Patent Application
20220313802
The present application is directed to compositions and methods for the treatment and prevention of prion disease.
Prion disease is a unique category of illness, affecting both animals and humans, in which the underlying pathogenesis is related to a conformational change of a normal, self-protein called PrP to a pathological and infectious conformer. There is a need in the art for compositions and methods for the treatment and prevention of prion disease. The present application is directed to overcoming these and other deficiencies in the art.
A first aspect of the present disclosure is directed to an isolated protein or peptide thereof comprising the amino acid sequence of:
wherein X2 is V or A; X3 is K or null; X4 is S or null; X5 is N or H; X6 is I, V, L or M; X8 is S, G, Y or C; X10 is I, L or M; X12 is V, A, or L; X16 is A, T, or V; X17 is T or M; X19 is S or T; X21 is V, I, M; X23 is L or F; X is R or W; X33 is G or null; X39 is G or null; X47 S or I; X55 is P or S; X39 is G or null; X60 is G or T; X76 is G or S; X84 is G or S; X92 is G or null; X93 is W or null; X94 is G or null; X95 is Q or null; X96 is P or null; X97 is H or null; X98 is G or null; X99 is G or null; X100 is G or null; X105 is G or S; X107 is G or null; X108 is T, A, or S; X110 is G, N, or S; X113 is N or G; X116 is S or N; X121 is N or S; X122 is M or L; X125 is V or M; X142 is M or L; X151 is L, M or I; X156 is N or S; X158 is Y or W; X168 is Y or H; X172 is N or E; X177 is R or K; X179 is V or M; X180 is D, N or S; X181 is Q, R or E; X183 is S or N; X184 is N or S; X187 is N, S, or T, X190 is H or R; X197 is V or I; X199 is Q or E; X216 is V, M or I; X218 is M or I; X221 is R or Q; X228 is I or V; X232 is Q, E, or R; X233 R, Q, or K; X235 is S or Y; X236 is Q or E; X238 is Y, S, F, or A; X239 is Y or Q; X240 is Q, D, or G; X241 is G or null; X242 R or null; X243 is R or K; X244 is G, S, or A; X245 is A or S; X246 is S or G; X247 is V, T, A, M or null; X248 is I, L, V; X249 is L or I; X252 is S or P; X256 is I or V; X261 is F or L; X264 is F or L; X268 is G or R; and wherein the isolated protein or peptide thereof does not comprise an amino acid sequence of a corresponding protein of any one of SEQ ID Nos: 2-34 or a corresponding peptide thereof.
Another aspect of the present disclosure relates to a polymer of one or more peptides of SEQ ID NO: 1 as defined herein, and vaccine compositions comprising the same.
Another aspect of the present disclosure relates to an attenuated bacterium transformed with a vector expressing one or more proteins or peptides thereof of SEQ ID NO: 1 as defined herein, and vaccine compositions comprising the same.
Another aspect of the present disclosure is directed to methods of inhibiting the onset of or treat prion disease in a mammalian subject that involves administering, to the mammalian subject, a vaccine composition or delivery vehicle comprising the vaccine composition as described herein in an amount effective to inhibit the onset of or treat the prion disease in the mammalian subject.
Another aspect of the present disclosure relates to animal bait or feed comprising a vaccine composition as described herein.
Another aspect of the present disclosure relates to an isolated polynucleotide sequence encoding the protein or peptide thereof of SEQ ID NO: 1 as described herein. The present disclosure also relates to vectors containing the isolated polynucleotide sequences and host cells comprising these vectors.
Another aspect of the present disclosure relates to an isolated antibody or epitope-binding fragment thereof, where the antibody or epitope-binding fragment thereof binds to an epitope of the protein or peptide thereof comprising the amino acid sequence of SEQ ID NO: 1 as disclosed herein.
Another aspect of the present disclosure relates to an isolated antibody or epitope-binding fragment thereof, where the antibody or epitope-binding fragment thereof binds to an epitope of a polymer of a peptide fragment of SEQ ID NO: 1 as disclosed herein.
Another aspect of the present disclosure relates to a method of inhibiting the onset of or treating prion disease in a mammalian subject. This method involves administering, to the mammalian subject, the antibody or epitope-binding portion thereof as described herein in an amount effective to inhibit the onset of or treat the prion disease in the mammalian subject.
The present disclosure is directed to a multi-species prion protein or peptide thereof, and vaccine compositions comprising the same that are useful for the vaccination and treatment of mammalian subjects at risk of having or having prion disease.
A first aspect of the present disclosure is directed to an isolated protein or peptide thereof comprising the amino acid sequence of:
wherein X2 is V or A; X3 is K or null; X4 is S or null; X5 is N or H; X6 is I, V, L or M; X8 is S, G, Y or C; X10 is I, L or M; X12 is V, A, or L; X16 is A, T, or V; X17 is T or M; X19 is S or T; X21 is V, I, M; X23 is L or F; X27 is R or W; X33 is G or null; X39 is G or null; X47 S or I; X55 is P or S; X59 is G or null; X60 is G or T; X76 is G or S; X84 is G or S; X92 is G or null; X93 is W or null; X94 is G or null; X95 is Q or null; X96 is P or null; X97 is H or null; X98 is G or null; X99 is G or null; X100 is G or null; X105 is G or S; X107 is G or null; X108 is T, A, or S; X110 is G, N, or S; X113 is N or G; X116 is S or N; X121 is N or S; X122 is M or L; X125 is V or M; X142 is M or L; X151 is L, M or I; X156 is N or S; X158 is Y or W; X168 is Y or H; X172 is N or E; X177 is R or K; X179 is V or M; X180 is D, N or S; X181 is Q, R or E; X183 is S or N; X154 is N or S; X187 is N, S, or T, X190 is H or R; X197 is V or I; X199 is Q or E; X216 is V, M or I; X218 is M or I; X221 is R or Q; X228 is I or V; X232 is Q, E, or R; X233 R, Q, or K; X235 is S or Y; X236 is Q or E; X238 is Y, S, F, or A; X239 is Y or Q; X240 is Q, D, or G; X241 is G or null; X242 R or null; X243 is R or K; X244 is G, S, or A; X245 is A or S; X246 is S or G; X247 is V, T, A, M or null; X248 is I, L, V; X249 is L or I; X252 is S or P; X256 is I or V; X261 is F or L; X264 is F or L; X268 is G or R. The isolated protein or peptide thereof does not comprise an amino acid sequence of a corresponding protein of any one of SEQ ID Nos: 2-34 or a corresponding peptide thereof.
The protein sequence of SEQ ID NO: 1 is derived from an alignment of prion protein sequences from 33 different species as shown in
In accordance with this aspect of the present disclosure the isolated protein or peptide thereof does not have the same sequence as any corresponding naturally occurring prion protein or peptide derived therefrom. A “corresponding protein” or “corresponding peptide thereof” refers to a protein or peptide thereof that is of the same length as the protein or peptide thereof of the present disclosure. Upon alignment of the isolated protein or peptide thereof of SEQ ID NO: 1 to any corresponding naturally occurring prion protein or peptide thereof (e.g., any one of SEQ ID Nos: 2-34, as per
In one embodiment, the isolated protein or peptide thereof, comprises the amino acid sequence of SEQ ID NO: 1 or a peptide thereof, wherein each of the one or more single amino acid variations, relative to any corresponding naturally occurring prion protein or peptide thereof, are independently selected from residues occurring in different species. For example, the isolated protein or peptide thereof may comprise sequence identity to a naturally occurring prion protein sequence at all but three amino acid residues. The variant residue at each of these three amino acid residue positions is selected from a different species, such that the resulting amino acid sequence is a compilation of at least three different prion sequences derived from three different species, rendering it a multi-species prion protein or peptide thereof.
