AU755574B2 - Synthetic peptide immunogens and antibodies thereto - Google Patents

Description

WO 01/00237 PCT/US00/17609 1 SYNTHETIC PEPTIDE IMMUNOGENS AND ANTIBODIES THERETO FIELD OF THE INVENTION The present invention generally relates to methods for selecting peptide immunogens to produce antibodies to a target protein which exhibits amino acid variation between species.

More specifically, the present invention relates to methods of selecting peptide immunogens and producing species-specific antibodies to a mammalian tissue factor, and antibodies produced by those methods.

DESCRIPTION OF RELATED ART Antibodies to protein antigens have been used for many years for numerous research and diagnostic purposes. Antibodies can be made to specific regions of a protein by injecting a small peptide portion of the protein (5-50 amino acids) which has been conjugated to an immunogenic carrier (such as keyhole limpet hemocyanin The immunized animal will recognize the peptide as an epitope of the immunogenic carrier. This approach has advantages over the more common methods involving the injection of the whole protein. For example, this approach allows antibody reagents to be made which bind to specific, predictable domains of the protein of interest. These antipeptide antibodies are useful for epitope mapping in structure/function studies, immunohistochemical localization, and immunoaffinity purification of the protein of interest. One particular protein of interest for this invention is mammalian tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein which plays a critical role in coagulation (blood clotting). Normal coagulation begins when vascular endothelium is damaged. Damaged vascular endothelium exposes TF to plasma, allowing TF to bind to factor VII, a serine protease zymogen. The binding of TF to factor VII, and subsequent activation to factor VIIa, is the primary step for coagulation initiation. Subsequent conformational changes and Ca2+ bridge formation allow the TF/FVIIa complex to bind to and to activate factor X. Activated WO 01/00237 PCT/US00/17609 2 factor X converts prothrombin into its active serine protease, thrombin. Thrombin converts fibrinogen into fibrin. Finally, fibrin fibers associate with one another to form a clot, or a platelet-fibrin network that prevents further bleeding.

The amino acid sequence of TF protein between mice, rabbits, and humans is highly conserved. Pawashe et al., 1991, Thromb.

Haemost. 66:315. The crystal structure of TF consists of two immunoglobulin-like domains with an extensive interdomain interface region which contains the binding site for factor VII.

Harlos et al., 1994, Nature 370:662.

TF is expressed in various tissues such as cardiac myocytes, renal glomeruli, the granular layer of the epidermis, the epithelium of oropharynx and vagina, and intestinal, urinary bladder, respiratory mucosa, and at tissue barriers between the body and the environment. Ruf et al., 1994, FASEB J. 8:385.

Tissue factor has also been implicated in the pathogenesis of various infectious, cardiovascular and neoplastic diseases.

Excessive coagulation, for example, leads to stroke and ischemia, resulting in infarction of tissues. Acute myeloblastic leukemias are known to express TF, resulting in disseminated intravascular coagulation (DIC), a disorder characterized by excessive bleeding due to rapid consumption of platelets and disruption of fibrin polymerization. Id. A better understanding of TF, therefore, allows researchers to design more effective therapies for controlling coagulation. Such control can potentially alleviate complications in patients suffering from these and other diseases.

Several antibodies have been made to TF. Most of these anti-TF antibodies were made to the whole TF protein. For example, monoclonal antibodies to native, factor VII-affinity purified human TF were used to study the essential regions of the extracellular domain of TF. Magdolen et al., 1998, Biological Chemistry 379:157. Other antibodies have been made to purified or recombinant TF from humans and rabbits. See, Taylor et al., 1991, Circulatory Shock 33:127; Morissey et el., 1988, Thrombosis Res. 52:247; Pawashe et al., 1994, Circulation Res.

74:56; U.S. Pat. App. 5,223,427. However, membrane-bound WO 01/00237 PCT/US00/17609 3 proteins such as TF are difficult to purify, especially in its native folding conformation. In another approach, monoclonal antibodies were made from mice injected with murine cultured cells expressing recombinant rabbit TF. Speidel et al., 1995, Circulation 92:3323.

Anti-peptide antibodies to TF has been made. In one case, polyclonal antibodies were made to human TF regions which allow them to inhibit TF binding to factor VII. Ruf et al., 1991, Biochem. J. 278:729. In another case, anti-peptide monoclonal antibodies were made to the C-terminal cytoplasmic domain of human TF. Carson et al., 1992, Blood Coagulation and Fibrinolysis 3:779.

