SEQ ID NO 1.

pT7H6UbiFXCysApoA (FIG. 6):

As described above, however, it was inserted after the sequence encoding the two glycine short-terms and before the apolipoprotein A-I sequence encoding the cysteine residue. The expressed and purified protein corresponds to SEQ ID NO 2.

pT7H6FXCysApoAI (FIG. 9):

This plasmid contains the following sequences:

Expression is governed by the T7 promoter.

H6: hexa-His affinity tag for purification of protein constructs

Fx: recognition sequence for Factor Xa

DNA encoding two Gly residues required for optimal cleavage by Factor Xa

DNA encoding cysteine residues

Apo AI: cDNA encoding amino acid residues 25-267 from human apolipoprotein A-I

The expressed and purified protein corresponds to SEQ ID NO 2.

Examples of additional plasmids for the expression of apolipoprotein constructs according to the invention are shown in FIGS. 10A-G with the corresponding amino acid sequences of the expressed and purified proteins disclosed in the appended sequence listing.

references

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Claims (111)

Apolipoprotein constructs for use as medicaments having the general formula: apo-A-X Wherein apo-A is an apolipoprotein component selected from apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, an analog or variant thereof, X is a heterologous moiety comprising at least one compound selected from amino acid, peptide, protein, carbohydrate and nucleic acid sequences, Provided that the construct consists of exactly two identical natural apolipoproteins, they are linked in series. The structure of claim 1 comprising a spacer between the apo-A component and X. 3. The structure of claim 2, wherein the spacer is a spacer peptide. The method of claim 3, wherein the spacer peptide comprises at least two amino acids, such as at least three amino acids, such as at least five amino acids, such as at least 10 amino acids, such as at least 15 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, For example at least 40 amino acids, such as at least 50 amino acids, such as at least 60 amino acids, such as at least 70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as at least 100 amino acids. The structure of claim 2, wherein the spacer is connected to the apo-A component and X via a covalent bond. The structure of claim 2, wherein the spacer is essentially non-immunogenic and / or prone to proteolytic cleavage and / or contains no cysteine residues at all. The structure of claim 2, wherein the three-dimensional structure of the spacer is linear or substantially linear. 3. The spacer peptide of claim 2, wherein the spacer peptide comprises the amino acid sequence GTKVHMK from tetranectin, the amino acid sequences PGTSGQQPSVGQQ and GTSGQ from ligation strand 3 from human fibronectin, PKPSTPPGSS, SGGTSGSTSGTGST, AGSSTGSSTAPGSTT or GGSGG from the mouse IgG3 Including the structures. The structure of claim 1, wherein component X is covalently linked to the N-terminal or C-terminal amino acid of apo-A. The structure of claim 1, wherein the apo-A component and X are linked by two covalent bonds. The structure of claim 1, wherein the apo-A component and X are linked via a C or N terminal S-S bridge, preferably a cysteine bridge. The construct of claim 1 wherein the protein or peptide comprises at least one mammalian protein. 13. The construct of claim 12 wherein the mammalian protein is a human protein. 13. The construct of claim 12, wherein the protein is non-immunogenic. 13. The heavy chain of claim 12, wherein the protein is engineered to be inactivated by albumin, more preferably serum albumin, a serine protease fragment of plasminogen or a catalytic triad, and a heavy chain of immunoglobulins. A construct comprising at least one protein selected from the constant bands of. 13. The construct of claim 12, wherein the protein comprises at least one amphoteric helix containing apolipoprotein. The structure of claim 1, wherein component X comprises at least one apolipoprotein A-I, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein E, analogs or variants thereof. 17. The construct of claim 1 and / or 16, wherein the analog or variant is capable of eliciting physiological responses substantially the same as apolipoprotein A-I, apolipoprotein A-II, and apolipoprotein A-IV. The structure of claim 1, wherein the amino acids constituting component X are cysteine residues. 20. The construct of claim 19 wherein the cysteine residue is linked N-terminally or C-terminally to the apo-A component. 13. The peptide of claim 12 wherein the peptide constituting component X comprises more than one amino acid, such as more than two amino acids, such as more than five amino acids, such as more than ten amino acids, such as more than 15 Amino acids, such as more than 20 amino acids, such as more than 30 amino acids, such as more than 40 amino acids, such as more than 50 amino acids, such as more than 75 amino acids, such as more than 100 amino acids, Eg more than 200 amino acids, such as more than 300 amino acids, such as more than 400 amino acids, such as more than 500 amino acids, such as more than 600 amino acids, such as more than 700 amino acids, such as 800 More than amino acids, such as more than 900 amino acids, such as more than 1000 amino acids, such as, 1250 In excess of amino acids such as amino acids in excess of 1500, for example, amino acids in excess of 2000, or for example structure, including an amino acid in excess of 2500. The structure of claim 1, wherein the oligomerization module is a dimerization module. The structure of claim 1, wherein the oligomerization module is a trimerization module. The structure of claim 1, wherein the oligomerization module is a tetramerization module. The structure of claim 1, wherein the oligomerization module is a multimerization module. The construct of claim 1 wherein the oligomerization module is from a non-peptide. 