In one embodiment, the isolated peptide as disclosed herein is at least 10 amino acid residues in length. In another embodiment, the isolated peptide as disclosed herein is at least 15 amino acid residues in length. In another embodiment, the isolated peptide disclosed herein is at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues in length.
In one embodiment, the isolate peptide as disclosed herein is no more than 50 amino acid residues in length. In another embodiment, the isolated peptide as disclosed herein is no more than 55 amino acid residues in length. In another embodiment, the isolated peptide as disclosed herein is no more than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 amino acid residues in length.
An exemplary isolated peptide of the present disclosure comprises the amino acid sequence of amino acid residues 52-75 of SEQ ID NO: 1. This peptide has the amino acid sequence of RYPX55QGGX59X60WGQPHGGGWGQPHGG (SEQ ID NO: 35). For avoidance of confusion, the residues of variability in this and all exemplary peptides disclosed herein are identified by their position in the full-length protein of SEQ ID NO: 1. Thus, as described supra, the amino acid residue at position X55 in the aforementioned exemplary peptide of SEQ ID NO: 35 is selected from P or S; X59 is selected from G or null; and X60 is selected from G or T. In one embodiment, the isolated peptide of the present disclosure comprises the amino acid sequence of RYPPPQGGGWGQPHGGGWGQPHGG (PrP I; SEQ ID NO: 41).
Another exemplary isolated peptide of the present disclosure comprises the amino acid sequence of amino acid residues 104-134 of SEQ ID NO: 1. This peptide has the amino acid sequence of QX105GX107X108HX110QWX113 KPX116KPKTX121X122KHX125AGAAAAGAV (SEQ ID NO: 36), where X105 is selected from G or S; X107 is selected from G or null; X108 is selected from T, A, or S; X110 is selected from G, N, or S; X113 is selected from N or G; X116 is selected from S or N; X121 is selected from N or S; X122 is selected from M or L; and X125 is selected from V or M. In one embodiment, the isolated peptide of the present disclosure comprises the amino acid sequence of
Another exemplary isolated peptide of the present disclosure comprises the amino acid sequence of amino acid residues 155-182 of SEQ ID NO: 1. This peptide has the amino acid sequence of GX156DX158EDRYYRENMX168RYPX172QVYYX177PX179X180X181Y (SEQ ID NO: 37), where X156 is selected from N or S; X158 is selected from Y or W; X168 is selected from Y or H; X172 is selected from N or E; X177 is selected from R or K; X179 is selected from V or M; X180 is selected from D, N or S; and X181 is selected from Q, R or E. In one embodiment, the isolated peptide of the present disclosure comprises the amino acid sequence of GSDYEDRYYRENMYRYPEQVYYRPMDRY (PrP IV; SEQ ID NO: 43).
Another exemplary isolated peptide of the present disclosure comprises the amino acid sequence of amino acid residues 132-159 of SEQ ID NO: 1. This peptide has the amino acid sequence of GAVVGGLGGYX142L GSAMSRP X151IHFGX156DX158E (SEQ ID NO: 38), where X142 is selected from M or L; X151 is selected from L, M or I; X156 is selected from N or S; X158 is selected from Y or W. In one embodiment, the isolated peptide of the present disclosure comprises the amino acid sequence of
Another exemplary isolated peptide of the present disclosure comprises the amino acid sequence of amino acid residues 203-233 of SEQ ID NO: 1. This peptide has the amino acid sequence of TTTTKGENFTETD X216KX218ME X221VVEQMCX228TQYX232X233 (SEQ ID NO: 39), where X216 is selected from V, M or I; X218 is selected from M or I; X221 is selected from R, Q, or null; X228 is selected from I or V; X232 is selected from Q, E, or R; and X233 is selected from R, Q, or K. In one embodiment, the isolated peptide of the present disclosure comprises the amino acid sequence of
Another exemplary isolated peptide of the present disclosure comprises the amino acid sequence of amino acid residues 176-204 of SEQ ID NO: 1. This peptide has the amino acid sequence of YX177PX179X180X181YX183X184QNX187 FVX190DCVNITX197KX199HTVTT (SEQ ID NO: 40), where X177 is selected from R or K; X179 is selected from V or M; X180 is selected from D, N or S; X181 is selected from Q, R or E; X183 is selected from S or N; X184 is selected from N or S; X187 is selected from N, S, or T, X190 is selected from H or R; X197 is selected from V or I; and X199 is selected from Q or E. In one embodiment, the isolated peptide of the present disclosure comprises the amino acid sequence of YRPMDEYSSQNSFVRDCVNITIKQHTVTT (PrPV; SEQ ID NO:46).
The amino acid residues of the isolated protein or peptides there as described herein may be in either L- or D-form. In one embodiment, the isolated protein or peptide thereof as described herein is comprised of D-form amino acid residues. In another embodiment, the isolated protein or peptide thereof as described herein is comprised of D-form amino acid residues. D-form peptides have a higher stability than L-form peptides in vivo. In another embodiment, the isolated protein or peptide thereof is N- or C-terminally coupled to a polylysine or polyaspartate segment. In another embodiment, the C-terminal residue of the isolated protein or peptide thereof is amidated.
The isolated protein or peptide thereof as described herein may further comprise an adapter sequence on its amino terminus, carboxy terminus, or both termini to ensure flexibility and proper conformation of the epitopes of the protein or peptide independently of their proximity to the amino or carboxy termini. Suitable adapter sequences for this purpose include, without limitation, short glycine-serine adapter sequences, such as -GSG or -GSGSG. In another embodiment, when the isolated protein or peptide thereof is polymerized as described herein, the isolated protein or peptide thereof should have a K (lysine) residue after the glycine at the amino end (GKSG) and/or before the glycine at the carboxyl end (GSKG) to maximize the polymerization with glutaraldehyde avoiding the amino-end Schiff base formation on a monomer versus the covalent link to another peptide.
Another aspect of the present disclosure is directed to polynucleotides encoding the isolated protein and peptides thereof as described herein. In one embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 1 or a peptide thereof. In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 35. In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 36. In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 37. In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 38. In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 39. In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 40.
In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 41 (PrP I). An exemplary polynucleotide encoding the amino acid sequence of SEQ ID NO: 41 has the nucleotide sequence of SEQ ID NO: 47.
In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 42 (PrP II). An exemplary polynucleotide encoding the amino acid sequence of SEQ ID NO: 42 has the nucleotide sequence of SEQ ID NO: 48.
In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 43 (PrP IV). An exemplary polynucleotide encoding the amino acid sequence of SEQ ID NO: 43 has the nucleotide sequence of SEQ ID NO: 49.
In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 44 (PrP III). An exemplary polynucleotide encoding the amino acid sequence of SEQ ID NO: 44 has the nucleotide sequence of SEQ ID NO: 50.
In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 45 (PrP VI). An exemplary polynucleotide encoding the amino acid sequence of SEQ ID NO: 45 has the nucleotide sequence of SEQ ID NO: 51.
In another embodiment, the polynucleotide encodes the amino acid sequence of SEQ ID NO: 46 (PrP V). An exemplary polynucleotide encoding the amino acid sequence of SEQ ID NO: 46 has the nucleotide sequence of SEQ ID NO: 52.