Antibodies made to proteins such as TF which have extensive homology between species would not be expected to exhibit species-specificity. This has been shown to be the case with TF, where monoclonal antibodies to human tissue factor cross-react and inhibit baboon TF. Taylor et al., 1991, Circulation Shock 33:127. This could be a problem, where the antibodies are used to diagnose diseases and are required to distinguish between two pathogenic species. Proteins such as TF with extensive sequence homology among vertebrates are also limited in the range of antibodies they would be expected to induce. Since there is little sequence difference between the protein from the species of interest and the same protein in the immunizing animal, the immune system of the immunizing animal would recognize the homologous regions of the injected protein as a "self" epitope and would thus only produce antibodies to immunodominant regions of the injected protein which are not homologous with the injected animal's analogous protein. For example, murine monoclonal antibodies to whole human TF could only be produced to regions of the human TF which are dissimilar to mouse TF. If antibodies are desired to many regions of the TF protein, for example for structure-function studies, the results would likely be disappointing. Therefore, there is a need for improved methods for making antibodies to proteins which show extensive amino acid homology between species, where the antibodies need to WO 01/00237 PCT/US00/1 7609 4 be species-specific or where antibodies are desired to several regions of vertebrate proteins.

SUMMARY OF THE INVENTION Among the objects of the invention may be noted the provision of methods for determining peptides of a target protein for use as immunogens in order to reliably produce antibodies which are species-specific. A more specific object of the invention is the provision of methods for determining regions of tissue factor for use as immunogens to produce antibodies which are species-specific. Another object of the invention is the provision of antibodies to tissue factor made using the above methods.

Briefly, therefore, the present invention is directed to a method for selecting a peptide immunogen of a target protein from a first species, wherein the target protein comprises an amino acid sequence which varies between the first species and a second species. The method comprises (a)identifying peptide regions of the amino acid sequence of the target protein from the first species which has a hydrophilicity value greater than 0, wherein the regions are 5-50 amino acids in length, and selecting a peptide immunogen from regions identified in step which have at least 1 nonhomologous amino acid between the first species and the second species.

The present invention is also directed to a method of making an antibody which is specific for a target protein of a first species, wherein the target protein comprises an amino acid sequence which varies between the first species and the second species. The method comprises selecting a peptide immunogen of the target protein by the above-described method, synthesizing the peptide immunogen, conjugating the peptide immunogen to an immunogenic carrier molecule to make a peptide-carrier antigen, and producing antibodies to the antigen.

Additionally, the present invention is directed to an antibody made by the above-described method.

Furthermore, the present invention is directed to a peptide which consists of a sequence from a tissue factor extracellular WO 01/00237 PCT/US00/17609 region from a first species which is 5-50 amino acids in length, has a hydrophilicity value greater than 0, and has at least 1 nonhomologous amino acid from a tissue factor from a second species.

The present invention is also directed to an antigen, and antibodies made to an antigen, where the antigen comprises an immunogenic carrier molecule conjugated to a peptide which consists of a sequence from a tissue factor extracellular region from a first species which is 5-50 amino acids in length, has a hydrophilicity value greater than 0, and has at least 1 nonhomologous amino acid from a tissue factor from a second species.

ABBREVIATIONS AND DEFINITIONS To facilitate understanding of the invention, a number of terms as used herein are defined below: As used herein, "TF" shall mean tissue factor.

As used herein, the term "species-specific", when used to describe an antibody, denotes that the antibody will bind to a protein of one species but not the same protein of another species. An antibody which binds to (or "reacts" or "recognizes") a protein from a first species but not the same protein from a second species is said to be species-specific to the protein from the first species, because it does not "crossreact" to the protein from the second species.

As used herein, the term "synthetic peptide" or "peptide" is a chain of 5 to 50 amino acids covalently linked to one another by peptide bonds.

As used herein, an "antigen" is a macromolecule to which antibodies are made. The antigen is usually injected into a vertebrate, which then elicits production of antibodies to regions ("epitopes") of the antigen.

As used herein, the term "immunogen" is a portion of an antigen to which antibodies are desired. An immunogen may be an epitope of the antigen or it may be comprised of several epitopes. More specifically, the term "immunogen" is applied herein to mean a peptide from a protein of interest which is WO 01/00237 PCT/US00/17609 6 conjugated to an immunogenic carrier molecule for the purpose of eliciting antibodies to the immunogen portion of the antigen.