24. The construct of claim 23 wherein the trimerization module comprises an amino acid sequence capable of mediation, trimerization, and alignment of interchain recognition of three polypeptide chains. The structure of claim 23, wherein the trimerized module is derived from tetranectin. The structure of claim 23, wherein the trimerization module comprises tetranectin trimerization module from tetranectin. The structure of claim 29, wherein the tetranectin trimerization module can form a stable complex with another tetranectin trimerization module. 31. The structure of claim 30, wherein the stable composite comprises a coiled coil structure. 32. The structure of claim 31, wherein the coiled coil structure is a triple alpha helical coiled coil. 33. The method of claim 27, wherein the trimerization module comprises two tetranectin trimerization modules joined by spacer portions, wherein the spacer portion causes both tetranectin trimerization modules to form part of the apolipoprotein structure. And a structure that allows participation in forming a complex with a third tetranectin trimerization module. 34. The construct of claims 28-33, wherein at least one tetranectin trimerization module is selected from human tetranectin, rat tetranectin, human, rat or shark cartilage C-type lectin. 35. The construct of claims 28-34, wherein the tetranectin trimerization module comprises a sequence having at least 68% identity with the consensus sequence of SEQ ID NO 12. The construct of claim 35, wherein the sequence identity with the consensus sequence is at least 75%, such as at least 81%, such as at least 87%, such as at least 92%. The method of claim 35, wherein the cysteine residue no. A structure wherein 50 is substituted by a serine residue, a threonine residue or a methionine residue. The method of claim 35, wherein the cysteine residue no. A structure wherein 50 is substituted with another amino acid residue. 36. The method according to claim 35, wherein the trimmerization module has at least 68% sequence identity, preferably at least 75%, such as at least 81%, such as at least 87%, such as at least 92%, with the Trip A module (SEQ ID NO 13). Having a structure. 24. The structure of claim 23 comprising a trimmerization module from a collectin neck region. The structure of claim 1, wherein the oligomerization module is presented after purification of the apolipoprotein structure. The structure of claim 1, further comprising at least one carbohydrate moiety. The structure of claim 1, further comprising an affinity tag. 44. The construct of claim 43 comprising a polyHis affinity tag. 44. The construct of claim 43 comprising an affinity tag selected from an antigenic tag, a GST tag. The method according to claim 1, wherein the half-life is equal to, preferably at least more than two times, more preferably more than at least three times, such as at least four times, the half-life of native Apo AI, A-II, or A-IV. , More preferably greater than at least 5 times, such as greater than 6 times, more preferably greater than at least 8 times, such as greater than 10 times. The structure of claim 1 having a higher binding affinity for cholesterol as compared to native Apo A-I, A-II, or A-IV. The method of claim 1, wherein the cuvlin, scavenger receptor class B, type 1 (SRB1), ATP-binding cassette 1 (ABC1), lecithin: cholesterol acyltransferase (LCAT), cholesteryl-ester transfer protein (CETP) ), A structure capable of binding to a receptor selected from phospholipid transport proteins (PLTP). The construct of claim 1 which does not actually give rise to an immune response in humans. The construct of claim 1 wherein the nucleic acid sequence comprises a DNA, RNA, PNA or LNA sequence. The construct of claim 1 which shares at least 70% sequence identity with any one of SEQ ID NO 2 to SEQ ID NO 11, or SEQ ID NO 14. The construct of claim 1 having an amino acid sequence that shares at least 70% sequence identity to any one of SEQ ID NO 3 to SEQ ID NO 11, or SEQ ID NO 14. 8. Apolipoprotein constructs having the general formula: apo-A-X Wherein apo-A is an apolipoprotein component selected from apolipoprotein AI, apolipoprotein AII, apolipoprotein AIV, analogs or variants thereof, X is a heterologous moiety selected from an oligomerization module and an apolipoprotein linked to a terminus. 54. The construct of claim 53, further comprising a spacer peptide between the apo-A component and X. 55. The method of claim 54, wherein the spacer peptide comprises at least two amino acids, such as at least three amino acids, such as at least five amino acids, such as at least 10 amino acids, such as at least 15 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, For example at least 40 amino acids, such as at least 50 amino acids, such as at least 60 amino acids, such as at least 70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as about 100 amino acids. 