United States Patent: 6,858,397
Issued: February 22, 2005
Inventors: Prusiner; Stanley B. (San Francisco, CA); Williamson; R. Anthony (San Diego, CA); Burton; Dennis R. (LaJolla, CA)
Assignee: The Regents of the University of California (Oakland, CA); The Scripps Research Institute (La Jolla, CA)
Appl. No.: 435602
Filed: May 9, 2003
Antibodies are disclosed which specifically bind to native PrPSc in situ. Preferred antibodies bind only to the native PrPSc of a particular species e.g., human, cow, sheep, pig, etc. Particularly preferred antibodies bind specifically to a particular isoform of human PrPSc. Preferred antibodies of the invention are (1) produced by phage display methodology, (2) bind specifically to native PrPSc, (3) neutralizes the infectivity of prions, (4) bind to PrPSc in situ and (5) bind 50% or more of PrPSc in a liquid flowable sample. Antibodies of the invention can be bound to a substrate and used to assay a sample (which has any PrPc denatured via proteinase K) for the presence of PrPSc of a specific species which PrPSc is associated with disease. Antibodies which specifically bind to human PrPSc can be labeled and injected carrying out an in vivo diagnostic test to determine if the human is infected with prions associated with disease. The antibodies are preferably produced using phage display technology wherein the genetic material in the phage expressing the antibody is obtained from a mammal with an ablated endogenous PrP protein gene and an endogenous chimeric PrP gene which mammal had been inoculated with PrPSc to induce antibody production.
Description of the Invention
FIELD OF THE INVENTION
This invention relates to methods for obtaining antibodies and assays for using such antibodies. More specifically, the invention relates to methods of obtaining antibodies which specifically bind to naturally occurring forms of PrPSc.
BACKGROUND OF THE INVENTION
Prions are infectious pathogens that cause central nervous system spongiform encephalopathies in humans and animals. Prions are distinct from bacteria, viruses and viroids. The predominant hypothesis at present is that no nucleic acid component is necessary for infectivity of prion protein. Further, a prion which infects one species of animal (e.g., a human) will not infect another (e.g., a mouse).
A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein ("PrP") [Bolton et al., Science 218:1309-11 (1982); Prusiner, et al., Biochemistry 21:6942-50 (1982); McKinley, et al., Cell 35:57-62 (1983)]. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPc is encoded by a single-copy host gene [Basler, et al., Cell 46:417-28 (1986)] and is normally found at the outer surface of neurons. Prion diseases are accompanied by the conversion of PrPC into a modified form called PrPSc. However, the actual biological or physiological function of PrPC is not known.
The scrapie isoform of the prion protein (PrPSc) is necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner, S. B., "Molecular biology of prion disease," Science 252:1515-1522 (1991). The most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith, J. and Wells, Microbiol. Immunol. 172:21-38 (1991)]. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek, D. C., Science 197:943-960 (1977); Medori et al., N. Engl. J. Med. 326:444-449 (1992)]. The presentation of human prion diseases as sporadic, genetic and infectious illnesses initially posed a conundrum which has been explained by the cellular genetic origin of PrP.
Most CJD cases are sporadic, but about 10-15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene [Hsiao et al., Neurology 40:1820-1827 (1990); Goldfarb et al., Science 258:806-808 (1992); Kitamoto et al., Proc. R. SOc. Lond. (In press) (1994)]. Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts [Brown et al., Lancet 340:24-27 (1992)]. Despite numerous attempts to link CJD to an infectious source such as the consumption of scrapie infected sheep meat, none has been identified to date [Harries-Jones et al., J. Neurol. Neurosurg. Psychiatry 51:1113-1119 (1988)] except in cases of iatrogenically induced disease. On the other hand, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism [Alpers, M. P., Slow Transmissible Diseases of the Nervous System, Vol. 1, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 66-90 (1979)].
The initial transmission of CJD to experimental primates has a rich history beginning with William Hadlow's recognition of the similarity between kuru and scrapie. In 1959, Hadlow suggested that extracts prepared from patients dying of kuru be inoculated into non-human primates and that the animals be observed for disease that was predicted to occur after a prolonged incubation period [Hadlow, W. J., Lancet 2:289-290 (1959)]. Seven years later, Gajdusek, Gibbs and Alpers demonstrated the transmissibility of kuru to chimpanzees after incubation periods ranging form 18 to 21 months [Gajdusek et al., Nature 209:794-796 (1966)]. The similarity of the neuropathology of kuru with that of CJD [Klatzo et al., Lab Invest. 8:799-847 (1959)] prompted similar experiments with chimpanzees and transmissions of disease were reported in 1968 [Gibbs, Jr. et al., Science 161:388-389 (1968)]. Over the last 25 years, about 300 cases of CJD, kuru and GSS have been transmitted to a variety of apes and monkeys.
The expense, scarcity and often perceived inhumanity of such experiments have restricted this work and thus limited the accumulation of knowledge. While the most reliable transmission data has been said to emanate from studies using non-human primates, some cases of human prion disease have been transmitted to rodents but apparently with less regularity [Gibbs, Jr. et al., Slow Transmissible Diseases of the Nervous System, Vol. 2, S. B. Prusiner and W. J. Hadlow, eds. (New York: Academic Press), pp. 87-110 (1979); Tateishi, et al., Prion Diseases of Humans and Animals, Prusiner, et al., eds. (London: Ellis Horwood), pp. 129-134 (1992)].