As used herein, the term "hydrophilic" when used in reference to amino acids refers to those amino acids which have polar and/or charged side chains. Hydrophilic amino acids include lysine, arginine, histidine, aspartate aspartic acid), glutamate glutamic acid), serine, threonine, cysteine, tyrosine, asparagine and glutamine. The terms "hydrophilicity value" or "Hydrophilicity index value" as used herein means a number value assigned to each amino acid from to where the larger numbers are more hydrophilic. The hydrophilicity values are calculated using the method of Hopp et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 76:3824. Also, a moving hydrophilicity average of these values for six amino acids have been calculated using the Hopp et al. method as described above.

As used herein, the term "hydrophobic" when used in reference to amino acids refers to those amino acids which have nonpolar side chains. Hydrophobic amino acids include valine, leucine, isoleucine, cysteine and methionine. Three hydrophobic amino acids have aromatic side chains. Accordingly, the term "aromatic" when used in reference to amino acids refers to the three aromatic hydrophobic amino acids phenylalanine, tyrosine and tryptophan.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a sequence homology comparison of the rabbit, human, and mouse tissue factor proteins. The figure was taken from Pawashe et al., 1991, Thromb. Haemost. 66:315.

FIGURE 2 is a list of candidate immunogenic peptides from rabbit tissue factor selected using the methods of the present invention.

FIGURE 3 is a list of candidate immunogenic peptides from human tissue factor selected using the methods of the present invention.

WO 01/00237 PCT/US00/17609 7 DETAILED DESCRIPTION OF THE INVENTION The contents of each of the references cited herein are herein incorporated by reference. The procedures disclosed herein which involve the molecular manipulation of nucleic acids are known to those skilled in the art. See generally Fredrick M.

Ausubel et al. (1995), "Short Protocols in Molecular Biology", John Wiley and Sons, and Joseph Sambrook et al. (1989), "Molecular Cloning, A Laboratory Manual", second ed., Cold Spring Harbor Laboratory Press, which are both incorporated by reference.

The present invention is directed to methods of selecting a peptide immunogen consisting of a region of a target protein from a first species, where the target protein has a different amino acid sequence in a second species. The immunogen is selected for the purpose of eliciting antibodies to the region of the target protein containing the peptide. The method comprises the following steps: the amino acid sequence of the target protein is analyzed for hydrophilicity, and regions from 5-50 amino acids long are identified which are hydrophilic, for example having a value greater than 0 using the method of Hopp et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 76:3824; and the hydrophilic amino acid regions of the target protein are compared to the same regions from the same protein of the second species, and a region is selected as the peptide immunogen which has at least 1 nonhomologous amino acid between the first species and the second species. Antibodies are then made to the selected immunogen after conjugating the immunogen to an immunogenic carrier molecule. Antibodies which are generated to the immunogen generally do not bind to the same protein of the second species.

The method of the current invention may be applied for the production of antibodies to any target protein in a species of interest to which there exists an analogous protein in another species which has an amino acid sequence which is different from the amino acid sequence in the target protein in the species of interest. Included are proteins from any prokaryotic, eukaryotic, or viral source, including but not limited to, WO 01/00237 PCT/US00/17609 8 vertebrates, invertebrates, protozoans, bacteria, fungi, and plants.

The amino acid sequences of the target protein and the analogous protein from the second species can be obtained from various sources, such as published material, as well as protein sequencing Edman degradation). The amino acid sequences may also be deduced from the genes encoding the target proteins which can be obtained from published sources or by cloning the genes encoding the target proteins from both species and determining the start codon by known methods.

The regions of the target protein which are hydrophilic are utilized because those regions tend to be more antigenic than hydrophobic regions. Hydrophilic peptide regions also tend to localize on the outer surface of the target protein, and are therefore more accessible for eliciting an antibody response and for being exposed to an antibody in in situ studies such as immunocytochemical investigations. Using hydrophilic peptides to produce immunogens also have the advantage of being more soluble in aqueous solution than hydrophobic peptides. This makes the procedure for conjugating the peptides to carrier proteins simpler with preferred conjugation procedures. Additionally, antigens comprising hydrophilic peptides are more soluble than antigens comprising hydrophobic peptides, making immunization procedures with the former antigen simpler.

