WO2018006092A1 - Composés d'amino acides de copolymères et leurs applications. - Google Patents

Composés d'amino acides de copolymères et leurs applications. Download PDF

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WO2018006092A1
WO2018006092A1 PCT/US2017/040598 US2017040598W WO2018006092A1 WO 2018006092 A1 WO2018006092 A1 WO 2018006092A1 US 2017040598 W US2017040598 W US 2017040598W WO 2018006092 A1 WO2018006092 A1 WO 2018006092A1
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composition
amino acid
polypeptides
amino acids
peptide sequence
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PCT/US2017/040598
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English (en)
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Eric Zanelli
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Declion Holdings Llc
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Priority to CA3029654A priority Critical patent/CA3029654A1/fr
Priority to AU2017290907A priority patent/AU2017290907A1/en
Priority to EP17821442.5A priority patent/EP3478316A1/fr
Publication of WO2018006092A1 publication Critical patent/WO2018006092A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the specific challenges may vary across applications, some of the challenges include (i) inducing antibodies against relevant target(s); (ii) preserving the normal functional protein where applicable; (iii) eliciting immunogenicity across a broad patient population; (iv) achieving efficacious antibody titer; (v) reaching target(s) in the brain; (vi) avoiding or minimizing a Thl/pro-inflammatory immunity and meningoencephalitis, where applicable; (vii) avoiding microhemorrhages and vasogenic edema; and (viii) significant costs associated with passive immunity.
  • compositions and methods that aim to address the limitations of existing technologies.
  • the present disclosure provides compositions comprising high complexity mixtures (e.g., mixtures of greater than at least 500 polypeptides) of polypeptides based on a template arrangement comprising one or more antigenic regions (ARs) and one or more random copolymer regions (RCRs) (e.g., compositions of the disclosure; amino acid polymer compositions of the disclosure).
  • such compositions are capable of inducing CCL22 release by monocytes and/or of inducing CD4+ T cell proliferation and/or of inducing specific antibodies, such as antibodies to an epitope within an antigenic region.
  • such compositions are capable of inducing antibodies against a relevant target (e.g., a target from which one or more ARs of the composition are derived; an antigen from which one or more ARs of the composition are derived).
  • a relevant target e.g., a target from which one or more ARs of the composition are derived; an antigen from which one or more ARs of the composition are derived.
  • the CCL22 release, CD4+ T-cell proliferation and/or antibody induction properties of the composition are better, as compared to (i) a single polypeptide based on one or more of the same AR and/or (ii) the same or similar polypeptide or polypeptide composition in the absence of RCRs and/or (iii) the same or similar polypeptide composition having a net charge or estimated net charge of less than 1 or less than 2.
  • the CCL22 release, CD4+ T-cell proliferation and/or antibody induction properties of the composition are better, as compared to (i) a single polypeptide based on one or more of the same AR and (ii) the same or similar polypeptide or polypeptide composition in the absence of RCRs.
  • the disclosure provides a composition comprising a mixture of at least 500 different polypeptides each having a length of between about 25 to 100 amino acids, wherein each polypeptide, such as at least 500 polypeptides, comprises one or more antigenic regions (each an AR) linked to one or more random copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one or more AR and the one or more RCR are arranged according to a linear template arrangement (together a "complex polypeptide mixture component").
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio (and/or by a relative molar input percentage) of the amino acids available for that position and is independently selected.
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid (E), or phenylalanine (F), and wherein, for each amino acid position of each RCR, the relative molar input percentage of A for each position is less than or equal to 65%, the relative molar input percentage of positively charged amino acids for each position is at least 35% and the relative molar input percentage of negatively charged amino acids for each position is less than or equal to 20%, and wherein the distribution of the amino acids at a given position of the one or more RCRs among the polypeptides is determined by a predetermined molar input ratio of the amino acids available for that position and is
  • polypeptides of the mixture comprise more than one RCR, amino acid content and molar input percentage of each RCR is independently selected.
  • the disclosure provides a composition comprising a mixture of at least 500 different polypeptides each having a length of between about 25 to 100 amino acids, wherein each polypeptide, such as at least 500 polypeptides, comprises one or more antigenic regions (each an AR) linked to two or more random copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one or more AR and the two or more RCRs are arranged according to a linear template arrangement (together a "complex polypeptide mixture component").
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides is determined by a pre-determined molar input ratio (or by a relative molar input percentage) of the amino acids available for that position and is independently selected.
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and wherein, for each amino acid position of the two or more RCRs, the relative molar input percentage of A for each position is less than or equal to 65%, the relative molar input percentage of positively charged amino acids for each position is at least 35% and the relative molar input percentage of negatively charged amino acids for each position is less than or equal to 20%, and wherein the distribution of the amino acids at a given position of the one or more RCRs among the polypeptides is determined by a predetermined molar input ratio of the amino acids available for that position and is
  • the disclosure provides a composition comprising a mixture of at least 500 different polypeptides each having a length of between about 25 to 100 amino acids, wherein each polypeptide, such as at least 500 polypeptides, comprises one or more antigenic regions (each an AR) linked to one or more random copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one or more AR and the one or more RCR are arranged according to a linear template arrangement (together a "complex polypeptide mixture component").
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio (and/or by a relative molar input percentage) of the amino acids available for that position and is independently selected.
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid, or phenylalanine (F), and wherein the distribution of the amino acids at a given position of the one or more RCRs among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position, and wherein, if the polypeptides of the mixture comprise more than one RCR, amino acid content and molar input percentage of each RCR is independently selected.
  • the ratio of the percentage of alanine to the percentage of lysine, on a molar basis, in the polypeptides of the composition having a length of between about 25 to 100 amino acids is greater than or equal to 1.5 and less than or equal to 5.0 (as expressed as a quotient, which ratio may also be expressed as a relative ratio of 1.5: 1 to 5.0: 1).
  • the disclosure provides a composition comprising a mixture of at least 500 different polypeptides each having a length of between about 25 to 100 amino acids, wherein each polypeptide, such as at least 500 polypeptides, comprises one or more antigenic regions (each an AR) linked to two or more random copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one or more AR and the two or more RCRs are arranged according to a linear template arrangement (together a "complex polypeptide mixture component").
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the peptides is determined by a pre-determined molar input ratio (and/or by a relative molar input percentage) of the amino acids available for that position and is independently selected.
  • each amino acid position of the two or more RCRs for each amino acid position of the two or more RCRs, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A),
  • polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and wherein the distribution of the amino acids at a given position of the one or more RCRs among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position, and wherein amino acid content and molar input percentage of each RCR is independently selected.
  • the ratio of the percentage of alanine to the percentage of lysine, on a molar basis, in the polypeptides of the composition having a length of between about 25 to 100 amino acids is greater than or equal to 1.5 and less than or equal to 5.0.
  • the disclosure provides a composition
  • a composition comprising a mixture of at least 500 different polypeptides each having a length of between about 25 to 100 amino acids, wherein each polypeptide, such as at least 500 polypeptides, comprises one or more antigenic regions (each an AR) linked to one or more random copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one or more AR and the one or more RCR are arranged according to a linear template arrangement (together a "complex polypeptide mixture component”), which composition has an estimated net charge of greater than 2.0 and less than 4.0 at pH7.
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio (and/or by a relative molar input percentage) of the amino acids available for that position and is independently selected.
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid (E), or phenylalanine (F), and wherein, for each amino acid position of each RCR, the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position of each RCR.
  • the disclosure provides a composition
  • a composition comprising a mixture of at least 500 different polypeptides each having a length of between about 25 to 100 amino acids, wherein each polypeptide comprises one or more antigenic regions (each an AR) linked to two or more random copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one or more AR and the two or more RCRs are arranged according to a linear template arrangement (together a "complex polypeptide mixture component"), which composition has an estimated net charge of greater than 2.0 and less than 4.0 at pH7.
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides is determined by a pre-determined molar input ratio (and/or by a relative molar input percentage) of the amino acids available for that position and is independently selected.
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and wherein, for each amino acid position of the two or more RCRs, the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position of each RCR.
  • one or more of the positively charged amino acids of an RCR may instead be a non-naturally occurring amino acid and/or one or more of the negatively charged amino acids of an RCR may instead be a non-naturally occurring amino acid.
  • the relative molar input percentage of A is between about 5% and 40%, between about 10% and 40%, between about 15%) and 40%, between about 10% and 35%, or between about 15% and 35% of the total input amino acid composition of the complex polypeptide mixture component. In certain embodiments, the relative molar input percentage of A is less than 30%, such as between 10 and 30%) or 15 and 30% of the total input amino acid composition of the complex polypeptide mixture. In certain embodiments, the relative molar input percentage of A is between 15 and 25%) of the total input amino acid composition. In certain embodiments, the relative molar input percentage of A is between 15 and 20% of the total input amino acid composition. In certain embodiments, the relative molar input percentage of A is between 20 and 25% of the total input amino acid composition.
  • the estimated net charge of the polypeptides of the composition having a length of between about 25 to 100 amino acids is greater than or equal to 2 at pH7. In certain embodiments of any of the foregoing or following, the estimated net charge is greater than or equal to 2 and less than or equal to 4 at pH7. In certain embodiments of any of the foregoing or following, the estimated net charge is greater than 2.1 and less than or equal to 4 at pH7.
  • At least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or greater than at least 90%) of the polypeptides in the complex polypeptide mixture based on the linear template arrangement are substantially full-length polypeptides or full-length polypeptides. In certain embodiments, at least 95% (or even greater) of the polypeptides in the complex mixture are full-length or substantially full-length polypeptides (based on the linear template arrangement).
  • amino acid sequence of at least one AR does not vary among the polypeptides in the composition. In certain embodiments of any of the foregoing or following, the amino acid sequence of at least one AR does not vary among the polypeptides in the composition, and the amino acid sequence of said AR comprises a sequence of amino acid positions corresponding to the base peptide sequence and the polypeptides in the composition have an amino acid at each position of the AR selected from an original amino acid found at the corresponding amino acid position of first base peptide sequence.
  • the amino acid sequence of at least one AR varies with respect to the base peptide sequence among the polypeptides of the composition, various polypeptides having an amino acid at each position independently selected from: an original amino acid found at the corresponding amino acid position of the base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid.
  • the polypeptides in the composition comprise two ARs (ARa and ARb), and each AR may be derived from the same or a different base peptide sequence, said different base peptide sequence a second base peptide sequence, and wherein ARa may be either N-terminal or C-terminal to ARb.
  • the full- length or substantially full-length polypeptides in the composition comprise two ARs (AR a and ARb), and each AR may be derived from the same or a different base peptide sequence, said different base peptide sequence a second base peptide sequence, and wherein ARa may be either N-terminal or C-terminal to ARb.
  • the polypeptides in the composition comprise two or more ARs (ARa, ARb, and AR m ), and each of these ARs may be derived from the same or a different base peptide sequence, and wherein m is an integer from 0-3.
  • the full-length or substantially full-length polypeptide in the composition comprise two or more ARs (ARa, ARb, and AR m ), and each of these ARs may be derived from the same or a different base peptide sequence, and wherein m is an integer from 0-3.
  • ARa and ARb are derived from a different base peptide sequence. In certain embodiments, all of the ARs are from the same antigen, but from a different base peptide sequence.
  • m is 1, such that the polypeptides in the composition, such as the substantially full-length polypeptides in the composition, each comprises three ARs.
  • the amino acid sequence of at least one AR does not vary among the polypeptides in the composition, and the amino acid sequence of said AR comprises a sequence of amino acid positions corresponding to the base peptide sequence and the polypeptides in the composition have an amino acid at each position of the at least one AR selected from an original amino acid found at the
  • the amino acid sequence of at least one AR varies with respect to its corresponding base peptide sequence among the polypeptides of the composition, various polypeptides having an amino acid at each position independently selected from: an original amino acid found at the corresponding amino acid position of the base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid.
  • the amino acid sequence of ARa and ARb varies independently with respect to the first and second base peptide sequence, respectively, among the polypeptides of the composition, various polypeptides having an amino acid at each position independently selected from: an original amino acid found at the corresponding amino acid position of the base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid.
  • At least one base peptide sequence and/or at least one AR is 9-30, 13-26, 15-20, or 13-20 amino acids in length. In certain embodiments, the foregoing applies to at least two, at least three, or three ARs and/or base peptide sequences, wherein the length of each is independently selected.
  • the polypeptides in the composition comprise two or more ARs (ARa, ARb, and AR m ), and each of these ARs may be derived from the same or a different base peptide sequence (a first, a second, and an mth base peptide sequence, respectively), and wherein m is an integer from 0-3, and wherein the length of each of the base peptides sequences and/or ARs is independently selected from 9-30, 13- 26, 15-20, or 13-20 amino acids.
  • the polypeptides in the composition comprise two RCRs (RCRa and RCRb), and each RCR may have the same or differing length and/or amino acid distribution, and wherein the distribution of amino acids at each position is independently selected.
  • the full-length polypeptides in the composition comprise two or more RCRs (RCRa, RCRb, and RCRn), and each of these RCRs may have the same or differing length and/or amino acid distribution, wherein the distribution of amino acids at each position is independently selected, and wherein n is an integer from 0-3.
  • RCRa and RCRb have the same amino acid distribution.
  • the polypeptides in the composition comprise three RCRs (RCRa, RCRb, and RCRc), and each RCR may have the same or differing length and/or amino acid distribution, and wherein the distribution of amino acids at each position is independently selected.
  • RCRa, RCRb, and RCRc have the same amino acid distribution.
  • the template In certain embodiments of any of the foregoing or following, the template
  • one or more of (a) an RCRa and ARa, (b) an AR a and RCRb, (c) an RCRa and ARb, (d) an RCRb and ARb, or (e) an RCRb and AR C , or (f) an RCRc and ARc are interconnected via a linker of at least one amino acid residues.
  • the polypeptides such as the substantially full-length polypeptides in the composition based on the template arrangement, comprise, at one or both ends, 1-8 positively charged residues selected independently, for each position, from Lysine (K), Arginine (R), or Histidine (H).
  • the polypeptides, such as the substantially full-length polypeptides in the composition based on the template arrangement comprise, at one terminus, 2-5 positively charged residues independently, for each position, selected from Lysine (K), Arginine (R), or Histidine (H).
  • one or more of these positively charged amino acids may be a non-naturally occurring positively charged amino acid.
  • the positively charged residue is the same at each position.
  • At least 80%, at least 90% or at least 95% of the polypeptides in the composition having a length of between 25 to 100 amino acids are of substantially the same length, such as full-length. In certain embodiments, at least 80% of said polypeptides in the composition having a length of between 25 to 100 amino acids are about 58-65 amino acids in length. In certain
  • polypeptides having a full-length of between 25 to 100 amino acids represents at least 90%, at least 95% or close to 100% of the composition.
  • the disclosure provides a method of manufacturing a composition by solid phase peptide synthesis.
  • the method comprises, in certain embodiments, selecting one or more antigens associated with a disease or condition; selecting one or more base peptide sequences based on said antigens; selecting, for each position of each AR and for each position of each RCR, one or more amino acid residues permissible at that position among the polypeptides in the composition and, for each possible permissible amino acid at each position, select a pre-determined molar input ratio of the amino acids available for that position; synthesizing the complex mixture of polypeptides, on a polypeptide-by-polypeptide basis, by solid phase peptide synthesis based on the available molar input ratio of permissible amino acids available for each position, wherein for each polypeptide in the complex mixture, synthesis is from C-terminus to N-terminus.
  • the C-terminal most amino acid residue for each polypeptide in the complex polypeptide mixture is Norleucine (Nle) or another non-naturally occurring amino acid.
  • the method or composition is such that at least 55% or at least 60% of the polypeptides in the complex polypeptide mixture that are based on the template arrangement are substantially full-length polypeptides. In certain embodiments, at least 80%, at least 90%, or at least 95% of the polypeptides in the complex polypeptide mixture that are based on the template arrangement are full-length or substantially full-length polypeptides
  • the relative molar input percentages of amino acids are, independently for each position of each of the RCRs, selected from 15% to 55% A, 35% to 75% K, R, or H, or combinations thereof, and 5% to 15% E or D, or combinations thereof. In other such embodiments, for each position of the one or more RCRs, the relative molar input
  • percentages of amino acids are, independently for each position of each of the RCRs, selected from 20% to 50% A, 40% to 70% K, R, or H, or combinations thereof, and 5% to 10% E or D, or combinations thereof.
  • the relative molar input percentage, for each position of and/or across each RCR, of A is less than or equal to 50%, such as 15%- 50%.
  • the relative molar input percentage, for each position of and/or across each RCR, of K (or R or H) is 50-70%).
  • the relative molar input percentages of amino acids are the same for each position of at least one RCR or for each RCR. In other such embodiments, the relative molar input percentages are
  • the molar input percentage at one position of an RCR varies versus the other positions. In some embodiments, the same pattern is repeated across the different RCRs.
  • the amino acid available at each position of at least one RCR or of each RCR is selected from A, K and E. In other such embodiments, the amino acid available at each position of at least one RCR or of each RCR is selected from A and K. In certain embodiments of any of the foregoing or following, the amino acid available at each position of each RCR is independently selected from (i) A, K and E; (ii) A and K; (iii) A, K and D; (iv) A and R; (v) A, R, and E; (vi) A, R and D; (vii) A and H; (viii) A, H and E; (ix) A, H and D.
  • the template In certain embodiments of any of the foregoing or following, the template
  • arrangement comprises two or more RCRs each of 3-7 amino acids in length with at least one AR interposed between two of the RCRs, and wherein the length of each RCR is
  • the two RCRs are 5, 6, or 7 amino acids in length.
  • the template In certain embodiments of any of the foregoing or following, the template
  • arrangement comprises three RCRs each of 3-7 amino acids in length with at least two ARs interposed, and wherein the length of each RCR is independently selected, and wherein the length of each AR is independently selected.
  • the three RCRs are 3, 4, or 5 amino acids in length.
  • the one or more ARs are or comprise base peptide sequences of 9-25 amino acids in length, and wherein, if two or more ARs are present, the length of each is independently selected.
  • the template sequence comprises two or more ARs, wherein: (i) the ARs are each based on the first base peptide sequence, or (ii) one of the ARs is based on the first base peptide sequence and another AR is based on a second base peptide derived from a second sequence of the antigen.
  • the antigen is associated with a protein conformational disorder affecting the central and/or peripheral nervous system.
  • the antigen is associated with a disease selected from Alzheimer's disease (AD); frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17); Dutch hereditary cerebral hemorrhage with amyloidosis (a.k.a cerebrovascular amyloidosis); congophilic angiopathy; Pick's disease; progressive supranuclear palsy; familial British dementia; Parkinson's disease (PD); Lewy body dementias; multiple system atrophy; Hallervorden-Spatz disease; amyotrophic lateral sclerosis (ALS); Huntington's disease (HD); spinocerebellar ataxia; neuronal intranuclear inclusion disease; hereditary dentatorubral-pallidoluysian atrophy; prion-related diseases such as scrapie, bovine spongiform encephalopathy, variant Creutzfeldt Jakob disease, Gerstmann- Straussler-Scheinker
  • hereditary cystatin c amyloid angiopathy dementia pugilistica; neurodegenerative diseases characterized by cerebral and nerve atrophy; spinal and bulbar muscular atrophy; hereditary systemic and cerebral amyloidosis; Finnish-type familial amyloidosis; senile systemic amyloidosis (a.k.a. senile cardiac amyloidosis); familial amyloid polyneuropathy; Type-2 diabetes, in particular pancreatic islet amyloidosis; dialysis-related amyloidosis (DRA); inflammation-associated reactive systemic amyloidosis (a.k.a.
  • AA amyloidosis AA amyloidosis); aortic medial amyloidosis; medullary carcinoma of the thyroid; hereditary renal amyloidosis; light chain associated amyloidosis, light chain deposition disease, light chain cast nephropathy, light chain cardiomyopathy; atrial amyloidosis; injection-localized amyloidosis; cystic fibrosis (CF); and sickle cell anemia.
  • the first base peptide sequence and/or any base peptide sequence comprises a sequence from an antigen selected from prion protein, amyloid beta precursor protein, ABri peptide, tau protein, alpha- synuclein, alpha-synuclein central fragment, SOD1, TDP-43, repeat-associated non-ATG (RAN)-translated peptides of the C90RF72 locus, islet amyloid polypeptide (a.k.a. amylin), prothymosin alpha, amino-terminal domain of androgen receptor protein, ataxin-1, DRPLA protein (a.k.a.
  • atrophin-1 calcitonin
  • cystatin c transthyretin
  • beta 2 microglobulin serum amyloid A protein
  • huntingtin exon I of huntingtin
  • immunoglobulin light chain variable domains insulin, lysozyme, alpha lactalbumin, monellin, ligand- and DNA-binding domains of androgen receptor protein
  • lactadherein lactadherein fragment (a.a. residue 245-294, a.k.a. medin)
  • gelsolin apolipoprotein Al
  • fibrinogen atrial natriuretic factor
  • the first base peptide sequence is a sequence selected from SEQ ID NOs: 1 through 20 or a portion thereof of at least 9 amino acid residues, or any of the other sequences provided in tables or the sequence listing herein.
  • one or more base peptide sequences comprise or permit variability based on one or more phosphorylated, nitrated, or acetylated amino acids.
  • at least one base peptide sequence comprises or permits variability based on at least one phosphorylated residue corresponding to a phosphorylation site in the native antigen that is associated with a pathological state.
