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Title:  Malaria polypeptides
United States Patent: 
7,125,958
Issued:  October 24, 2006

Inventors:  Wahlgren; Mats (Stocksund, SE), Barragan; Antonio (Huddinge, SE), Carlson; Johan (Stockholm, SE), Qijun; Chen (Stockholm, SE), Fernandez; Victor (Stockholm, SE)
Assignee: 
Karolinska Innovations AB (Stockholm, SE)
Appl. No.: 
09/508,967
Filed: 
September 18, 1998
PCT Filed: 
September 18, 1998
PCT No.: 
PCT/SE98/01675
371(c)(1),(2),(4) Date: 
April 07, 2000
PCT Pub. No.: 
WO99/15557
PCT Pub. Date: 
April 01, 1999


 

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Abstract

The present invention relates to carbohydrates capable of acting as receptors for malaria antigens present on the surfaces of malaria infected erythrocytes. The receptors according to the invention comprises negatively charged glycosaminoglycan-like moities, preferably sulphated. The invention also relates to novel malaria polypeptides capable of acting as ligands in relation to the receptors according to the invention. The invention also encompasses the use thereof as medicaments, pharmaceutical compositions containing the same as well as antibodies directed against said new ligands.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Thus, in a first aspect, the present invention relates to a carbohydrate, which exhibits at least one negatively charged glycosaminoglycan-like moiety and thereby is capable of essentially specific binding to a malaria erythrocyte membrane protein or an analogue thereof. Accordingly, the carbohydrate according to the invention may be used as a receptor to bind ligands, which are expressed on the surface of cells infected by malaria, such as erythrocytes infected by Plasmodium falciparum. In a preferred embodiment, said glycosaminoglycan moiety is sulfated and more specifically, it may be a heparan sulfate moiety. Most preferably, the carbohydrate according to the invention is fucoidan, or a functional fragment thereof. Further, the invention also relates to any functionally equivalent analogues of the above described carbohydrates.

Indeed, it has been shown in the prior art that P. falciparum-rosettes may be disrupted by low concentrations of heparin, an effect that is immediate and reversible (Carlson, J., H. P. Ekre, H. Helmby, J. Gysin, B. M. Greenwood, and M. Wahlgren. 1992. Disruption of Plasmodium falciparum erythrocyte rosettes by standard heparin and heparin devoid of anticoagulant activity. Am, J. Trop. Med. Hyg. 46, 595 602, Rowe, A., A. R. Berendt, K. Marsh, and C. I. Newbold. 1994. Plasmodium falciparum: a family of sulfated glycoconjugates disrupts erythrocyte rosettes. Exp. Parasitol. 79, 506 16). However, prior to the present invention, it has not been possible to explain the mechanism or basis for this phenomena and, accordingy, it has not been possible to create any such advantageous and useful receptors as the present receptor carbohydrate until now. Thus, for the first time, the present invention discloses receptors capable of essentially specific binding of such malaria proteins.

The glycosaminoglycans, or GAGs, being the receptor carbohydrate according to the invention, are composed of repeated units of sulfated or acetylated di-saccharides and are present in the human body bound to a protein-core in the form of proteoglycans (PG) (Yanagisha, M. and V. Hascall. 1992. Cell surface heparan sulfate proteoglycans. J. Biol. Chem. 267, 9451 9454). Heparan sulfate and chondroitin sulfate are GAGs exposed at all human cell-surfaces, but they are diverse and vary from cell-to-cell and maybe within the same cell due to secondary modifications of the extensive carbohydrate chains (deacetylations, O-sulfations etc.). Heparin is only found in mast-cells and is characterized by a higher degree of sulfation and epimerization than heparan sulfate. However, heparan sulfate and heparin are similar due to heparin-like stretches also found in heparan sulfate (Faham, S., E. R. Hileman, R. J. Fromm, J. R. Lindardt, and C. D. Rees. 1996. Heparin structure and interactions with basic fibroblast growth factor. Science, 271, 1116 1120).

HS and heparin, both members of the heterogeneous GAG family, are composed of alternating glucosamine and uronic acid residues. The glucosamine can be either N-sulfated or N-acetylated and can contain an O-sulfate ester at C-6. The uronic acid exists as either a glucoronic or an iduronic acid epimer and may be O-sulfated at C-2. In contrast to heparin chains which are extensively sulfated, HS chains are less extensively modified and the sulfate groups are unevenly distributed over the chains due to incompleteness of modification in each step of RS biosynthesis (Lindahl et al. 1994) Consequently, HS and heparin are similar due to highly sulfated, heparin-like stretches found in HS.