A hydrophilicity analysis provides a determination of the moving average of the polar portion, preferably a hexapeptide, of the protein. The hydrophilic epitopes of tissue factor may be determined by the method of Hopp et al., 1981, Proc. Natl. Acad.

Sci. U.S.A. 76:3824. Computer software, such as MacVector from International Biotechnologies, Inc., are also available to analyze hydrophilicity of a target protein. Using the Hopp method where hydrophilicity values for individual amino acids range from -3.4 (for tryptophan) to 3.0 (for lysine, glutamic acid, aspartic acid, and arginine), the hydrophilicity is preferably determined by averaging the mean hexapeptide hydrophilicity value over the length of the peptide. Peptides WO 01/00237 PCT/US00/1 7609 9 may be selected which have a hydrophilicity greater than 0.

Preferred peptides have a hydrophilicity greater than 0.2.

After selecting hydrophilic peptides from the regions of interest in the target protein, the sequence of each peptide is compared with the analogous sequence in the same protein from the second species. To make this comparison, the amino acid sequences of the target protein and the same protein from the second species are preferably aligned by 3-dimensional structural similarity. The structural similarity between the two sequences can be determined by comparing predicted 3-dimensional structure which can be determined by a number of computer programs, or by determining the crystal structure of both of the proteins by known methods (see, Harlos et al., 1994, Nature 370:662 for the crystal structure of TF). However, 3-dimensional structural similarity can generally be ascertained by aligning the sequences of the two proteins by introducing gaps in one or the other sequence to minimize mismatches. This task can also be performed by a number of well-known computer programs (see, Altschul et al., 1997, Nucl. Acids Res. 25:3389). See Figure 1 for such an alignment of mouse, rabbit, and human TF.

The aligned sequences of the candidate hydrophilic peptides are compared for sequence homology. Peptides which have at least 1 nonhomologous amino acid between the target protein and the analogous protein from the second species may be used as immunogens according to the present invention. Preferably, the candidate hydrophilic peptides have at least 3 nonhomologous amino acids between the target protein and the analogous protein from the second species. The nonhomologous amino acids of the candidate hydrophilic peptides may or may not be consecutive in the peptide chain. Preferably, the homology between the two peptide sequences is less than 75%. Even more preferably the hQmology between the two peptide sequences is less than 60%. As the sequence homology between the two peptide sequences decreases, the species-specific properties of the antibodies generated from the immunogen of the protein of the first species increases, thereby decreasing the possibility that the antibody will cross-react with the same protein of the second species.

WO 01/00237 PCT/US00/17609 After the peptide immunogen is selected, it is synthesized in preparation for conjugation to the carrier molecule.

Synthesis of the peptide can be by any convenient method, for example by translating a nucleic acid sequence encoding the peptide in a suitable recombinant host cell, E. coli, then purifying the peptide by routine methods reversed phase HPLC). However, the peptide is preferably synthesized using chemical methods, preferably solid phase methods. The preferred method for this employs Fmoc chemistry (Fields et al., 1990, Int.

J. Pept. Protein Res. 35:161; Wellings et al., 1997, Meth.

Enzymol. 289:44). Preferably, the peptide is selected with a terminal amino acid residue suitable for coupling to the carrier with a crosslinking reagent, or synthesized with an additional amino acid residue at the terminus for this purpose. Examples of such amino acids are tyrosine, for a bis-diazobenzidine (BDB)activated carrier; aspartic acid, glutamic acid, or a free carboxy terminus, for a l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)-activated carrier; or cysteine, lysine, or a free amino terminus, for a m-maleimidobenzoyl-Nhydrosuccinamide (MBS)-activated carrier. Preferred is cysteine and MBS carrier activation.

After the peptide immunogen is synthesized, it is conjugated to a carrier molecule. The carrier is preferably an antigenic, soluble protein. Preferably, the carrier is selected which would not elicit antibodies which would bind to antigens in the first or second species. A preferred carrier is keyhole limpet hemocyanin (KLH).

Alternatively, the peptide immunogen can be conjugated to a resin-polylysine carrier, to follow the multiple antigenic peptide strategy. See, McClean et al.,1991, J. Immunol.

Meth. 137:149.

Production of the antigen is achieved by conjugating the peptide immunogen to the carrier molecule. This conjugation preferably proceeds by activation of the carrier, by BDB, EDC, or, preferably, MBS, then addition of the peptide.