The isolated protein or peptide thereof as described supra may be linked in-frame to an adjuvant polypeptide. The adjuvant polypeptide can be any adjuvant polypeptide known in the art, including, but not limited to, cholera toxin B, flagellin, human papillomavirus L1 or L2 protein, herpes simplex glycoprotein D (gD), complement C4 binding protein, TL4 ligand, influenza HA, Gb3, and IL-1β. The isolated protein or peptide thereof as described herein may be linked directly to the adjuvant polypeptide or coupled to the adjuvant by way of a short linker sequence. Suitable linker sequences include glycine-rich (e.g. G3-5) or serine-rich (e.g., GSG, GSGS, GSGSG, GSNG) linker sequences or other flexible immunoglobulin linkers as disclosed in U.S. Pat. No. 5,516,637 to Huang et al, which is hereby incorporated by reference in its entirety.
In another embodiment, the isolated protein or peptide thereof as described supra may be conjugated to an immunogenic carrier molecule. The immunogenic carrier molecule can be covalently or non-covalently bonded to the protein or peptide thereof. Suitable immunogenic carrier molecules include, but are not limited to, serum albumins, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, thyroglobulin, pneumococcal capsular polysaccharides, CRM 197, immunoglobulin molecules, alum, and meningococcal outer membrane proteins. Other suitable immunogenic carrier molecules include T-cell epitopes, such as tetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis B surface antigen, pertussis, toxoid, diphtheria toxoid, measles virus F protein, Chlamydia trachomatis major outer membrane protein, Plasmodium falciparum: circumsporozite T, P. falciparum CS antigen, Schistosoma mansoni triose phosphate isomersae, Escherichia coli TraT, and Influenza virus hemagluttinin (HA). Other suitable immunogenic carrier molecules include promiscuous T helper cell epitopes which are derived from hepatitis B virus, Bordetella pertussis, Clostridium tetani, Pertusaria trachythallina, E. coli, Chlamydia trachomatis, Diphtheria, P. falciparum, and Schistosoma mansoni (see U.S. Pat. No. 6,906,169 to Wang; U.S. Patent Application Publication No. 20030068325 to Wang, and WO/2002/096350 to Wang, which are hereby incorporated by reference in their entirety). Yet other suitable carriers include T-helper cell epitopes derived from tetanus toxin, cholera toxin B, pertussis toxin, diphtheria toxin, measles virus F protein, hepatitis B virus surface antigen, C. trachomitis major outer membrane protein, P. falciparum circumsporozoite, S. mansoni triose phosphate isomerase, or E. coli TraT (see WO01/42306 to Chain, which is hereby incorporated by reference in its entirety).
The isolated protein and peptide thereof of the present disclosure can be linked to immunogenic carrier molecules by chemical crosslinking. Techniques for linking a peptide immunogen to an immunogenic carrier molecule include the formation of disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue). These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein, and an amide linkage through the epsilon-amino on a lysine, or other free amino group in other amino acids. A variety of such disulfide/amide-forming agents are described by Jansen et al., “Immunotoxins: Hybrid Molecules Combining High Specificity and Potent Cytotoxicity,” Immun Rev 62:185-216 (1982), which is hereby incorporated by reference in its entirety. Other bifunctional coupling agents form a thioether rather than a disulfide linkage. Many of these thio-ether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, and 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
Another aspect of the present disclosure relates to a polymer of the isolated protein or peptide thereof of SEQ ID NO: 1 as disclosed herein. Polymerization of the isolated protein or peptide thereof alone or conjugated to an adjuvant polypeptide or immunogenic carrier molecule can be achieved using standard techniques known in the art. In one embodiment, the protein or peptide thereof is polymerized by a reaction with a cross linking reagent. Suitable cross-linking reagents include, but are not limited to glutaraldehyde and 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC). Alternatively, the isolated protein or peptide thereof can be polymerized by cysteine oxidation induced disulfide cross linking.
In one embodiment, a polymer as disclosed herein is a homopolymer, which is made of repeating units (i.e., monomers) of a single isolated protein or peptide thereof of SEQ ID NO: 1 as described herein. A monomer of a polymer as described herein is a single isolated protein or peptide thereof of SEQ ID NO: 1. In another embodiment, a polymer as disclosed herein is a heteropolymer, which is made of repeating monomers of two or more different isolated proteins or peptides thereof of SEQ ID NO: 1.
In one embodiment, the polymer as disclosed herein is made of repeating units of the peptide having the amino acid sequence of SEQ ID NO: 35. In another embodiment, the polymer as disclosed herein is made of repeating units of the peptide having the amino acid sequence of SEQ ID NO: 36. In another embodiment, the polymer as disclosed herein is made of repeating units of the peptide having the amino acid sequence of SEQ ID NO: 37. In another embodiment, the polymer as disclosed herein is made of repeating units of the peptide having the amino acid sequence of SEQ ID NO: 38. In another embodiment, the polymer as disclosed herein is made of repeating units of the peptide having the amino acid sequence of SEQ ID NO: 39. In another embodiment, the polymer as disclosed herein is made of repeating units of the peptide having the amino acid sequence of SEQ ID NO: 40. In another embodiment, the polymer as disclosed herein is comprised of repeating units of two or more peptides selected from the peptides of SEQ ID Nos: 35-40.
In one embodiment, the polymer as described herein is made via a controlled glutaraldehyde crosslinking reaction. Polymers made in this manner, including homopolymers and heteropolymers comprise, monomers (e.g., peptides as described herein) that are each covalently linked to another monomer of the polymer by a glutaraldehyde molecule as described in Goni et al., PLoS One. 5(10):e13391 (2010) and Goni et al., Sci Rep. 7(1):9881 (2017), which are hereby incorporated by reference in their entirety.
Another aspect of the present disclosure relates to a vaccine composition comprising one or more polymers as described herein and a pharmaceutically acceptable carrier. The polymers of the vaccine composition may be homopolymers, heteropolymers, or a mixture thereof. In one embodiment, the vaccine composition comprises a single polymer. In another embodiment, the vaccine composition comprises two or more different polymers. In another embodiment, the vaccine composition comprises three of more different polymers. In another embodiment, the vaccine composition comprises up to six different polymers.
In one embodiment, the vaccine composition comprises a polymer of the peptide having the amino acid sequence of SEQ ID NO: 35, alone or in combination with a different polymer. In another embodiment, the vaccine composition comprises a polymer of the peptide having the amino acid sequence of SEQ ID NO: 36, alone or in combination with a different polymer. In another embodiment, the vaccine composition comprises a polymer of the peptide having the amino acid sequence of SEQ ID NO: 37, alone or in combination with a different polymer. In another embodiment, the vaccine composition comprises a polymer of the peptide having the amino acid sequence of SEQ ID NO: 38, alone or in combination with a different polymer. In another embodiment, the vaccine composition comprises a polymer of the peptide having the amino acid sequence of SEQ ID NO: 39, alone or in combination with a different polymer. In another embodiment, the vaccine composition comprises a polymer of the peptide having the amino acid sequence of SEQ ID NO: 40, alone or in combination with a different polymer. In another embodiment, the vaccine composition as disclosed herein comprises one or more heteropolymers, where each heteropolymer comprises two or more peptides selected from the peptides of SEQ ID Nos: 35-40.