In general, micro-heterogeneity within the structure of GAGs modulates their binding and biological activities (Casu 1991). Their binding properties are usually associated with their anionic sulfate and carboxyl groups. Increasing charge density and molecular weight is often associated with stronger protein binding, but this is not a strict rule (Ruoslahti 1989). Thus, a general increase in binding properties can be achieved by chemical oversulfation (Casu 1991). Most GAGs express their biological properties through interactions with plasma and tissue components (Jackson et al. 1991; Kjellen et al. 1991) and their ubiquitous distribution on cell surfaces make them well suited for microbial attachment.

In a particular embodiment, the receptor carbohydrate, or said functional equivalent thereof, is more specifically capable of essentially specific binding to at least one of the segments denoted binding sites of the amino acid sequence according to SEQ ID NO:1, wherein a novel variant of Plasmodium falciparum erythrocyte membrane protein (PfEMP1) is disclosed. Even though it is more fully discussed elsewhere in this application, especially in connection with the ligand polypeptide below, it should be understood that said binding segments are sequences, which, according to the present invention, have been found to bind specifically and strongly to glycosaminoglycan-like moieties on receptor substances, such as the ones present on most cells or the present polypeptide. Thus, in a specific embodiment, the receptor carbohydrate according to the invention is capable of binding to an amino terminal part, and preferably an essential part, of the binding segments indicated in SEQ ID NO:1. Consequently, the protein disclosed in SEQ ID NO:1 may be regarded as a ligand, whereas the carbohydrate defined as above may be regarded as the receptor therefore. Most preferably, the receptor binds to an essential part of said sequence or any sequence having a substantial identity or similarity with SEQ ID NO:1.

The carbohydrate receptor according to the present invention may be prepared by fractionation from animal cells or by conventional methods, which are well known in the art (see e.g. Binkley: Modern Carbohydrate Chemistry, Marcel Dekker, New York, 1988, with references; and U.S. Pat. Nos. 5,308,460 and 5,532,147).

In another aspect, the present invention relates to a carbohydrate as defined above for use as a medicament as well as the use of said receptor as a medicament. Such a medicament is effective in that it is capable of effectively dissolving the rosettes formed by erythrocytes infected by a malaria parasite, as described more detailed in the introductory section of this application. Thereby, a medicament comprising the receptor carbohydrate according to the invention in a suitable form will be effective in dissolving and preventing the occlusion of blood vessels, especially in cerebral malaria. Accordingly, the invention also encompasses the use of said carbohydrate in the manufacture of a medicament against malaria, preferably against P. falciparum malaria Thus, in one embodiment, the invention relates to a pharmaceutical composition comprising a carbohydrate according to the invention ill a pharmaceutically or veterinary acceptable carrier. Other additives or excipients which are deemed suitable for the specific application may also be present in such a composition. Further, the present invention also relates to any other substance, which has been achieved by use of parts or all of the novel polypeptide defined in SEQ ID NO:1, or, alternatively, by use of a carbohydrate according to the invention as a lead substance, for use as medicaments, for use in the manufacture of pharmaceutical preparations and pharmaceutical compositions comprising such a substance together with a carrier. Methods for producing such substances, which are functionally equivalent to the present carbohydrates, will be discussed in more detail below.

One more aspect relating to the above defined carbohydrate, or malaria protein receptor substance, is a method of treating a patient suffering from a malaria infection, preferably a P. falciparum infection. Such a method comprises administering to the patient of an effective amount of the pharmaceutical composition described in detail elsewhere in the present description.

Another aspect of the present invention is a polypeptide, which is capable of acting as a ligand in relation to the receptor carbohydrate described above. Said ligand polypeptide originates from a malaria erythrocyte membrane protein and may be denoted a novel PfEMP1-variant.

Thus, by single-cell RT-PCR, the present inventor has identified a novel PfEMP1-variant of a resetting parasite. Clusters of GAG-binding motifs have been identified in the sequence, denoted binding sites in the sequence listing below. In addition, it has been shown that the recombinant form of the novel PfEMP1-variant, or ligand polypeptide, according to the invention, adheres to solid-phase heparin, to heparan sulfate on the erythrocyte surface, and disrupts rosettes. Further, naturally formed rosettes also seem to be mediated by binding to the same GAG.