The antigen is then used to produce an antibody which binds to the immunogen. The antibody may be a mono-specific antibody.

WO 01/00237 PCT/US00/17609 11 The monospecific antibody may be a monoclonal antibody produced, for example, by the method of Galfre et al., 1977, Nature 266:550. Alternatively, the monospecific antibody may be a recombinant antibody produced, for example, by the method of Lowman et al., 1991, Biochemistry 30:10832.

The antibody can also be a polyclonal antibody. The polyclonal antibody can be prepared, for example, by immunizing a mammal such as a mouse, goat, sheep, or rabbit with the peptidecarrier antigen and subsequently isolating the serum therefrom to obtain an antiserum that contains the polyclonal antibodies.

The antibody is preferably characterized by determining its ability to bind to the immunogen and the target protein by, e.g., ELISA, hemagglutination, fluorescent antibody binding to tissue, western blot, or any other suitable procedure. Tissue factor antibodies made by the invention method can be tested, for reactivity to the immunogen, and to recombinant and/or native TF from the first and second species, or other species.

The peptide antigens, immunogens, and antibodies of the present invention can be used in various applications. Among other uses, the peptide antigens can be employed to purify the antibodies as noted above. The synthetic peptides of the present invention can be used to test antibody specificity as noted above. The antigens can also be used in inhibition assays. For example, a peptide from TF can be added to, thromboplatin assays to determine if it can inhibit TF function. Such work could help characterize TF structure and function.

Applications of antibodies of the present invention include epitope mapping in structure/function studies, immunohistochemical localization, and immunoaffinity purification of the target protein. For example, anti-TF antibodies can be used in a thromboplastin assays to determine if the antibody can inhibit TF function. These antibodies can also be used to study other aspects of TF, such as characteristics of the TF/substrate complex.

The antibodies of the present invention can also be used in diagnostic assays for antigens, particularly where it is required WO 01/00237 PCT/US00/17609 12 that the antibodies do not cross-react with antigens from another.

species assays for a particular bacteria in food, etc.)

EXAMPLES

Example 1 Identification of Peptide Immunogens to Rabbit and Human Tissue Factor The sequences of rabbit, human, and mouse tissue factor were have been described (Pawashe et al., 1991, Thromb Haemost.

66:315). A hydrophilicity analysis was performed using the method of Hopp et al., 1981, Proc. Natl. Acad. Sci. U.S.A.

76:3824. The analysis was performed using the moving averages function on the Excel computer spreadsheet program. Briefly, each amino acid of the rabbit and human sequence was assigned a value from 3.0 to where the larger numbers are more hydrophilic. A moving hydrophilicity average of each hexapeptide was established. A mean hydrophilicity average of the hexapeptide averages was then established for regions of the proteins with predominantly positive hexapeptide averages.

Regions with mean hexapeptide averages above 0.2 were selected as candidate immunogens for further evaluation. Figures 2 and 3 shows these candidate immunogens.

The candidate immunogens were next evaluated for homology to the mouse sequence. The human and rabbit sequence was aligned with the mouse sequence as in Figure 1. The candidate human and rabbit immunogens were then compared to the aligned mouse sequence and immunogens were selected which have more than 3 nonhomologous amino acids from the analogous mouse sequence.

The selected immunogens are shown as the candidate immunogens of Figures 2 and 3 which have 3 or more mismatches.

Eight of the ten candidate immunogens from rabbit TF and eight of the eleven candidate immunogens from human TF. All of those immunogens are useful for producing anti-TF antibodies. However, the most effective immunogens would be those which have a higher mean hydrophilicity value and a larger number of mismatches.

WO 01/00237 PCT/US00/17609 13 Example 2 Production and Characterization of Polyclonal Antibodies to Rabbit Tissue Factor Polyclonal antibodies were made to two regions of rabbit TF.

The 12 residue peptide TTGFPEEPPFRN from position 84 through (84T-95S), located in the loop connecting domains 1F and 1G and oriented toward the putative top of the molecule (Harlos et al., 1994, Nature 370:662), demonstrated the most amino acids mismatches of the candidate immunogens (Figure The 13 residue peptide VIPSRKRKQRSPE from position 190 through 202 (190V-202E), located within the 2F-2G loop constrained by a disulfide bond, had the highest hydrophilicity index (Figure 2).