  • the antigen is associated with a pathogenic infection.
  • the pathogen is an intracellular pathogen.
  • the pathogen is selected from viruses or bacteria.
  • the pathogen is of the genus selected from Chlamydia, Rickettsia, Coxiella, Mycobacterium, Francisella, Listeria, Salmonella, Brucella, Legionella, Nocardia, Rhodococcus, Yersinia, and Neisseria.
  • the pathogen is human papilloma virus (HPV).
  • the one or more base peptide sequences comprise or permit variability based on one or more non-natural amino acids.
  • the ratio of the molar input percentage of Alanine to that of Lysine, in the total amino acid input concentration of the composition is between 1.25 to 2.5 (as expressed as a quotient, which can also be expressed as 1.25: 1 to 2.5: 1).
  • the ratio of the percentage of alanine to the percentage of lysine, on a molar basis, across the polypeptides of the composition having a length of between about 25 to 100 amino acids is greater than or equal to 2.5 and less than or equal to 7. Such a ratio refers to the output.
  • the percentage of alanine, on a molar basis, across the polypeptides of the composition having a length of between about 25 to 100 amino acids is greater than or equal to 12 and less than or equal to 30. In certain embodiments, this refers to the output.
  • the percentage of lysine, on a molar basis, across the polypeptides of the composition having a length of between about 25 to 100 amino acids is greater than or equal to 4 and less than or equal to 9. In certain embodiments, this refers to the output.
  • the length of each or of at least 60% of the polypeptides in the composition based on the template arrangement (and template sequence) is about 40-80 residues. In certain embodiments, the length of each or of at least 60% of the polypeptides in the composition based on the template arrangement is about 45-65 residues.
  • the conserved substitution comprises replacing an original amino with a similar or interchangeable amino acid defined according to amino acid similarity. In certain embodiments, at least one conserved substitution is selected from an amino acid present in a naturally occurring variant of the antigen. In certain embodiments, the conserved substitution comprises replacing an original amino acid with a phosphorylated or nitrated form of that amino acid. In certain embodiments, one or more RCRs have an amphipathic alpha-helical structure.
  • the disclosure provides a pharmaceutical composition comprising an amino acid copolymer composition of the disclosure and one or more pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical composition comprises an adjuvent.
  • the disclosure provides a method for prophylactic or therapeutic treatment of a protein conformational disorder or a pathogenic infection, comprising administering to a subject in need thereof an effective amount of a composition according to the disclosure, wherein the antigen is an antigen associated with the protein conformational disorder or the pathogenic infection.
  • the disclosure provides a kit comprising a composition according to the disclosure and instructions for use in the treatment of a protein conformational disorder.
  • the disclosure provides a method of eliciting or otherwise promoting a release of a Th2-associated cytokine or chemokine from monocytes, comprising contacting the monocytes with a composition according to the disclosure, wherein the Th2-associated cytokine or chemokine is selected from: IL-4, IL-5, IL-6, IL-10, IL-13, CCL17, and CCL22.
  • the disclosure provides a method of eliciting or otherwise promoting a CCL22 release from monocytes, comprising contacting the monocytes with a composition according to the disclosure.
  • the disclosure provides a method of eliciting or otherwise promoting CD4+ T cell proliferation, comprising contacting peripheral blood mononuclear cells with a composition according to the disclosure.
  • the disclosure provides a method of eliciting or otherwise promoting CD8+ T cell proliferation, comprising contacting peripheral blood mononuclear cells with a composition according to the disclosure.
  • the disclosure provides a kit comprising a composition of the disclosure and instructions for use in the treatment of a protein conformational disorder or pathogenic infection.
  • the disclosure provides a method of targeting a phosphorylated or nitrated site on a target antigen, comprising administering to a subject or contacting a sample with a composition of the disclosure or a pharmaceutical composition according to the disclosure.
  • the disclosure provides a method for generating antibodies comprising the steps of (i) administering a composition of the disclosure to an animal; and (ii)(a) isolating antibodies immunoreactive with said composition from said animal, or (ii)(b) isolating cells that produce antibodies immunoreactive with said composition from said animal, and then isolating antibodies immunoreactive with said composition from said isolated cells.
  • the method further comprises administering the antibodies so isolated or contacting cells or tissues with said antibodies.
  • compositions of the disclosure described using any one or more structural and/or functional features, can be used in any of the in vitro or in vivo methods described herein. Moreover, compositions may be screened using methods described herein to identify compositions having suitable characteristics.
  • Figure 1 shows a schematic representation of a high-complexity amino acid copolymer composition of the disclosure.
  • polypeptides in the composition are based on the following template arrangement: 2 RCRs (RCRa and RCRb ) associated with 2 ARs (ARa and ARb), in an arrangement of RCRa-ARa-RCRb-ARb.
  • the RCRs and ARs are associated and may be associated directly (e.g., they are contiguous) or via a linker (e.g., via an amino acid residue that is neither part of the RCR nor part of the AR).
  • the polypeptides may include additional sequence at the N- or C-terminus, such as a tag to facilitate detection or manufacture.
  • the template arrangement, template sequence and input amino acid percentages permitted at each position influence the possible sequences of the particular individual polypeptides within the composition. This is depicted in Figure 1 by the output polypeptides.
  • Each output peptide will comprise a specific amino acid sequence within each of the RCRs and ARs, and thus, will have a specific amino acid sequence influenced by the linear template arrangement, input amino acids, and input percentages (e.g. molar input percentage).
  • the RCRs of individual peptides of the mixture are depicted as: RCRai(SEQ ID NO: 46), RCRa2(SEQ ID NO: 47), RCR a 3 (SEQ ID NO: 48) (e.g., the N- terminal RCR (RCRa) of peptides 1, 2 and 3, respectively, of the mixture) and RCRbi (SEQ ID NO: 49), RCRb2(SEQ ID NO: 50), RCRb3 (SEQ ID NO: 51) (e.g., the second RCR (RCRb) of peptides 1, 2 and 3, respectively, of the mixture).
  • ARs of individual peptides of the mixture are depicted as: ARai(SEQ ID NO: 40), AR a2 (SEQ ID NO: 41), ARa3 (SEQ ID NO: 42) (e.g., the N-terminal AR (ARa) of peptides 1, 2 and 3, respectively, of the mixture) and AR b i(SEQ ID NO: 39), AR b2 (SEQ ID NO: 39), AR b 3 (SEQ ID NO: 39) (e.g., the second AR (ARb) of peptides 1, 2 and 3, respectively, of the mixture).
  • Figure 2 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to C-terminal portions of Tau (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-CO 16.
  • Figure 2 discloses SEQ ID NO: 52.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as a plurality of at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 52 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific variability depicted in Figure 2 and SEQ ID NO: 52.
  • the amino acid at each position can be K/A/E, and for particular positions within the ARs, the amino acid at each position varies, as depicted and described.
  • Figure 3 shows another example of a schematic corresponding to the design of another amino acid copolymer composition with antigenic specificity to C-terminal portions of Tau (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C016.C).
  • Figure 3 as modified by the description provided in the example, discloses SEQ ID NO: 60.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as a plurality of at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 60 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 3 and SEQ ID NO: 60.
  • the amino acid at each position can be K/A/E, and for particular positions within the ARs, the amino acid at each position varies, as depicted and described.
  • FIG. 4 shows the effect of DP-CO 16.B and DP-CO 16. C on a monocyte assay using RAW cells.
  • DP-CO 16. A (which is an amino acid copolymer composition lacking any RCRs but with an AR spanning the same region of Tau as the two ARs present in DP-CO 16.B and DP-CO 16.C), COPAXONE ® , and single phosphorylated (Phospho Peptide Cnt) and non- phosphorylated (Non-Phospho Peptide Cnt) control peptides spanning the same region of Tau as the two ARs present in DP-CO 16.B and DP-CO 16. C were also assayed for comparison. This assay can be readily used to confirm that activity of other compositions.
  • Figures 5A and 5B show the effect of DP-C016.A, DP-C016.B, DP-C016.C,
  • FIG. 5 A shows flow cytometry data after staining with anti-CD3 and anti-CD4.
  • Figure 5B is a graphical representation of the proliferation index of CD4+ T-cells when stimulated with DP-CO 16.
  • a PBS-only negative control and an anti-CD3/CD28 positive control were also included for comparison.
  • Figures 6A and 6B show the immunogenic effects of DP-CO 16. A, DP-CO 16.B and DP-C016.C.
  • Figure 6A shows the splenocyte recall response when splenocytes from mice injected with DP-C016.A, DP-C016.B or DP-C016.C are restimulated with the respective immunogen.
  • Figure 6B shows the results of antibody ELISA to measure the IgGl response against respective immunogen.
  • Figure 7 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to human a-synuclein (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C003.A).
  • Figure 7 discloses SEQ ID NO: 53.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 53 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Nle Norleucine
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 7 and SEQ ID NO: 53.
  • Figure 8 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to human a-synuclein (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C003.B).
  • Figure 8 discloses SEQ ID NO: 54.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 54 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Nle Norleucine
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 8 and SEQ ID NO: 54
  • Figure 9 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to human a-synuclein (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C003.C).
  • Figure 9 discloses SEQ ID NO: 55.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 55 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Nle Norleucine
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 9 and SEQ ID NO: 55
  • Figure 10 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to human a-synuclein (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C003.D).
  • Figure 10 discloses SEQ ID NO: 56.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 56 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Nle Norleucine
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 10 and SEQ ID NO: 56
  • Figure 11 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to C-terminal portions of Tau (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C016.E).
  • Figure 11 discloses SEQ ID NO: 57.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 57 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Nle Norleucine
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 11 and SEQ ID NO: 57
  • Figure 12 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to C-terminal portions of Tau (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C016.F).
  • Figure 12 discloses SEQ ID NO: 58.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 58 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Nle Norleucine
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 12 and SEQ ID NO: 58
  • Figure 13 shows tau isoforms, domains, and potential phosphorylation sites for targeting when designing an amino copolymer composition.
  • Figure 14 shows the effect of DP-C016.B, DP-C016.D, DP-C016.E, DP-C016.F, and
  • COPAXONE ® on CD4+ T-cell proliferation in a PBMC CFSE assay.
  • Figure 15A, 15B, and 15C show the immunogenic effects of DP-C016.C, DP-C016.F, or IF A alone as measured by antibody -ELIS A.
  • Figure 15A shows the anti-DP-C016 and anti-PHFl total IgG response on Day 29.
  • Figure 15B shows the anti-pS422 and anti- recombinant Tau antibody titers.
  • Figure 15C shows the anti-PHF-tau antibody titers and the anti-PHF-Tau total IgG response on Day 29.
  • Figure 15D shows the binding of antibodies induced by DP-C016.C or DP-C016.F to human Alzheimer's disease brain tissue. Antibodies are shown to specifically bind to neurofibrillary tangles (NFT), Neuropil threads, and beta-amyloid plaques.
  • NFT neurofibrillary tangles
  • Neuropil threads and beta-amyloid plaques.
  • Figure 16 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to HPV L2 (e.g., an example of a composition of the disclosure referred to as DP-C024.1).
  • Figure 16 discloses SEQ ID NO: 59.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 59 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Figure 17 shows an example of MALDI-TOF-MS results of two different preparations of an amino acid copolymer composition with antigenic specificity to Tau (e.g., an example of a composition of the disclosure referred to as DP-CO 16. F). The relative purity of the amino acid copolymer composition is calculated on the right.
  • Figure 18 shows the full MALDI-TOF spectrum of DP-CO 16.F recovered from PLA particles.
  • the DP-CO 16. F was previously loaded onto Polylactic Acid (PLA) particles, particles which protect foreign antigens (Ags) from degradation and dilution, for 4 months.
  • PVA Polylactic Acid
  • Figure 19A shows CD4+ T-cell proliferation assays wherein healthy donor PBMC were cultured in the presence of 7.5 ⁇ DP-C016.F.
  • Figure 19B shows CD8+ T-cell proliferation assays wherein healthy donor PBMC were cultured in the presence of 7.5 ⁇ DP-C016.F.
  • Figure 19C shows that DP-CO 16.F loaded onto PLA particles significantly induced a CD4+ T-cell response.
  • PLA particles (90 and 260 ug/mL final concentration in the culture medium) significantly boosted the CD4+ T-cell proliferation against DP-CO 16.F at 0.25 uM and 0.75 uM.
  • the PLA particles alone were cytotoxic at higher concentrations (860 ug/mL).
  • the final concentrations of PLA particles were 860, 260, and 90 ⁇ g/mL in the cell culture wells containing 2.5, 0.75, and 0.25 ⁇ of DP-C016.F, respectively.
  • Figure 19D shows 0.25 ⁇ DP-CO 16.F loaded onto PLA particles shows a significant increase in CD4+ and CD8+ T-cell response as compared to DP-C016.F only diluted in PBS.
  • Figure 20A shows the results of an antibody ELISA against PFIF-tau, which are paired helical filaments otherwise defined as hyper-phosphorylated tau oligomers isolated from AD patients' brain autopsies, and full-length non-phosphorylated recombinant Tau (recTau) in mice immunized with DP-C016.F at 0.3 or 1.2mg/kg either loaded onto PLA particles or emulsified in Incomplete Freund's Adjuvant (IF A).
  • Figure 20A also shows that high titers of anti-PHF-tau antibodies are induced by DP-C016.F at 1.2mg/kg emulsified in IFA.
  • Figure 20B shows the results of an antibody ELISA against a short linear peptide covering PHF1 (pS396/pS404) and pS422 epitopes in mice immunized with DP-C016.F at 0.3 mg/kg or 1.2 mg/kg either loaded onto PLA particles or emulsified with IFA.
  • Figure 20B also shows that the highest titers of anti-PHFl antibodies are induced by DP-C016.F, 1.2 mg/kg emulsified in IFA.
  • Figure 20C shows the antibody response on day 168 as compared to day 28 in mice treated with DP-C016.F 0.3 mg/kg loaded onto PLA particles.
  • the graph shows the results of the antibody ELISA against a short linear peptide covering PHF1, a short peptide covering pS422, PHF-tau isolated from AD patients' autopsies, and recombinant Tau (recTau), respectively.
  • Figure 20D shows the effect of DP-CO 16.
  • F loaded onto PLA particles on hind limb clasping as a measure of neurodegeneration and motor impairment in J PL3 mice.
  • the graph depicts the correlation between anti-PFIFl antibody titer and hind limb clasping in JNPL3 mice treated with DP-CO 16.F (boxes) or hind limb clasping in all the mice in the study, treated with DP-CO 16. F and non-treated controls (black circles).
  • Figure 21 A shows the effect of DP-C016.F, 0.8 mg/kg emulsified in IF A or DP- C016.F, 0.8 mg/kg loaded onto PLA particles on the production of anti-immunogen antibodies which were detected using both anti-IgGl and anti-IgG2a isotype secondary antibodies, in separate reactions.
  • Figure 21B shows the effect of DP-C016.F, 0.8 mg/kg emulsified in IF A or DP- C016.F, 0.8 mg/kg loaded onto PLA particles on the production of anti-Tau antibodies which were detected using both PHF-tau and recombinant Tau (recTau) as antigens, in separate reactions.
  • Figure 21C shows the effect of DP-C016.F, 0.8 mg/kg emulsified in IF A, DP-C016.F, 0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on total soluble tau content in ug/mg of total proteins in the cortex, hindbrain and hippocampus of J PL3 mice.
  • Figure 21D shows the effect of DP-C016.F, 0.8 mg/kg emulsified in IF A, DP-C016.F, 0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on the relative percentages of hyperphosphorylated soluble tau, using 3 monoclonal antibodies PHFl, CP 13 and RZ3 specific for pS396/pS404, pS202 and pT231 respectively, in the hindbrain of JNPL3 mice.
  • Figure 2 IE shows the effect of DP-CO 16.F, 0.8 mg/kg emulsified in IF A, DP-CO 16.F,
  • Figure 21F shows the effect of DP-C016.F, 0.8 mg/kg emulsified in IF A, DP-C016.F,
  • Figure 21G shows the effect of DP-C016.F, 0.8 mg/kg emulsified in IF A, DP-C016.F,
  • Figure 21H shows the effect of DP-C016.F, 0.8 mg/kg emulsified in IF A, DP-C016.F, 0.8 mg/kg loaded onto PLA particles or PLA particles alone (vehicle) on hind limb clasping, and its correlation with total soluble tau in the cortex of JNPL3 mice.
  • Figure 22 shows an example of a schematic corresponding to the design of an amino acid copolymer composition with antigenic specificity to human a-synuclein (e.g., an example of a composition of the disclosure, compositions based on this template arrangement and amino acid input referred to as DP-C003.E).
  • Figure 22 discloses SEQ ID NO: 61.
  • An example of compositions of the disclosure is a composition comprising a plurality of polypeptides (such as at least 500 different polypeptides) comprising on amino acid sequence set forth in SEQ ID NO: 61 (in the presence or absence of a terminal Norleucine (Nle) or similar non-naturally occurring amino acid).
  • Nle Norleucine
  • a plurality of the individual polypeptides within the composition are complex and differ in sequence based on the position-specific sequence variability depicted in Figure 22 and SEQ ID NO: 61.
  • compositions that are, in certain embodiments, capable of inducing or otherwise stimulating a Th2 chemokine (e.g., CCL22 and/or CCL17) release by monocytes and/or of inducing CD4+ T cell proliferation and/or of inducing antibodies against a relevant target (e.g., a target from which one or more ARs of the composition are derived).
  • a relevant target e.g., a target from which one or more ARs of the composition are derived.
  • compositions of the disclosure are capable of inducing CD8+ T cell proliferation.
  • the Th2 chemokine (e.g., CCL22 and/or CCL17) release, CD4+ T-cell proliferation and/or antibody induction properties of the composition are better, as compared to (i) a single polypeptide based on one or more of the same AR and/or (ii) the same or similar polypeptide or polypeptide composition in the absence of RCRs2.
  • such compositions are capable of stimulating CCL22 release by monocytes.
  • such compositions additionally or alternatively, are capable of stimulating CCL17 release by monocytes.
  • the compositions of the disclosure do not require in vivo priming to induce strong T-cell proliferation in vitro.
  • the presence of the RCRs imparts and/or contributes to numerous functional benefits to the compositions (e.g. CCL22 release from monocytes, broad T cell proliferation, activation of monocytes, increased induction of antibody titers, etc.). These and other benefits of RCRs can be readily seen in the examples.
  • compositions of the disclosure are useful in numerous methods in vitro and in vivo.
  • compositions of the disclosure provide an improved approach to immunotherapy and may be suitable, for example, for studying disease mechanism, for raising antibodies, for promoting an anti-inflammatory Th2 immunity, for promoting or otherwise stimulating Th2 chemokine (e.g., CCL22 and/or CCL17) release by monocytes, for promoting CD4+ T-cell proliferation, for promoting CD8+ T-cell proliferation, and/or in methods of treating various diseases or conditions related to targets, portions of which are the basis for the ARs of the composition of the disclosure (e.g. protein conformational disorders, pathogenic infections, conditions amenable to treatment via an immunotherapy, etc.).
  • Th2 chemokine e.g., CCL22 and/or CCL17
  • amino acid copolymer compositions of the disclosure comprise novel mixtures of polypeptides having high complexity (e.g., compositions of greater than 500, greater than 1 x 10 3 , greater than 1 x 10 4 , greater than 1 x 10 5 , greater than 1 x 10 6 , or greater than 1 x 10 8 polypeptides based on the same linear template arrangement).
  • compositions may be manufactured using solid-phase peptide synthesis, such as in a single solid-phase peptide synthesis step, wherein the polypeptides of the mixture have one or more antigenic regions from a target (e.g., referred to as a protein or antigen) related to the disease or condition of interest that are presented in association with one or more (e.g., 1, 2, 3, or more than 3) short random copolymer regions (RCRs).
  • a target e.g., referred to as a protein or antigen
  • RCRs short random copolymer regions
  • immunotherapeutics e.g., significant improvements versus those obtained in the absence of RCRs; surprising in view of the state of the art.
  • the presence of the RCRs imparts and/or contributes to numerous functional benefits to the compositions (e.g. CCL22 release from monocytes, broad T cell proliferation, activation of monocytes, increased induction of antibody titers, etc.).
  • CCL22 release from monocytes broad T cell proliferation, activation of monocytes, increased induction of antibody titers, etc.
  • These and other benefits of RCRs can be readily seen in the examples.).
  • the disclosure contemplates that, in certain embodiments, when an RCR of 3 or even 4 residues is used, the template arrangement includes 3 such RCRs (e.g., for a total RCR length of 9-12 residues). As elsewhere, the amino acid percentage make-up can vary across the RCR as described throughout. On the other hand, in certain embodiments, the disclosure contemplates that when larger RCRs are used, such as RCRs of 8 or 9 residues, two RCRs are used - although 1 or 3 RCRs can also be used. For RCRs of 5 or 6 or 7 residues in length, in certain embodiments, the disclosure contemplates that two or three RCRs are used.
  • compositions of the disclosure include one or more RCRs, each of which is short (e.g., 3-15 amino acid residues, such as 3-14 residues or 3-11 residues or 3-9 residues or even just 3-7 residues or three to six residues) and corresponds to a mixture of alanine and positively charged amino acids at every position of the RCR.
  • the length of composition of each RCR is independently selected.