The infrequent transmission of human prion disease to rodents has been cited as an example of the "species barrier" first described by Pattison in his studies of passaging the scrapie agent between sheep and rodents [Pattison, I. H., NINDB Monograph 2, D. C. Gajdusek, C. J. Gibbs Jr. and M. P. Alpers, eds. (Washington, D.C.: U.S. Government Printing), pp. 249-257 (1965)]. In those investigations, the initial passage of prions from one species to another was associated with a prolonged incubation time with only a few animals developing illness. Subsequent passage in the same species was characterized by all the animals becoming ill after greatly shortened incubation times.
The molecular basis for the species barrier between Syrian hamster (SHa) and mouse was shown to reside in the sequence of the PrP gene using transgenic (Tg) mice [Scott, et al., Cell 59:847-857 (1989)]. SHaPrP differs from MoPrP at 16 positions out of 254 amino acid residues [Basler, et al., Cell 46:417-428 (1986); Locht, et al., Proc. Natl. Acad. Sci. USA 83:6372-6376 (1986)]. Tg(SHaPrP) mice expressing SHaPrP had abbreviated incubation times when inoculated with SHa prions. When similar studies were performed with mice expressing the human, or ovine PrP transgenes, the species barrier was not abrogated, i.e., the percentage of animals which became infected were unacceptably low and the incubation times were unacceptably long. Thus, it has not been possible, for example in the case of human prions, to use transgenic animals (such as mice containing a PrP gene of another species) to reliably test a sample to determine if that sample is infected with prions. The seriousness of the health risk resulting from the lack of such a test is exemplified below.
More than 45 young adults previously treated with HGH derived from human pituitaries have developed CJD [Koch, et al., N. Engl. J. Med. 313:731-733 (1985); Brown, et al., Lancet 340:24-27 (1992); Fradkin, et al., JAMA 265:880-884 (1991); Buchanan, et al., Br. Med. J. 302:824-828 (1991)]. Fortunately, recombinant HGH is now used, although the seemingly remote possibility has been raised that increased expression of wtPrPC stimulated by high HGH might induce prion disease [Lasmezas, et al., Biochem. Biophys. Res. Commun. 196:1163-1169 (1993)]. That the HGH prepared from pituitaries was contaminated with prions is supported by the transmission of prion disease to a monkey 66 months after inoculation with a suspect lot of HGH [Gibbs, Jr., et al., N. Engl. J. Med. 328:358-359 (1993)]. The long incubation times associated with prion diseases will not reveal the full extent of iatrogenic CJD for decades in thousands of people treated with HGH worldwide. Iatrogenic CJD also appears to have developed in four infertile women treated with contaminated human pituitary-derived gonadotrophin hormone [Healy, et al., Br. J. Med. 307:517-518 (1993); Cochius, et al., Aust. N. Z. J. Med. 20:592-593 (1990); Cochius, et al., J. Neurol. Neurosurg. Psychiatry 55:1094-1095 (1992)] as well as at least it patients receiving dura mater grafts [Nisbet, et al., J. Am. Med. Assoc. 261:1118 (1989); Thadani, et al., J. Neurosurg. 69:766-769 (1988); Willison, et al., J. Neurosurg. Psychiatric 54:940 (1991); Brown, et al., Lancet 340:24-27 (1992)]. These cases of iatrogenic CJD underscore the need for screening pharmaceuticals that might possibly be contaminated with prions.
Recently, two doctors in France were charged with involuntary manslaughter of a child who had been treated with growth hormones extracted from corpses. The child developed Creutzfeldt-Jakob Disease. (See New Scientist, Jul. 31, 1993, page 4). According to the Pasteur Institute, since 1989 there have been 24 reported cases of CJD in young people who were treated with human growth hormone between 1983 and mid-1985. Fifteen of these children have died. It now appears as though hundreds of children in France have been treated with growth hormone extracted from dead bodies at the risk of developing CJD (see New Scientist, Nov. 20, 1993, page 10.) Prior attempts to create PrP monoclonal antibodies have been unsuccessful (see Barry and Prusiner, J. of Infectious Diseases Vol. 154, No. 3, Pages 518-521 (1986). Thus there is a need for an assay to detect compounds which result in disease. Specifically, there is a need for a convenient, cost-effective assay for testing sample materials for the presence of prions which cause CJD. The present invention offers such an assay.
SUMMARY OF THE INVENTION
Antibodies of the invention will specifically bind to a native prion protein (i.e., native PrPSc) in situ with a high degree of binding affinity. The antibodies can be placed on a substrate and used for assaying a sample to determine if the sample contains a pathogenic form of a prion protein. The antibodies are characterized by one or more of the following features (1) an ability to neutralize infectious prions, (2) will bind to prion proteins (PrPSc) in situ i.e., will bind to naturally occurring forms of a prion protein in a cell culture or in vivo and without the need to treat (e.g., denature) the prion protein, and (3) will bind to a high percentage of the PrPSc form (i.e. disease form) of prion protein in a composition e.g., will bind to 50% or more of the PrPSc form of the prion proteins. Preferred antibodies are further characterized by an ability to (4) bind to a prion protein of only a specific species of mammals e.g., bind to human prion protein and not prion protein of other mammals.
An important object is to provide antibodies which bind to native prion protein (PrPSc).
Another object is to provide antibodies which specifically bind to epitopes of prion proteins (PrPSc) of a specific species of animal and not to the prion protein (PrPSc) of other species of animals.