Both peptides are unique to the rabbit TF sequence when analyzed by the BLASTP search engine with an E value of 100. These two peptides were synthesized using standard FMOC chemistry. An additional cysteine was added to the carboxy terminal. The peptides were then conjugated to MBS-activated KLH by standard methods. For each peptide immunogen, three goats were immunized with the peptide-KLH antigens. Antisera collected from the goats were then tested for immunoreactivity in a microtiter plate format in which unconjugated peptides, rabbit recombinant TF, human recombinant TF, and a Triton extract of rabbit brain acetone powder (RBAP) were the coated antigens. The plates were probed with rabbit anti-goat IgG alkaline phosphatase conjugate.

Each antisera demonstrated specific immunoreactivity to its respective peptide immunogen. All antisera cross-reacted with rabbit recombinant TF. Two antisera, one to each of the peptides, also demonstrated reactivity to natural rabbit TF in the RBAP extracts.

Other features, objects and advantages of the present invention will be apparent to those skilled in the art. The explanations and illustrations presented herein are intended to acquaint others skilled in the art with the invention, its principles, and its practical application. Those skilled in the art may adapt and apply the invention in its numerous forms, as may be best suited to the requirements of a particular use.

Accordingly, the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention.

With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, we note that unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that we intend each of those words to be so interpreted in construing the foregoing description and/or the following claims.

e *e EDITORIAL NOTE NO: 77493/98 Sequence listing pages 1-5 is part of the description.

The claims are to follow.