  • the RCRs also include, in a lesser proportion, an amount of negatively charged amino acid to, for example, increase solubility and/or to promote an alpha-helical configuration and/or a lesser proportion of certain other amino acids like phenylalanine.
  • the short random repeats increase the immunogenicity of the compositions and/or induce an immune response and, as provided herein, can be provided in association with one or more ARs of interest (e.g., ARs relevant to any of a range of diseases or conditions).
  • the RCRs and other portions of the composition may be designed for the whole composition to have an estimated net charge at pH7, such as to have an estimated net charge above 1 or even above 2, such as from 2 to 4.
  • estimated net charge of the whole composition may be less positive or even negative.
  • the RCR compositions are selected to increase the net charge of the composition versus the same ARs in the absence of the RCRs.
  • the net positive charge at pH7 of the composition may be negative, but less negative than an equivalent composition containing identical ARs without the RCRs.
  • compositions of the disclosure are, in certain embodiments, capable of inducing a safe, anti-inflammatory Th2 immunity (e.g., safe in comparison to other compositions which may induce a significant pro-inflammatory Thl immunity). In other embodiments, compositions of the disclosure are capable of inducing Thl immunity. In certain embodiments, a safe, anti-inflammatory Th2 immunity (e.g., safe in comparison to other compositions which may induce a significant pro-inflammatory Thl immunity). In other embodiments, compositions of the disclosure are capable of inducing Thl immunity. In certain embodiments, capable of inducing a safe, anti-inflammatory Th2 immunity (e.g., safe in comparison to other compositions which may induce a significant pro-inflammatory Thl immunity). In other embodiments, compositions of the disclosure are capable of inducing Thl immunity. In certain embodiments, a safe, anti-inflammatory Th2 immunity (e.g., safe in comparison to other compositions which may induce a significant pro-inflammatory Thl immunity). In other embodiments, compositions of the disclosure are capable of
  • compositions of the disclosure are capable, such as with or without also inducing T cell proliferation, of producing antibodies against an antigen of interest (e.g., an antigen associated with a disease or condition suitable for intervention via an antigen of interest).
  • an antigen of interest e.g., an antigen associated with a disease or condition suitable for intervention via an antigen of interest
  • compositions of the disclosure may be referred to as immunotherapeutics or polypeptide vaccines.
  • an advantage of the compositions of the disclosure is the ability to induce an immune response against an antigen and certain variants of an antigen, or across a range of particular variants or strains, and/or against more than one epitope of a target. This may be useful for providing compositions that both promote a more robust immune response and are effective across a broader population.
  • composition of the disclosure can be designed to target hyper-phosphorylated Tau (e.g., a design in which one or more ARs, such as two ARs, are based on a base peptide sequence corresponding to a portion of hyper-phosphorylated Tau, optionally, where a position in the AR is selected from an amino acid or the
  • compositions of the disclosure are capable of inducing production of specific antibodies against many variants of phosphorylated Tau proteins found in oligomers and paired helical filaments (PHFs) and can be used to treat various tauopathies such as but not limited to Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy.
  • the compositions of the disclosure may be capable of inducing specific antibodies that inhibit early phosphorylation and accumulation of toxic precursor tau oligomers.
  • the compositions of the disclosure target glial tau pathology in astrocytes.
  • the compositions of the disclosure target glial tau pathology in microglia.
  • compositions and methods of the present disclosure overcome many of the challenges currently faced in designing polypeptide vaccines/therapeutics and outlined above in the Background section.
  • the compositions of the disclosure comprise mixtures of polypeptides based on a linear template arrangement and comprising one or more RCRs and one or more ARs.
  • the composition has a complexity (e.g., is composed of at least a certain number of different polypeptides, such as more than 500) where the polypeptides in the composition are related by sequence and the complexity and variability is based on the permitted level of sequence differences at each position, as well as the pre-determined molar input percentages of the amino acids permitted at each position.
  • compositions of the disclosure may also be described based on amino acid output (e.g., percentage of one or more particular amino acids, on a molar basis, in the amino acid composition).
  • compositions of the disclosure are mixtures of polypeptides based on a linear template arrangement.
  • the polypeptides comprise one or more random copolymer regions (RCRs) linked (directly or via an amino acid linker) to one or more antigenic regions (ARs).
  • RCRs random copolymer regions
  • ARs antigenic regions
  • additional amino acid sequences may be present at the N- or C-terminus, such as additional positively charge residues.
  • compositions of the disclosure may be described based on a linear template arrangement and template sequence, and optionally, on amino acid input ratios/percentages at each position or in total and/or on output ratios and/or on estimated net charge at pH7.
  • the antigenic regions (ARs) of the composition promote the induction of antibodies against the relevant disease targets (e.g., against antigens/protein typically associated with a disease or condition), optionally, while preserving the normal functional protein (if present).
  • compositions of the disclosure are capable of promoting or otherwise stimulating Th2 chemokine (e.g., CCL22 and/or CCL17) release by monocytes (e.g., CCL22 release by monocytes) and/or promoting CD4+ T-cell proliferation (e.g., CD4+ T-cell proliferation among human peripheral blood mononuclear cells), such as via the RCRs and/or the combined activity of the ARs and RCRs.
  • Th2 chemokine e.g., CCL22 and/or CCL17
  • monocytes e.g., CCL22 release by monocytes
  • CD4+ T-cell proliferation e.g., CD4+ T-cell proliferation among human peripheral blood mononuclear cells
  • compositions of the disclosure do not require in vivo priming to induce strong T-cell proliferation in vitro.
  • compositions of the disclosure exhibit epitope promiscuity, and are thus suitable for eliciting immunogenicity in patients with different HLA phenotypes.
  • the amino acid copolymer compositions of the present disclosure improve upon existing designs and comprise mixtures of polypeptides made of one or more short random copolymer regions (RCRs) linked to one or more antigenic regions (ARs).
  • the compositions of the disclosure contain a mixture of polypeptides containing 3 RCRs of 3, 4, or 5 amino acids each, linked to two or more ARs.
  • the compositions of the disclosure contain a mixture of polypeptides containing 2 RCRs of 3, 4, 5, 6, or 7 amino acids each, linked to one or more ARs.
  • the disclosure contemplates the use of slightly longer RCRs, such as RCRs of 8 or 9 residues.
  • compositions of the disclosure can be synthesized by solid phase peptide synthesis, such as in a single manufacturing step.
  • compositions of the disclosure contain a mixture of polypeptides wherein at least 55% or at least 60% of the polypeptides in the complex mixture based on the linear template arrangement are substantially full-length polypeptides. In other embodiments, the percentage of such full-length or substantially full-length polypeptides is at least 75% or at least 80%.
  • the first amino acid at the C-terminal position of each polypeptide in the mixture that is based on the template arrangement is Norleucine (Me).
  • the first amino acid at the C-terminal position is Nle or another non-naturally occurring amino acid.
  • Compositions of the disclosure have numerous beneficial properties, including having combined B- and T-cell determinants.
  • the antigenic regions (ARs) of the compositions of the present disclosure incorporate alanine to confer epitope promiscuity and, in certain embodiments, to improve solubility.
  • the random copolymer regions incorporate alanine, positively charged residues, and optionally, negatively charged residues for monocyte activation and broad T- cell proliferation (and optionally a negatively charged and/or certain other residues).
  • the improvement in one or more properties of compositions of the disclosure containing one or more short RCRs versus single polypeptides or mixtures of polypeptides that are similar to compositions of the disclosure but lack RCRs was surprising. In other words, it was unexpected that the introduction of such short, random sequences would instill the desirable properties observed, such as adjuvant-like properties.
  • compositions of the disclosure comprising one or more RCRs have an estimated net charge (e.g., based on predicted net charge of full-length polypeptides and given input percentages (e.g. molar input percentages) and amino acid distributions) of greater than 2 (e.g., 2.1 or even about 3) at pH7. In some embodiments the estimated net charge is greater than 2.0 and less than 4.0 at pH7.
  • the use of RCRs in compositions of the disclosure increases the estimated net charge of the composition, versus a composition of the same complex ARs without RCRs, the estimated net charge at pH7 can be less than 2. Accordingly, the disclosure contemplates the use of the RCRs to increase the estimated net charge of the composition at pH7 and this can be modulated to improve solubility of the composition.
  • the ARs may have a negative charge at pH7, and the RCR compositions increase the net positive charge at pH7 of the composition as a whole (although not necessarily to a positive charge). It will be understood that the impact of the RCRs is not merely on charge, as described herein, as compositions of the disclosure have numerous functional features indicative of their use as a vaccine and improved functional properties owing to the addition of the RCRs.
  • the estimated net charge is a proxy for the predicted charge (e.g., estimated charge) at pH7 and is based on the net charge of the ARs and RCRs within the linear template arrangement.
  • the estimated net charge for a composition of the disclosure is based on the template sequence where: (i) the net charge of the ARs, is calculated based on the base peptide sequence without the inclusion of potential variation at each amino acid position of the AR, (ii) the net charge of the RCRs, is calculated for a hypothetical polypeptide in which for every 1 position in which a positively charged amino acid is assumed to occur, alanine is assumed to occur in two positions (e.g., if template arrangement and template sequence for a polypeptide mixture includes 1 RCR of 3 amino acid residues in length, estimated net charge is calculated based on the assumption that the RCR contains 1 positively charged residue and two alanines); and (iii) and additional amino acids outside of the ARs and RCRs and present in the design are included (e.g., a 4 residue N-
  • compositions of the disclosure have one or more of the following advantages: (i) ability to target multiple antigenic targets and adaptability across targets and disease indications; (ii) sustained immunogenicity; (iii) anti-inflammatory properties; (iv) antigen-independent T-cell help; (v) IgG4 antibody response; (vi) ULA promiscuity; (vii) intrinsic adjuvant properties that eliminate/decrease the need for the addition of a strong adjuvant or carrier protein for therapeutic efficacy; (viii) no/decreased risk of neutralizing antibodies; (ix) no IV administration; (x) cost-effectiveness; (xi) satisfactory safety profile; (xii) PK/PD correlation; (xiii) improved solubility; (ix) net positive charge at pH7 (e.g., preferably net charge or estimated net charge of greater than 2 or of greater than 2 and less than 4).
  • pH7 e.g., preferably net charge or estimated net charge of greater than 2 or of greater than 2 and less than 4
  • compositions may vary depending on the nature of the ARs.
  • the compositions do have an increase in net charge versus that of a similar composition based on the complex ARs alone.
  • Improved properties of the compositions of the disclosure may be measured versus, for example, a similar composition having the same or similar ARs but in the absence of RCRs.
  • the presence of the RCRs imparts and/or contributes to numerous functional benefits to the compositions (e.g. CCL22 release from monocytes, broad T cell proliferation, activation of monocytes, increased induction of antibody titers, etc.).
  • polypeptide mixture compositions of the disclosure designed as described herein, have numerous advantageous immunological properties, while also, in certain embodiments, inducing antigen-specific antibodies (e.g., antibodies against targets consistent with the ARs).
  • the compositions of the disclosure are, in certain embodiments, able to stimulate Th2 chemokine (e.g., CCL22 and/or CCL17) release from monocytic cells and promote broad CD4+ T-cell proliferation.
  • Monocyte activation and T-cell proliferation activities of the compounds demonstrate their intrinsic adjuvant properties and their Th2 immunity profile.
  • compositions according to the disclosure that target the C-terminal region of Tau (e.g., ARs correspond to base peptides derived from human Tau-F), induced anti-hyper-phosphorylated tau antibodies, while promoting CCL22 release from monocytes and promoting CD4+ T-cell proliferation.
  • ARs correspond to base peptides derived from human Tau-F
  • induced anti-hyper-phosphorylated tau antibodies while promoting CCL22 release from monocytes and promoting CD4+ T-cell proliferation.
  • incorporation of RCRs of 3-15 amino acids in length is applicable to any target of interest (e.g., a target for which the generation of antibodies, such as to use for study or as an immunotherapy, would be useful), and not just targets limited to protein conformational disorders. Exemplary suitable targets are described herein. Moreover, further description of the linear template arrangements, RCRs, and ARs is described herein.
  • the disclosure contemplates compositions based on any combination of structural and/or functional features described herein. Any such compositions may be made, for example, based on the methods described herein. Any such compositions may be used in methods, in vitro and/or in vivo.
  • compositions of the disclosure provides methods of making (e.g., manufacturing) compositions of the disclosure, and any such compositions may be so described based on any one or combination of features or embodiments described herein.
  • the disclosure provides a composition made by a method of manufacture, comprising synthesizing a high complexity polypeptide composition based on a template arrangement, as described herein.
  • composition comprising:
  • each polypeptide is manufactured based on a template arrangement comprising one or more antigenic regions (each an AR) linked to two or more random copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one or more ARs and the two or more RCRs are arranged according to a linear template arrangement (together a "complex polypeptide mixture component"), wherein at least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of lysine (K), arginine (R) or histidine (H), and, optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and wherein, for each amino acid position of the two or more RCRs, the relative molar input percentage of A for each position is less than or equal to 65%, the relative molar input percentage of positively charged amino acids for each position is at least 35% and the relative molar input percentage of
  • compositions of the disclosure may include or be described based on any of the features of the compositions of the disclosure provided herein.
  • composition comprising:
  • each polypeptide is manufactured based on a template arrangement comprising one or more antigenic regions (each an AR) linked to two or more random copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one or more AR and the two or more RCRs are arranged according to a linear template arrangement (together a "complex polypeptide mixture component"),
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the
  • amino acid position of the first base peptide sequence alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the peptides is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected,
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and wherein the distribution of the amino acids at a given position of the one or more RCRs among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position, and wherein amino acid content and molar input percentage of each RCR is independently selected, wherein the ratio of the percentage of alanine to the percentage of lysine, on a molar basis, in the polypeptides of the composition having a length of between about 25 to 100 amino acids (the output ratio) is greater than or equal to 1.5 and less than or equal to 5.0.
  • compositions of the disclosure may include or be described based on any of the features of the compositions of the disclosure provided herein.
  • composition comprising:
  • each polypeptide is manufactured based on a template arrangement comprising one or more antigenic regions (each an AR) linked to one or more random copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one or more AR and the one or more RCR are arranged according to a linear template arrangement (together a "complex polypeptide mixture component"),
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected,
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid (E), or phenylalanine (F), and wherein, for each amino acid position of each RCR, the relative molar input percentage of A for each position is less than or equal to 65%, the relative molar input percentage of positively charged amino acids for each position is at least 35% and the relative molar input percentage of negatively charged amino acids for each position is less than or equal to 20%, and wherein the distribution of the amino acids at a given position of the one or more RCRs among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position, and wherein, if the polypeptides of the mixture comprise more than one RCR, amino acid content and molar input percentage
  • compositions of the disclosure may include or be described based on any of the features of the compositions of the disclosure provided herein.
  • composition comprising:
  • each polypeptide is manufactured based on a template arrangement compriseing one or more antigenic regions (each an AR) linked to one or more random copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one or more AR and the one or more RCR are arranged according to a linear template arrangement (together a "complex polypeptide mixture component"),
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the
  • amino acid position of the first base peptide sequence alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected,
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid, or phenylalanine (F), and wherein the distribution of the amino acids at a given position of the one or more RCRs among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position, and wherein, if the polypeptides of the mixture comprise more than one RCR, amino acid content and molar input percentage of each RCR is independently selected, and
  • the ratio of the percentage of alanine to the percentage of lysine, on a molar basis, in the polypeptides of the composition having a length of between about 25 to 100 amino acids is greater than or equal to 1.5 and less than or equal to 5.0 (as expressed as a quotient, which ratio may also be expressed as a relative ratio of 1.5: 1 to 5.0: 1).
  • compositions of the disclosure may include or be described based on any of the features of the compositions of the disclosure provided herein.
  • composition comprising:
  • each polypeptide is manufactured based on a template arrangement comprising one or more antigenic regions (each an AR) linked to one or more random copolymer regions of 3-15 amino acids in length (each an RCR), wherein the one or more AR and the one or more RCR are arranged according to a linear template arrangement (together a "complex polypeptide mixture component"), which composition has an estimated net charge of greater than 2.0 and less than 4.0 at pH7,
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid (E), or phenylalanine (F), and wherein, for each amino acid position of each RCR, the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position of each RCR
  • compositions of the disclosure may include or be described based on any of the features of the compositions of the disclosure provided herein.
  • composition comprising:
  • each polypeptide is manufactured based on a template arrangement comprising one or more antigenic regions (each an AR) linked to two or more random copolymer regions of 3-7 amino acids in length (each an RCR), wherein the one or more AR and the two or more RCRs are arranged according to a linear template arrangement (together a "complex polypeptide mixture component"), which composition has an estimated net charge of greater than 2.0 and less than 4.0 at pH7,
  • At least one AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence derived from an antigen associated with a disease and for each amino acid position of said base peptide sequence, each polypeptide has an amino acid independently selected from one or more of: an original amino acid found at the
  • amino acid position of the first base peptide sequence alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and wherein the distribution of the amino acids at a given position among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected,
  • each polypeptide has an amino acid selected from (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, optionally, (iii) at least one of aspartic acid (D) or glutamic acid (E), and wherein, for each amino acid position of the two or more RCRs, the distribution of the amino acids at a given position among the polypeptides in the mixture is determined by a pre-determined molar input ratio of the amino acids available for that position and is independently selected for each position of each RCR.
  • the foregoing composition may include or be described based on any of the features of the compositions of the disclosure provided herein.
  • the RCRs are 8 or 9 residues in length
  • the template arrangement comprises at least two RCRs or two RCRs.
  • the RCRs are 3 or 4 residues in length
  • the template arrangement comprises three RCRs.
  • the RCRs are 5, 6, or 7 amino acids in length
  • the template arrangement comprises two or three RCRs.
  • compositions comprising a plurality of polypeptides based on any of the template arrangements and designs disclosed herein, such as in the figures and sequence listing.
  • compositions comprise a plurality of related polypeptides (such as at least 500 or more than 500 polypeptides) each based on the sequences provided.
  • the linear sequence of the template arrangements provided in the examples describe the variety of polypeptides in the complex compositions, and thus, can be used to describe that composition, as described herein.
  • Such polypeptides may be used and formulated, just as any other compositions of the disclosure, as described herein, including with an adjuvant and/or for the various routes of delivery provided herein.
  • compositions of the disclosure may be described based on any combination of one or more features disclosed herein (e.g., sequence, template arrangement, molar input percentage, amino acid selection, ARs, base peptide sequence(s), antigen, number of RCRs, etc.).
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions, and including interactions such as salt bridges and water bridges.
  • HLA human immunoglobulin A
  • patient refers to an animal, preferably a mammal, including humans as well as livestock and other veterinary subjects. In certain embodiments, the patient is a human.
  • peptide polypeptide
  • protein protein
  • peptide and “peptidomimetic” are used interchangeably herein. These terms refer to unmodified amino acid chains, and also include minor modifications, such as phosphorylations, glycosylations and lipid modifications.
  • peptide and “peptidomimetic” are not mutually exclusive and include substantial overlap.
  • a "peptidomimetic” includes any modified form of an amino acid chain, such as a phosphorylation, capping, fatty acid modification and including unnatural backbone and/or side chain structures. As described below, a peptidomimetic comprises the structural continuum between an amino acid chain and a non-peptide small molecule. Peptidomimetics generally retain a recognizable peptide-like polymer unit structure. Thus, a peptidomimetic may retain the function of binding to a HLA protein forming a complex, which activates autoreactive T cells in a patient suffering from an autoimmune disease.
  • substantially full-length refers to a polypeptide that varies in length by less than 5% versus a comparator.
  • a substantially full-length polypeptide produced following solid phase synthesis would be a polypeptide that is anywhere from 57 amino acids to 63 amino acids.
  • polypeptides in the composition are, in certain embodiments, full-length (based on a particular template arrangement, as described herein).
  • the method is based on synthesis using particular input molar percentages of particular amino acid residue(s) at each position, including each position of each RCR and each position of each AR.
  • This can be readily described for any of the compositions of the disclosure, and thus, the disclosure contemplates that products by process and methods of manufacture are similarly described based on the characteristics of the compositions described herein.
  • the disclosure provides complex compositions of greater than 500 polypeptides manufactured based on any of the template arrangements of RCRs and ARs, as provided herein, and based on any of the molar input percentages and amino acid distributions and rules described herein.
  • amino acid residue is known in the art. In general the abbreviations used herein for designating the amino acids and the protective groups are based on
  • amino acids used in the application of this disclosure are those naturally occurring amino acids found in proteins, or the naturally occurring anabolic or catabolic products of such amino acids, which contain amino and carboxyl groups.
  • Particularly suitable amino acid side chains include side chains selected from those of the following amino acids: glycine, alanine, valine, cysteine, leucine, isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and tryptophan.
  • amino acid residue further includes analogs, derivatives and congeners of any specific amino acid referred to herein, as well as C-terminal or N-terminal protected amino acid derivatives (e.g. modified with an N-terminal or C-terminal protecting group).
  • the present disclosure contemplates the use of amino acid analogs wherein a side chain is lengthened or shortened while still providing a carboxyl, amino or other reactive precursor functional group for cyclization, as well as amino acid analogs having variant side chains with appropriate functional groups).
  • the subject compound can include an amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1- methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid.
  • amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenylalanine, 5-hydroxytryptophan, 1- methylhistidine, 3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyric acid.