The vaccine composition comprising one or more polymers described herein may further contain, in addition to the polymers, other pharmaceutically acceptable components (see R
Vaccine compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized sepharose, agarose, cellulose), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
The vaccine composition of the present invention can further contain an adjuvant. An “adjuvant” as used herein is a substance that augments, stimulates, activates, or potentiates an immune response against a prion protein at either the cellular or humoral level. The adjuvant may be conjugated or cross-linked to the immunogen. Alternatively, the adjuvant is not conjugated to the prion protein of SEQ ID NO: 1, peptide thereof, or polymer thereof, but is added as an exogenous adjuvant/emulsion formulation which maximizes the immune response to the protein, peptide thereof, or polymer thereof. Suitable adjuvants include, without limitation, aluminum salt, cholera toxin subunit B, heat labile enterotoxin, flagellin, Freund's complete adjuvant, Freund's incomplete adjuvant, lysolecithin, pluronic polyols, polyanions, an oil-water emulsion, dinitrophenol, iscomatrix, influenza HA, Gb3, and liposome polycation DNA particles. In one embodiment, the adjuvant is one that maximizes the mucosal immune response. Preferred adjuvants are those which are shown to promote mucosal immunity with minimal or at least acceptable side effects.
One class of preferred adjuvants is aluminum salts, such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulating agents such as MPL or 3-DMP, QS-21, flagellin, polymeric or monomeric amino acids such as polyglutamic acid or polylysine, or pluronic polyols. Oil-in-water emulsion formulations are also suitable adjuvants that can be used with or without other specific immunostimulating agents such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn- -glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) Theramide™, or other bacterial cell wall components). A suitable oil-in-water emulsion is MF59 (containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton Mass.) as described in WO90/14837 to Van Nest et al., which is hereby incorporated by reference in its entirety. Other suitable oil-in-water emulsions include SAF (containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion) and Ribi™ adjuvant system (RAS; containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components). Another class of preferred adjuvants is saponin adjuvants, such as Stimulon™ (QS-21) or particles generated therefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other suitable adjuvants include incomplete or complete Freund's Adjuvant (IFA), cytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), lysolecithin, tumor necrosis factor (TNF), and liposome polycation DNA particles. Such adjuvants are generally available from commercial sources.
In another embodiment, the vaccine composition is packaged into a delivery vehicle. Suitable delivery vehicles include, but are not limited to biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, micelles, and cochleates. Cochleates are stable phospholipid calcium precipitates distinct from liposomes, which provide protection for vaccines from harsh acid and degradative environments thereby allowing efficient delivery by mucosal routes (Mannino and Gould-Fogerite, Pharm. Biotechnol., 6:363-87 (1995), which is hereby incorporated by reference in its entirety).
Another aspect of the present disclosure is directed to an attenuated microorganism transformed with a vector expressing one or more proteins or peptides thereof of SEQ ID NO: 1 as disclosed herein.
An “attenuated” microorganism is an organism with reduced virulence (infectivity). Because of their reduced virulence, attenuated microorganisms are suitable for use as antigen delivery vectors (sometimes with adjuvant properties) in vaccines. Methods for attenuating microorganisms are well known in the art. See, e.g., Chabalgoity et al., Vaccine, 19:460-469 (20000; Pasetti et al. Clin Immunol 92:76-89 (1999) and Hoiseth et al. Nature, 291:238-239 (1981), which are hereby incorporated by reference in their entirety. In one embodiment, the attenuated microorganism is an attenuated bacterium. Suitable attenuated bacterium include, without limitation, an attenuated strain of Salmonella, e.g., an attenuated strain of S. typhimurium, such as LVR01 (Chabalgoity et al., Vaccine, 19:460-469 (2000), which is hereby incorporated by reference in its entirety), LVR03 (a mouse-adapted derivative of LVR01), or SL3261 (Hoiseth et al., Nature, 291:238-239 (1981), Salmonella typhi CVD908-htrA (Tacket et al., Infect Immun., 68(3): 1196-1201 (2000), or Salmonella typhi Ty21a (Pasetti et al., Clin Immunol 92:76-89 (1999), which is hereby incorporated by reference in its entirety). Another suitable attenuated bacterium for use in accordance with the present disclosure is an attenuated strain of Shigella (see e.g., Chatfield et al., Vaccine, 10:53-60 (1992), which is hereby incorporated by reference in its entirety), such as WRSS1 (see e.g., Kotloff et al., Infect. Immun., 70:2016-21 (2002), which is hereby incorporated by reference in its entirety).
In one embodiment, the attenuated bacterium comprises a vector expressing the one or more proteins or peptides of interest, where each protein or peptide is expressed as single units from the vector. In other words, the vector comprises a single nucleotide sequence expressing a single protein or peptide of interest. In another embodiment, the attenuated bacterium comprises a vector expressing tandemly repeated units of the protein or peptide of interest. In this embodiment, the vector comprises a nucleotide sequence comprising tandem repeats (e.g., 2-4 repeats) encoding repeating units of the peptide or protein of interest.
In one embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 1 or a peptide thereof. In one embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 35. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 36. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 37. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 38. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 39. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 40. In another embodiment, the attenuated bacterium comprises a vector expressing the two or more peptide selected from the peptides of SEQ ID Nos: 35-40.
In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 41. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 42. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 43. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 44. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 45. In another embodiment, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 46. In another embodiment, the attenuated bacterium comprises a vector expressing the two or more peptide selected from the peptides of SEQ ID Nos: 41-46.
In some embodiments, the attenuated bacterium comprises a vector expressing the peptide having the amino acid sequence of SEQ ID NO: 1 or a peptide thereof, or any of the amino acid sequences of SEQ ID Nos: 35-46 as described above, where the expressed protein or peptide is conjugated to an immunostimulatory peptide which provides a strong T-helper cell (Th) response, including epitopes from potent immunogens such as tetanus toxin, pertussis toxin, the measles virus F protein, and the hepatitis B virus surface antigen (HBsAg), as disclosed in U.S. Pat. No. 5,843,446, which is hereby incorporated by reference. The Th epitopes selected to be conjugated to the protein or peptide thereof as described herein are preferably capable of eliciting T helper cell responses in large numbers of individuals expressing diverse MHC haplotypes. These epitopes function in many different individuals of a heterogeneous population and are considered to be promiscuous Th epitopes. Promiscuous Th epitopes provide an advantage of eliciting potent antibody responses in most members of genetically diverse population groups.
Method of producing the attenuated bacterium transformed with a vector expressing a protein or peptide thereof of SEQ ID NO: 1 can be carried out as described in U.S. Pat. No. 8,685,718 to Wisniewski et al.; Goñi et al., “Mucosal Vaccination Delays or Prevents Prion Infection via an Oral Route,” Neurosci. 133:413-421 (2005); Goñi et al., “High Titers of Mucosal and Systemic Anti-PrP Antibodies Abrogate Oral Prion Infection in Mucosal-Vaccinated Mice,” Neurosci. 153: 679-86 (2008); and Goñi et al., “Mucosal Immunization with an Attenuated Salmonella Vaccine Partially Protects White-Tailed Deer from Chronic Wasting Disease,” Vaccine 33:726-733 (2015), which are hereby incorporated by reference in their entirety, using conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. The general genetic engineering tools and techniques, including transformation and expression, the use of host cells, vectors, expression systems, etc., are well known in the art. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. (1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994), which are hereby incorporated by reference in its entirety.
Another aspect of the present disclosure is directed to a vaccine composition comprising the attenuated bacterium as described herein containing a vector expressing a protein or peptide thereof of SEQ ID NO: 1, and a pharmaceutically acceptable carrier.
In one embodiment, the vaccine composition comprises a single attenuated bacterium, where the bacterium is transformed with a vector expressing a protein or SEQ ID NO: 1 or a peptide thereof as described above. In another embodiment, the composition comprises two or more types of attenuated bacteria, each type of attenuated bacterium transformed with a vector expressing a different protein or peptide thereof of SEQ ID NO: 1, e.g., one or more peptides selected from SEQ ID Nos: 35-46. In another embodiment, the composition comprises 2, 3, 4, 5, or 6 types of attenuated bacteria, each type of attenuated bacterium transformed with a vector expressing a different protein or peptide thereof of SEQ ID NO: 1.