55. The structure of claim 54, wherein the spacer is connected to X with the apo-A component through a covalent bond. 55. The structure of claim 54, wherein the spacer is essentially non-immunogenic, or does not tend to be proteolytic, or contains no cysteine residues. 55. The structure of claim 54, wherein the three dimensional structure of the spacer is linear or substantially linear. 55. The spacer peptide of claim 54, wherein the spacer peptide is the amino acid sequence GTKVHMK from tetranectin, the amino acid sequences PGTSGQQPSVGQQ and GTSGQ from ligation strand 3 from human fibronectin, PKPSTPPGSS, SGGTSGSTSGTGST, AGSSTGSSTAPGSTT or GGSGG from the mouse IgG3. Including the structures. 54. The construct of claim 53, wherein component X comprises at least one amphoteric helix containing apolipoprotein. 54. The construct of claim 53, wherein the apolipoprotein comprises at least one apolipoprotein A-I, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein E, analogs or variants thereof. 62. The construct of claim 53 or 61, wherein the analog or variant is capable of eliciting a physiological response that is substantially the same as apolipoprotein A-I, apolipoprotein A-II, and apolipoprotein A-IV. 54. The structure of claim 53, wherein the oligomerization module is a dimerization module. 54. The structure of claim 53, wherein the oligomerization module is a trimerization module. 54. The structure of claim 53, wherein the oligomerization module is a tetramerization module. 54. The structure of claim 53, wherein the oligomerization module is a multimerization module. 54. The construct of claim 53 wherein the oligomerization module is from a non-peptide such as a nucleic acid sequence comprising, for example, a DNA, RNA, PNA, or LNA sequence. 65. The construct of claim 64, wherein the trimerization module comprises an amino acid sequence capable of mediation, trimerization, and alignment of interchain recognition of three polypeptide chains. 65. The construct of claim 64, wherein the trimerization module is derived from tetranectin. 65. The structure of claim 64, wherein the trimerization module comprises tetranectin trimerization module from tetranectin. The structure of claim 70, wherein the tetranectin trimerization module is a tetranectin trimerization module capable of forming a stable complex with another tetranectin trimerization module. 72. The structure of claim 71, wherein the stable composite comprises a coiled coil structure. The structure of claim 72, wherein the coiled coil structure is a triple alpha helical coiled coil. 74. The method of claims 69-73, wherein the trimerization module comprises two tetranectin trimerization modules joined by spacer portions, wherein the spacer portion causes both tetranectin trimerization modules to form part of the apolipoprotein structure. And a structure that allows participation in forming a complex with a third tetranectin trimerization module. 75. The construct of claim 69-74, wherein the at least one tetranectin trimerization module is selected from human tetranectin, rat tetranectin, human, rat or shark cartilage C-type lectin. 76. The construct of claim 69-75 wherein the tetranectin trimerization module comprises a sequence having at least 68% identity with the consensus sequence of SEQ ID NO 12. 77. The construct of claim 76 wherein the sequence identity with the consensus sequence is at least 75%, such as at least 81%, such as at least 87%, such as at least 92%. 77. The method of claim 76, wherein the cysteine residue no. A structure wherein 50 is substituted by a serine residue, a threonine residue or a methionine residue. 77. The method of claim 76, wherein the cysteine residue no. A structure wherein 50 is substituted with another amino acid residue. 77. The process of claim 76, wherein the trimmerization module has at least 68% sequence identity, preferably at least 75%, such as at least 81%, such as at least 87%, such as at least 92%, with the Trip A module (SEQ ID NO 13) Having a structure. 65. The construct of claim 64 comprising a trimmerization module from a collectin neck region. 54. The structure of claim 53, further comprising at least one carbohydrate moiety. 54. The construct of claim 53 further comprising an affinity tag selected from affinity tags selected from among polyHis affinity tags, antigenic tags, and GST tags. 55. The construct of claim 53 having an amino acid sequence that shares at least 70% sequence identity with any one of SEQ ID NOs to SEQ ID NOs 11, or SEQ ID NOs 14. A nucleic acid comprising a nucleotide sequence encoding the apolipoprotein structure as defined in claims 1 to 84. 86. The method of claim 85, wherein at least 70% of the sequence of any one of SEQ ID NO 2 to SEQ ID NO 11 or SEQ ID NO 14, preferably of SEQ ID NO 3 to SEQ ID NO 11 or SEQ ID NO 14 Nucleic acid encoding an amino acid sequence that shares sequence identity. 86. The nucleic acid of claim 85 wherein the coding sequence is operabaly linked to regulatory sequences for expression of the protein construct. 87. A vector comprising the nucleic acid of claim 85 or 87. A transformed host cell comprising the nucleic acid sequence defined in claim 85 or 87. 