  • amino acid analog such as, for example, cyanoalanine, canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine, dihydroxy-phenyla
  • amino acids used in the polypeptide compositions of the present disclosure may exist in particular geometric or stereoisomeric forms.
  • amino acids used in the polypeptide compositions of the present disclosure may exist in particular geometric or stereoisomeric forms.
  • the amino acids used to form the subject polypeptide mixtures are (L)-isomers, although (D)-isomers may be included in the polypeptides such as at non-anchor positions or in the case of peptidomimetic versions of the polypeptides.
  • Naturally occurring variations are allelic variations, isomeric and species differences of functionally equivalent proteins, naturally occuring amino acid modifications, whether or not incorporated while synthesis or post- synthesis (i.e. post-translation modification in vivo and post-synthesis modification in vitro) such as preformed phosphorylations, preformed nitrations, preformed glycosylations, methylation, modification by deamination, modification by deimination, modification by fatty acids (such as myristoylation), modified amino acid side chains including modification to produce amino acid analogs as described in paragraph defining "amino acid residue", cross-linking such as disulfide bonds, and other known modifications.
  • synthesis or post- synthesis i.e. post-translation modification in vivo and post-synthesis modification in vitro
  • preformed phosphorylations i.e. post-translation modification in vivo and post-synthesis modification in vitro
  • preformed glycosylations methylation
  • modification by deamination modification by deimination
  • modification by fatty acids such as
  • Prevent means to delay or preclude the onset of, for example, one or more symptoms, of a disorder or condition.
  • Treatment means at least lessening the severity or ameliorating the effects of, for example, one or more symptoms, of a disorder or condition.
  • Treatment regimen encompasses therapeutic, palliative and prophylactic modalities of administration of one or more compositions of the disclosure.
  • a particular treatment regimen may last for a period of time at a particular dosing pattern, which will vary depending upon the nature of the particular disease or disorder, its severity and the overall condition of the patient, and may extend from once daily, or more preferably once every 36 hours or 48 hours or longer, to once every month or several months.
  • structure-activity relationship refers to the way in which altering the molecular structure of drugs alters their interaction with a receptor, enzyme, etc.
  • the present disclosure is based on the discovery that antigenic sequences can be modified by linking them to short cationic polypeptides in high-complexity mixtures to elicit a robust immune response.
  • the compositions of the disclosure have the further advantage of being able to stimulate the generation of specific antibodies against an antigen and, in certain embodiments, against variants of the antigen (including against all significant variants of the antigen).
  • the amino acid copolymer compositions of the disclosure comprise novel mixtures of polypeptides having high complexity (e.g., compositions of greater than 500 polypeptides based on the same linear template arrangement).
  • the compositions may be manufactured using solid-phase peptide synthesis, such as in a single solid-phase peptide synthesis step, wherein the polypeptides of the mixture have a portion based on one or more antigenic sequences from a target (e.g., referred to as a protein or antigen) related to the disease or condition of interest that are presented in association with one or more short random copolymer repeats (RCRs).
  • a target e.g., referred to as a protein or antigen
  • the disclosure is based on the surprising finding that the use of one or more short RCRs, such as use of 2, 3 or more than 3 RCRs, provides significant and surprising improvements in properties consistent with the use of the compositions as immunotherapeutics (e.g., significant improvements versus those obtained in the absence of RCRs; surprising in view of the state of the art).
  • the compositions of the disclosure contain a mixture of polypeptides made of 3 RCRs of 3, 4, or 5 amino acids each, linked to one or more ARs.
  • the length of each RCR may be the same or independent from that of the other RCRs.
  • compositions of the disclosure contain a mixture of polypeptides made of 2 RCRs of 3, 4, 5, 6, 7, 8, or 9, such as 5, 6, 7, 8, or 9 amino acids each, linked to one or more ARs.
  • the length of each RCR may be the same or independent from that of the other RCRs.
  • the compositions of the disclosure contain a mixture of polypeptides wherein at least 55% or at least 60% of the polypeptides in the complex mixture based on the linear template arrangement are full-length or substantially full-length polypeptides.
  • compositions of the disclosure include one or more RCRs, each of which is short (e.g., 3-15 amino acid residues, such as 3- 12 residues or 3-9 residues or 3-7 residues or 3-6 residues or 3-4 residues) and corresponds to a mixture of alanine and positively charged amino acids at every position of the RCR.
  • RCRs each of which is short (e.g., 3-15 amino acid residues, such as 3- 12 residues or 3-9 residues or 3-7 residues or 3-6 residues or 3-4 residues) and corresponds to a mixture of alanine and positively charged amino acids at every position of the RCR.
  • the RCRs also include, in a lesser proportion, an amount of negatively charged amino acid to, for example, increase solubility and/or to promote an alpha-helical
  • the short random copolymer repeats may increase the immunogenicity of the compositions and/or induce an immune response, such as a T cell response and, as provided herein, can be provided in association with one or more ARs of interest (e.g., ARs relevant to any of a range of diseases or conditions).
  • ARs of interest e.g., ARs relevant to any of a range of diseases or conditions.
  • Each composition of the disclosure is a high-complexity mixture (e.g. >500 different polypeptides) of related polypeptides based on a linear template arrangement of one or more antigenic regions (each an AR, such as AR a , ARb...and so on) linked, directly or indirectly, to one or more random copolymer regions (each an RCR, such as RCR a , RCRb... and so on).
  • a linear template arrangement refers to the arrangement of ARs and RCRs in a linear sequence that is common to all of the full-length polypeptides in a composition and upon each of the complex mixture of polypeptides of the composition is based.
  • an antigenic region refers to a portion having an amino acid sequence based on a sequence derived from an antigen of interest (a base peptide derived from a target of interest).
  • an antigenic region is anywhere from about 9-60 amino acids long and could be based on any relevant portion of a protein of interest that is associated with a disease or condition.
  • an antigenic region may be based on a portion of a protein that is associated with a disease or condition, such as a portion containing an immunodominant determinant.
  • amino acid sequence of an AR may remain constant amongst the polypeptides of the composition, i.e. each polypeptide of the composition may have the same amino acid sequence as the base peptide in that region.
  • amino acid sequence of an AR may be a source of complexity, such that the specific sequence of the AR may vary across the polypeptides of the composition, i.e. the
  • polypeptides may differ from the base peptide and from one another at one or more amino acid positions in that region.
  • an amino acid at each position may be selected from an original amino acid found at the corresponding amino acid position of the first base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid.
  • the template AR refers to the sequence and permissible amino acid substitutions present across all of the polypeptides in the mixture, and are referred to generically as AR (or antigenic region) or using letters to specifically refer to a particular AR present in a linear template arrangement (ARa, ARb, ARn, where n is an integer from 0-3).
  • the ARs of that specific polypeptide are depicted using numbers (AR a i, ARa2, ARa3 . . . . ARa5oo ... etc., where the first AR of each of the specific polypeptides 1-500 of the composition are thus represented).
  • a “random copolymer region” or “RCR” as used herein refers to short random repeats (e.g. anywhere between 3-15 amino acids long, such as 3-14 amino acids long or 3-12 or 3-9 or 3-7 amino acids long or three to six amino acids) generated from mixtures (at each position) of alanine, at least one positively charged amino acid (e.g. lysine, arginine, histidine, non-naturally occurring positively charged residue), and optionally, at least one negatively charged amino acid (e.g. aspartic acid or glutamic acid).
  • the RCR may optionally further comprise phenylalanine.
  • the RCR may optionally further comprise one or more amino acids that promote an alpha-helical secondary structure (e.g. methionine or leucine).
  • alpha-helical secondary structure e.g. methionine or leucine.
  • RCR random copolymer region
  • Exemplary RCRs are each 3, 4, 5, 6, 7, 8, or 9 amino acids in length.
  • the template RCR refers to the sequence and permissible amino acid substitutions present across the polypeptides in the mixture, and are referred to genetically as RCR (or random copolymer region) or using letters to specifically refer to a particular RCR present in a linear template arrangement (e.g., RCR a , RCRb, RCRm, where m is an integer from 0-3; e.g., there may be 1, 2, 3, 4, or 5 RCRs).
  • RCRai, RCRa2, RCRa3 . . . . RCRasoo ... etc. where the first RCR of each of the specific polypeptides 1-500 of the composition are thus represented).
  • exemplary numbers of RCRs are two or three, although more and fewer are contemplated.
  • each polypeptide such as the full-length or substantially full-length
  • polypeptides in the composition will comprise one or more antigenic regions based on the one or more template antigenic regions (each an ARxy, wherein x corresponds to the position of the AR in the linear template arrangement and describes that template, and wherein y identifies the individual polypeptide, e.g. a composition with 500 different polypeptides will have a first AR that is identified as AR a i, ARa, ARa3. . . ARasoo for each polypeptide of the composition) and one or more random copolymer regions (each an RCRxy, wherein x corresponds to the position of the RCR in the linear template arrangement and wherein y identifies the individual polypeptide, e.g. a composition with 500 different polypeptides could have a first RCR that is identified as RCRai, RCRa2, RCR a 3. . RCRasoo in each polypeptide of the composition).
  • each an ARxy wherein x corresponds to the position of the AR in the
  • the linear template arrangement of polypeptides in the composition comprises: two RCRs and 2 ARs configured as RCRa-ARa-RCRb-ARb.
  • the complexity of the composition derives from the diversity of input amino acids provided for certain positions along the length of the polypeptides, the diversity of output amino acids, and the diversity in polypeptide length.
  • the random copolymer regions (RCRs) of each polypeptide in the composition are made of A, K, or E, at each position.
  • the distribution of A, K, and E at each position of a random copolymer region will depend on the relative molar input percentages of A, K, and E (not shown in Figure 1).
  • the first antigenic region (ARa) in the example is based on base peptide sequence "STLYA" and the second antigenic region (ARb) is based on base peptide sequence "HIQRW".
  • multiple input amino acids are provided for certain positions along the first antigenic region to permit variability at those positions across the specific polypeptides in the composition, while only the base peptide residue is provided for each position of the second antigenic region. Distribution at each position will depend on the relative molar input percentage (or relative molar input ratio) of each possible amino acid at that position of the AR.
  • Each output polypeptide comprises a specific amino acid sequence within each of the
  • RCRs and ARs will have a specific amino acid sequence influenced by the linear template arrangement, input amino acids, input and output percentages.
  • the RCRs of individual polypeptides of the composition are depicted as: RCRai, RCRa2, RCRa3 (e.g., the first RCR (RCRa) of polypeptides 1, 2 and 3, respectively, of the composition) and RCRbi, RCRb2, RCRb3 (e.g., the second RCR (RCRb) of polypeptides 1, 2 and 3, respectively, of the mixture).
  • the ARs of individual polypeptides of the composition are depicted as: ARai, ARa2, ARa3 (e.g., the first AR (AR a ) of polypeptides 1, 2 and 3, respectively, of the composition) and ARbi, ARb2, ARb3 (e.g., the first AR (ARb) of polypeptides 1, 2 and 3, respectively, of the composition).
  • the amino acids provided in the composition for position 1 of ARa include (i) S, the original amino acid at the corresponding position of the base peptide, (ii) A, and (iii) T, a conserved substitution for S.
  • positions 2 and 5 of AR a only T or only A, the original amino acids at the corresponding positions of the base peptide are provided.
  • position 3 of ARa L, the original amino acid at the corresponding position of the base peptide, and M, a conserved substitution for L, are provided.
  • position 4 of AR a Y, the original amino acid at the corresponding position of the base peptide, and A are provided.
  • the polypeptides of the composition may differ from each other in their first antigenic sequence (e.g., the first antigenic region of polypeptide 1 (ARai) has the sequence ATLAA, the first antigenic region of polypeptide 2 (AR A 2) has the sequence TTMYA, the first antigenic region of polypeptide 3 (ARB) has the sequence STMYA. Since only the base peptide residue is provided for each position of the second antigenic region, the second antigenic region remains constant amongst the polypeptides of the composition (e.g., the second antigenic regions of polypeptides 1-3, designated by ARbi, ARb2, and ARb3, respectively, all have the sequence HIQRW).
  • the distribution of amino acids at each position of the antigenic regions depends on the relative molar input percentages of amino acids provided for that position and on the molar output percentages for that position (not shown on Figure 1).
  • the schematic in Fig. 1 is for illustrative purposes only and is not to be construed as limiting the full scope of the disclosure.
  • the relative molar input percentage of A is between about 5% and 65%, between about 10% and 65%, between about 15% and 65%, between about 10% and 35%, or between about 15% and 35% of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar input percentage of alanine is between about 15-65% of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar input percentage of alanine is between about 20-35% of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar input percentage of alanine is between about 20-30%) of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments, the relative molar input percentage of alanine is between about 10-25% of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments, the relative molar input percentage of alanine is between about 15-30% of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar input percentage of alanine is less than 50% and greater than 10%, less than 40% and greater than 10%, or less than 30% and greater than 15% of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments, the relative molar input percentage of alanine is 10-25% or 15- 25% of the total input amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar output percentage of A is between about 5% and 75%), between about 10% and 75%, between about 15% and 75%, between about 10% and 35%), or between about 15% and 35% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar output percentage of alanine is between about 15-75% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar output percentage of alanine is between about 20-35% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar output percentage of alanine is between about 20-30%) of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments, the relative molar output percentage of alanine is between about 10-25% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments, the relative molar output percentage of alanine is between about 15-30% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar output percentage of lysine (K) is between about 1% and 40%, between about 3% and 40%, between about 5% and 40%, between about 5% and 20%, between about 5% and 15%, between about 5% and 10%, between about 7% and 15%), between about 6% and 8% or between about 15% and 35% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs, and inclusive of the end points of these ranges.
  • the relative molar output percentage of alanine is about 1-40% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • the relative molar output percentage of alanine is about 10%-40%, or about 10%-35%, or about 12%- 30%), or about 7-15% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs. In some embodiments, the relative molar output percentage of alanine is between about 5-40% of the total output amino acid composition of the polypeptide mixture, i.e., across all the RCRs and ARs.
  • any of the foregoing molar output percentages of lysine may referred to the molar output percentage of any other single positively charged amino acid, such as arginine, histidine, or a non- naturally occurring positively charged amino acid, or may refer to the percentage of all positively charged amino acid residues present (e.g., R+K+H).
  • the relative molar output ratio of the percentage of A to the percentage of K, on a molar basis is greater than or equal to 1.5 and less than or equal to 5.0, between about 1 and 5, between about 1 and 4, between about 1.5 and 5.5, between about 1.5 and 6, or between about 1.5 and 3. In some embodiments, the relative output ratio of the percentage of A to the percentage of K, on a molar basis is between about 1 and 4.5. In some embodiments, the relative output ratio of the percentage of A to the percentage of K, on a molar basis is between about 1.5 and 3.5. In some embodiments, the relative output ratio of the percentage of A to the percentage of K, on a molar basis is between about 1 and 7.
  • the relative output ratio of the percentage of A to the percentage of K, on a molar basis is between about 1 and 6. In some embodiments, the relative output ratio of the percentage of A to the percentage of K, on a molar basis is between about 1.5 and 6.5. Note that the foregoing relative molar output ratio is expressed as a quotient. An output ratio of percentage of A to the percentage of K expressed as a quotient of 1.5 can interchangeably be expressed as 1.5: 1.
  • any of the foregoing relative molar output ratio of the percentage of A to the percentage of K may have the K be replaced with other positively charged amino acids (i.e. arginine (R) or histidine (H)).
  • the relative ratio can also be expressed as a quotient.
  • a relative ratio of of A:K of 4: 1 is equivalent to a quotient of 4.
  • any of the foregoing output ratios may refer to the molar output percentage of any other single positively charged amino acid, such as arginine, histidine, or a non-naturally occurring positively charged amino acid, or may refer to the percentage of all positively charged amino acid residues present (e.g., R+K+H).
  • the input ratios of amino acids may be adjusted in view of the ratio of alanine to positively charged amino acids across the entire composition (RCRs and ARs).
  • the molar input ratio of A:(K+R+H) in the total amino acid concentration of the composition is about 1.25: 1 to 2.5: 1.
  • the molar input ratio of A:(K+R+H) in the total amino acid concentration of the composition is about 1 : 1,
  • the molar input ratio of A:(K+R+H) in the total amino acid concentration of the composition is about 1 : 1 to 1.25: 1, 1.25: 1 to 1.5: 1, 1.5: 1 to 1.75: 1, 1.75: 1 to 2: 1, 2: 1 to 2.25: 1, or 2.25: 1 to 2.5: 1.
  • the relative ratio can also be expressed as a quotient. For example, a relative ratio of A:(K+R+H) of 2.5: 1 is equivalent to a quotient of 2.5, and this quotient may also be referred to as the relative ratio.
  • the polypeptides comprise two or more RCRs and two or more ARs.
  • the two or more RCRs may be of the same or differing lengths and/or amino acid distributions (e.g., content and/or input percentages (e.g. molar input percentages)).
  • the combined length of the two or more RCRs is less than 20 amino acids (e.g., the combined length of RCRa + RCRb is less than 20 amino acid residues).
  • the two or more ARs may be based on the same base peptide or different base peptides. In some
  • the different base peptides are from the same antigen. In some embodiments, the different base peptides are from different antigens.
  • the one or more random copolymer regions and the one or more antigenic regions can be arranged in any order, with respect to each other, in the linear template arrangement.
  • the regions are associated (e.g., directly or indirectly interconnected), thus giving rise to contiguous polypeptides.
  • at least one antigenic region is interposed between them.
  • no antigenic region is interposed between them.
  • the linear template arrangement is an alternating string of random copolymer regions and antigenic regions, wherein there are two or more random copolymer regions and two or more antigenic regions.
  • the linear template arrangement comprises RCRa-ARa-RCRb-ARb or AR a - RCRa-ARb-RCRb. In some embodiments, the linear template arrangement comprises RCR a - ARa-RCRb-ARb-RCRc-ARc or ARa-RCRa-ARb-RCRb-ARc-RCRc or ARa-RCRa-ARb-RCRb- ARc In some embodiments, the linear template arrangement comprises RCRa-ARa-RCRb or ARa-RCRa-ARb.
  • the length of each of the polypeptides in the composition i.e., the length across all of the RCRs and ARs for a single polypeptide, is about 40-80 residues. In some embodiments, length of each of the polypeptides in the composition, i.e., the length across all of the RCRs and ARs, is about 40-100 or 50-100 residues. In some embodiments, length of each of the polypeptides in the composition, i.e., the length across all of the RCRs and ARs, is about 30-80, 30-60, 40-60, 40-75, 45-65, or 45-60 residues. In some
  • the length of each polypeptide in the composition i.e., the length across each polypeptide, varies between 1-100 residues. In some embodiments, the length of each polypeptide in the composition, i.e., the length across each polypeptide, varies between 40-80 residues. In some embodiments, the length of each polypeptide in the composition, i.e., the length across each polypeptide, varies between 40-65 residues.
  • the complexity of a composition of the disclosure is greater than 5 x 10 2 different polypeptides (e.g., polypeptides that differ in sequence at at least one position but that are related based on linear template arrangement and the characteristics of the RCRs and ARs). In certain embodiments, the complexity of the composition is greater than 1 x 10 4 different polypeptides. In certain embodiments, the complexity of the composition is greater than 1 x 10 6 different polypeptides. In some embodiments, the complexity of a composition of the disclosure is greater than 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , or 1 x 10 10 .
  • Designing an amino acid copolymer composition of the disclosure comprises selecting a target (e.g., an antigen), and selecting one or more base peptide sequences from that target for use as the ARs.
  • a target e.g., an antigen
  • a linear template arrangement of the one or more ARs and one or more RCRs is also selected.
  • the length, sequence composition and input percentage (e.g. molar input percentage) for the RCRs are also selected.
  • compositions of the disclosure arises from defined substitution rules for one or more positions of the antigenic regions and from the variation and input percentage (e.g. molar input percentage) amongst the random copolymer regions of the polypeptides.
  • the composition is synthesized by applying a set of synthesis rules that define the amino acid variations along the length of the linear template arrangement and the input ratio of occurrence of introduction of such amino acid residues at any given position.
  • the compositions of the disclosure are not synthesized as single polypeptides, but are synthesized as mixtures of multiple related polypeptides, related to one another and based on the linear template arrangement, the overall composition of which is reproducible and consistent with the rules of synthesis that were applied.
  • the overall composition of amino acids that make up the antigenic regions may be modified via the introduction of different, related amino acids at one or more positions, such introduction made in accordance with a defined set of rules.
  • the antigenic regions are not modified and the diversity comes from the random copolymer regions alone.
  • the result is a mixture of related polypeptides useful in and of itself as a therapeutic, and which is useful to induce production of antibodies that react with specificity with the known sequence, but are not easily elicited by a simple immunization using the known sequence.
  • the mixture of amino acids for a given position in the polypeptide is defined by a ratio one to another. Prior to starting the synthesis, such ratio is determined for each position along the linear template arrangement.
  • a limitation of solid phase peptide synthesis is that large polypeptides may be more difficult to synthesize due to incomplete coupling reactions.
  • each resulting complex polypeptide mixture may contain some percentage of polypeptides that are less than full-length (e.g., polypeptides which comprise, for example, only about 75% or 85% of the full-length polypeptide based on the original template arrangement).