A vaccine composition comprising the attenuated bacterium transformed with a vector expressing a protein or peptide as described herein may comprise about 1×109, 1×1010, 1×1011, 1×1012, 1×1013, 1×1014, or >1×1014 CFU/ml in a pharmaceutically acceptable carrier, such as sterile PBS.
The vaccine composition may further comprise one or more adjuvants. In one embodiment, the one or more adjuvant is one that promotes or enhances mucosal immune response. Suitable adjuvants for inclusion in the vaccine compositions described herein include, without limitation, enterotoxin of E. coli; aluminum salts (e.g., aluminum hydroxide that also provides an alkaline environment to facilitate entry into the intestines of the recipient when used as a supplement in an oral immunization); and molecular adjuvants such as CpG DNA, i.e., an oligodeoxynucleotide that has been shown to be an inducer of innate immunity.
The vaccine compositions described herein, whether they comprise the isolated protein, peptide thereof, polymer thereof, or attenuated bacterium expressing the protein or peptide thereof, can be prepared by conventional methods known in the art for the preparation of pharmaceutically acceptable compositions which can be administered to subjects (see e.g., Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, Pa., USA 1985), which is hereby incorporated by reference in its entirety). The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. For oral administration, the vaccine can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl-pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art.
Preparations for oral administration can also take the form of, for example, solutions, syrups, emulsions or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts (e.g., aluminum hydroxide), flavoring, coloring and sweetening agents as appropriate.
Another aspect of the present disclosure relates to a method of inhibiting the onset of or treat an existing prion disease in a mammalian subject. This method involves administering, to a mammalian subject in need thereof, a vaccine composition as described herein. In one embodiment, the vaccine composition comprises the isolated protein or peptide thereof derived from SEQ ID NO: 1. Exemplary peptides of a vaccine composition include the peptide of SEQ ID Nos: 35-46. As described herein, the isolated protein or peptide thereof may itself be antigenic, may be linked to an adjuvant polypeptide and/or may be encapsulated in a delivery vehicle as described herein. In another embodiment, the vaccine composition comprises one or more polymers of the isolated protein or peptide thereof derived from SEQ ID NO: 1 alone or coupled to an adjuvant polypeptide. Exemplary compositions comprise one or more polymers of a peptide having an amino acid sequence of any one or more of SEQ ID Nos: 35-46. In another embodiment, the vaccine composition comprises one or more attenuated bacteria transformed with a vector expressing one or more isolated proteins or peptides thereof of SEQ ID NO: 1, e.g., any one or more of SEQ ID Nos: 35-46 (alone or coupled to an adjuvant polypeptide). In accordance with this aspect of the disclosure, the vaccine composition is administered to the mammalian subject in an amount effective to inhibit the onset of prion disease is said mammalian subject.
In accordance with this aspect of the disclosure, the vaccine compositions as described herein can be formulated for administration by parenteral, mucosal, topical, intravenous, oral, subcutaneous, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment. The most typical route of administration is subcutaneous although others can be equally effective. The next most common is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. Intravenous injections as well as intraperitoneal injections, intra-arterial, intracranial, or intradermal injections are also effective in generating an immune response.
In one embodiment, the vaccine composition is formulated for mucosal delivery, i.e., absorption via the oral, intragastric, intranasal, rectal õr intraocular mucosa to achieve systemic distribution. Methods for mucosal administration of the vaccine compositions as described herein are known in the art, see e.g., U.S. Pat. No. 8,685,718 to Wisniewski, Goñi et la., “Mucosal Immunization with an Attenuated Salmonella Vaccine Partially Protects White-Tailed Deer from Chronic Wasting Disease,” Vaccine 35(5):726-33 (2015), and Goñi et al., “Mucosal Vaccination Delays or Prevents Prior Infection via an Oral Route,” Neuroscience 133(20): 413-21 (2005), which are hereby incorporated by reference in their entirety.
Mammalian subjects which are at risk for exposure to prions or for developing prion disease that are suitable for treatment in accordance with the methods described herein, include, without limitation, humans, deer, elk, cows, sheep, buffalo, bison, moose, hamsters, camel, dromedary, vicuña, alpaca, llama, guanaco, gorilla, chimpanzee, Greater Kudu, wild felines in general, mice, and rats.
As referred to herein, “prion disease” generally refers to a neurodegenerative disease cause by a prion, i.e., a proteinaceous infection particle that causes misfolding of the prion protein. Prion diseases in non-human mammals that can be treated or prevented by employing the methods and compositions described herein include, without limitation, bovine spongiform encephalopathy, chronic wasting disease, scrapie, feline spongiform encephalopathy, transmissible mink encephalopathy, exotic ungulate encephalopathy, or any form of camelid spongiform encephalopathy. Prion diseases in human subjects that can be treated or prevented by employing the methods and compositions described herein include, without limitation, sporadic or familial Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomnia, and Kuru.
In accordance with the methods described herein, immunotherapy regimens which produce distinct and detectable immune responses following the administration of the fewest number of doses, ideally only one dose, are employed. Specific administration schedules and dosages of the vaccine compositions described herein can be readily determined by the ordinary skilled artisan. The immunization protocol is designed to primarily induce mucosal immunity to the prion protein. The vaccine can be administered in solid form for oral administration or in liquid form. Liquid forms may be administered to a subject either orally or intranasally. For example, unit dosage forms can be administered in intranasal form via topical use of suitable intranasal vehicles.
For example, the vaccines can be mucosally administered as a single dose or divided into multiple doses for administration. In one embodiment, vaccine is administered at least three times at spaced apart intervals, e.g., once weekly, once every 10 days, and once every two weeks. In another embodiment, a single vaccine dose is administered at day 0, and a booster vaccine administered about one week, two weeks, one month, or 2 months later. The booster vaccine can be the same as or different from the initial vaccine composition. For example, in one embodiment, a vaccine composition comprising an attenuated bacterium transformed to express one or more proteins or peptides thereof of SEQ ID NO:1 is initially administered to induce an immune response. The booster vaccine composition that is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or >10 weeks after the initial vaccine administration is a vaccine composition comprising one or more polymers of the protein and peptides thereof as described herein. In another embodiment, a vaccine composition comprising a cocktail of attenuated bacterium, each transformed to express one or more different proteins or peptides thereof of SEQ ID NO:1 is initially administered to induce an immune response. The booster vaccine composition that is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or >10 weeks after the initial vaccine administration is a vaccine composition comprising the same or different cocktail of attenuated bacterium, each transformed to express one or more different proteins or peptides thereof of SEQ ID NO:1.
For administration to wild animals such as deer, elk, bison, camels, etc., the vaccine can be mixed with food, and the food placed in a suitable container in an area where the target population lives using bait and baiting systems as have been developed for free-ranging animals for delivery of oral rabies vaccine (where the vaccine is mixed with food the animal likes to eat. (Knobel et al., “Development of a bait and baiting system for delivery of oral rabies vaccine to free-ranging African wild dogs (Lycaon pictus),” J. Wildl. Dis., 38:352-62 (2002), which is hereby incorporated by reference in its entirety.
Another aspect of the present disclosure is directed to animal bait or feed comprising any of the vaccine compositions described herein.