In an advantageous embodiment, the ligand polypeptide according to the invention comprises at least about 300 amino acids of the amino acid sequence according to SEQ ID NO:1. Preferably, the ligand comprises an amino terminal sequence of said sequence, or an analogue thereof. More preferably, the ligand according to the invention comprises about 400 500 amino acids, and most preferably about 400 amino acids (DBL-1), such as about 423 amino acids, thereof. In addition, the ligand polypeptide according to the invention is capable of essentially specific binding to a negatively charged glycosaminoglycan-like moiety. In the preferred embodiment thereof, the ligand polypeptide according to the invention is capable of binding any receptor carbohydrate according to the invention and described above, In a particular embodiment thereof, the polypeptide according to the invention comprises essentially all of the sequence according to SEQ ID NO:1, wherein the complete amino acid sequence of FCR3S1.2 PfEMP1 is shown. The location of potential GAG binding motifs are shown in pink. Motifs no. 4, 5 and 9, 10 (aa 221 232 and 533 549, respectively) are seen as a single stretch as they are located next to each other (see methods section for description of identification of GAG-binding motifs). These sequence data are available from GenBank under accession number of AF003473.

The weight of the polypeptide according to the invention will depend on the length of the amino acid sequence as discussed above but may be about 100 300 kDa, preferably about 280 kDa. Further, the present invention also relates to any biologically active fragments of the herein disclosed polypeptides and proteins.

Another aspect of the invention is a method of pr paring a ligand polypeptide as defined herein, which comprises the steps of (1) the inserting into an expression vector of a nucleic acid encoding said ligand polypeptide or functionally equivalent analogue thereof; (2) the transfection of a host cell capable of expressing said nucleic acid with said expression vector to express said polypeptide; and (3) the recovery of the expressed polypeptide and advantageously purification thereof to a biologically pure form. Expression vectors, host cells, process parameters etc. are easily chosen by someone skilled in this area.

Accordingly, a further aspect of the invention is vectors on host cells useful in the above disclosed method and comprising nucleic acids encoding a polypeptide according to the invention.

In one particular embodiment of the invention, the polypeptide may be cloned using DNA amplification methods, such as the polymerase chain method (PCR) (see e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbour, N.Y., 1989; Berger & Kiel, Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, Calif., 1987; Co et al (1992) J. Immunol. 148:1149). Thus, for example, the nucleic acid sequence, which will be disclosed in more detail below, or subsequence is PCR amplified using a sense primer containing one restriction site and an antisense primer containing another restriction site. This will produce a nucleic acid encoding the desired sequence or subsequence having terminal restriction sites. This nucleic acid can then easily be ligated into a vector having appropriate corresponding restriction sites. Suitable PCR primers are easily chosen by one of skill in the art based on the sequence to be expressed. Appropriate restriction sites can also be added by site-directed mutagenesis (see Gillman & Smith (1979) Gene, 8: 81 97; Roberts et al. (1987) Nature 328: 731 734).

The nucleic acids according to the invention, which are described in more detail below, may be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast and various higher eucaryotic cells, such as the COS, CHO and HeLa cell lines and myeloma cell lines. The recombinant protein gene will be operably linked to appropriate expression control sequences for each host. For E. coli, this includes a promoter, such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eucaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences. The plasmids can be transferred into the chosen host cell by well-known methods, such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as amp, gpt, neo and hyg genes.

Once expressed, the recombinant polypeptides according to the invention may be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see generally R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutcher, Methods in Enzymology vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y. (1990)). Substantially pure compositions are preferred, such as at least about 90 95% homogeneity, and most preferred, 98 99% homogeneity. Once purified, partially or to the homogeneity as desired, the polypeptides according to the invention may be used, e.g. as immunogens for antibodyproduction.

As one of skill in this field would recognize, after chemical synthesis, biological expression, or purification, the proteins, polypeptides or fission proteins according to the present invention may possess a conformation substantially different from the native conformations of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (see e.g. Debinski et al. (1993) J. Biol. Chem., 268; 14065 14070; Kreitman and Pastan (1993) Bioconj. Chem., 4:581 585; and Buchner et al. (1992) Anal. Biochem., 205:263 270).

One of skill would recognize that modifications can be made to the present polypeptides and proteins without diminishing their biological activity, e.g. in order to facilitate cloning, expression or incorporation of the targeting molecule into a fusion protein. Such modifications are well known and any thus modified peptides and proteins are understood to be encompassed within the scope of the present invention as defined by the appended claims.