  • compositions of the disclosure comprising very high percentages of full length polypeptides (based on the template arrangement) are provided, such as compositions in which greater than 75%, greater than 80%, greater than 85%), greater than 90%, or even 95% or greater than 95% of the polypeptides in the complex mixture are full length.
  • polypeptides by solid phase peptide synthesis may result in a preferential incorporation of alanine (A) over lysine (K) in the polypeptide.
  • A alanine
  • K lysine
  • the resulting polypeptide mixture comprises a multiplicity of related polypeptide sequences.
  • the polypeptide compositions of the present disclosure have a net positive charge. In some embodiments, the polypeptide compositions of the present disclosure have a net charge that is greater than or equal to 2 at pH7. In some embodiments, the polypeptide compositions of the present disclosure have a net charge that is between 2 and 4 at pH7. In some embodiments, the polypeptide compositions of the present disclosure have a net charge that is greater than 2.1 at pH7. In some embodiments, the polypeptide compositions of the present disclosure have an increased estimated net charge versus a similar composition based on ARs of the same complexity but in the absent of the RCRs.
  • Positively charged proteins may have such attributes as being more soluble and more immunogenic.
  • the net charge of the protein affects protein solubility, and in turn, the immunogenicity of the polypeptide composition.
  • proteins, which carry a positive charge have an improved solubility profile.
  • Numerous factors in the base peptide sequence may influence the net charge of a polypeptide composition at pH7. These factors in the base peptide sequence may include the length and distribution along the copolymers of the RCR motifs, the alanine content, the lysine content, and the arginine content. Additionally, the output ratio of alanine, lysine, and arginine may influence the net charge of the polypeptide composition at pH7.
  • net charge at pH7 may be negative and glutamic acid (Glu) residues might provide alpha-helix configurations and the use of such compositions is also provided for, as described herein.
  • Glu glutamic acid
  • compositions described herein have sustained release
  • compositions described herein also act as intrinsic T-cell adjuvants and exhibit HLA promiscuity.
  • the compositions promote the release of Th2 chemokines and also promoted CD4+ T cell proliferation.
  • the compositions promote the release of a Th2- associated cytokine or chemokine from monocytes, wherein the Th2-associated cytokine or chemokine is selected from at least one of: IL-4, IL-5, IL-6, IL-10, IL-13, CCL17, and CCL22.
  • the compositions promote the release of CCL22.
  • the compositions promote the release of CCL17.
  • compositions of the disclosure comprise mixtures (e.g., greater than 500) of polypeptides with one or more random copolymer regions (e.g., amino acid copolymer compositions of the disclosure).
  • a "random copolymer region" or “RCR” refers to short random repeats (e.g. anywhere between 3-15 amino acids long, such as 3-14 amino acids long or 3-7 or 3-6 or 3-4 amino acids long) generated from mixtures of alanine, at least one positively charged amino acid (e.g. lysine, arginine, or histidine), and optionally, at least one negatively charged amino acid (e.g. aspartic acid or glutamic acid).
  • the RCR may optionally further comprise phenylalanine. In some embodiments, the RCR may optionally further comprise one or more amino acids that promote an alpha-helical secondary structure in the repeats (e.g. methionine or leucine). The contribution of the various residues to polypeptides in the composition is determined based on, for example, the molar input percentage of each amino acid residues at each position of the RCR.
  • Each polypeptide in a composition of the disclosure has one or more random copolymer regions corresponding to the random copolymer regions of the linear template arrangement (each an RCRxy, wherein x corresponds to the position of the RCR in the linear template arrangement and wherein y identifies the individual polypeptide, e.g. a composition with 500 different polypeptides could have a first RCR that is identified as RCRal, RCRa2, RCRa3...RCRa500 in each polypeptide of the mixture).
  • the distribution of the amino acids at a given position of the one or more random copolymer regions among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position.
  • an RCR (such as each RCR) is 3-15 or 3-14 amino acids long. In some embodiments, the RCRs are 5-15 amino acids long. In some embodiments, the RCRs are 3-12 or 3-10 amino acids long. In some embodiments, the RCRs are 3-8 or 3-9 amino acids long. In some embodiments, the RCRs are 3-7, 7-9, 9-11, 11-13, or 13-15 amino acids long. In some embodiments, the RCRs are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In certain embodiments, where the linear template arrangement comprises more than one RCR, the length of each is independently selected and may be the same or different.
  • the total length of the RCRs is less than 15 amino acids in length.
  • the linear template arrangement comprises two RCRs, one of which is 8 amino acids long and one of which is 9 amino acids long
  • the total length of the RCRs across the linear template arrangement is less than 20 amino acids.
  • the length of the RCRs in each individual polypeptide of the composition is less than 15 amino acids.
  • the linear template arrangement is based on one RCR, and thus, polypeptides in the composition comprise one RCR. In some embodiments, the linear template arrangement is based on two or more RCRs, and thus polypeptides in the composition comprise two or more random copolymer regions. In some embodiments, the polypeptides comprise three or more random copolymer regions. In some embodiments, the polypeptides comprise four, or five random copolymer regions. In some embodiments, the two or more RCRs each have the same length and amino acid distribution (e.g., amino acids and molar input percentage). In some embodiments, the random copolymers regions have the same or differing length and/or amino acid distribution.
  • the template arrangement comprises two RCRs (or optionally three RCRs) and the RCRs are 8 or 9 (or more than 9) residues in length, as illustrated in the examples. In other embodiments, the 2 RCRs are 5, 6, 7, 8 or 9 residues in length (as illustrated in the examples). In some embodiments, the template arrangement comprises three RCRs (or optionally more) and the RCRs are 3 or 4 or 5 or 6 residues in length, as illustrated in the examples.
  • the amino acids available for each position of an RCR include (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid (E), or phenylalanine (F).
  • the polypeptides of the compositions will have a mixture of (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid (E), or phenylalanine (F).
  • a random polypeptide composed of (i) A and (ii) at least one of K, arginine (R) or histidine (H), and, (iii) optionally, at least one of aspartic acid (D), glutamic acid (E), or phenylalanine (F).
  • a positively charged residue in an RCR may be a non-naturally occurring, positively charged residue.
  • one or more random copolymer regions of the composition are cationic in nature and/or have an amphipathic alpha-helical structure.
  • the relative molar input percentage of A for each position of at least one random copolymer region is less than or equal to 65% (but greater than or equal to 30%)
  • the relative molar input percentage of positively charged amino acids for each position is at least 35% (but less than or equal to 70%) and the relative molar input percentage of negatively charged amino acids for each position is less than or equal to 20%.
  • the relative molar input percentage of alanine for each position of a random copolymer region is 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, or 60-65%. In some embodiments, the relative molar input percentage of alanine for each position of a random copolymer region is 35-45%, 45-55%o, or 55-65%). In some embodiments, the relative molar input percentage of positively charged amino acids (K, R or H) for each position is 30-35%), 40-45%), 45-50%), 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75-80%.
  • the relative molar input percentage of positively charged amino acids (K, R or H) for each position is 35- 75%o. In some embodiments, the relative molar input percentage of positively charged amino acids (K, R or H) for each position is 30-80% or 40-70% or 50-70%. In some embodiments, only K is provided as the positively charged amino acid. In some embodiments, only R is provided as the positively charged amino acid. In some embodiments, a combination of K and R, K and H, or R and H are provided as the positively charged amino acids. In some embodiments, the relative molar input percentage of negatively charged amino acids for each position is 0-5%, 5-10%, 10-15%), or 15-20%).
  • the relative molar input percentage of negatively charged amino acids for each position is 0-10%, 5-15%, 10-20%>, or 5-20%).
  • only E is provided as the negatively charged amino acid.
  • only D is provided as the negatively charged amino acid.
  • a combination of D and E is provided as the negatively charged amino acids.
  • the input percentages of the various amino acids for each position add up to a 100%>. It will be understood that where the input percentages of A, K/R/H, and optionally D/E specified in a composition do not add up to a 100%>, the remaining percentage is made up of one or more optional amino acids such as F, M, or L or any other amino acid that assists in the
  • the relative molar input percentage of F at one or more positions of a random copolymer region is l-3%>, 3-5%, 1-5%, 5-7%, or 7-10%.
  • the relative molar input percentages of amino acids are the same for each position of a random copolymer region.
  • the relative molar input percentages of amino acids may be different for each position or for one or more positions of a random copolymer region.
  • the relative molar input percentages of amino acids are the same for each position across a given RCR, but may vary between two RCRs.
  • the composition comprises more than two RCRs, and each RCR may have the same or differing length and/or amino acid distribution.
  • one or more random copolymer regions are composed of A and K.
  • the amino acids available at each position of the RCR are A and K, each provided at a given input percentage.
  • the random copolymer regions are composed of A, K, and E.
  • the random copolymer regions are composed of (i) A, K, E and F; (ii) A, K and D; (iii) A, K, D and F; (iv) A and R; (v) A, R, and E; (v) A, R, E and F; (vi) A, R and D; and (vii) A, R, D and F.
  • the composition such as the substantially full-length polypeptide, comprises more than two RCRs, and each RCR may have the same amino acid composition and/or amino acid distribution (e.g., the linear template arrangement comprises more than two RCRs).
  • the composition, such as the substantially full-length polypeptide comprises more than two RCRs, and each RCR may have the same or differing length and/or amino acid composition.
  • the composition, such as the substantially full- length polypeptide comprises three RCRs (e.g., the linear template arrangement comprises three RCRs.)
  • the relative molar input percentages of amino acids for each position of a random copolymer region are 45% to 55% A, 35% to 50% K or R, and 5% to 20%) E or D. In some embodiments, relative molar input percentages of amino acids for each position of a random copolymer region are 45-50% A, 40-45% K or R, and 5-15% E or D. In some embodiments, relative molar input percentages of amino acids for each position of a random copolymer region are 45-50% A, 40-45% K or R, and 10% E or D. In some embodiments, the relative molar input percentages of amino acids for each position of a random copolymer region are 50% A, 40% K, and 10% E.
  • the relative molar input percentages of amino acids for each position of a random copolymer region are 30-80% K, 15-55% A, and 5-15% E. In some embodiments, the relative molar input percentages of amino acids for each position of a random copolymer region are 35-75%) K, 15-50%) A, and 5-15% E. In some embodiments, the relative molar input percentages of amino acids for each position of a random copolymer region are 35-75%) K, 15-55%) A, and 5-15%) E.
  • the relative molar input percentage of amino acids for each position of a random copolymer region are 35%-75% K (e.g., 45%, 50%, 55%, 60%, 65%, 70%), 15-50% A (e.g., 20%, 25%, 30%, 35%, 40%)), and 0-15% E (e.g., 0, 5%, 10%).
  • K e.g., 45%, 50%, 55%, 60%, 65%, 70%
  • 15-50% A e.g., 20%, 25%, 30%, 35%, 40%
  • 0-15% E e.g., 0, 5%, 10%
  • the relative molar input percentages of amino acids are the same for each position of a random copolymer region. In some embodiments, the relative molar input percentages of amino acids are the different for each position of a random copolymer region. In some embodiments, the composition comprises more than two RCRs, and each RCR may have the same or differing length and/or amino acid distribution.
  • the relative molar input percentages of amino acids for each position of a random copolymer region are 45-55% A, and 45-55%) K, with the total adding up to 100%). In some embodiments, the relative molar input percentages of amino acids for each position of a random copolymer region are 40-65%) A, and 35-60%) K, with the total adding up to 100%>. In other embodiments, the total of A and K adds up to less than 100%>, and the RCR comprises relative molar input percent of D or E of 0-20% and/or of F of 0- 10%). In certain embodiments of any of the foregoing, the RCR may comprise R instead of K.
  • the relative molar input percentages of amino acids for each position of a random copolymer region are 45-55%) A, 35-45%> K, and 5-15% E, with the total adding up to 100%>. In some embodiments, the relative molar input percentages of amino acids for each position of a random copolymer region are 40-65%) A, 35-60%) K, and 5-10% E, with the total adding up to 100%. In other embodiments, the total of A and K and E adds up to less than 100%, and the RCR comprises relative molar input percent of F of 0-10%. In certain embodiments of any of the foregoing, the RCR may comprise R instead of K and/or D instead of E.
  • the relative molar input percentage of A at each position of the RCR and/or across the RCR is less than or equal to 55%, such as less than or equal to 50%). In some embodiments, the relative molar input percentage of K at each position of the RCR and/or across the RCR is greater than or equal to 35%, such as greater than or equal to 40% or 50%.
  • the molar input ratio of A:(K+R+H) in the total amino acid concentration of an RCR is about 0.8: 1 to 2: 1. In some embodiments, the molar input ratio of A:(K+R+H) in the total amino acid concentration of an RCR is about 0.75: 1, 0.8: 1, 0.9: 1, 1 : 1, 1.25: 1, 1.35: 1, 1.5: 1, 1.75 : 1, or 1.8: 1 or 2: 1. In some embodiments, the molar input ratio of A:(K+R+H) in the total amino acid concentration of an RCR is about 0.75: 1 to 1 : 1, 1.1 : 1 to 1.25: 1, 1.25: 1 to 1.5: 1, 1.5: 1 to 1.75: 1, or 1.75: 1 to 2: 1.
  • the molar input ratio of A:K in the total amino acid concentration of an RCR is about 0.8: 1 to 2: 1, with no R or H being present. In some embodiments, the molar input ratio of A:R in the total amino acid concentration of an RCR is about 0.8: 1 to 2: 1, with no K or H being present. In some embodiments, the molar input ratio of A:(K+R) in the total amino acid concentration of an RCR is about 0.75: 1, 0.8: 1, 0.9: 1, 1 : 1, 1.25: 1, 1.35: 1, 1.5: 1, 1.75: 1, or 1.8: 1 or 2: 1.
  • the molar input ratio of A:(K+R) in the total amino acid concentration of an RCR is about 0.75: 1 to 1 : 1, 1.1 : 1 to 1.25: 1, 1.25: 1 to 1.5: 1, 1.5: 1 to 1.75: 1, or 1.75: 1 to 2: 1.
  • the ratio can also be expressed as a quotient.
  • a ratio of A:(K+R+H) of 2: 1 is equivalent to a ratio of 2, when expressed as a quotient.
  • the molar output ratio of A:(K+R+H) in the total amino acid concentration of the composition is about 1.5 : 1 to 7: 1. In some embodiments, the molar output ratio of A:(K+R+H) in the total amino acid concentration of the composition is about 7: 1, 6.5: 1, 6: 1, 5.5: 1, 5: 1, 4: 1, 3 : 1, 2.5: 1, 2: 1, or 1.5: 1. In some embodiments, the molar output ratio of A:(K+R+H) in the total amino acid concentration of the composition is about 6.75: 1, 6.25: 1, 5.75: 1, 5.25: 1, 4.75: 1, 4.5: 1, 3.75: 1, 2.25: 1, 1.75: 1, and 1.25: 1.
  • the molar output ratio of A:K in the total amino acid concentration of the composition is about 7: 1, 6.5: 1, 6: 1, 5.5: 1, 5: 1, 4: 1, 3 : 1, 2.5: 1, 2: 1, or 1.5: 1.
  • the molar output ratio of A:K in the total amino acid concentration of the composition is about 6.75: 1, 6.25: 1, 5.75: 1, 5.25: 1, 4.75: 1, 4.5: 1, 3.75: 1, 2.25: 1, 1.75: 1, and 1.25: 1.
  • These ratios may interchangeably be expressed as a quotient.
  • a ratio of A:(K+R+H) of 1.5: 1 is equivalent to a ratio of 1.5, expressed as a quotient.
  • compositions of the disclosure comprise the polypeptide sequences disclosed in SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61.
  • compositions comprising a plurality of polypeptides (such as at least or more than 500 different polypeptides) comprising the amino acid sequence set forth in any of SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NO: 61.
  • polypeptides such as at least or more than 500 different polypeptides
  • compositions of the disclosure comprise high complexity mixtures (e.g., greater than 500, greater than 1 x 10 3 , greater than 1 x 10 4 , greater than 1 x 10 5 , greater than 1 x 10 6 , or greater than 1 x 10 8 ) of polypeptides with one or more antigenic regions (ARs) in addition to one or more random copolymer regions (RCRs).
  • ARs antigenic regions
  • RCRs random copolymer regions
  • a polypeptide sequence useful for this purpose is a polypeptide sequence that is associated with a disease or condition of interest.
  • the base peptide sequence can be derived from viral, bacterial or parasitic proteins known to be associated with a pathogenic infection of a mammal.
  • the base peptide sequences can be derived from proteins known to be associated with protein conformational disorders.
  • a polypeptide sequence useful for this purpose may be related to an immune response in a mammal.
  • These polypeptide sequences are derived from, for example, partial sequences of any protein or antigen associated with the disease or condition of interest, or empirically derived polypeptide sequences, such as through screening of a library created by combinatory chemistry.
  • an "antigenic region” or “AR” refers to a sequence based on a sequence derived from an antigen of interest (a base peptide).
  • Each polypeptide in the mixture will comprise one or more ARs based on the one or more template ARs (each an ARxy, wherein x corresponds to the position of the AR in the linear template arrangement and wherein y identifies the individual polypeptide, e.g. a composition with 500 different polypeptides will have a first AR that is identified as ARal, ARa2, ARa3... ARa500 in each polypeptide of the mixture).
  • an AR is anywhere between 8-60 amino acids long and could be based on any relevant portion of a protein of interest that is associated with a disease or condition.
  • an AR is at least 8 amino acids, at least 10 amino acids, at least 12 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, or at least 30 amino acids (but in each case less than 80 amino acids long).
  • an AR is between 8-30, 8-25, 10-25, 10- 30, 10-40, 10-50, 10-60, 10-80, 15-25, 15-30, 20-30, 20-40, 20-50, 20-60, or 20-80 amino acids long.
  • An AR may remain constant amongst the polypeptides of the composition, i.e. each polypeptide of the composition has the same amino acid sequence as the base peptide in that region or an AR may vary across the polypeptides of the composition, i.e. the
  • polypeptides may differ from the base peptide and from one another at one or more amino acid positions.
  • polypeptides is determined by a pre-determined molar input ratio and molar output ratio of the amino acids available for that position (Fig. 1).
  • These variations of the target polypeptides may induce generation of antibodies that are different from those induced by the target polypeptides, but may cross- react sufficiently with the target polypeptides.
  • these related polypeptides may be desirable and/or required to identify an antibody that may not be induced by an epitope of the original sequence.
  • single polypeptides may not be very immunogenic and strong adjuvant and/or carrier proteins are frequently used to increase the antibody response.
  • Single polypeptides are also limited in terms of MHC binding and only bind to specific HLA alleles. The compositions of the instant disclosure overcome these hurdles.
  • one or more ARs of the composition are synthesized with one of several amino acids at one or more amino acid positions. In one aspect, one or more ARs are not varied, i.e., the compositions are synthesized with just one amino acid at each amino acid position.
  • the amino acid to be incorporated into a particular position of a polypeptide is selected from one or more of: an original amino acid found at the corresponding amino acid position of the corresponding base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid, and the distribution of the amino acids at a given position among the polypeptides is determined by a pre-determined molar input ratio of the amino acids available for that position.
  • the amino acid sequence of at least one AR does not vary among the polypeptides in the composition, and the amino acid sequence of said AR comprises a sequence of amino acid positions corresponding to a first base peptide sequence and the polypeptides in the composition have an amino acid at each position of the AR selected from an original amino acid found at the corresponding amino acid position of the first base peptide sequence.
  • the amino acid sequence of at least one AR varies with respect to the first base peptide sequence among the polypeptides of the composition, various polypeptides having an amino acid at each position selected from one or more of: an original amino acid found at the corresponding amino acid position of the base peptide sequence, alanine (A), lysine (K), arginine (R), or an amino acid serving as a conserved substitution for the original amino acid.
  • the first amino acid at the C- terminal position is a Norleucine (Nle).
  • the first amino acid at the N- terminal position is Nle.
  • the first amino acid at the C-terminal position or the N-terminal position is a non-naturally occurring amino acid.
  • the polypeptides comprise two or more ARs. In some embodiments, the polypeptides comprise three or more ARs. In some embodiments, the polypeptides comprise four, five, six, seven, or eight ARs. In some embodiments, the two or more ARs are each derived from the same base peptide sequences. In some embodiments, the ARs are each derived from different base peptide sequences, with the different base peptide sequences being derived from the same or different antigens. In some embodiments, two or more of the ARs share a base peptide sequence that differs from the base peptide sequence(s) of the other antigenic regions. In some embodiments, the lengths of the different ARs are independently selected. In some embodiments, at least one RCR is interposed between two of the ARs. In some embodiments, two or more ARs based on the same or different base peptide sequences are arranged consecutively.
  • one or more AR includes little or no complexity (e.g., the input amino acids correspond to the base peptide sequence).
  • Ala is present at one or more positions of one or more ARs, but the input percentage of Ala is kept relatively low, such as less than or equal to 20%, less than or equal to 15% or less than or equal to 10% at each position (independently selected at each position), other than
  • positions where Ala is the native amino acid at that position (where the input percentage is independently selected at each position). This helps keep the overall molar input percentage of Ala across ARs and RCRs less than, for example in certain embodiments, 50%), less than 45%>, less than 40%>, less than 35%>, less than 30%>, less than 25%>, or less than or equal to 20% (e.g., 10-25% or 15-25%, etc.).
  • a base peptide sequence that is derived from an antigen of interest can be modified in one or more ways with respect to the corresponding sequence of the original antigen.