WO 01/00237 WO 0100237PCTIUSOOII7609 SEQUENCE LISTING <110> Goldford, Marc Triscott, Mark X <120> SYNTHETIC PEPTIDE <130> sgmd834O.1 <140> <141> <150> 60/141,699 <151> 1999-06-30 <160> 11 <170> Patentln Ver. 2.1 <210> 1 <211> 86 <212> PRT <213> Rabbit <400> 1 Ala Asp Thr Thr Gly Arg 1 5 Phe Lys Thr Ile Leu Glu Tyr Thr Val Gin Ile Ser Phe Leu Thr Ala Giu Thr 50 Asp Val Gly Gin Thr Tyr 70 Asn Gly Asn Thr Thr Gly IMMUNOGENS AND ANTI80DIES THERETO Ala Trp Thr Giu 55 Met Tyr Giu Arg 40 Cys Ala Asn Pro 25 Leu Asp Arg Leu 10 Lys Giu Leu Val1 Thr Ser Asn Thr Leu 75 Trp, Ile Trp Asp Ser Lys Asp Lys Giu Tyr Ser Ile Ser Val Pro Thr Ile Lys Val1 Ala Asn Val Cys Lys Arg <210> 2 <211> 141 <212> PRT <213> Rabbit <400> 2 Phe Pro Giu Giu Pro Pro Phe Arg Asn Ser Pro Giu Phe Thr Pro Tyr 1 5 .10 Leu Asp Thr His Leu Gly Gin Pro Thr Ile Gin Ser Phe Giu Gin Vai 25 WO 01/00237 PCT/US00/17609 2 Gly Thr Lys Leu Asn Val Thr Val Gin Asp Ala Arg Thr Leu Val Arg 40 Arg Asn Gly Thr Phe Leu Ser Leu Arg Ala Val Phe Gly Lys Asp Leu 55 Asn Tyr Thr Leu Tyr Tyr Trp Arg Ala Ser Ser Thr Gly Lys Lys Thr 70 75 Ala Thr Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu 90 His Thr Cys Phe Ser Val Gin Ala Val Ile Pro Ser Arg Lys Arg Lys 100 105 110 Gin Arg Ser Pro Glu Ser Leu Thr Glu Cys Thr Ser Arg Glu Gin Gly 115 120 125 Arg Ala Arg Glu Met Phe Phe Ile Ile Gly Ala Val Val 130 135 140 <210> 3 <211> 14 <212> PRT <213> Rabbit <400> 3 Val Val Ala Leu Leu Ile Ile Val Leu Ser Val Thr Val Tyr 1 5 <210> 4 <211> <212> PRT <213> Rabbit <400> 4 Lys Cys Arg Lys Ala Arg Ala Gly Pro Ser Gly Lys Glu Ser Ser Pro 1 5 10 Leu Asn Ile Ala <210> <211> 83 <212> PRT <213> Human <400> Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser 1 5 -10 Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Lys Pro Val His 25 WO 01/00237 WO 0100237PCTUSOO/1 7609 Gin Val Lys Cys Val Lys Ala Gly Tyr Thr Val Gin Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser 40 Phe Tyr Thr Thr Asp Thr Giu Cys Asp Leu Thr Asp Giu Ile 55 Asp Val Lys Gin Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro 70 75 Asn <210> 6 <211> 6 <212> PRT <213> Human <400> 6 Val Giu Ser Thr Gly Ser 1 <210> 7 <211> 154 <212> PRT <213> Human <400> 7 Ala Gly Glu Pro Leu Tyr Giu 1 5 Giu Thr Asn Leu Gly Gin Pro Thr Lys Val Asn Vai Thr Val Asn Asn Thr Phe Leu Ser Leu 55 Tyr Thr Leu Tyr Tyr Trp, Lys 65 70 Lys Thr Asn Thr Asn Giu Phe Tyr Cys Phe Ser Val Gin Ala 100 Lys Ser Thr Asp Ser Pro Val 115 Phe Arg Giu Ile Phe Tyr Ile 130 135 Asn Thr Glu 40 Arg Ser Leu Vai Giu 120 Ile Ser Ile 25 Asp Asp Ser Ile Ile 105 Cys Giy Pro 10 Gin Giu Val1 Ser Asp 90 Pro Met Al a Giu Ser Arg Phe Ser 75 Vai Ser Gly Val Phe Phe Thr Gly Gly Asp Arg Gin Val 140 Thr Glu Leu Lys Lys Lys Thr Gin 125 Phe Pro Gin Vai Asp Lys Giy Val Lys Vali Tyr Val1 Arg Leu Thr Giu Asn Gly Val1 Leu Gly Arg Ile Aia His Arg Giu Ile WO 01/00237 PCTIUSOO/1 7609 4 Ile Leu Val Ile Ile Leu Ala Ile Ser Leu 145 150 <210> 8 <211> 22 <212> PRT <213> Human <400> 8 Ile Ile Lys Cys Arg Lys Ala Gly Val Gly Gin Ser Trp Lys Glu Asn 1 5 10 Ser Pro Leu Asn Val Ser <210> 9 <211> <212> PRT <213> Mouse <400> 9 Gly Ile Pro Glu Lys 1 <210> <211> 240 <212> PRT <213> Mouse <400> Ala Phe Asn Leu Thr Trp Ile Ser Thr Asp Phe Lys Thr Ile Leu Glu 1 5 10 Trp Gin Pro Lys Pro Thr Asn Tyr Thr Tyr Thr Val Gin Ile Ser Asp 25 Arg Ser Arg Asn Trp Lys Asn Lys Cys Phe Ser Thr Thr Asp Thr Glu 40 Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Thr Trp Ala Tyr Glu 55 Ala Lys Val Leu Ser Val Pro Arg Arg Asn Ser Val His Gly Asp Gly 70 75 Asp Gin Leu Val Ile Asn Gly Glu Glu Pro Pro Phe Thr Asn Ala Pro 85 90 Lys Phe Leu Pro Tyr Arg Asp Thr Asn Leu Gly Gin Arg Val Ile Gin 100 105 110 Gin Phe Glu Gin Gin Gly Arg Lys Leu Asn Val Val Val Lys Asp Ser 115 120 125

I

WO 01/00237 WO 0100237PCTIUSOOI17609 Leu Phe 145 Thr Val Ser Glu Val1 225 Thr 130 Gly Gly Giu Arg Asn 210 Val Leu Lys Lys Glu Lys 195 Trp Leu Val1 Asp Lys Gly 180 Thr Ser Leu Arg Leu Thr 165 Val1 Asn Lys Ala Lys Gly 150 Asn Ser Gin Phe Thr 230 Asn 135 Tyr Ile Tyr Asn Leu 215 Ile Gly Ile Thr Cys Ser 200 Gly Phe Thr Ile Asn Phe 185 Pro Glu Ile Phe Thr Thr 170 Phe Gly Thr Ile Leu Tyr 155 Asn Val Ser Leu Leu 235 Thr 140 Arg Glu Gin Ser Ile 220 Leu Leu Leu Phe Aia Thr 205 Ile Ser Arg Gly Ser Met 190 Val1 Val Ile Gin Ser Ile 175 Ile Cys Gly Ser Val Ser 160 Asp Phe Thr Ala Leu 240 <210> 11 <211> 19 <212> PRT <213> Mouse <400> 11 Cys Lys Arg Arg Lys Asn Arg Ala Gly Gin Lys Giy Lys Asn Thr Pro 1 5 10 Ser Arg Leu