Further, the present invention also relates to vectors and recombinant host cells comprising the sequence according to SEQ ID NO:1 or any biologically active fragment thereof. In addition, the invention also encompasses any isolated cell comprising the sequence according to SEQ ID NO:1 as an endogenous gene, which has been manipulated by gene activation, that is, wherein additional regulatory sequences have been introduced in order to increase the expression of the native coding sequence (see e.g. U.S. Pat. No. 5,578,461 and U.S. Pat. No. 5,641,670). The culture of cells according to the present invention is well known in the art (see e.g. Freshney, Culture of Animal Cells, A Manual of Basic Technique, third ed., Wiley-Liss, New York (1994) and the references cited therein for a general guide to the culture of cells).

As indicated above, the present invention also relates to nucleic acids encoding the polypeptides according to the invention as well as to any nucleic acid capable of specific hybridization, under stringent conditions, to such a nucleic acid. The nucleic acids according to the invention thus include, but are not limited to, DNAs, RNAs, an mRNA transcript, a cDNA reverse transcribed from a mRNA, and RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA or subsequences of any of these nucleic acids and may be prepared by any suitable method, which is easily chosen by someone skilled in this field. Such methods include, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method (Narang et al., Meth. Enymol. 68:90 99 (1979)); the phosphodiester method (Brown et al., Meth. Enzymol. 68: 109 151 (1979)); the diethylphpsphoramidite method (Beaucage et al., Tetra. Lett., 22:1859 1862 (1981)); and the solid support method (U.S. Pat. No. 4,458,066).

Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence or by polymerisation with a DNA polymerase using the single strand as a template. The DNA sequence obtained by chemical synthesis is limited in length by the technique used, however, longer sequences are easily obtained by the ligation of such shorter sequences to the desired length. Alternatively, subsequences may be cloned and the appropriate subsequences cleaved using suitiable restriction enzymes. The fragments may then be ligated to produce DNA sequences of the desired size.

A nucleic acid according to the invention may e.g. be used in the above method for preparing a polypeptide, as a probe etc. For the use thereof as a probe, it is often desirable to label the sequence with a detectable label. The label may be incorporated during the amplification step in the preparation of the nucleic acid, e.g. by PCR, by transcription amplification using a labelled nucleotide incorporations a label in the transcribed nucleic acid or, alternatively, by direct addition thereof, such as by nick-translation or any other suitable method Detectable labels suitable for use according to the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means and methods for the detection thereof are well known to those of skill in the art.

In an alternative aspect of the invention, the polypeptide is produced in the form of a recombinant fusion protein, which comprises said polypeptide, comprising suitable glycosaminglycan-like moieties, fused to another protein or polypeptide. Such a polypeptide comprising fusion protein may be more advantageous for certain applications than the polypeptide per se. In a preferred embodiment of the invention, the fusion proteins are synthesized using recombinant nucleic acid methodology. Generally, this involves creating a nucleic acid that encodes the receptor-targeted fission molecule, placing the nucleic acid in an expression cassette under the control of a suitable promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein. Techniques sufficient to guide one of skill through such procedures are found in e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2.sup.nd ed., vol. 1 3, Cold Spring Harbor Laboratory, 1989; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, vol. 152, Academic Press, Inc., San Diego, Calif.; and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols (Greene Publishing Associates, Inc. And John Wiley & Sons, Inc.) (1994 Supplement). Alternatively, the fusion protein is produced by any method for chemical synthesis well known in the art. Thus, a fusion protein according to the invention may comprise any polypeptide, antibody or biologically active fragment thereof according to the present invention. The effects of exemplary fusion proteins on rosetting is e.g. discussed in the Experimental section of this specification, see e.g. FIG. 4.