Another object is to provide monoclonal antibodies which specifically bind to prion proteins (PrPSc) associated with disease, (e.g., human PrPSc) which antibodies do not bind to denatured PrP proteins not associated with disease (e.g., human PrPC).
Still another object is to provide specific methodology to allow others to generate a wide range of specific antibodies characterized by their ability to bind one or more types of prion proteins from one or more species of animals.
Another object of the invention is to provide an assay for the detection of PrPSc forms of PrP proteins.
Another object of the invention is to provide an assay which can specifically differentiate prion protein (PrPSc) associated with disease from PrPSc not associated with disease.
Another object is to detect prions which specifically bind to native PrPSc of a specific species such as a human, cow, sheep, pig, dog cat or chicken.
An advantage of the invention is that it provides a fast, efficient cost effective assay for detecting the presence of native PrPSc in a sample.
A specific advantage is that the assay can be used as a screen for the presence of prions (i.e., PrPSc) in products such as pharmaceuticals (derived from natural sources) food, cosmetics or any material which might contain such prions and thereby provide further assurances as to the safety of such products.
Another advantage is that the antibodies which can be used with a protease which denatures PrPc thereby providing for a means of differentiating between infectious (PrPSc) and non-infectious forms (PrPSc) of prions.
Yet another advantage of the invention is that antibodies of the invention are characterized by their ability to neutralize the infectivity of naturally occurring prions e.g., neutralize PrPSc.
Another advantage is that antibodies of the invention will bind to (PrPSc) prion proteins in situ, i.e., will bind to naturally occurring (PrPSc) prions in their natural state in a cell culture or in vivo without requiring that the prion proteins be particularly treated, isolated or denatured.
Another advantage is that the prion proteins of the invention will bind to a relatively high percentage of the infectious form of the prion protein (e.g., PrPSc) for example bind to 50% or more of the PrPSc form of prion proteins in a composition.
An important feature of the invention is that the methodology makes it possible to create a wide variety of different prion protein antibodies with the same or individually engineered features which features may make the antibody particularly suitable for uses such as (1) prion neutralization to purify a product, (2) the extraction of prion proteins and (3) therapies.
A feature of the invention is that it uses phage display libraries in the creation of the antibodies.
Another feature of the invention is that the phage are genetically engineered to express a specific binding protein of an antibody on their surface.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
General Aspect of the Invention
The core of the invention is an antibody which specifically binds to a PrPSc protein and preferably binds to a native non-denatured PrPSc protein in situ with an affinity of 107 moles/liter or more, preferable 108 moles/liter or more of a single species (e.g., human) and more preferably binds only to human PrPSc and not denatured fragments of human PrPC). The antibody may bind to all proteins coded by the different mutations and/or polymorphisms of the PrP protein gene. Alternatively, a battery of antibodies (2 or more different antibodies) are provided wherein each antibody of the battery specifically binds to protein coded by a different mutation or polymorphism of the PrP gene. The antibody can be bound to support surface and used to assay a sample in vitro for the presence of a particular type of human PrPSc. The antibody can also be bound to a detectable label and injected into an animal to assay in vivo for the presence of a particular type of native PrPSc.
Although there are known procedures for producing antibodies from any given antigen practice has shown that it is particularly difficult to produce antibodies which bind to certain proteins e.g., PrPSc. The difficulty with obtaining antibodies to PrPSc relates, in part, to its special and unknown qualities. By following procedures described herein antibodies which bind native PrPSc in situ have been obtained and others may follow the procedures described here to obtain other antibodies to PrPSc and to other proteins for which it is difficult to generate antibodies.
To produce antibodies of the invention it is preferable to begin with inoculating a host mammal with prion proteins i.e., infectious PrPSc. The host mammal may be any mammal and is preferably a host mammal of the type defined herein such as a mouse, rat, guinea pig or hamster and is most preferably a mouse. The host animal is inoculated with prion proteins which are endogenous to a different species which is preferably a genetically diverse species. For example a mouse is inoculated with human prion proteins. Preferably, the host mammal is inoculated with infectious prion proteins of a genetically diverse mammal. For example, a mouse is inoculated with human PrPSc. Using a normal host mammal in this manner it is possible to elicit the generation of some antibodies. However, when a hosts animal includes a prion protein gene and is inoculated with prions from a genetically diverse species antibodies will, if at all, only be generated for epitopes which differ between epitopes of the prion protein of the host animal and epitopes of the genetically diverse species. This substantially limits the amount of antibodies which might be generated and decreases the ability to find an antibody which selectively binds to an infectious form of a prion protein and does not bind to denatured fragments of a non-infectious form. Thus, unless one is attempting to generate antibodies which differentiate between prion proteins of different species it is preferable to begin the antibody production process using a mammal which has an ablated prion protein gene i.e., a null PrP gene abbreviated as Prnp0/0. Accordingly, the invention is generally described in connection with the use of such "null" mammals and specifically described in connection with "null mice."
Antibodies are produced by first producing a host animal (e.g., a mouse) which has its endogenous PrP gene ablated, i.e., the PrP gene rendered inoperative. A mouse with an ablated PrP gene is referred to as a "null mouse". A null mouse can be created by inserting a segment of DNA into a normal mouse PrP gene and/or removing a portion of the gene to provide a disrupted PrP gene. The disrupted gene is injected into a mouse embryo and via homologous recombination replaces the endogenous PrP gene.
The null mouse is injected with prions in order to stimulate the formation of antibodies. Further, injections of adjuvants and prions are generally used to maximize the generation of antibodies.