Claims (31)

1. 2. The method of claim 1, wherein the hydrophilicity value is greater than 0.2.
2. 3. The method of claim i, wherein the level of homology between the two species is less than
3. 4. The method of claim i, wherein the level of homology between the two species is less than The method of claim 1, wherein the first and second species are selected from the group consisting of human, rabbit, and mouse.
4. 6. A method of claim i, wherein the peptide immunogen has at least 3 non-homologous amino acids between the first species and the second species.
5. 7. The method of claim 1, wherein the target protein is tissue factor.
6. 8. The method of claim 7, wherein the target protein is rabbit tissue factor. WO 01/00237 PCT/US00/17609 16
7. 9. The method of claim 7, wherein the target protein is human tissue factor. A method of making an antibody which is specific for a target protein of a first species, wherein the target protein comprises an amino acid sequence which varies between the first species and a second species, the method comprising: selecting a peptide immunogen of the target protein by the method of claim 1; synthesizing the peptide immunogen; conjugating the peptide immunogen to an immunogenic LO carrier molecule to make a peptide-carrier antigen; producing antibodies to the antigen.
8. 11. The method of claim 10, wherein the peptide antigen is synthesized by recombinant DNA methods.
9. 12. The method of claim 10, wherein the peptide antigen is synthesized using solid phase chemical methods.
10. 13. The method of claim 10, wherein the immunogenic carrier molecule is keyhole limpet hemocyanin.
11. 14. The method of claim 10, wherein the antibodies are monospecific. The method of claim 14, wherein the antibodies are monoclonal antibodies.
12. 16. The method of claim 10, wherein the antibodies are polyclonal.
13. 17. The method of claim 16, wherein the antibodies are made in a mouse, goat, rabbit, or sheep. WO 01/00237 PCT/US00/17609 17
14. 18. The method of claim 17, wherein the antibodies are made in a goat.
15. 19. An antibody made by the method of claim A peptide which consists of a sequence from a tissue factor extracellular region from a first species which is 5-50 amino acids in length, has a hydrophilicity value greater than 0, and has at least 1 nonhomologous amino acids from a tissue factor from a second species.
16. 21. The peptide of claim 20, wherein the hydrophilicity value is greater than 0.2.
17. 22. The peptide of claim 20, wherein the homology between the two species is less than
18. 23. The peptide of claim 20, wherein the homology between the two species is less than
19. 24. The peptide of claim 20 wherein the peptide has at least 3 non-homologous amino acids from a tissue factor from a second species. The peptide of claim 20, wherein the first species is selected from the group consisting of human, rabbit, and mouse.
20. 26. The peptide of claim 25, wherein the first species is rabbit and the second species is mouse.
21. 27. The peptide of claim 25, wherein the first species is human and the second species is mouse.
22. 28. The peptide of claim 26, selected from the group consisting of 36I-42N, 49L-63K, 84T-95S, 124T-133K, 157R-164K, 174L- 179K, 190V-202E, 205T-214R, and 243R-253E. WO 01/00237 PCT/US00/17609 18
23. 29. The peptide of claim 28, wherein the peptide is 84T-95S or 190V-202E. The peptide of claim 27, selected from the group consisting of 37Q-44D, 84E-95E, 124N-135R, 158W-169K, 176L-181K, 194P- 204D, and 208E-216E.
24. 31. The peptide of claim 30, wherein the peptide is 84E-95E, 124N-135R, 158W-169K, or 194P-204D.
25. 32. An antigen comprising an immunogenic carrier molecule conjugated to the peptide of claim
26. 33. The antigen of claim 32, wherein the carrier molecule is keyhole limpet hemocyanin.
27. 34. An antibody made to the antigen of claim 32. The antibody of claim 34, wherein the antibody is monospecific.
28. 36. The antibody of claim 35, wherein the antibody is a monoclonal antibody.
29. 37. The antibody of claim 34, wherein the antibody is part of a polyclonal antibody preparation.
30. 38. The antibody of claim 37, wherein the antibody is from a mouse, goat, rabbit, or sheep.
31. 39. The antibody of claim 38, wherein the antibody is from a goat.