The present invention also relates to a polypeptide according to the invention for use as a medicament as well as the use thereof in the manufacture of a medicament. Such a medicament is effective in that it is capable of effectively dissolving the rosettes formed by erythrocytes infected by a malaria parasite, as described more detailed in the introductory section of this application. Thereby, a medicament comprising the polypeptide according to the invention, per se or in the form of a fusion protein, will be effective in dissolving and preventing the occlusion of blood vessels, esecially in cerebral malaria. In addition, the above described rosettes have also been shown to be associated with other severe complications of malaria (Carlson, J., et al, Lancet 336, 1457 1460 (1990); Treutiger et al, Am. J. Trop. Med. Hyg. 46, 503 510 (1992); and Rowe, A et al, Inf. Immun 63, 2323 2326 (1995)). Thus, a medicament comprising a polypeptide according to the invention is also effective in the treatment of such other conditions. Accordingly, the invention also encompasses the use of said polypeptide in the manufacture of a medicament against malaria, preferably P. falciparum malaria, and/or other severe complications of malaria. Consequently, in a further aspect, the invention relates to a pharmaceutical preparation comprising said polypeptide or fusion protein in a pharmaceutically or veterinary acceptable carrier, e.g. an aqueous carrier, such as buffered saline or the like. These solutions are sterile and generally free of any undesirable matter. They may be sterilized by conventional, well known sterilization techniques. The pharmaceutical preparations according to the invention may be used in any suitable method of administration, such as in parenteral, topical, oral or even local administration for prophylactic and/or therapeutic treatment and may be administered in a variety of unit dosage forms depending on the administration method chosen. Further, the pharmaceutical compositions according to the invention may comprise any pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate etc. Actual methods for preparing the appropriate administrable compositions will be known or apparent to those skilled in this field and are described in more detail in e.g. Remington's Pharmaceutical Science, 15.sup.th ed., Mack Publishing Company, Easton, Pa. (1980). (For a brief review of methods for drug delivery, see Langer, Science 249:1527 1533 1990)).

Accordingly, another aspect of the present invention is a method of treating a patient suffering from a malaria infection by the administration of the present polypeptide or a fusion protein or, alternatively, preventing malaria in a subject at risk of being infected thereof. It is to be understood, that in the present context, the term "preventing" refers to the prevention of a disorder, such as malaria, or the symptoms thereof, by use of a medicament as well as such a prevention by use of a vaccine composition. Thus, the present invention encompass preventive or prophylactic treatments and medicaments used therein, which treatments will include drugs that actively participate in the prevention of a particular disorder, such as malaria or the symptoms thereof. Further, the invention also encompases treatments that elicit a preventive response from a patient, such as an immunological response in the case of vaccination. Consequently, "a medicament for the prevention of malaria" includes also includes vaccine compositions. The method comprises administering to said patient of an effective amount of the herein described pharmaceutical composition, said amount being dependent on the severity of the disease and the general state of the health of the patient. Preferably, the malaria infection treated by the method according to the invention is a P. falciparum infection.

In the preferred embodiment, the vaccine compositions according to the invention will include a molecule according to the invention, such as a polypeptide, in an immunologically effective amount together with a suitable pharmaceutically acceptable carrier. In this context, examples of suitable carriers are e.g. thyroglobulin, albumins, such as human serum albumin, tetanus toxoid, polyamino acids, such as poly(D-lysine; D-glutamic acid), influenza, hepatitis B virus core protein as well as other carriers well known to those skilled in the art. The vaccines may also include a physiologically tolerable diluent, such as water, buffered water, buffered saline, saline and they may further also include any suitable adjuvant, such as incomplete Freunds' adjuvant, aluminium phosphate, aluminium hydroxide, alum, ammonium hydroxide in case of human pateient, etc. The immune response of the patient may include generation of antibodies, activation of cytotoxic T-lymphocytes against cells expressing the polypeptides or other mechanisms known within this field. (See e.g. Paul, Fundamental Immunology, 2.sup.nd ed., Raven Press; Sedagh et al., Proc. Natl. Acad. Sci. (1994) 91:9866 9877; U.S. Pat. No. 4,722,848; and Langford, C. L. et al., 1986, Mol. Cell. Biol. 6:3191 3199.)

Another highly interesting and advantageous aspect of the present invention is the use of the polypeptide as defined above as a model substance for identifying substances binding to a malaria erythrocyte membrane protein, such as the novel variant disclosed in SEQ ID NO:1, or analogues thereof. Thereby, new receptor substances effective in the treatment and/or prevention of malaria infections may be isolated from suitable environments or bulks comprising the same.

More specifically, a polypeptide according to the invention, or a fragment thereof is used in the design of an organic compound, which is modeled to resemble the three dimensional structure of the amino acid residues of said polypeptide. Such a design of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property. In relation to mimetics, or mimics, as they are sometimes denoted, see for example Shikman A. R. and Cunningham M. W., J. Immunol. 152(9):4375 (1994); Vaughan et al., Xenotransplantation 3:18 23 (1996); and Koogman et al, Dean (1994), BioEssays, 683 687; Cohen and Shatzmiller (1993), J. Mol. Graph., 11: 166 173; Wiley and Rich (1993), Med. Res. Rev., 13:327 384; Moore (1994), Trends Pharmacol. Sci., 15:124 129; Hruby (1993), Biopolymers, 33: 1073 1082; Bugg et al. (1993), Sci. Am., 269:92 98.