The mouse is then sacrificed and bone marrow and spleen cells are removed. The cells are lysed, RNA is extracted and reversed transcribed to cDNA. Antibody heavy and light chains (or parts thereof) and then amplified by PCR. The amplified cDNA library may be used as is or after manipulation to create a range of variants and thereby increase the size of the library.
An IgG phage display library is then constructed by inserting the amplified cDNA encoding IgG heavy chain and the amplified cDNA encoding a light chain into a phage display vector (e.g., a pComb3 vector) such that one vector contains a cDNA insert encoding a heavy chain fragment in a first expression cassette of the vector, and a cDNA insert encoding a light chain fragment in a second expression cassette of the vector.
Ligated vectors are then packaged by filamentous phage M13 using methods well known in the art. The packaged library is then used to infect a culture of E. coli, so as to amplify the number of phage particles. After bacterial cell lysis, the phage particles are isolated and used in a panning procedure.
The library created is panned against a composition containing prions. Antibody fragments which selectively bind to PrPSc e.g., human PrPSc are then isolated.
Specifics of a PrP Protein
The major component of purified infectious prions, designated PrP 27-30, is the proteinase K resistant core of a larger native protein PrPSc which is the disease causing form of the ubiquitous cellular protein PrPC. PrPSc is found only in scrapie infected cells whereas PrPC is present in both infected and uninfected cells implicating PrPSc as the major, if not the sole, component of infectious prion particles. Since both PrPC and PrPSc are encoded by the same single copy gene, great effort has been directed toward unraveling the mechanism by which PrPSc is derived from PrPC. Central to this goal has been the characterization of physical and chemical differences between these two molecules. Properties distinguishing PrPSc from PrPC include low solubility (Meyer, et al 1986 PNAS), poor antigenicity (Kascack, J. Virol 1987; Serban D. 1990) protease resistance (Oesch, et al 1985 Cell) and polymerization of PrP 27-30 into rod-shaped aggregates which are very similar, on the ultrastructural and histochemical levels, to the PrP amyloid plaques seen in scrapie diseased brains (Prusiner, et al Cell 1983). By using proteinase K it is possible to denature PrPC but not PrPSc. To date, attempts to identify any post-transitional chemical modifications in PrPC that lead to its conversion to PrPSc have proven fruitless (Stahl, et al 1993 Biochemistry). Consequently, it has been proposed that PrPC and PrPSc are in fact conformational isomers of the same molecule.
Conformational description of PrP using conventional techniques has been hindered by problems of solubility and the difficulty in producing sufficient quantities of pure protein. However, PrPC and PrPSc are conformationally distinct. Theoretical calculations based upon the amino acid sequences of PrPs from several species have predicted four putative helical motifs in the molecule. Experimental spectroscopic data would indicate that in PrPC these regions adopt .alpha.-helical arrangements, with virtually no .beta.-sheet (Pan, et al PNAS 1993). In dramatic contrast, in the same study it was found that PrPSc and PrP 27-30 possess significant .beta.-sheet content, which is typical of amyloid proteins. Moreover, studies with extended synthetic peptides, corresponding to PrP amino acid residues 90-145, have demonstrated that these truncated molecules may be converted to either .alpha.-helical or .beta.-sheet structures by altering their solution conditions. The transition of PrPC to PrPSc requires the adoption of .beta.-sheet structure by regions that were previously .alpha.-helical.
In general, scrapie infection fails to produce an immune response, with host organisms being tolerant to PrPSc from the same species. Polyclonal anti-PrP antibodies have though been raised in rabbits following immunization with large amounts of SHaPrP 27-30 (Bendheim, et al PNAS 1985, Bode, et al J. Gen. Virol. 1985). Similarly, a handful of anti-PrP monoclonal antibodies have been produced in mice (Kascack, et al, J. Virol. 1987, Barry, et al, J. Infect. Dis. 1986). These antibodies are able to recognize native PrPC and denatured PrPSc from both SHa and humans equally well, but do not bind to MoPrP. Unsurprisingly, the epitopes of these antibodies were mapped to regions of sequence containing amino acid differences between SHa- and MoPrP (Rogers, et al, J. Immunol. 1993).
It is not entirely clear as to why antibodies of the type described in the above cited publications will bind to PrPC but not to PrPSc. Without being bound to any particular theory it is suggested that such may take place because epitopes which are exposed when the protein is in the PrPC conformation are unexposed or partially hidden in the PrPSc configuration--where the protein is relatively insoluble and more compactly folded together. It is pointed out that stating that an antibody binds to PrPC but not to PrPSc is not correct in absolute terms (but correct in commonly accepted terms) because some minimal binding to PrPSc may occur. For purposes of the invention an indication that no binding occurs means that the equilibrium or affinity constant Ka is 106 l/mole or less. Further, binding will be recognized as existing when the Ka is at 107 l/mole or greater preferably 108 l/mole or greater. The binding affinity of 107 l/mole or more may be due to (1) a single monoclonal antibody (i.e., large numbers of one kind of antibodies) (2) a plurality of different monoclonal antibodies (e.g., large numbers of each of five different monoclonal antibodies) or (3) large numbers of polyclonal antibodies. It is also possible to use combinations or (1)-(3).