  • One or more amino acid residues present in the original antigen may be absent in a base peptide sequence.
  • One or more amino acids of the base peptide can be modified in accordance with an inflammatory or oxidative environment, for example, with nitration, nitrosylation, or phosphorylation.
  • One or more amino acids of the base peptide can be modified by acetylation.
  • One or more additional amino residues, not present in the corresponding sequence of the original antigen, e.g. one or more cysteine residues may be introduced into the base peptide sequence.
  • the base peptide sequence comprises a Norleucine (Nle) residue.
  • the base peptide sequence may comprise one or more non- naturally occurring amino acids or amino acid derivatives.
  • the amino acid derivative is pyroglutamate.
  • disulfide bridges between cysteine residues of individual polypeptides are generated.
  • the post-translation formation of the cysteine bridge is performed using the reduction of a disulfide bridge between two cysteine residues.
  • reducing due to the presence of disulfide bond reductases e.g. thioredoxins and glutaredoxins are generated.
  • Peptide sequences with some significance to a disease state or an adverse reaction may be identified through experimental investigation of a relevant epitope. These sequences may include non-naturally occurring peptide sequences that proved to be useful in treating a disease or a condition, an example found in the international patent application publication WO 2006/031727, US Pat. No. 6,930,168 and the related scientific publication Stern et al, Proc. Nat. Acad. Sci. USA, 2005, 102: 1620-25.
  • epitopes or antigenic determinants are empirically determined by identifying candidate sequences by positional scanning of synthetic combinatorial peptide libraries (see, for example, D. Wilson et al, above; R. Houghten et al, above; Hernandez et al, Eur J Immunol, 2004, 34:2331-41), or by making overlapping polypeptide sequences of the entire protein of interest, and testing those polypeptides for immune reactivity (using, for example, any readout assay useful for such purposes, described in Current Protocols in Immunology Edited by John E Coligan, Ada M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strober NIH, John Wiley & Sons) in an in vitro or in vivo assay system appropriate for the disease and species the epitope is sought for.
  • Each amino acid position is kept constant or subjected to change based on a defined set of rules.
  • one or more ARs of the composition are synthesized with one of several amino acids at one or more amino acid positions.
  • the amino acid to be incorporated to a particular position of a polypeptide is selected from one or more of: an original amino acid found at the corresponding amino acid position of the
  • the amino acid to be incorporated into a particular position of a polypeptide is the original amino acid found at the corresponding amino acid position of the corresponding base peptide sequence.
  • the amino acid to be incorporated into a particular position of a polypeptide is selected from the original amino acid found at the corresponding amino acid position of the corresponding base peptide sequence and alanine (A). In some embodiments, the amino acid to be incorporated to a particular position of a polypeptide is selected from the original amino acid found at the corresponding amino acid position, alanine (A), and lysine (K). In some embodiments, the amino acid to be incorporated to a particular position of a polypeptide is selected from the original amino acid found at the corresponding amino acid position, and lysine (K).
  • the amino acid to be incorporated to a particular position of a polypeptide is selected from the original amino acid found at the corresponding amino acid position of the corresponding base peptide sequence, alanine (A), and a conserved substitution.
  • the amino acid to be incorporated to a particular position of a polypeptide is selected from the original amino acid found at the corresponding amino acid position of the corresponding base peptide sequence, and a conserved substitution.
  • only the original amino acid found at the corresponding amino acid position of the corresponding base peptide sequence is provided for one or more, or all of the amino acid positions of a polypeptide.
  • an amino acid to be incorporated at a particular position some proportion of the time is phosphorylated (e.g., either the original amino acid found at the corresponding position is or can be phosphorylated, or such phosphorylated amino acid is deemed a conservative substitution).
  • an amino acid to be incorporated at a particular position some proportion of the time is nitrated (e.g., either the original amino acid found at the corresponding position is or can be nitrated, or such nitrated amino acid is deemed a conservative substitution).
  • a probable set of additional related polypeptides may be generated using modeling and prediction algorithms described in readily available references, for example WO 2000/042559, align and analyze the predicted binding of these probable epitopes using available prediction methods described in, for example, WO 2005/103679, WO 2002/073193 and WO 99/45954.
  • amino acid substitutions at one or more positions of the base peptide are determined as follows: select from the polypeptides having the highest predicted activity /binding, take 40% of the predicted sequences and acquire the percentage of any given amino acid at each position. Use those percentages to create the rules for amino acid incorporation into the synthesis scheme.
  • incorporation of alanine and/or other substitutions into the polypeptides of the mixture may increase immunogenicity by creating artificial T- and B-cell epitopes and HLA/MHC promiscuity.
  • a "conserved substitution" in the context of the antigenic region may refer to a conservative substitution as understood by the skilled artisan.
  • an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another
  • a conserved substitution in the context of an antigenic region is selected according to the methods of rational comparison and findings of similarity described in Kosiol et al., J. Theoretical Biol., 2004, 228:97-106. Amino acids are grouped together in a matrix, referred therein as PAM replacement matrix.
  • a conserved substitution is based on amino acid similarity and means the relationship of those amino acids grouped together according to Kosiol, based on the characteristics of the residues such as size, charge, hydrophobicity, etc.
  • a conserved substitution can be selected in accordance with the methods set forth in US Patent
  • a conserved substitution may also be selected in accordance with the differences in amino acid composition at a given position between species.
  • a conserved substitution may be an orthologous substitution, such as a substitution present in an ortholog of the gene encoding the protein of interest.
  • amino acids can be changed in accordance with the differences at a given position between individual examples within the same species.
  • a conserved substitution refers to a modified form of the amino acid.
  • a conserved substitution refers to a phosphorylated form of the amino acid.
  • a conserved substitution refers to a nitrated form of the amino acid.
  • the amino acids can be modified in accordance with an inflammatory or oxidative environment, for example, with nitration, or phosphorylation. Alternatively, the amino acids can be modified by acetylation.
  • a conserved substitution is an isomer of the amino acid.
  • the amino acids can be changed or modified in order to promote, delay, accelerate or inhibit amlyoidogenesis.
  • a conserved substitution includes a small, polar, or charged amino acid. In some embodiments, a
  • “conserved substitution” includes the absence of an amino acid at a given position.
  • a conserved substitution provided for a particular position of an antigenic region is are defined according either: (1) to a rational comparison and finding of similarities of relevant characteristics of the original amino acid found at the corresponding amino acid position of the base peptide sequence with those of the substitute residue, (2) in accordance with the differences at a given position between species, (3) in accordance with the differences at a given position within individuals of the same species, or (4) to a comparison of reported experimental results on the relative activities of actual polypeptides having slight variations from the base sequence, or (5) is a conservative substitution.
  • the substitute residues defined in any of these approaches are termed "conserved substitution" herein.
  • a comparison of experimental results showing the relative activities of polypeptides having slight variations from the base sequence can also be used as a basis for the rule for substitution.
  • the sequences of the polypeptides responsible for observed changes are aligned and the type and percent presence of the new amino acid are noted. If there is more than one amino acid substitution at any given position of the polypeptide, the frequency of occurrence of an amino acid and the magnitude of activity change compared to the original sequence are taken into account to determine the order of prevalent substitution. Examples of the overall process leading up to the rule generation for amino acid copolymer synthesis can be found using libraries ⁇ Molec. Immunol. 40: 1047-1055; Molec. Immunol. 40: 1063-74; J
  • Such an assay system can be either an in vitro or in vivo system, and can comprise adaptive or innate immune reactivity.
  • Readouts for the assay system can be the up- or down-regulation of the status of the activation state of a protein, a change in the localization of a protein, the expression of the mRNA encoding for the protein, the relative concentration of a protein, changes in the generation of specific cell types, changes in cellular phenotype, changes in cellular activation, changes in cell number, changes in organ size or function, changes in animal behavior or phenotype.
  • the base peptides can be derived from any protein or antigen of interest that is associated with a disease or condition of interest (e.g., a target).
  • a disease or condition of interest e.g., a target
  • the compositions of the disclosure are broadly applicable to numerous diseases and indications, for example, by selecting ARs corresponding to portions of a target of interest.
  • the antigen e.g., target of interest
  • the antigen is associated with a protein conformational disorder.
  • the antigen is prion protein, amyloid beta precursor protein, ABri peptide, Tau protein, alpha-synuclein, alpha-synuclein central fragment, SOD1, TDP-43, repeat-associated non-ATG (RAN)-translated peptides of the C90RF72 locus, islet amyloid polypeptide (a.k.a. amylin), prothymosin alpha, amino- terminal domain of androgen receptor protein, ataxin-1, DRPLA protein (a.k.a.
  • the antigen is Tau.
  • the antigen is alpha-synuclein. In some embodiments, the antigen is Abeta. In some embodiments, the antigen is SOD1. In some embodiments, the antigen is TDP-43. In some embodiments, the antigen is RAN peptides of the C90RF72 locus. In some embodiments, the antigen is associated with a pathogenic infection. In some embodiments, the antigen is a bacterial protein. In some embodiments, the antigen is a viral protein. In some embodiments, the antigen is a protein from a parasitic agent. In some embodiments, the antigen is a protein from human
  • the antigen is a protein from human immunodeficiency virus (HIV). In some embodiments, the antigen is a protein from human papillomavirus (HPV).
  • the amino acid sequences of exemplary targets are provided in the sequence listing. Base peptide sequences for use in deriving an AR can be selected from, for example, these targets. Exemplary base peptide sequences suitable as ARs are also provided in the sequence listing.
  • the antigen is associated with one of the diseases or conditions described in this disclosure and the compositions of the disclosure are useful in treating or preventing one of the diseases or conditions described below.
  • Suitable uses include, for example, to study the disease or condition with which the antigen is associated, to raise antibodies reactive with a target (e.g., antigen), to identify other binders of the target, to induce CD4+ T-cell proliferation in vitro or in vivo, to induce a Th2 immune response in vitro or in vivo, and the like.
  • a target e.g., antigen
  • PCDs protein conformational disorders
  • AD Alzheimer's disease
  • PD Parkinson disease
  • amyotrophic lateral sclerosis ALS
  • frontotemporal dementia and parkinsonism linked to chromosome 17 FTDP-17
  • progressive supranuclear palsy DAA
  • reactive amylosis Type-2 diabetes
  • cystic fibrosis CF
  • Huntington's disease HD
  • Creutzfeldt- Jakob disease CJD
  • related disorders and systemic and cerebral hereditary amyloidosis.
  • globular proteins that undergo fibrillogenesis include transthyretin, beta 2 microglobulin, serum amyloid A protein, Ig light chains, insulin, human lysozyme, alpha lactalbumin, and monellin.
  • natively unfolded proteins that undergo fibrillogenesis include amyloid beta protein, tau protein, alpha-synuclein, amylin, and prothymosin alpha.
  • fibrillogenesis the protein relevant to PCD pathology is in a naturally folded conformation and in monomelic or defined oligomeric forms, each polypeptide comprising a mixture of alpha-helices, some beta-sheets, and random coils.
  • fibrillogenesis the protein is aggregated, and the polypeptide has adopted an altered conformation, i.e. mostly a beta- pleated sheet conformation. The conformational changes of the polypeptides and aggregation appear to coincide, but the cause and effect of conformational change and aggregation, and the sequence of events, remain to be elucidated.
  • the fibrillogenesis is a crystallization-like process: after a "seed" of oligomers forms, an aggregate grows over time through self-association.
  • the protein may take an altered conformation because the aggregate exists and serves as a template, or it may take the altered conformation because of other factors, but once in that conformation, easily participates in fibrillogenesis.
  • another proposal hypothesizes that the conformational alterations alone may not cause or promote aggregation, and there is a factor that induces the
  • Such underlying factors that promote or induce structural changes in the protein include inflammatory or oxidative environments, nitration, phosphorylation, pH, or metal ion exposure (high concentrations of copper ions can induce the oligomerization of ⁇ 2 microglobulin monomers, which in turn leads to fibril formation (Eakin et al., Biochemistry 2004, 43, 7808-7815)).
  • a treatment mode with an assumption that fibrillogenesis is caused by the beta-sheet conformation will attempt to inhibit the beta-sheet formation.
  • a treatment mode may aim to inhibit aggregation by various means.
  • An illustration of the former approach includes an attempt to inhibit the formation of, or to break, beta-sheets, using polypeptides.
  • polypeptides are designed from the sequences of areas of proteins most likely involved in the process of nucleation and aggregation, such as the hydrophic core of amyloid-beta, a polypeptide intimately involved in the pathology of Alzheimer's disease.
  • An illustration of the latter approach is an attempt to manipulate protein conformation and prohibit nucleation and subsequent formation of amyloids, or, "amyloidogenesis,” by creating mini-chaperone polypeptides from outside of the beta-sheet regions.
  • the antibodies that are desirable recognize and specifically bind to proteins of certain altered conformation.
  • the difficulty lies in the fact that these proteins exist as normal parts of the patient's system, were it not for the altered conformation that they are in.
  • these proteins may not elicit strong natural immune responses in the afflicted individuals, and it may be difficult to elicit an immune response (thus to raise antibodies) using the native sequence of the target protein in other subjects of the same species, or in an individual with similar immunological profile, which is often desirable due to the lower probability of adverse immunological reaction.
  • a protein relevant to a PCD may have the same primary structure, whether in a non-pathological condition or in pathological condition. Without the ability to distinguish, the antibody intended for therapeutic purposes may adversely affect the patient by eliminating or interfering with the normal, functioning protein. Thus, a high specificity towards the particular conformation, or series of alterations, is required.
  • abnormal hyperphosphorylation of Tau is a hallmark of AD and several other related neurodegenerative disorders, called tauopathies. Hyperphosphorylation of Tau causes conformational changes and leads to the formation of pathogenic species such as paired helical filaments (PHF) and neurofibrillary tangles (NFT).
  • PHF paired helical filaments
  • NFT neurofibrillary tangles
  • Effects of Tau hyperphosphorylation include dissociation of Tau from microtubules, toxicity, and synaptic dysfunction.
  • a method of targeting variants of hyperphosphorylated Tau in a specific manner will lead to efficient treatment.
  • the present disclosure provides compositions uniquely suitable to treating and studying PCDs.
  • one or more base peptide sequences that form the basis for the antigenic regions of the amino acid copolymer compositions of the disclosure are derived from an antigen relevant to the pathology of protein conformational disorders affecting the central and/or peripheral nervous system, selected from the group consisting of: Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP- 17), Dutch hereditary cerebral hemorrhage with amyloidosis (a.k.a cerebrovascular amyloidosis), congophilic angiopathy; Pick's disease, progressive supranuclear palsy; familial British dementia; Parkinson's disease (PD), Lewy-body related diseases, multiple system atrophy, Hallervorden-Spatz disease; amyotrophic lateral sclerosis (ALS); Huntington's disease (HD); spinocerebellar ataxia; neuronal intranuclear inclusion disease; hereditary dentator
  • a base peptide sequence is derived from an antigen relevant to the pathology of protein conformational disorders affecting multiple organs or organs other than the central nervous system, selected from the group consisting of: spinal and bulbar muscular atrophy; hereditary systemic and cerebral amyloidosis, Finnish-type familial amyloidosis; senile systemic amyloidosis (a.k.a. senile cardiac amyloidosis), familial amyloid polyneuropathy; Type-2 diabetes, in particular pancreatic islet amyloidosis; dialysis-related amyloidosis (DRA); inflammation-associated reactive systemic amyloidosis (a.k.a.
  • AA amyloidosis AA amyloidosis); aortic medial amyloidosis; medulary carcinoma of the thyroid; hereditary renal amyloidosis; light chain associated amyloidosis, light chain deposition disease, light chain cast nephropathy, light chain cardiomyopathy; atrial amyloidosis; injection-localized amyloidosis; cystic fibrosis (CF); sickle cell anemia, and other disorders wherein
  • fibrillogenesis is observed in the affected organs or tissues.
  • Examples of natively unfolded proteins and polypeptides, and those suspected to be natively unfolded, that undergo fibrillogenesis, and therefore associated with protein conformational disorders and that may be use as the source sequences of the base peptides for the preparation of a composition of the disclosure include: prion protein and its fragments, amyloid beta protein and its fragments, ABri peptide, tau protein, alpha-synuclein and its central fragment, islet amyloid polypeptide (a.k.a. amylin), exon I of huntingtin, prothymosin alpha, amino-terminal domain of androgen receptor protein, ataxin-1, DRPLA protein (a.k.a. atrophin-1), and calcitonin.
  • Examples of globular proteins that undergo fibrillogenesis and therefore associated with protein conformational disorders and that may be use as the source sequences of the base peptides for the preparation of a composition of the disclosure include: cystatin c, transthyretin, beta 2 microglobulin, serum amyloid A protein and its fragments, huntingtin and its fragments (including exon I of huntingtin), imunoglobulin light chain variable domains, insulin, lysozyme (in particular human lysozyme), alpha lactalbumin, and monellin, ligand- and DNA-binding domains of androgen receptor protein, lactadherein and more specifically its fragments (for example, a.a. residue 245-294, a.k.a. medin), gelsolin, apolipoprotein Al, fibrinogen and its fragments, and atrial natriuretic factor. Fragments of all the proteins in this paragraph may also be used as source sequences.
  • Alzheimer's disease pathology correlates strongly with the presence of a 4 kDa amyloid beta ( ⁇ ) peptide that is enzymatically cleaved from the Amyloid precursor protein (APP) by ⁇ -secretase and ⁇ -secretase.
  • ⁇ peptides are 40 amino acids long, and designated ⁇ 40, ⁇ 4 ⁇ , ⁇ - 4 ⁇ , or, having varied amino terminal, ⁇ ⁇ -4 ⁇ .
  • Studies have indicated that the fibrillar form of ⁇ - 4 ⁇ stimulates the microglia, which cell type is currently thought to play an important role in the pathogenesis of Alzheimer's disease. (Jekabsone, A. et al., J.
  • the peptide sequence of ⁇ - 4 ⁇ is shown as SEQ ID NO: 1 in Table I.
  • ⁇ -42 which is a minor fraction of plaque-forming ⁇ , is thought to contribute to the initiation of the formation of fibrillar ⁇ .
  • This "long form" of the peptide is described as SEQ ID NO: 2 in Table 1. Therefore, a base peptide sequence of an antigenic region is derived from such an ⁇ peptide, exemplified by SEQ ID NO: 2.
  • the base peptide sequence may also be derived from a shorter peptide, i.e.
  • a base peptide sequence of an antigenic region is a portion of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the base peptide sequence is modified with respect to the above sequences to include one or more nitrated, acetylated or phosphorylated residues, one or more insertions or deletions, or non-natural amino acids.
  • PD Parkinson's Disease
  • Lewy Bodies a-Synuclein is the most abundant protein in Lewy Bodies, and appears to be an important mediator, perhaps even a causal factor, of toxicity in PD. Thus, reduction of toxic ⁇ -Synuclein is thought to be beneficial to PD patients.
  • SEQ ID NO: 3 The sequence of one such mouse a- Synuclein polypeptide, derived from the C-terminal region of the full-length protein, is shown as SEQ ID NO: 3 in Table 1.
  • the base peptide sequence of an antigenic region is derived from, for example, SEQ ID NO: 3, or a portion thereof.
  • the base peptide sequence is derived from a fragment comprising amino acids 121-137 of human a-Synuclein
  • the a-Synuclein fragment (121-137) sequence is substituted at positions 121 and 122 in different species, tri- nitrated at each Y (tyrosine) position, and/or phosphorylated at S129.
  • the base peptide sequence is derived from a fragment most closely corresponding to amino acids 123-137 of human ⁇ -Synuclein, the fragment of which is set forth in SEQ ID NO: 36 (GSE A YEMP SEEGYQD YE; SEQ ID NO: 36).
  • the base peptide sequence is derived from a fragment comprising amino acids 121- 137 of human ⁇ -Synuclein, and this fragment is set forth in SEQ ID NO: 37
  • the base peptide sequence is modified with respect to the above sequences to include one or more nitrated, acetylated or phosphorylated residues, one or more insertions or deletions, or non-natural amino acids.
  • a portion thereof corresponds to a contiguous portion or fragment of the antigen of interest. In some embodiments, "a portion thereof refers to a portion of an antigen that is at least 6 amino acid residues, at least 8, at least 9, at least 10 amino acid residues, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, or at least 30 amino acids. In some embodiments, "a portion thereof refers to a portion or fragment of an antigen between 8-20, 9-30, 10-30, 10-40, 10-60, 10-80, 20-30, 20-40, 20-50, 20-60, 20- 80, or 20-100 amino acids long. In certain embodiments, a portion of an antigen or target of interest is suitable for use as a base peptide sequence, and thus, forms the basis of an AR.
  • tauopathies e.g. Alzheimer's disease (AD), FTDP-17, progressive supranuclear palsy (PSP), PSP-like tauopathy, etc.
  • AD Alzheimer's disease
  • FTDP-17 progressive supranuclear palsy
  • PSP progressive supranuclear palsy
  • PSP-like tauopathy etc.
  • Hyperphosphorylation of Tau particularly in the C-terminal region, leads to the formation of pathological PHFs and NFTs and it is desirable to design and manufacture compositions that target all significant variants of phosphorylated Tau protein found in early oligomers, PHFs and NFTs.
  • An immune response capable of eliminating toxic species of Tau will slow down disease progression in patients suffering from a tauopathy.