In brief, the particular parts of the compound that are critical and/or important are firstly determined. This can be done systematically by varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore". Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.

In a specific embodiment, the three dimensional structure of the ligand and its binding partner are modelled This can be especially useful where the ligand and/or binding partner (receptor) change conformation on binding, allowing the model to take account of this in the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic(s) found by this approach can then be screened to see whether or not they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing. Accordingly, the use of the present polypeptide ligand and/or carbohydrate binding partner as models to identify optimal pharmaceutically active substances, preferably for preventing and/or treating malaria, is encompassed by the present invention as are any substances identified thereby, which fulfills the herein desired criteria.

In another aspect, the invention relates to diagnostic and screening assays, wherein e.g. the polypeptides according to the invention are used for the detection of antibodies or wherein antibodies as disclosed below are used to detect PfEMP1 or fragments thereof. Further, nucleic acids may be detected in biological samples. (For a review of diagnostic immunoassay procedures, see e.g. Basic and Clinical Immunology, 7.sup.th ed., Stites, D., and Terr, A., 1991.)

One further aspect of the invention is an antibody, which is specifically immunoreactive with a ligand polypeptide according to the present invention. Such an antibody may be used to formulate another pharmaceutical composition together with suitable pharmaceutically and/or veterinary acceptable carriers, excipients etc. Accordingly, the present invention also relates to such antibody compositions as well as to methods of treating and/or preventing malaria infection in a patient. Such methods comprises administer to said patient of an effective amount of the pharmaceutical composition defined above. Most preferably, the infection to be treated and/or prevented is a P. falciparum infection.

Thus, antibodies are raised to the polypeptides of the present invention, including individual, ellelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these polypeptides in either their native configuarions or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill. (In this context, see e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y. 1993.) While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).

A number of immunogens are used to produce antibodies specifically reactive with polypeptides according to the invention. Recombinant or synthetic polypeptides according to the invention of 8 15, preferably 10, amino acids in length, or greater, are the preferred polypeptide immunogen (antigen) for the production of monoclonal or polyclonal antibodies.

Methods of producing polyclonal antibodies are known to those of skill in the art. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lan (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY.

Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified polypeptides, or screened for agonistic or antagonistic activity, e.g., activity mediated through a suppressor of fused protein. Specific monoclonal and polyclonal antibodies will usually bind with a K.sub.D of at least about 0.1 mM, more usually at least about 50 .mu.M, and most preferably at least about 1 .mu.M or better.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g. Stites et al. (eds) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., and references cited therin; Harlow and Lane, supra; Goding (1986) Monoclonal Antiboides: Principles and Practice (2nd ed.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature 256: 495497.

Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse et al. (1989) Science 246: 1275 1281; and Ward, et al. (1989) Nature 341 544-546; and Vaughan et al. (1996) Nature Biotechnology, 14: 309 314).

Advantageously, the polypetides and antibodies according to the invention are labelled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels, and conjugation techniques are known and are reported extensively in both the scientific and patent literature. See for example patents teaching the use of such labels, including U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

In a specific embodiment of the invention, the present antibodies are used for affinity chromatography in isolating polypeptides. Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles such as agarose. Sephadex, or the like, where a cell lysate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified polypeptides are released.

The antibodies can be used to screen expression libraries for particular expression products. Usually the antibodies in such a procedure are labelled with a moiety allowing easy detection of presence of antigen by antibody binding.

Antibodies raised against the present polypeptides can also be used to raise antiidiotypic antibodies. These are useful for detecting or diagnosing the various pathological conditions related to the presence of the respective antigens.

The antibodies of this invention can also be administered to an organism (e.g., a human patient) for therapeutic purposes. A large numer of methods of generating chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 4,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and 4,975,369). For references regarding human antibodies, see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, for review).

Human antibodies of the present invention are may e.g. be produced initially in trioma cells. The general approach for producing human antibodies by trioma technology has been described by Ostberg et al. (1983) Hybridoma 2: 361 367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al. U.S. Pat. No. 4,634,666.

As someone skilled in this field easily realises, the receptor carbohydrates, the ligand polypeptide and the antibodies according to the invention are also useful for a wide variety of diagnostic purposes, such as in immuno assays. Methods, other reagents, amounts etc are easily determined by someone skilled in the area on the basis of the information given in this application (for immuno assays, see e.g. Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbour Publications, New York).
 


Claim 1 of 6 Claims

1. An isolated polypeptide comprising SEQ ID NO: 1.

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