84. A method for producing an apolipoprotein structure as defined in claims 1 to 84, comprising the following steps. Incubating the transformed host cell in the presence of promoting expression of the protein construct according to claims 1 to 84, Obtaining and recovering the protein construct, Optionally, the protein structure is further modified. 84. A method for preparing an apolipoprotein structure as defined in claims 1 to 84, comprising the following steps: Chemically synthesizing at least one oligomerization module, and then Connecting the module with at least one apolipoprotein, an apolipoprotein analog or an apolipoprotein variant, After obtaining the apolipoprotein structure, The resulting apolipoprotein structure is isolated, Optionally further modifying the structure. 84. A method for preparing an apolipoprotein structure as defined in claims 1 to 84, comprising the following steps: Cultivating the transformed host cell under conditions that promote expression of the apolipoprotein or apolipoprotein analog or apolipoprotein variant encoded by the nucleic acid fragment, and then Covalently linking said apolipoprotein or apolipoprotein analog or apolipoprotein variant to a heterologous moiety, Obtaining apolipoprotein structures, The resulting apolipoprotein structure is isolated, Optionally further modifying the structure. 84. A method for preparing an apolipoprotein structure as defined in claims 1 to 84, comprising the following steps: -Culturing the transformed host cell under conditions that facilitate expression of the oligomerization module encoded by the nucleic acid fragment, and then Covalently linking said module with at least one apolipoprotein, an apolipoprotein analog or an apolipoprotein variant, Obtaining apolipoprotein structures, After isolation of the resulting apolipoprotein structure, Optionally further modifying the structure. Use of an apolipoprotein structure as defined in claims 1 to 50 in the manufacture of a pharmaceutical composition. 95. The use of claim 94, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, adjuvant, additive, such as phospholipid, cholesterol or triglyceride. 98. The pharmaceutical composition of claim 94 or 95, wherein the pharmaceutical composition is intravenous, intraarterial, intramuscular, percutaneous, pulmonary, subcutaneous, intradermal, intradural, or through cheeks, anus, vagina, conjunctiva, or intranasal tissue, or Use inoculated or implanted into a tissue, such as tumor tissue, or administered orally. 95. The composition of claim 94, wherein the composition contains at least 50 mg of the apolipoprotein structure to the subject per week, preferably at least 100 mg / week, such as at least 250 mg / week, such as at least 500 mg / week, such as at least 750 mg. A composition containing in an amount per week, such as at least 1000 mg / week, such as at least 1250 mg / week, such as at least 1500 mg / week, such as at least 2000 mg / week, such as at least 2500 mg / week, such as at least 5000 mg / week Use comprising administering. 95. The use of claim 94 for administration of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or up to 10 days. 95. The method of claim 94, wherein at least 10 mg / kg body weight, such as at least 20 mg / kg body weight, such as at least 30 mg / kg body weight, such as at least 40 mg / kg body weight, such as at least 50 mg / kg body weight, such as at least 60 mg / kg body weight, such as at least 70 mg / kg body weight, such as at least 75 mg / kg body weight, such as at least 90 mg / kg body weight, such as at least 100 mg / kg body weight, such as at least 125 mg / kg body weight, such as at least 150 mg / kg body weight For example at least 200 mg / kg body weight, such as at least 250 mg / kg body weight, such as at least 300 mg / kg body weight, such as at least 400 mg / kg body weight, such as at least 500 mg / kg body weight, such as at least 600 mg / kg body weight, such as Use in an amount of at least 700 mg / kg body weight, such as at least 800 mg / kg body weight, such as at least 900 mg / kg body weight, such as at least 1000 mg / kg body weight. 95. The use of claim 94, wherein the dosage of the pharmaceutical composition is administered once a week. 95. The use of claim 94, wherein the pharmaceutical composition is administered once every two weeks, or once every three weeks, or once every four weeks. 95. The use of claim 94 for the treatment and / or prevention of atherosclerosis. 95. The use of claim 94 for removing endotoxins. 95. The use of claim 94 for treating angina. 95. The use of claim 94 for treating myocardial infarction. 95. The use of claim 94 for treating plaque angina. 95. The use of claim 94 for treating unstable angina. 95. The use of claim 94 for use in the treatment of arterial stenosis, such as carotid artery stenosis, claudication, or cerebral artery stenosis. Use of a nucleic acid sequence as defined in claims 85-87 for gene therapy, wherein the DNA sequence encoding the apolipoprotein structure is used to transfect or infect at least one cell population. . 109. The use of claim 109, wherein the at least one cell terminal comprises macrophages. 109. The use of claim 109, wherein the at least one cell population comprises liver cells.