Preferred antibodies will bind 50% or more of the PrPSc in a sample. However, this may be accomplished by using several different antibodies as per (1)-(3) above. It has been found that an increased number of different antibodies is more effective in binding a larger percentage of the PrPSc in a sample as compared to the use of a single antibody. For example, the use of six copies of a single antibody "Q" might bind 40% of the PrPSc in a sample. Similar results might be obtained with six copies of antibody "R" and "S". However, by using two copies each of "Q", "R" and "S" the six antibodies will bind over 50% of the PrPSc in a sample. Thus, a synergistic effect can be obtained by combining combinations of two or more antibodies which bind PrPSc i.e., by combining two or more antibodies which have a binding affinity Ka for PrPSc of 107 l/mole or more. Thus combination of D4, R2, 6D2, D14, R1 and R10 and/or related antibodies can provide synergistic results.
Antibody/Antigen Binding Forces
The forces which hold an antigen and antibody together are in essence no different from non-specific interactions which occur between any two unrelated proteins i.e., other macromolecules such as human serum albumin and human transferrin. These intermolecular forces may be classified into four general areas which are (1) electrostatic; (2) hydrogen bonding; (3) hydrophobic; and (4) Van der Waals. Electrostatic forces are due to the attraction between oppositely charged ionic groups on two protein side-chains. The force of attraction (F) is inversely proportional to the square of the distance (d) between the charges. Hydrogen bonding forces are provided by the formation of reversible hydrogen bridges between hydrophilic groups such as --OH, --NH2 and --COOH. These forces are largely dependent upon close positioning of two molecules carrying these groups. Hydrophobic forces operate in the same way that oil droplets in water merge to form a single large drop. Accordingly, non-polar, hydrophobic groups such as the side-chains on valine, leucine and phenylalanine tend to associate in an aqueous environment. Lastly, Van der Waals are forces created between molecules which depend on interaction between the external electron clouds.
Further information regarding each of the different types of forces can be obtained from "Essential Immunology" edited by I. M. Roitti (6th Edition) Blackwell Scientific Publications, 1928. With respect to the present invention useful antibodies exhibit all of these forces. It is by obtaining an accumulation of these forces in larger amounts that it is possible to obtain an antibody which has a high degree of affinity or binding strength to the PrP protein and in particular an antibody which has a high degree of binding strength to PrPSc in situ.
Measuring Antibody/Antigen Binding Strength
The binding affinity between an antibody and an antigen can be measured which measurement is an accumulation of a measurement of all of the forces described above. Standard procedures for carrying out such measurements exist and can be directly applied to measure the affinity of antibodies of the invention for PrP proteins including native PrPSc in situ.
One standard method for measuring antibody/antigen binding affinity is through the use of a dialysis sac which is a container comprised of a material which is permeable to the antigen but impermeable to the antibody. Antigens which are bound completely or partially to antibodies are placed within the dialysis sac in a solvent such as in water. The sac is then placed within a larger container which does not contain antibodies or antigen but contains only the solvent e.g., the water. Since only the antigen can diffuse through the dialysis membrane the concentration of the antigen within the dialysis sac and the concentration of the antigen within the outer larger container will attempt to reach an equilibrium. After placing the dialysis sac into the larger container and allowing for time to pass towards reaching an equilibrium it is possible to measure the concentration of the antigen within the dialysis sac and within the surrounding container and then determine the differences in concentration. This makes it possible to calculate the amount of antigen which remains bound to antibody in the dialysis sac and the amount which disassociates from the antibody and diffuses into the surrounding container. By constantly renewing the solvent (e.g., the water) within the surrounding container so as to remove any antigen which is diffused thereinto it is possible to totally disassociate the antibody from antigen within the dialysis sac. If the surrounding solvent is not renewed the system will reach an equilibrium and it is possible to calculate the equilibrium constant (K) of the reaction i.e., the association and disassociation between the antibody and antigen. The equilibrium constant (K) is calculated as an amount equal to the concentration of antibody bound to antigen within the dialysis sac divided by the concentration of free antibody combining sites times the concentration of free antigen. The equilibrium constant or "K" value is generally measured in terms of liters per mole. The K value is a measure of the difference in free energy (deta g) between the antigen and antibody in the free state as compared with the complexed form of the antigen and antibody. When using the phage display methodology described below the antibodies obtained have an affinity or K value of 107 mole/liter or more.
As indicated above the term "affinity" describes the binding of an antibody to a single antigen determinate. However, in most practical circumstances one is concerned with the interaction of an antibody with a multivalent antigen. The term "avidity" is used to express this binding. Factors which contribute to avidity are complex and include the heterogeneity of the antibodies in a given serum which are directed against each determinate on the antigen and the heterogeneity of the determinants themselves. The multivalence of most antigens leads to an interesting "bonus" effect in which the binding of two antigen molecules by an antibody is always greater, usually many fold greater, than the arithmetic sum of the individual antibody links. Thus, it can be understood that the measured avidity between an antiserum and a multivalent antigen will be somewhat greater than the affinity between an antibody and a single antigen determinate.
Null PrP Mice to make Antibodies
The present invention circumvents problems of tolerance and more efficiently generates panels of monoclonal antibodies capable of recognizing diverse epitopes on Mo and other PrPs in part using mice with both alleles of the PrP gene (Prnp) are ablated (Prnp0/0) (Bueler, et al, 1992). These PrP-deficient mice (or null mice), are indistinguishable from normal mice in their develop and behavior. These null mice are resistant to scrapie following intracerebral inoculation of infectiou [MpPrPSc ] MoPrPSc (Bueler, et al, 1993 Cell; Prusiner, et al, PNAS 1993). In addition Prnp0/0 mice will develop IgG serum titers against Mo-, SHa and human PrP following immnunization with relatively small quantities of purified SHaPrP 27-30 in adjuvant (Prusiner, et al, PNAS 1993). After allowing sufficient time to generate antibodies the immunized Prnp0/0 mice were sacrificed for hybridoma production in the conventional manner. Fusions derived from these mice did secrete PrP specific antibody. However, these hybridomas would not secrete PrP specific antibodies for more than a few hours. In view of the somewhat limited success a different approach was taken.