Another aspect of the present disclosure relates to an isolated antibody or epitope-binding portion thereof, wherein the antibody or epitope-binding portion thereof binds to an epitope of a protein or peptide thereof comprising the amino acid sequence of:
wherein X2 is V or A; X3 is K or null; X4 is S or null; X5 is N or H; X6 is I, V, L or M; X8 is S, G, Y or C; X10 is I, L or M; X12 is V, A, or L; X16 is A, T, or V; X17 is T or M; X19 is S or T; X21 is V, I, M; X23 is L or F; X27 is R or W; X33 is G or null; X39 is G or null; X47 S or I; X55 is P or S; X59 is G or null; X60 is G or T; X76 is G or S; X84 is G or S; X92 is G or null; X93 is W or null; X94 is G or null; X95 is Q or null; X % is P or null; X97 is H or null; X98 is G or null; X99 is G or null; X100 is G or null; X105 is G or S; X107 is G or null; X108 is T, A, or S; X110 is G, N, or S; X113 is N or G; X116 is S or N; X121 is N or S; X112 is M or L; X125 is V or M; X142 is M or L; X151 is L, M or I; X156 is N or S; X158 is Y or W; X168 is Y or H; X172 is N or E; X177 is R or K; X179 is V or M; X180 is D, N or S; X181 is Q, R or E; X183 is S or N; X184 is N or S; X187 is N, S, or T, X190 is H or R; X197 is V or I; X199 is Q or E; X216 is V, M or I; X218 is M or I; X221 is R or Q; X228 is I or V; X232 is Q, E, or R; X233 R, Q, or K; X235 is S or Y; X236 is Q or E; X238 is Y, S, F, or A; X239 is Y or Q; X240 is Q, D, or G; X241 is G or null; X242 R or null; X243 is R or K; X244 is G, S, or A; X245 is A or S; X246 is S or G; X247 is V, T, A, M or null; X248 is I, L, V; X249 is L or I; X252 is S or P; X256 is I or V; X261 is F or L; X264 is F or L; X268 is G or R; and wherein the protein or peptide thereof does not comprise an amino acid sequence of a corresponding protein of any one of SEQ ID Nos: 2-34 or a corresponding peptide thereof.
In one embodiment, the antibody or epitope-binding portion thereof is raised against the protein or peptide thereof of SEQ ID NO: 1.
Another aspect of the present disclosure relates to an isolated antibody or epitope-binding portion thereof, wherein said antibody or epitope-binding portion thereof binds to a polymer of a protein or peptide thereof, wherein said protein or peptide thereof of the polymer comprises the amino acid sequence of:
wherein X2 is V or A; X3 is K or null; X4 is S or null; X5 is N or H; X6 is I, V, L or M; X8 is S, G, Y or C; X10 is I, L or M; X12 is V, A, or L; X16 is A, T, or V; X17 is T or M; X19 is S or T; X21 is V, I, M; X23 is L or F; X27 is R or W; X33 is G or null; X39 is G or null; X47 S or I; X55 is P or S; X59 is G or null; X60 is G or T; X76 is G or S; X84 is G or S; X92 is G or null; X93 is W or null; X94 is G or null; X95 is Q or null; X96 is P or null; X97 is H or null; X98 is G or null; X99 is G or null; X100 is G or null; X105 is G or S; X107 is G or null; X108 is T, A, or S; X110 is G, N, or S; X113 is N or G; X116 is S or N; X121 is N or S; X122 is M or L; X125 is V or M; X142 is M or L; X151 is L, M or I; X156 is N or S; X158 is Y or W; X168 is Y or H; X172 is N or E; X177 is R or K; X179 is V or M; X180 is D, N or S; X181 s is Q, R or E; X183 is S or N; X184 is N or S; X187 is N, S, or T, X190 is H or R; X197 is V or I; X199 is Q or E; X216 is V, M or I; X218 is M or I; X221 is R or Q; X228 is I or V; X232 is Q, E, or R; X233 R, Q, or K; X235 is S or Y; X236 is Q or E; X238 is Y, S, F, or A; X239 is Y or Q; X240 is Q, D, or G; X241 is G or null; X242 R or null; X243 is R or K; X244 is G, S, or A; X245 is A or S; X246 is S or G; X247 is V, T, A, M or null; X248 is I, L, V; X249 is L or I; X252 is S or P; X256 is I or V; X261 is F or L; X264 is F or L; X268 is G or R; and
wherein the protein or peptide thereof does not comprise an amino acid sequence of a corresponding protein of any one of SEQ ID Nos: 2-34 or a corresponding peptide thereof.
In one embodiment, the antibody or epitope-binding portion thereof is raised against a polymer of a protein or peptide thereof of SEQ ID NO: 1.
As used herein, “epitope” refers to the antigenic determinant of the isolated protein or peptide thereof that is recognized by the isolated antibody. The epitope recognized by the antibody of the present invention may be a linear epitope, i.e. the primary structure of the amino acid sequence of the isolated protein or peptide thereof. Preferably, the linear epitope recognized by the isolated antibody of the present invention comprises a non-naturally occurring amino acid sequence, i.e., is not a sequence found within any of SEQ ID Nos: 2-34. Alternatively, the epitope recognized by the isolated antibody or epitope binding portion thereof is a non-linear or conformational epitope, i.e. the tertiary or quaternary structure of a polymerized protein or peptide thereof as described herein. More preferably, the non-linear or conformational epitope recognized by the isolated antibody or epitope-binding portion thereof is a conformational epitope that is common or shared with one or more, or all, prion proteins. Accordingly, the isolated antibody of the present invention has antigenic specificity for a shared conformational epitope common to all pathological prion proteins known in the art.
An isolated antibody of the present disclosure encompasses any immunoglobulin molecule that specifically binds to a linear or conformational epitope of an isolated protein or peptide thereof of SEQ ID NO: 1 as defined herein. As used herein, the term “antibody” is meant to include intact immunoglobulins derived from natural sources or from recombinant sources, as well as immunoreactive portions (i.e., antigen binding portions) of intact immunoglobulins. The antibodies of the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), antibody fragments (e.g. Fv, Fab and F(ab)2), as well as single chain antibodies (scFv), chimeric antibodies and humanized antibodies (Ed Harlow and David Lane, U
Antibodies of the present disclosure may also be synthetic antibodies. A synthetic antibody is an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. Alternatively, the synthetic antibody is generated by the synthesis of a DNA molecule encoding and expressing the antibody of the invention or the synthesis of an amino acid specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
Methods for monoclonal antibody production may be carried out using the techniques described herein or other well-known in the art (M
The antibody-secreting lymphocytes are then fused with myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is achieved by standard and well-known techniques, for example, by using polyethylene glycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation of Specific Antibody-Producing Tissue Culture and Tumor Lines by Cell Fusion,” Eur J Immunol 6:511 (1976), which is hereby incorporated by reference in its entirety). The immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and have good fusion capability. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody.
Alternatively, the antibodies described herein can be prepared by any of a variety recombinant DNA techniques using isolated polynucleotides, vectors, and host cells. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies via conventional techniques, or via transfection of antibody genes, heavy chains and/or light chains into suitable bacterial or mammalian cell hosts, in order to allow for the production of antibodies, wherein the antibodies may be recombinant. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium. Transfecting the host cell can be carried out using a variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., by electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the antibodies described herein in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is sometimes preferable, and sometimes preferable in mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
As noted above, exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216-4220 (1980), which is hereby incorporated by reference in its entirety). Other suitable mammalian host cells include, without limitation, NS0 myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown.
Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present disclosure. For example, it may be desirable to transfect a host cell with DNA encoding functional fragments of either the light chain and/or the heavy chain of an antibody described herein. Recombinant DNA technology may also be used to remove some or all the DNA encoding either or both of the light and heavy chains that is not necessary for binding to the antigens of interest. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies described herein.
In one embodiment, the sequence of the polynucleotide molecules encoding the antibodies and binding fragments or the proteins and peptides thereof of SEQ ID NO: 1 described herein are modified using gene editing technology. Suitable gene editing technology and systems include, for example, zinc finger nucleases (“ZFNs”) (Urnov et al., “Genome Editing with Engineered Zinc Finger Nucleases,” Nat. Rev. Genet. 11: 636-646 (2010), which is hereby incorporated by reference in its entirety), transcription activator-like effector nucleases (“TALENs”) (Joung & Sander, “TALENs: A Widely Applicable Technology for Targeted Genome Editing,” Nat. Rev. Mol. Cell Biol. 14: 49-55 (2013), which is hereby incorporated by reference in its entirety), clustered regularly interspaced short palindromic repeat (“CRISPR”)-associated endonucleases (e.g., CRISPR/CRISPR-associated (“Cas”) 9 systems) (Wiedenheft et al., “RNA-Guided Genetic Silencing Systems in Bacteria and Archaea,” Nature 482:331-338 (2012); Zhang et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems,” Science 339(6121): 819-23 (2013); and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based Methods for Genome Engineering,” Cell 31(7):397-405 (2013), which are hereby incorporated by reference in their entirety). Gene editing modifications can be employed for humanization, class-switch recombination, and/or antibody fragment production (see e.g., Cheong et al., “Editing of Mouse and Human Immunoglobulin Genes by CRISPR-Cas9 System,” Nature Comm. 7: 10934 (2016), and Flisikowska et al., “Efficient Immunoglobulin Gene Disruption and Targeted Replacement in Rabbit Using Zinc Finger Nucleases,” PLOS One 6(6): e21045 (2011), which are hereby incorporated by reference in their entirety.
The antibodies and antibody binding fragments are recovered and purified from recombinant cell cultures by known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be used for purification.
The monoclonal antibody of the present disclosure can be a humanized antibody. Humanized antibodies are antibodies that contain minimal sequences from non-human (e.g., murine) antibodies within the variable regions. Such antibodies are used therapeutically to reduce antigenicity and human anti-mouse antibody responses when administered to a human subject. In practice, humanized antibodies are typically human antibodies with minimal to no non-human sequences. A human antibody is an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.
An antibody can be humanized by substituting the complementarity determining region (CDR) of a human antibody with that of a non-human antibody (e.g., mouse, rat, rabbit, hamster, etc.) having the desired specificity, affinity, and capability (Jones et al., “Replacing the Complementarity-Determining Regions in a Human Antibody With Those From a Mouse,” Nature 321:522-525 (1986); Riechmann et al., “Reshaping Human Antibodies for Therapy,” Nature 332:323-327 (1988); Verhoeyen et al., “Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science 239:1534-1536 (1988), which are hereby incorporated by reference in their entirety). The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability.
Human antibodies can be produced using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See e.g., Reisfeld et al., M
Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the isolated protein or peptide thereof as described herein or a polymer of such isolated protein or peptide thereof as described herein subcutaneously to rabbits (e.g., New Zealand white rabbits), goats, sheep, swine, or donkeys which have been bled to obtain pre-immune serum. The antigens can be injected in combination with an adjuvant. The rabbits are bled approximately every two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. Polyclonal antibodies are recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. This and other procedures for raising polyclonal antibodies are disclosed in Ed Harlow and David Lane, U
In addition to whole antibodies, the present invention encompasses binding portions of such antibodies. Such binding portions include the monovalent Fab fragments, Fv fragments (e.g., single-chain antibody, scFv), and single variable VH and VL domains, and the bivalent F(ab′)2 fragments, Bis-scFv, diabodies, triabodies, minibodies, etc. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in James Goding, M
Also suitable for use in the present invention are antibody fragments engineered to bind to intracellular proteins, i.e. intrabodies. Accordingly, an intrabody can be used to bind selectively to an epitope of a pathological prion protein within a cell. Intrabodies are generally obtained by selecting a single variable domain from variable regions of an antibody having two variable domains (i.e., a heterodimer of a heavy chain variable domain and a light chain variable domain). Single chain Fv fragments, Fab fragments, ScFv-Ck fusion proteins, single chain diabodies, VH-CH1 fragments, and even whole IgG molecules are suitable formats for intrabody development (Kontermann R. E., “Intrabodies as Therapeutic Agents,” Methods 34:163-70 (2004), which is here by incorporated by reference in its entirety).
Intrabodies having antigen specificity for a conformational epitope of an prion protein can be obtained from phage display, yeast surface display, or ribosome surface display. Methods for producing libraries of intrabodies and isolating intrabodies of interest are further described in U.S. Published Patent Application No. 20030104402 to Zauderer and U.S. Published Patent Application No. 20050276800 to Rabbitts, which are hereby incorporated by reference in their entirety. Methods for improving the stability and affinity binding characteristics of intrabodies are described in WO2008070363 to Zhenping; Contreras-Martinez et al., “Intracellular Ribosome Display via SecM Translation Arrest as a Selection for Antibodies with Enhanced Cytosolic Stability,” J Mol Biol 372(2):513-24 (2007), which are hereby incorporated by reference in their entirety.
It may further be desirable, especially in the case of antibody fragments, to modify the antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis).
Antibody mimics are also suitable for use in accordance with the present invention. A number of antibody mimics are known in the art including, without limitation, those known as monobodies, which are derived from the tenth human fibronectin type III domain (10Fn3) (Koide et al., “The Fibronectin Type III Domain as a Scaffold for Novel Binding Proteins,” J Mol Biol 284:1141-1151 (1998); Koide et al., “Probing Protein Conformational Changes in Living Cells by Using Designer Binding Proteins: Application to the Estrogen Receptor,” Proc Natl Acad Sci USA 99:1253-1258 (2002), each of which is hereby incorporated by reference in its entirety); and those known as affibodies, which are derived from the stable alpha-helical bacterial receptor domain Z of staphylococcal protein A (Nord et al., “Binding Proteins Selected from Combinatorial Libraries of an alpha-helical Bacterial Receptor Domain,” Nature Biotechnol 15(8):772-777 (1997), which is hereby incorporated by reference in its entirety).
Another aspect of the present disclosure is directed to a pharmaceutical composition comprising an antibody or epitope-binding portion thereof and a pharmaceutically acceptable carrier.
Other aspects of the present disclosure relate to methods of preventing the onset of a prion disease in a mammalian subject and method of treating a mammalian subject having a prion disease. This method involves administering to the mammalian subject an antibody or epitope-binding portion or a pharmaceutical composition containing the same as described herein. In one embodiment, the subject is at risk for acquiring a prion disease and is thus, administered the antibody or epitope-binding portion thereof or a pharmaceutical composition containing the same in an amount effective to prevent the onset of the prion disease. In another embodiment, the subject has prion disease, and, thus, is administered the antibody or epitope-binding portion thereof or a pharmaceutical composition containing the same in an amount effective to treat the prion disease in the subject. In accordance with this aspect of the invention, the antibody or pharmaceutical composition containing the antibody is administered in an amount effective to generate passive immunity in the mammalian subject against one or more pathological prion proteins, thereby facilitating the clearance of the pathological prion protein from the subject. Suitable mammalian subjects and prion conditions that can be treated in accordance with this aspect of the present disclosure are described supra.