  • PFIF-Tau is made of phosphorylated Tau peptides interacting through MTBD R2 and R3 repeats. Normally, the N- and C- terminal regions of Tau inhibit aggregation. However, phosphorylation of the N- and C- terminal regions (at the AT8 and PHFl sites) neutralizes the inhibitory effect.
  • the PHF-1 site includes S396 and S404 of Tau-F.
  • a base peptide of an antigenic region is derived from the
  • a base peptide sequence can be derived from a partial sequence of residues 388-441 of Tau-F (SEQ ID NO: 6). In some embodiments a base peptide sequence is derived from residues 388-408 of Tau-F (SEQ ID NO: 7) or a portion thereof. In some embodiments a base peptide sequence is derived from residues 417-441 of Tau-F (SEQ ID NO: 8) or a portion thereof. In some embodiments, a base peptide sequence is derived from residues 390-408 of Tau-F (SEQ ID NO: 9) or a portion thereof.
  • a base peptide sequence is derived from residues 418- 441 of Tau-F (SEQ ID NO: 10) or a portion thereof. In some embodiments, a base peptide sequence is derived from residues 414-438 of Tau-F (SEQ ID NO: 31). In some embodiments, a base peptide sequence is derived from residues 414-429 of Tau-F (SEQ ID NO: 32). In some embodiments, a base peptide sequence is derived from residues 424-438 of Tau-F (SEQ ID NO: 33). In some embodiments, one or more of the phosphorylation sites are
  • the antibodies produced are capable of decreasing intracellular Tau. In some embodiments, the antibodies produced are capable of decreasing extracellular Tau. In certain embodiments, the disclosure provides a method of decreasing the presence of intracellular Tau, such as Tau present in the hindbrain, cortex, or hippocampus. In certain embodiments, the disclosure provides a method of decreasing the presence of extracellular Tau, such as Tau present in the hindbrain, cortex, or hippocampus. In certain embodiments, the disclosure provides a method of decreasing the presence of soluble Tau.
  • the disclosure provides a method of decreasing the presence of insoluble Tau. In certain embodiments, the disclosure provides a method of decreasing the presence of aggregated Tau. In certain embodiments on any of the foregoing, the decrease in Tau is in the hindbrain or hippocampus. In certain embodiments, the compositions of the disclosure are capable of inducing specific antibodies that inhibit early phosphorylation and accumulation of toxic precursor tau oligomers. In some
  • compositions of the disclosure target glial tau pathology in astrocytes. In some embodiments, the compositions of the disclosure target glial tau pathology in microglia.
  • Exemplary amino acid copolymer compositions designed according to the methods described herein have been shown to induce a specific Th2 immune response capable of eliminating toxic species of Tau.
  • tauopathies may include, but are not limited to, AD, FTDP-17, PSP, PSP-like tauopathy, corticobasal degeneration (CBD), pseudobulbar palsy, traumatic brain injury, chronic traumatic
  • CTE encephalopathy
  • Pick's disease Niemann-Pick disease type C
  • postencephalitic parkinsonism dementia pugilistica
  • chronic traumatic encephalopathy lytico-Boding disease
  • ganglioglioma gangliocytoma
  • subacute sclerosing panencephalitis meningioangiomatosis
  • lead encephalopathy tuberous sclerosis
  • Hallervorden-Spatz disease and lipofuscinosis.
  • a base peptide sequence of an antigenic region is derived from a protein sequence relevant to prion-diseases.
  • SEQ ID NO: 13 is human prion protein sequence.
  • a relevant base peptide may be derived from partial sequences of SEQ ID NO: 13.
  • Various species' prion sequences are disclosed by Harmeyer, S. et al, J Gen Virol. 79(Pt 4):937-45 (1998), the entirety of which is incorporated herein by reference.
  • the amino acid variations by species can be used to design the substituting amino acids.
  • the base peptide sequence is modified with respect to the above sequences to include one or more nitrated, acetylated or phosphorylated residues, one or more insertions or deletions, or non-natural amino acids.
  • a base peptide sequence of an antigenic region is derived from superoxide dismutase I (SODl).
  • SODl mutation is known to have causal relationship with the pathology of some forms of familial ALS. It has been reported that the antisera raised against a mutant form of SODl, human G93A SODl recombinant protein, had protective effect on a mouse model of ALS carrying G37R mutant SODl (line 29), which overexpress human SODl protein by 4-fold higher than endogenous mouse SODl .
  • SOD1 protein sequence is SEQ ID NO: 14. Therefore, in one embodiment, a base peptide sequence is derived from SEQ ID NO: 14 or a portion thereof.
  • a base peptide sequence of an antigenic region is derived from TAR DNA-binding protein 43 (TDP-43).
  • TDP-43 proteinopathies consisting of several neurodegenerative diseases including frontotemporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS), are characterized by the presence of inclusion bodies made of hyperphosphorylated and poly-ubiquitinated full-length and truncated TDP-43. TDP-43 abnormalities also occur in a subset of Alzheimer's disease patients. Therefore, in one embodiment, a base peptide sequence of an antigenic region is derived from SEQ ID NO: 18 or a portion thereof.
  • a base peptide sequence of an antigenic region is derived from dipeptide repeat proteins (DPR) translated from the GGGGCC repeats upstream of the chromosome 9 orf 72 (C9orf72) coding region.
  • DPR dipeptide repeat proteins
  • C9orf72 chromosome 9 orf 72
  • FTLD familial frontotemporal lobar degeneration
  • a base peptide sequence of an antigenic region is derived from RAN-translated peptides of the C9orf72 locus and comprises a poly- (Gly-Ala) dipeptide repeat sequence.
  • a base peptide sequence of an antigenic region comprises a poly-(Gly-Pro) dipeptide repeat sequence.
  • a base peptide sequence of an antigenic region comprises a poly-(Gly-Arg) dipeptide repeat sequence.
  • the base peptide sequences derived from the RAN- translated dipeptide repeats of the C9orf72 locus are 10-20 amino acid residues in length. In some embodiments, the base peptide sequences derived from the RAN-translated dipeptide repeats of the C9orf72 locus are 10-15, 10-25, or 10-30 amino acid residues in length.
  • Misfolded protein also plays a role in Huntington's disease, a genetic disorder caused by the pathological expansion of a polyglutamine (polyQ) tract in the huntingtin (htt) protein (SEQ ID NO: 15), resulting in neurodegeneration and premature death of the afflicted individual.
  • polyQ polyglutamine
  • htt huntingtin
  • a single-chain antibody that binds to an epitope formed by the N-terminal 17 amino acids of htt (Lecerf, J.-M. et al, P roc Natl Acad Sci USA. 98(8): 4764-4769 (2001) SEQ ID NO: 5) has been shown to reduce symptoms in a Drosophila model of Huntington's disease. (Wolfgang, W.J.
  • a base peptide sequence is derived from SEQ ID NO: 5.
  • a base peptide sequence of an antigenic region is derived from a portion of SEQ ID NO: 15.
  • DRA Dialysis-related Amyloidosis
  • DRA may be caused by different forms of blood filtration, such as haemodialysis, hemofiltration, or Continuous Ambulatory Peritoneal Dialysis (CAPD).
  • CPD Continuous Ambulatory Peritoneal Dialysis
  • B2M beta-2-microglobulin
  • Conformational isomers of B2M have been observed in a clinical setting (Uji et al. Nephron Clin Pract 2009; l l l :cl73-cl81).
  • B2M is part of the human leukocyte antigen (HLA) class I molecule, and has a prominent beta-pleated structure characteristic of amyloid fibrils. B2M is known to circulate as an unbound monomer distributed in the extracellular space. B2M undergoes fibrillogenesis to form amyloid deposits in a variety of tissues. This deposition causes renal failure, which causes an increase in synthesis and release of B2M, exacerbating the condition.
  • a protein the base sequence of which is used for preparation of a composition of the disclosure is beta 2 microglobulin (SEQ ID NO: 12) and fragments thereof.
  • SEQ ID NO: 12 beta 2 microglobulin
  • An exemplary fragment of B2M is that spanning amino acid residues 21- 40, SEQ ID NO: 11 in Table 1, useful as a base peptide for DRA.
  • the composition for example, a composition used to treat or diagnose DRA
  • AGE advanced glycation end
  • AGE products are a heterogeneous group of carbohydrate molecules formed by non- enzymatic glycation and oxidative reactions between reducing sugars and protein amino groups.
  • AGE-modification of B2M is often observed in DRA patients, and appears to contribute to the pathology of DRA.
  • the author observed imidazolone, ⁇ - (carboxymethyl)lysine (CML), and pentosidine modifications.
  • CML carboxymethyl
  • AGE-B2M interacts with mononuclear phagocytes (MPs), cells important in the pathogenesis of inflammatory arthropathy. This interaction prompts the MPs to secrete elevated levels of TNFa and interleukin-1, worsening inflammation (Rashid et al., IMAJ 2006;8:36-39).
  • MPs mononuclear phagocytes
  • an AR or base peptide sequence comprises or consists of all or a portion of a sequence set forth in the tables below or as described above.
  • Table 1 Examples of peptides associated with protein conformational disorders
  • Protein TGSIDMVDSPQLAT Human Tau-F 414-429 of conformational LA SEQ ID No. 17 disorders;
  • Protein QLATLADEVSASL Human Tau-F 424-438 of conformational AKRRR (SEQ ID SEQ ID No. 17 disorders; NO: 33)
  • the infection of a host by an infectious pathogen is a complex event comprising a series of coordinated events in which the pathogen attempts to evade both the host's innate and adaptive immune systems. In their attempts to replicate and survive, the invading pathogens cause damage to the host. The destruction generally takes the form of cell death, either from pathogen entry into the cell or from endo/exo-toxins produced by the pathogen, as well as the induction of host cellular responses that have the ability to cause further tissue damage, scarring or hypersensitivity.
  • the decline in infectious disease-related mortalities decreased 8.2% per year (Armstrong, GL et al, JAMA 1999, 281 :61-6,), the death rates due to infectious disease have been increasing since the 1980s.
  • infectious diseases comprise two distinct but interconnected aspects: (1) the various organisms attempting to invade a host, each different class of infectious agents having distinct means by which they attempt to evade the host's immune system, and (2) the host's immune response to the invading pathogen.
  • Humans have warded off infectious diseases in both of these aspects, in the first aspect, by preventing the access of infectious agents (e.g. by sanitary habits) and in the second aspect, by assisting and boosting the immune system by, for example, vaccination.
  • polypeptide-based vaccines are specific to epitopes (also called antigenic determinants) that are the precise moieties within antigenic materials to interact with immune system components, intended to boost immune reactivity, and are administered using methods designed to excite immune function.
  • epitopes also called antigenic determinants
  • epitope/polypeptide-based approach is the variability with which the human MHC class I and II receptors bind the polypeptides.
  • HIV human immunodeficiency virus
  • cytomegalovirus cytomegalovirus
  • severe acute respiratory syndrome coronavirus as well as bacteria such as Pseudomonas aeruginosa, Neisseria gonorrhea, or Mycobacterium tuberculosis or parasitic diseases such as malaria or hookworm disease.
  • bacteria such as Pseudomonas aeruginosa, Neisseria gonorrhea, or Mycobacterium tuberculosis or parasitic diseases such as malaria or hookworm disease.
  • the current licensed vaccines are produced in eggs, and make use of half-century old technology (Ben-Yedidia 2007, above).
  • infectious agents, bacteria, and parasitic diseases are harder to treat using the inactive pathogen vaccine approach because of the organism's ability to evade host detection.
  • the HIV or the flu virus has the ability to alter its immune profile multiple times in the amount of time less than a calendar year, progressively marginalizing the effectiveness of immunity gained by previous infection and/or even the most recently created vaccine.
  • the invading pathogens have acquired by natural selection the ability to quickly create variability in the relevant immunogenic epitope.
  • Examples of viral mutants generating escape variants that avoid immune detection have been reported for Hepatitis B, influenza, and HIV (Tabor, E, Journal of Med. Virol, 2006, 78:S43-S47; Daniels, RS et al., EMBO Journal 1987, 6: 1459-65; D'Costa, S et al, AIDS Res Hum Retroviruses 2001, 17: 1205-9).
  • This variation progressively dampens the utility of single-epitope vaccines.
  • a simple mixture of several soluble polypeptide epitopes faced certain manufacturing issue of soluble polypeptide mixtures, such as difficulties in delivering a consistent ratio and quantity of each of the polypeptides in the mixture.
  • prophylactically effective may be cryptic, or they may change so frequently as to render a developed vaccine ineffective and irrelevant. Rapidly evolving viruses would mean the number of people carrying the virus with same immunological profile becomes small, fragmenting the patient population against which a vaccine may be effective. Moreover, chronic infections caused by agents such as HIV, hepatitis C virus (HCV), and human papillomavirus (HPV) may require a different approach to immune elicitation due to the fact that they evolved to evade immune systems and are poor antigens.
  • HIV hepatitis C virus
  • HPV human papillomavirus
  • a base peptide sequence of an antigenic region may be derived from an HIV protein such as, but not limited to, gag, protease, reverse transcriptase, gpl20, nef peptides and lipopeptide, gp41, or env proteins or portions thereof.
  • a base peptide sequence of an antigenic region may be derived from a
  • Hepatitis C protein such as, but not limited to, glycoprotein E1/E2 or a portion thereof.
  • a base peptide sequence of an antigenic region is derived from epitopes associated with human papilloma virus (HPV).
  • HPV human papilloma virus
  • the target antigen is based on an HPV protein and the base peptide and/or AR corresponds to a portion of that HPV protein.
  • Exemplary such proteins that may be the target from which the base peptide is derived include, for example, LI and L2.
  • HPV is a non-enveloped double stranded DNA virus, the genome of which encodes two protein types: 'Early proteins' (El, E2, E4, E5, E6, and E7) which regulate the replication of viral DNA and 'Late proteins' (LI and L2) which are the structural components of the viral capsid.
  • Targets for compositions of the disclosure may be based on structural components or other components of the virus. More than 170 different types of HPV have been identified.
  • CervarixTM a bivalent vaccine which is effective in preventing HPV types 16 & 18 and
  • GardasilTM a quadrivalent vaccine effective in preventing HPV 6, 11, 16, and 18. Both the Gardasil and Cervarix vaccine are made from purified virus-like particles (VLPs) of the VLPs.
  • LI capsid protein The LI protein is highly HPV type specific, and thus does not provide broad protection against the full range of HPV type infections. Another limitation of targeting LI is that LI proteins are only expressed during the initial infection with the virus. Thus, vaccines targeting LI are ineffective once the infection becomes established and systemic.
  • HPV associated targets may be the basis of compositions of the disclosure.
  • base peptides based on or derived from, for example, L2 may provide protection against more HPV types.
  • L2 although more similar mechanistically to using LI, based vaccination approaches may provide protection against additional HPV types.
  • a base peptide sequence is derived from residues most closely corresponding to 13-40 of HPV L2 protein (SEQ ID NO: 34).
  • SEQ ID NO: 34 residues most closely corresponding to 13-40 of HPV L2 protein.
  • base peptides may be derived from any portion of a HPV L2 protein.
  • an AR or base peptide sequence comprises or consists of all or a portion of a sequence set forth in the tables below or as described above
  • one or more base peptide sequences that form the basis for the antigenic regions of the amino acid copolymer compositions of the disclosure are derived from an antigen associated with a pathogenic infection.
  • the pathogen is a virus, bacteria, or a parasite.
  • the pathogen is virus selected from HIV, porcine reproductive and respiratory syndrome (PRRS), foot-and-mouth diseasese, or HSV.
  • the pathogen is a bacterium selected from Salmonella, Mycobacterium (e.g., M.
  • the pathogen is a parasite such as a protozoan selected from a trypanosome (e.g., Trypanosoma brucei, Trypanosoma cruzi, Leishmanid), ox Plasmodium (e.g., P. falciparum, P. vivax, P. knowlesi, P. malariae, P. ovale, P. brasilianum, P. cynomolgi, P. inui, P. rhodiani, P.
  • a protozoan selected from a trypanosome (e.g., Trypanosoma brucei, Trypanosoma cruzi, Leishmanid), ox Plasmodium (e.g., P. falciparum, P. vivax, P. knowlesi, P. malariae, P. ovale, P. brasilianum, P. cynomolgi, P. inui, P. rhodiani,
  • the pathogen is a parasite such as a helminth (e.g. monogeneans, cestodes, trematodes, nematodes).
  • the antigens are derived from an intracellular pathogen.
  • the pathogen is of the genus selected from Chlamydia, Rickettsia, Coxiella, Mycobacterium, Francisella, Listeria, Salmonella, Brucella, Legionella, Nocardia,
  • the peptide sequences are derived from SEQ ID Nos. 21-29 or portions thereof.
  • the one or more base peptide sequences that form the basis for the antigenic regions of the amino acid copolymer compositions of the disclosure are derived from an antigen associated with any of the pathogen disclosed herein. The peptide sequences of these proteins and epitopes have been extensively investigated and are readily available to one skilled in the art. V. Polypeptide Synthesis Methods
  • the synthesis is done in multiple steps from C-terminus to N-terminus by the Solid Phase Peptide Synthesis (SPPS) approach using Fmoc protected amino acids.
  • the first amino acid at the C-terminal position may be a Norleucine (Nle) covalently coupled to the resin support.
  • SPPS is based on sequential addition of protected amino acid derivatives, with side chain protection where appropriate, to a polymeric support (bead).
  • the base-labile Fmoc group is used for N-protection. After removing the protecting group (via piperidine hydrolysis) the next amino acid mixture is added using a coupling reagent (TBTU).
  • Fmoc may be cleaved and an amino acid is randomly selected from the amino acid solution described in the linear template arrangement for each position. After the final amino acid is coupled, the N-terminus is acetylated.
  • the resulting polypeptides (attached to the polymeric support through the C-terminus) are cleaved with TFA to yield the crude polypeptide. During this cleavage step, all of the side chains protecting groups are also cleaved. After precipitation with diisopropyl ether, the solid is filtered and dried. The resulting polypeptides are analyzed and stored at 2-8°C.
  • each resulting complex polypeptide mixture may contain some percentage of polypeptides that are less than full-length or less than
  • substantially full-length e.g., polypeptides which comprise, for example, only about 75% or 85% of the full-length polypeptide based on the original template arrangement.
  • these shorter polypeptides, if any, in a composition are generally truncated from the N-terminus (e.g., missing a more N-terminal portion). That said, such truncated polypeptides, when present, can be minimized or eliminated by either post synthesis purification steps or by increasing synthesis times or other methods described briefly herein.
  • compositions of the disclosure comprising very high percentages of full length polypeptides (based on the template arrangement) are provided, such as compositions in which greater than 75%, greater than 80%, greater than 85%), greater than 90%, or even 95% or greater than 95% of the polypeptides in the complex mixture are full length.
  • full-length or substantially full- length polypeptides may be separated from the mixture.
  • high complexity mixtures of polypeptides may comprise truncated polypeptides based on a portion of the complete template arrangement, or may be enriched for a higher percentage of full-length or substantially full-length polypeptides based on the template arrangement.
  • longer coupling times or the addition of redundant coupling reactions may be used. Accordingly,
  • compositions comprising greater than 90% or even greater than 95% full-length polypeptide can be obtained.
  • any polypeptide synthesis method that allows synthesis incorporating more than one amino acid species at a controlled ratio in any given position of the
  • polypeptide sequence is suitable for use with this disclosure.
  • the polypeptides of a composition according to the disclosure may be peptidomimetics or include unnatural or modified amino acids, necessitating adaptation to allow addition of such chemical species to the polymers synthesized up to that point.
  • the synthesis may include unnatural amino acids, or amino acid analogs.
  • the polypeptides of the compositions of the disclosure are comprised of naturally occurring and synthetic derivatives, for example, selenocysteine.
  • Amino acids further include amino acid analogs.
  • An amino acid "analog" is a chemically related form of the amino acid having a different configuration, for example, an isomer, or a D-configuration rather than an L-configuration, or an organic molecule with the approximate size and shape of the amino acid, or an amino acid with modification to the atoms that are involved in the peptide bond, so as to be protease resistant when polymerized in a polypeptide.
  • the polypeptides of the compositions can be composed of stereoisomers (e.g., D-amino acids, Nle, Nva, Cha, Orn, Hie, Chg, Hch, or Har) of the twenty conventional amino acids, unnatural amino acids such as ⁇ -, ⁇ -di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for the polypeptides of the present disclosure.
  • stereoisomers e.g., D-amino acids, Nle, Nva, Cha, Orn, Hie, Chg, Hch, or Har
  • unnatural amino acids such as ⁇ -, ⁇ -di substituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be suitable components for the polypeptides of the present disclosure.
  • unconventional amino acids include (i.e., are not limited to): 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ -trimethyllysine, ⁇ -N- acetyllysine, O-phosphoserine, N-acetyl serine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, ⁇ - ⁇ -methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline).
  • the polypeptides include Norleucine (Nle) at the C-terminus.
  • the polypeptides include analogs of phosphorylated amino acids.
  • the analog is (a,a-difluoroalkyl)phosphonate analog of phosphoserine, phosphothreonine or allo-threonine.
  • the analog is (a,a-difluoromethylene) phosphonate mimic of phosphoserine (pCF 2 Ser).
  • the analog is p-aminobenzylphosphonate or p-aminobenzylphosphonate or derivates thereof.