Combinatorial antibody library technology, i.e., antigen based selection from antibody libraries expressed on the surface of M13 filamentous phage, offers a new approach to the generation of monoclonal antibodies and possesses a number of advantages relative to hybridoma methodologies which are particularly pertinent to the prion problem (Huse, et al, 1989; Barbas, et al, 1991; Clackson, et al, 1991; Burton and Barbas, 1994). The present invention uses such technology to provide PrP-specific monoclonal antibodies from phage antibody libraries prepared from MoPrP-immunized Prnp0/0 mice. The invention provides the first monoclonal antibodies recognizing MoPrP in situ and demonstrates the application of combinatorial libraries for cloning specific antibodies from null mice. The general methodologies involved in creating large combinatorial libraries using phage display technology is described and disclosed in U.S. Pat. No. 5,223,409 issued Jun. 29, 1993 which patent is incorporated herein by reference to disclose and describe phage display methodology.
The invention is largely described herein with respect to null mice i.e., FVB mice with both alleles of the PrP gene ablated. However, other host animals can be used and preferred host animals are mice and hamsters, with mice being most preferred in that there exists considerable knowledge on the production of transgenic animals. Possible host animals include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats), Oryctolagus (e.g. rabbits), and Mesocricetus (e.g. hamsters) and Cavia (e.g., guinea pigs). In general mammals with a normal full grown adult body weight of less than 1 kg which are easy to breed and maintain can be used.
The genetic material which makes up the PrP gene is known for a number of different species of animals (see Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992). Further, there is considerable homology between the PrP genes in different mammals. For example, see the amino acid sequence of mouse PrP compared to human, cow and sheep PrP in FIGS. 2, 3 and 4 wherein only the differences are shown. Although there is considerable genetic homology with respect to PrP genes, the differences are significant in some instances. More specifically, due to small differences in the protein encoded by the PrP gene of different mammals, a prion which will infect one mammal (e.g. a human) will not normally infect a different mammal (e.g. a mouse). Due to this "species barrier", it is not generally possible to use normal animals, (i.e., animal which have not had their genetic material related to PrP proteins manipulated) such as mice to determine whether a particular sample contains prions which would normally infect a different species of animal such as a human. The present invention solves this problem by providing antibodies which bind to native PrPSc proteins of any species of animal for which the antibody is designed.
Pathogenic Mutations and Polymorphisms
There are a number of known pathogenic mutations in the human PrP gene. Further, there are known polymorphisms in the human, sheep and bovine PrP genes. The following is a list of such mutations and polymorphisms:
Pathogenic human Human Sheep Bovine mutations Polymorphisms Polymorphisms Polymorphisms 2 octarepeat Codon 129 Codon 171 5 or 6 insert Met/Val Arg/Glu octarepeats 4 octarepeat Codon 219 Codon 136 insert Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert 7 octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102 Pro-Leu Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178 Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210 Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala
The DNA sequence of the human, sheep and cow PrP genes have been determined allowing, in each case, the prediction of the complete amino acid sequence of their respective PrP proteins. The normal amino acid sequence which occurs in the vast majority of individuals is referred to as the wild-type PrP sequence. This wild-type sequence is subject to certain is characteristic polymorphic variations. In the case of human PrP (SEQ ID NO:2), two polymorphic amino acids occur at residues 129 (Met/Val) and 219 (Glu/Lys). Sheep PrP (SEQ ID NO:4) has two amino acid polymorphisms at residues 171 and 136, while bovine PrP (SEQ ID NO:3) has either five or six repeats of an eight amino acid motif sequence in the amino terminal region of the mature prion protein. While none of these polymorphisms are of themselves pathogenic, they appear to influence prion diseases. Distinct from these normal variations of the wild-type PrP proteins, certain mutations of the human PrP gene which alter either specific amino acid residues of PrP or the number of octarepeats have been identified wich segregate with inherited human prion diseases.
In order to provide further meaning to the above chart demonstrating the mutations and polymorphisms, one can refer to the published sequences of PrP genes. For example, a chicken, bovine, sheep, rat and mouse PrP gene are disclosed and published within Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992). The sequence for the Syrian hamster is published in Basler et al., Cell 46:417-428 (1986). The PrP gene of sheep is published by Goldmann et al., Proc. Natl. Acad. Sci. USA 87:2476-2480 (1990). The PrP gene sequence for bovine is published in Goldmann et al., J. Gen. Virol. 72:201-204 (1991). The sequence for chicken PrP gene is published in Harris et al., Proc. Natl. Acad. Sci. USA 88:7664-7668 (1991). The PrP gene sequence for mink is published in Kretzschmar et al., J. Gen. Virol. 73:2757-2761 (1992). The human PrP gene sequence is published in Kretzschmar et al., DNA 5:315-324 (1986). The PrP gene sequence for mouse is published in Locht et al., Proc. Natl. Acad. Sci. USA 83:6372-6376 (1986). The PrP gene sequence for sheep is published in Westaway et al., Genes Dev 8:959-969 (1994). These publications are all incorporated herein by reference to disclose and describe the PrP gene and PrP amino acid sequences.