For passive immunization with a composition comprising an antibody or epitope-binding portion thereof as described herein, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg of the host body weight. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated. Antibody is usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of antibody to polymerized first or second peptides or fusion peptides in the patient. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
PRNP Sequencing. Genomic DNA was purified from 200 μL of whole blood using commercial kits (Illustra G&E, or Purelink Genomic DNA mini spin kit, Invitrogen). Polymerase chain reaction (PCR) was performed with forward primer: 5′-CACAGCAGATATAAGTCATCATGGTG-3′ (SEQ ID NO:53) and reverse primer 5′-GGTAAAGATGATTAAGAAGATGATGAAAACAGGAAGG-3′ (SEQ ID NO:54). These primers amplify the Codificant region of the PRNP gene and were designed based on PRNP gene sequences from different species available in GenBank. PCR was carried out in 50 μl reaction mixtures containing 50 ng of purified DNA, 0.2 μM of each dNTP, 15 pmol of each primer, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 3.0 mM MgCl2, and 2.5 U of Taq DNA Polymerase (Invitrogen). Cycling conditions after initial denaturation at 94° C. for 3 minutes were 35 cycles of denaturation (94° C., 30 seconds), annealing (48° C., 30 seconds) and extension (72° C., 45 seconds), and a final extension at 72° C. for 5 minutes. PCR fragments (˜850 bp) were purified using Pure link PCR purification kit (Invitrogen) and sequenced directly (using the primers described above) or cloned into pGEM-T easy vector (Promega) and then sequenced (using primers sp6 and t7). Sequencing was carried out using an 3500×1 Genetic Analyzer (Applied Biosystems) by INTA's Genomic Unit (Biotechnology Institute, INTA, Buenos Aires, Argentina).
The nucleotide sequences of newly sequenced PRNP genes are set forth below.
Peptide Synthesis and Polymerization. The peptides shown in Table 1 were synthesized and used for Electron Microscopy in saline, after controlled polymerization with glutaraldehyde (using the procedure outlined below), and after hetero-polymerization with Cholera Toxin B (CTB) on a controlled reaction with glutaraldehyde (
RYPPQGGGWGQPHGGGWGQPHGG
QGGSHSQWNKPSKPKTNLKHMAGAAAAGAV
GAVVGGLGGYMLGSAMSRPIIHFGNDWE
GSDYEDRYYRENMYRYPEQVYYRPMDRY
YRPMDEYSSQNSFVRDCVNITIKQHTVTT
TTTTKGENFTETDIKMMERVVEQMCVTQYER
The peptides were subjected to controlled polymerization using the following protocol. The peptides were dissolved at 3 mg/ml, in 100 mM borate buffer saline (BBS), pH 7.4. Fresh 1% glutaraldehyde in BBS was prepared and added to the peptide to a final 5 mM glutaraldehyde concentration and incubated in an Eppendorf block at 800 rpm at 56° C. for 16 hrs. The solution was then quenched with 0.5 M glycine to make the solution 100 mM in glycine. After five minutes the solution was diluted 1:3 with BBS, dialyzed extensively against 200 volumes and three changes of 2 mM BBS overnight at 4° C., aliquoted, and lyophilized.
Plasmid Construction. Expression plasmid pTECH2 and hybridized oligonucleotide pairs (forward and reverse), having the nucleic acid sequences shown in Table 2, were each digested with BamHI and HindIII and then ligated together to form the expression vectors pTECH2-PrP I, -PrP II, -PrP III, -PrP IV, -PrP V, and -PrP VI, containing a single copy of the cloned peptide (x1), as shown in
For construction of plasmids expressing tandem copies of the cloned peptides (x2), unique PstI, XbaI, and SbeI sites were used (
CGTTACCCTCCGCAGGGCGGTGGCT
CTT
GCT
CGC
CAGGGTGGCAGCCATAGCCAATGG
CGC
GGCGCCGTGGTCGGTGGCCTGGGTG
CGC
GGTAGCGATTACGAAGATCGTTATT
CGC
TATCGTCCGATGGATGAATACTCCA
AAGCTT
GCT
CGC
ACCACGACCACGAAAGGCGAGAAC
CGC
Recombinant fusion vectors expressing a single copy (x1) or two tandem copies (x2) of the PrP I, -II, -II, -IV, -V, and -VI peptides as C-terminal fusion to tetanus toxin fragment C in a live aroC attenuated vaccine strain of Salmonella LVR01 were created (as shown in
Prp multi-species peptides I, II, III, IV, V, and VI (as described supra) were expressed in attenuated Salmonella LVR01 (as described supra). Immunoblotting showed the presence of multi-species PrP peptides I-VI in the supernantants of the vaccine ready corresponding Salmonellas and the expression shown in the lysates of the same vaccine ready Salmonellas (
The vaccine ready Salmonella LVR01 multi-species PrP I-VI were kept for 7 days at 4° C. and at room temperature (RT) for 36 hours before inoculation. The viability of the single or combined vaccines, measured as the percentage of bacteria determined by light microscopy in a Neubauer camera that showed some Brownian movement within 2 minutes of the observation; was between 87 and 95%, which makes all preparations suitable for scale production and for use combined with food in confined environments or as bait in the open field.
Antisera against Salmonella show the vaccine attenuated bacteria to be located in lymphoid follicles of the crevices and in epithelial cells of a mouse inoculated with the attenuated Salmonella vaccine 20 hours prior (
Multi-species peptides I-VI were expressed in vaccine attenuated Salmonella LVR01 either as a single copy (x1) or two tandem copies (x2), or synthesized and control polymerized with glutaraldehyde. Transgenic mice were inoculated weekly three times, twice with the Salmonella vaccine and the last one with the corresponding control polymerized synthetic peptides in sterile saline and alum (Al(OH)3), as described in Goni et al., “Mucosal Immunization With an Attenuated Salmonella Vaccine Partially Protects White-Tailed Deer From Chronic Wasting Disease,” Vaccine 33:726-33 (2015), which is hereby incorporated by reference in its entirety. Transgenic mice included KO PrP (knock out for PrP), Hu PRP (transgenic for human PrP), Sh PrP (transgenic for sheep PrP), Elk PrP (transgenic for Elk-Deer PrP), and All PrP (including the wild type mouse background PrP). Control animals were inoculated with the same attenuated Salmonella LVR01 carrying the TETc fragment alone and the last boost with sterile saline-alum vehicle alone.
Immunoblots of different prion disease samples (human, deer-CWD, sheep-scrape) were run on 12.5% SDS-PAGE gel and transferred onto nitrocellulose and incubated at room temperature for one hour with mouse plasma diluted 1:1000 in TBS-T, from samples obtained from the corresponding animals 10 days after the third inoculation (second boost). The secondary one hour incubation was performed with antisera anti-mouse GAM (IgG-IgA-IgM) conjugated to horse radish peroxidase. The blots were developed by ECL and recorded on X-ray films (
At the same time, samples from feces of the corresponding animals were collected, homogenized and diluted 1:1000 to be used as primary antibody on similar blots; the secondary antibody was specific anti-mouse IgA-HRP to detect the most important antibody that could be produced in the intestinal mucosa (
All methodologies used were similar to the ones detailed in Goni et al., “High Titers of Mucosal and Sytemic anti-PRP Antibodies Abrogate Oral Prion Infection in Mucosal-Vaccinated Mice,” Neuroscience 153(3):679-86 (2008).
This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 62/846,250, filed May 10, 2019, which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant numbers NS47433 and NS073502 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/032326 | 5/11/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62846250 | May 2019 | US |