"Strains" of Human Prions
Studies in rodents have shown that prion strains produce different patterns of PrPSc accumulation [Hecker et al., Genes & Development 6:1213-1228 (1992); DeArmond et al., Proc. Natl. Acad. Sci. USA 90:6449-6453 (1993)]; which can be dramatically changed by the sequence of PrPSc [Carlson et al., Proc. Natl. Acad. Sci. USA in press (1994)]. The molecular basis of prion diversity has for many years been attributed to a scrapie specific nucleic acid [Bruce et al., J. Gen. Virol. 68:79-89 (1987)] but none has been found [Meyer et al., J. Gen. Virol. 72:37-49 (1991); Kellings et al., J. Gen. Virol. 73:1025-1029 (1992)]. Other hypotheses to explain prion strains include variations in PrP Asn-linked sugar chains [Hecker et al., Genes & Development 6:1213-1228 (1992)] and multiple conformers of PrPSc [Prusiner, S. B., Science 252:1515-1522 (1991)]. The patterns of PrPSc in Tg(MHu2M) mice were remarkably similar for the three inocula from humans dying of CJD.
The patterns of PrPSc accumulation in the brains of inoculated Tg(MHu2M) mice were markedly different for RML prions and Hu prions. However, RML prion inocula containing MoPrPSc stimulated the formation of more MoPrPSc while Hu prion inocula containing HuPrPCJD triggered production of MHu2MPrPSc. The distribution of neuropathological changes characterized by neuronal vacuolation and astrocytic gliosis is similar to the patterns of PrPSc accumulation in the brains of Tg(MHu2M) mice inoculated with RML prions or Hu prions.
Standardized Prion Preparation
Standardized prion preparations may be produced in order to test assays of the invention and thereby improve the reliability of the assay. Although the preparation can be obtained from any animal it is preferably obtained from a host animal which has brain material containing prions of a test animal. For example, a transgenic mouse containing a human prion protein gene can produce human prions and the brain of such a mouse can be used to create a standardized human prion preparation. Further, in that the preparation is to be a "standard" it is preferably obtained from a battery (e.g., 100; 1,000, or more animals) of substantial identical animals. For example, 100 mice all containing a very high copy number of human PrP genes (all polymorphisms and mutations) would spontaneously develop disease and the brain tissue from each could be combined to make a useful standardized prion preparation.
Standardized prion preparations can be produced using any of modified host mammals of the type described above. For example, standardized prion preparations could be produced using mice, rats, hamsters, or guinea pigs which are genetically modified so that they are susceptible to infection with prions which prions would generally only infect genetically diverse species such as a human, cow, sheep or horse and which modified host mammals will develop clinical signs of CNS dysfunction within a period of time of 350 days or less after inoculation with prions. The most preferred host mammal is a mouse in part because they are inexpensive to use and because a greater amount of experience has been obtained with respect to production of transgenic mice than with respect to the production of other types of host animals. Details regarding making standardized prion preparation are described in U.S. Patent application entitled "Method of Detecting Prions in a Sample and Transgenic Animal Used For Same" filed Aug. 31, 1995, Ser. No. 08/521,992 and U.S. Patent application entitled "Detecting Prions In A Sample And Prion Preparation And Transgenic Animal Used For Same", Attorney Docket No 06510/056001, filed Jul. 30, 1996, both of which applications are incorporated herein by reference.
Once an appropriate type of host is chosen, such as a mouse, the next step is to choose the appropriate type of genetic manipulation to be utilized to produce a standardized prion formulation. For example, the mice may be mice which are genetically modified by the insertion of a chimeric gene of the invention. Within this group the mice might be modified by including high copy numbers of the chimeric gene and/or by the inclusion of multiple promoters in order to increase the level of expression of the chimeric gene. Alternatively, hybrid mice of the invention could be used wherein mice which have the endogenous PrP gene ablated are crossed with mice which have a human PrP gene inserted into their genome. There are, of course, various subcategories of such hybrid mice. For example, the human PrP gene may be inserted in a high copy number an/or used with multiple promoters to enhance expression. In yet another alternative the mice could be produced by inserting multiple different PrP genes into the genome so as to create mice which are susceptible to infection with a variety of different prions, i.e., which generally infect two or more types of test animals. For example, a mouse could be created which included a chimeric gene including part of the sequence of a human, a separate chimeric gene which included part of the sequence of a cow and still another chimeric gene which included part of the sequence of a sheep. If all three different types of chimeric genes were inserted into the genome of the mouse the mouse would be susceptible to infection with prions which generally only infect a human, cow and sheep.
After choosing the appropriate mammal (e.g., a mouse) and the appropriate mode of genetic modification (e.g., inserting a chimeric PrP gene) the next step is to produce a large number of such mammals which are substantially identical in terms of genetic material related to prions. More specifically, each of the mice produced will include an identical chimeric gene present in the genome in substantially the same copy number. The mice should be sufficiently identical genetically in terms of genetic material related to prions that 95% or more of the mice will develop clinical signs of CNS dysfunction within 350 days or less after inoculation and all of the mice will develop such CNS dysfunction at approximately the same time e.g., within +30 days of each other.
Once a large group e.g., 50 or more, more preferably 100 or more, still more preferably 500 or more of such mice are produced. The next step is to inoculate the mice with prions which generally only infect a genetically diverse mammal e.g., prions from a human, sheep, cow or horse. The amounts given to different groups of mammals could be varied. After inoculating the mammals with the prions the mammals are observed until the mammals exhibit symptoms of prion infection e.g., clinical signs of CNS dysfunction. After exhibiting the symptoms of prion infection the brain or at least a portion of the brain tissue of each of the mammals is extracted. The extracted brain tissue is homogenized which provides the standardized prion preparation.
As an alternative to inoculating the group of transgenic mice with prions from a genetically diverse animal it is possible to produce mice which spontaneously develop prion related diseases. This can be done, for example, by including extremely high copy numbers of a human PrP gene into a mouse genome. When the copy number is raised to, for example, 100 or more copies, the mouse will spontaneously develop clinical signs of CNS dysfunction and have, within its brain tissue, prions which are capable of infecting humans. The brains of these animals or portions of the brain tissue of these animals can be extracted and homogenized to produce a standardized prion preparation.
The standardized prion preparations can be used directly or can be diluted and tittered in a manner so as to provide for a variety of different positive controls. More specifically, various known amounts of such standardized preparation can be used to inoculate a first set of transgenic control mice. A second set of substantially identical mice are inoculated with a material to be tested i.e., a material which may contain prions. A third group of substantially identical mice are not injected with any material. The three groups are then observed. The third group, should, of course not become ill in that the mice are not injected with any material. If such mice do become ill the assay is not accurate probably due to the result of producing mice which spontaneously develop disease. If the first group, injected with a standardized preparation, do not become ill the assay is also inaccurate probably because the mice have not been correctly created so as to become ill when inoculated with prions which generally only infect a genetically diverse mammal. However, if the first group does become ill and the third group does not become ill the assay can be presumed to be accurate. Thus, if the second group does not become ill the test material does not contain prions and if the second group does become ill the test material does contain prions.
By using standardized prion preparations of the invention it is possible to create extremely dilute compositions containing the prions. For example, a composition containing one part per million or less or even one part per billion or less can be created. Such a composition can be used to test the sensitivity of the antibodies, assays and methods of the invention in detecting the presence of prions.
Prion preparations are desirable in that they will include a constant amount of prions and are extracted from an isogeneic background. Accordingly, contaminates in the preparations will be constant and controllable. Standardized prion preparations will be useful in the carrying out of bioassays in order to determine the presence, if any, of prions in various pharmaceuticals, whole blood, blood fractions, foods, cosmetics, organs and in particular any material which is derived from an animal (living or dead) such as organs, blood and products thereof derived from living or dead humans. Thus, standardized prion preparations will be valuable in validating purification protocols where preparations are spiked and reductions in teeter measured for a particular process.
As indicated above and described further below in detailed examples it is possible to use the methodology of the invention to create a wide range of different antibodies. i.e., antibodies having different specific features. For example, antibodies can be created which bind only to a prion protein naturally occurring within a single species and not bind to a prion protein naturally occurring within other species. Further, the antibody can be designed so as to bind only to an infectious form of a prion protein (e.g., PrPSc) and not bind to a non-infectious form (e.g., PrPC). A single antibody or a battery of different antibodies can then be used to create an assay device. Such an assay device can be prepared using conventional technology known to those skilled in the art. The antibody can be purified and isolated using known techniques and bound to a support surface using known procedures. The resulting surface having antibody bound thereon can be used to assay a sample in vitro to determine if the sample contains one or more types of antibodies. For example, antibodies which bind only to human PrPSc can be attached to the surface of a material and a sample can be denatured via proteinase K. The denatured sample is brought into contact with the antibodies bound to the surface of material. If no binding occurs it can be deduced that the sample does not contain human PrPSc.
Antibodies of the invention are also characterized by their ability to neutralize prions. Specifically, when antibodies of the invention are allowed to bind to prions the infectivity of the prion is eliminated. Accordingly, antibody compositions of the invention can be added to any given product in order to neutralize any infectious prion protein within the product. Thus, if a product is produced from a natural source which might contain infectious prion proteins the antibodies of the invention could be added as a precaution thereby eliminating any potential infection resulting from infectious prion proteins.
The antibodies of the invention can be used in connection with immunoaffinity chromatography technology. More specifically, the antibodies can be placed on the surface of a material within a chromatography column. Thereafter, a composition to be purified can be passed through the column. If the sample to be purified includes any prion protein which binds to the antibodies those prion proteins (PrPSc) will be removed from the sample and thereby purified.
Lastly, the antibodies of the invention can be used to treat a mammal. The antibodies can be given prophylactically or be administered to an individual already infected with infectious prion proteins such infection having been determined by the use of the assay described above. The exact amount of antibody to be administered will vary depending on a number of factors such as the age, sex, weight and condition of the patient. Those skilled in the art can determine the precise amount by administering antibodies in small amounts and determining the effect and thereafter adjusting the dosage. It is suggested that the dosage can vary from 0.01 mg/kg to about 300 mg/kg, preferably about 0.1 mg/kg to about 200 mg/kg, more preferably about 0.2 mg/kg to about 20 mg/kg in one or more dose administrations daily, for one or several days. Preferred is administration of the antibody for 2 to 5 or more consecutive days in order to avoid "rebound" of prion infectivity occurring.
Claim 1 of 7 Claims
What is claimed is:
1. A method of determining PrPSc infection in an animal, comprising:
extracting tissue from an animal;
contacting the tissue with an antibody characterized by its ability to bind to native PrPSc in situ wherein the antibody binds to a form of PrPSc specific to the animal wherein the antibody binds to said PrPSc with a binding affinity Ka of 107 l/mol or more; and
determining if the antibody has bound to PrPSc ;
wherein presence of PrPSc in the tissue is indicative of PrPSc infection.