  • polypeptides of the compositions according to the present disclosure can be composed of L- or D-amino acids or mixtures thereof.
  • L-amino acids occur in most natural proteins.
  • D-amino acids are commercially available and can be substituted for some or all of the amino acids used to make polypeptides of the present disclosure.
  • the present disclosure contemplates polypeptides containing both D- and L-amino acids, as well as polypeptides consisting essentially of either L- or D-amino acids.
  • the polypeptides of the present disclosure include such linear polypeptides that are further modified by substituting or appending different chemical moieties.
  • such modification is at a residue location and in an amount sufficient to inhibit proteolytic degradation of the polypeptides in a subject.
  • the amino acid modification may be the presence of at least one proline residue in the sequence; the residue is present in at least one of carboxy- and amino termini; further, the proline can be present within four residues of at least one of the carboxy- and amino-termini.
  • the amino acid modification may be the presence of a D-amino acid.
  • the subject polypeptides are peptidomimetics.
  • Peptidomimetics are compounds based on, or derived from, polypeptides and proteins.
  • the peptidomimetics of the present disclosure typically can be obtained by structural modification of one or more native amino acid residues, e.g., using one or more unnatural amino acids, conformational restraints, isosteric replacement, and the like.
  • the subject peptidomimetics constitute the continuum of structural space between peptides and non-peptide synthetic structures.
  • Such peptidomimetics can have such attributes as being non-hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the corresponding polypeptides), increased specificity and/or potency.
  • peptide analogs of the present disclosure can be generated using, for example,
  • benzodiazepines e.g., see Freidinger et al. in "Peptides: Chemistry and Biology," G.R.
  • the polypeptides of the composition are synthesized in a single manufacturing step.
  • different template regions of the polypeptide composition are synthesized separately and coupled by peptide ligation (e.g., an RCR and an AR can be synthesized separately and coupled together by peptide ligation).
  • one or more RCRs are synthesized separately and couple to a recombinant protein or antigen of interest or portion thereof to generate an amino acid copolymer of the composition.
  • the peptide ligation is Native Chemical Ligation or bis(2-sulfanylethyl)amino (SEA) Native Peptide Ligation.
  • the molecular weight of a polypeptide composition can be adjusted during polypeptide synthesis or after the polypeptides have been synthesized.
  • the synthetic conditions or the amounts of amino acids are adjusted so that synthesis stops when the polypeptide reaches the
  • polypeptides with the desired molecular weight can be obtained by any available size selection procedure, such as chromatography of the polypeptides on a molecular weight sizing column or gel, and collection of the molecular weight ranges desired.
  • the present polypeptides can also be partially hydrolyzed to remove high molecular weight species, for example, by acid or enzymatic hydrolysis, and then purified to remove the acid or enzymes.
  • polypeptides with a desired molecular weight may be prepared by a process which includes reacting a protected polypeptide with hydrobromic acid to form a trifluoroacetyl-polypeptide having the desired molecular weight profile.
  • the reaction is performed for a time and at a temperature, which is predetermined by one or more test reactions.
  • the time and temperature are varied and the molecular weight range of a given batch of test polypeptides is determined.
  • the test conditions which provide the optimal molecular weight range for that batch of polypeptides, are used for the batch.
  • a trifluoroacetyl-polypeptide having the desired molecular weight profile can be produced by a process which includes reacting the protected polypeptide with hydrobromic acid for a time and at a temperature predetermined by test reaction.
  • the trifluoroacetyl- polypeptide with the desired molecular weight profile is then further treated with an aqueous piperidine solution to form a low toxicity polypeptide having the desired molecular weight.
  • a test sample of protected polypeptide from a given batch is reacted with hydrobromic acid for about 10-50 hours at a temperature of about 20-28°C.
  • the best conditions for that batch are determined by running several test reactions.
  • the protected polypeptide is reacted with hydrobromic acid for about 17 hours at a temperature of about 26°C.
  • specific glycogenated forms of a polypeptide composition are generated.
  • the post-translational modification of a polypeptide composition is performed using glycogen synthase, or alternatively using chemical complexation techniques well known in the art.
  • specific glycosylated forms of a polypeptide composition are generated.
  • the glycosylation is selected from one or more of N-linked glycosylation, O-linked glycosylation, phospho-serine glycosylation, C-mannosylation, and glypiation.
  • the post-translational modification of a polypeptide is performed using glycosyl transferases, or alternatively using chemical complexation techniques well known in the art.
  • specific nitrated forms of a polypeptide composition are generated.
  • specific nitrosylated forms of a polypeptide composition are generated.
  • S-nitrosylation of a polypeptide composition are generated.
  • disulfide bridges between cysteine (disulfide bridge) residues of individual polypeptides are generated.
  • the post-translation formation of the cysteine bridge is performed using the reduction of a disulfide bridge between two cysteine residues.
  • reducing due to the presence of disulfide bond reductases e.g. thioredoxins and glutaredoxins are generated.
  • the net charge on a protein can be either positively (cation) or negatively (anion) charged depending on both the amino acid composition of the protein and the pH conditions.
  • the net charge of the protein may affect protein solubility, and in turn, the immunogenicity of the polypeptide composition. Accordingly, depending on the nature of the ARs, the RCRs can, in addition to their other specific immunogenic properties, be used to increase the estimated net charge of the composition as a whole.
  • the output of the polypeptide composition may vary from input due to several factors.
  • One of these factors is the synthesis of the polypeptide composition by solid phase peptide synthesis (SPSS).
  • SPSS can lead to two types of variation in the output of the polypeptide composition: (1) polypeptides with varied lengths due to incomplete polypeptide synthesis (which, as noted here, can be controlled) and (2) variation in the relative ratio of amino acid composition at each position due to a preferential incorporation of a particular amino acid (which can be evaluated based on output ratios, as described herein).
  • SPSS Solid Phase Peptide Synthesis
  • compositions of the disclosure comprising very high percentages of full length polypeptides (based on the template arrangement) are provided, such as compositions in which greater than 75%, greater than 80%, greater than 85%, greater than 90%, or even 95% or greater than 95% of the polypeptides in the complex mixture are full length.
  • the variations in polypeptide length may be evaluated using a myriad of techniques.
  • MALDI-TOF-MS analysis may be used to evaluate the percentage of full-length proteins found within the copolymer composition.
  • MALDI-TOF-MS may be used to determine the mass/charge (m/z) value of various copolymers within the copolymer composition.
  • F template are about 63 amino acids in length and have a mass of about 6900 Daltons. The peaks which occur prior to the 6900 Dalton peak are due to synthesis of polypeptides based on this template arrangement but which are shorter due to incomplete protein synthesis.
  • the relative ratio between the masses of polypeptides in the composition having a substantially full-length polypeptide sequence and the masses of the polypeptides having masses indicative of a less than full-length sequence it may be determined at between which positions peptide synthesis was disrupted.
  • the resulting composition synthesized had a purity of about 53% (e.g., calculated at about 52.61%).
  • Purity in this case, refers to the percentage of polypeptides in the composition based on the template arrangement that are full-length or substantially full-length
  • polypeptides are full-length or substantially full-length polypeptides.
  • the purity may be increased by separating the substantially full-length polypeptides from the copolymer mixture. This may be achieved by a multitude of protein purification methods based on size, which may include but is not limited to techniques such as SDS-polyacrylamide gel electrophoresis, liquid chromatography, or gel filtration chromatography. Furthermore, the substantially full-length polypeptide purity may be increased by modifying the synthesis protocol based on the copolymer template and incomplete polypeptide synthesis information gathered from MALDI-TOF-MS analysis.
  • Example 12 and Figure 17 MALDI-TOF analysis was used to determine that incomplete synthesis was occurring between positions corresponding to positions 16-17 and positions 24-25 of the DP-CO 16.F polypeptide.
  • modifications to the synthesis protocol such as the strategic addition of coupling reactions at particular positions to improve the coupling efficiency and generate a higher yield in each step, longer coupling times or the addition of redundant coupling reactions may be used.
  • synthesis of polypeptides by SPSS may result in a preferential incorporation of alanine (A) over lysine (K) in the polypeptide.
  • A alanine
  • K lysine
  • the A/K input ratio and A/K output ratio may be determined by using a combination of the information gained from the MALID-TOF-MS analysis and amino acid analysis (AAA).
  • Amino acid analysis is a technique, which can be used to determine the amino acid composition of each individual amino acid in a polypeptide. Amino acid analysis can take advantage of an internal standard to which the individual amino acid may be compared to determine the molar concentration of each amino acid.
  • Norleucine may provide an internal standard for amino acid analaysis. As discussed above, solid phase peptide synthesis synthesizes each polypeptide in a C-terminus to N-terminus direction.
  • Norleucine may be covalently coupled to the resin support and thus incorporated into the C-terminal most position of each polypeptide. Considering that SPSS proceeds in a C-terminus to N-terminus direction, and Norleucine will be in the C-terminal most position, it can be assumed that one Norleucine will be incorporated into each polypeptide. Thus, Norleucine may be used as an internal standard to determine the composition of the individual polypeptides. The output analysis of amino acid that may be obtained from a copolymer composition is shown in the Table below.
  • DP-CO 16.B represents analysis of one preparation of the DP-CO 16.
  • DP-CO 16.F represents analysis of one preparation of the DP-CO 16.
  • Glx Due to the hydrolysis solution used during amino acid analysis, the concentrations of glutamine and glutamic acid may not be differentiated and is indicated as Glx.
  • concentrations of asparagine and aspartic acid may not be differentiated and is indicated as Asx.
  • lysine may be counter-selected against and alanine may be preferentially selected during some SPSS coupling reactions. This results in a variation of the A/K input ratio and the A/K output ratio across the copolymer compositions.
  • the method is drawn to increasing the diversity of antibodies generated to react with a ligand. Further, the method is drawn to overcoming the problem of creating antibodies against ligands with low immunogenicity. Still further, the method is drawn to overcoming problems relating to generating antibodies having reactivities to only a single species.
  • the instant disclosure comprises a method of creating antibody reagents for use in research studies.
  • the instant disclosure comprises a method of creating antibody reagents for use as diagnostic tools.
  • the instant disclosure further comprises a method for the generation of antibodies useful as therapeutic agents for the treatment of disease.
  • antibodies may be produced in vivo, i.e., the compositions for stimulating antibody production may be used as vaccines. Immunization steps of all the representative methods described below can be modified for in vivo use of the immunogens of the present disclosure as vaccines.
  • Antibodies are produced by designing and synthesizing the amino acid copolymer compositions as described above, creating antibodies by introducing the compositions into an in vivo setting, or alternatively introducing the compositions into an in vitro setting, or still alternatively contacting the composition with a system of maintaining the connection between antibody phenotype and genotype such as phage display, determining the activity of the generated antibodies by contacting the antibodies with the native molecule of interest, selecting antibodies having desired activity, such activity being either of a higher affinity antibody, or alternatively a lower affinity antibody, a single species reactivity, or alternatively a multi-species reactivity, a single-molecule of interest reactivity or alternatively a multi-molecule reactivity.
  • the instant disclosure also comprises a process for producing antibodies that are therapeutically or prophylactically useful in the treatment of various diseases or conditions (e.g. protein conformational disorders or pathogenic infections), or useful for use as research reagents, and as diagnostic tools for such diseases or conditions, by eliciting immune responses using a composition comprising directed epitope polypeptide mixtures.
  • various diseases or conditions e.g. protein conformational disorders or pathogenic infections
  • diagnostic tools for such diseases or conditions e.g. protein conformational disorders or pathogenic infections
  • composition comprising directed epitope polypeptide mixtures.
  • composition comprising antibodies thus produced.
  • the method of the instant disclosure also encompasses an augmentation of the paratopes associated with an antibody response to an antigen of interest.
  • the method of the instant disclosure further encompasses the generation of novel functioning antibodies having antigen binding properties that elicit a varied amount of downstream consequences to the binding event.
  • the method comprises the steps of selecting a protein relevant to the disease or condition of interest (e.g. a protein conformational disorder, a pathogenic infection, etc.), determining relevant epitopes within the protein known or suspected to be closely associated with the disease or condition, selecting the relevant antigenic region(s), selection the number and/or arrangement of one or more random copolymer regions, designing directed permutations of the antigenic regions and random copolymer regions so as to create an expanded yet related series of polypeptides, performing solid phase synthesis thus creating a high-complexity polypeptide mixture, using the polypeptide mixture collectively as a set of antigens by placing the mixture in contact with a means of antibody generation, determining the activity of the generated antibodies, selecting antibodies having the desired activity, and utilizing the antibody as a single species reagent, multi-species reagent, single species diagnostic, multi-species diagnostic, or alternatively as a therapeutic.
  • a protein relevant to the disease or condition of interest e.g. a protein conformational disorder,
  • the means of antibody generation is, for example, an animal to be immunized by the polypeptide mixture and cells from such an animal (e.g. spleen cells from a mouse for monoclonal antibody production), a phage display library, or a B cell library.
  • the instant disclosure encompasses methods of producing antibodies, the methods comprising: selecting the relevant antigenic region(s), selection the number and/or arrangement of one or more random copolymer regions, designing directed permutations of the antigenic regions and random copolymer regions so as to create an expanded yet related series of polypeptides, performing solid phase synthesis thus creating a high-complexity polypeptide mixture, preparing the polypeptide mixture as a
  • another aspect of the present disclosure is a composition comprising antibodies generated against an amino acid copolymer composition as described above, wherein the base peptide sequence of at least one antigenic region is derived from a sequence of a protein known to be associated with a disease or condition (e.g. protein conformation disorder, pathogenic infection, etc.).
  • the base peptide sequence is derived from a protein known to be associated with a protein conformational disorder. More particularly, such protein is known to form an aggregate or fibril.
  • antibodies thus generated are specific to the pathological conformation of such protein.
  • the base peptide sequence is derived from a protein known to be associated with a pathogenic infection.
  • the antibodies are modified antibodies having an engineered Fc region, wherein the engineered Fc region confers favorable pharmacodynamic profiles.
  • the Fc region enhances clearance of antibody-antigen complex.
  • the Fc region is not immunogenic to the subject.
  • modified antibodies may be created after antibodies with certain desired (complementarity determining regions) are identified, by replacing chemically or by molecular biological means the Fc region with an IgA, IgG, IgE, IgM, or IgD region.
  • the antibodies are humanized antibodies having desired CDRs (complementarity determining regions), such CDRs having been identified using the compositions of the disclosure or antibodies having such CDRs having been generated using the compositions of the disclosure.
  • Humanized antibodies may be made according to any means known in the art, including CDR grafting and the introduction of point mutations to reduce immunogenicity.
  • the antibodies are single chain variable fragment (scFv), either engineered from an identified antibody, or generated using a phage display library and other means and screened for desired antibodies using the compositions of the disclosure. Methods of scFv production and phage display are known in the art.
  • the antibodies may also have a detectable label, such as a radiolabel, an enzymatic label, or a fluorescent label.
  • the fluorescent label is selected from the group consisting of Texas Red, phycoerythrin (PE), cytochrome c, and fluorescent isothiocyanate (FITC).
  • labels such as biotin followed by streptavidin-alkaline phosphatase (AP), horseradish peroxidase (HRP) are contemplated.
  • AP streptavidin-alkaline phosphatase
  • HRP horseradish peroxidase
  • This disclosure also provides antibodies with at least 70%, 80%, 90%, 95%, or 99% amino acid sequence identity to the antibodies described above.
  • Antibodies in general have well characterized structure-activity relationships, and one of skill in the art would be well aware that certain mutations would be unlikely to disrupt the antigen-binding function of an antibody. For example, conservative substitutions in the constant region would be unlikely to disrupt antigen binding, while substitutions in the CDRs would be more likely to disrupt antigen binding.
  • An aspect of the disclosure is a composition
  • a composition comprising a scaffold or support material to which antibodies are attached, which antibodies are generated against an amino acid copolymer composition as described above, wherein the base peptide sequence is derived from a sequence of a protein known to be associated with a protein conformational disorder.
  • the scaffold is a membrane compatible with haemodialysis. Membranes for haemodialysis are typically semi-permeable, allowing for water and some dissolved solutes to pass through. The membranes can have different pore sizes and are thus categorized as low-flux or high-flux.
  • Membranes can be made from a variety of materials, including cellulose acetate, polyarylethersulfone, polyamide, polyvinylpyrrolidone, polycarbonate, and polyacrylonitrile.
  • the antibodies are conjugated to such membrane. This will allow for removal of specified proteins at while haemodialysis is carried out. This process is useful, inter alia, for treating removing amyloid forms of B2M and treating DRA.
  • the antibodies are conjugated to a resin, such as CN-Br agarose resin (for example CN-Br Sepharose® (Pharmacia), to create an immunoaffinity resin.
  • compositions of the disclosure also referred to as amino acid copolymer compositions of the disclosure.
  • Such compositions comprise a plurality of polypeptides having the same template arrangement and comprising one or more RCRs and one or more ARs. Sequence complexity is contributed by the RCRs and/or ARs, such that the composition comprises a mixture of polypeptides that are closely related by sequence.
  • compositions of the disclosure may be administered in vitro or in vivo. Any of the compositions of the disclosure, described based on any combination of structural and/or functional features provided herein, may be used in any of the methods described herein. Compositions of the disclosure may be administered in vitro or in vivo alone, or in combination with one or more other agents. In certain embodiments, the one or more other agent is an adjuvant. Although in other embodiments, an additional adjuvant is not required (e.g., the method does not include use of an adjuvant other than a composition of the disclosure).
  • compositions of the disclosure are suitable for use as an immunotherapy.
  • the compositions of the disclosure are useful in the treatment of a disease or condition that would benefit from induction of an antigen-specific immune response (e.g. protein conformational disorders, pathogenic infections, etc.) and/or from stimulation of the immune system.
  • an antigen-specific immune response e.g. protein conformational disorders, pathogenic infections, etc.
  • the amino acid copolymer compositions of the disclosure can be any suitable for use as an immunotherapy.
  • the amino acid copolymer composition generates a Th2 immune posture.
  • Dynamic administration of amino acid copolymer compositions of the disclosure is comprised of any combination of dose, regimen, route of administration, and/or formulation. This dynamic immunomodulation provides for increased effectiveness at any of the multiple stages of a disease within a particular patient, as well as the ability to treat multiple, pathogenic antigenic-determinant unrelated diseases more effectively.
  • the disclosure provides methods for the treatment or prevention of a disease in a subject, preferably in a human, which subject is afflicted with or is suspected to be afflicted with the disease.
  • One embodiment of the present disclosure is a method for prophylactically treating a subject at risk of developing a pathogenic infection by administering an amino acid copolymer composition of the disclosure.
  • One embodiment of the present disclosure is a method for prophylactically treating a subject at risk of developing a protein conformational disorder by administering an amino acid copolymer composition of the disclosure.
  • One embodiment of the present disclosure is a method for therapeutically treating a subject at risk of developing a protein conformational disorder by administering an amino acid copolymer composition.
  • a subject at risk is identified by, for example, determining the genetic susceptibility to a protein conformational disorder by testing for alleles of HLA that are associated with such disorder, and/or based on familial history, or other genetic markers that correlate with such disorder.
  • many patients receiving dialysis or other form of blood filtration are at risk for developing dialysis related amyloidosis (DRA), especially if the blood filtration is performed over a long period of time, such as more than 3, 5, 7, or 10 years.
  • DUA dialysis related amyloidosis
  • subjects that are asymptomatic but show biochemical markers of a protein conformational disorder are at risk of developing such disorder.
  • One aspect of the disclosure provides methods of treating or preventing a disease, the method comprising administering to said subject a dosing regimen of an effective amount of an amino acid copolymer composition of the disclosure for the amelioration of a disease treatable with the amino acid copolymer composition, said effective amount delivered to said subject at time intervals greater than 24 hours, 36 hours, or more preferably greater than 48 hours.
  • a related aspect of the disclosure provides a method for the treatment of a subject in need thereof, comprising administering to said subject a dosing regimen of an effective amount of an amino acid copolymer composition of the disclosure for the amelioration of a disease treatable with the amino acid copolymer composition, said effective amount delivered to the subject using a sustained-release formulation which administers the amino acid copolymer composition over a period of at least 2 days, at least 4 days, or at least 6 days, wherein the effective amount is an amount that is effective if delivered daily.
  • an amino acid copolymer composition of the disclosure is administered encapsulated in poly(lactide) microparticles, poly(lactide-co-glycolide) microparticles, liposomes, archaeosome adjuvants, mucosal adjuvants, polyphosphazenes.
  • an amino acid copolymer composition of the disclosure is encapsulated in poly(lactide) microparticles.
  • Compositions of the disclosure may be administered with an adjuvant - either co-formulated or administered in separate formulations at the same or different time.
  • an amino acid copolymer composition of the disclosure is formulated and/or administered with alum as an adjuvant.
  • an amino acid copolymer composition of the disclosure is formulated and/or administered with aluminum hydroxide or aluminum phosphate.
  • One aspect of the disclosure is the administration of an amino acid copolymer composition of the disclosure to a subject in need there of, as described above, in
  • the additional therapeutically active agents may treat the same or related disease as the amino acid copolymer composition, or may be intended to treat an undesirable side effect of administration of the amino acid copolymer composition, such as to reduce swelling at a site of intradermal injection.
  • the other therapeutic agents enhance the activity of the amino acid copolymer compositions.
  • Such additional therapeutic agents are, by way of example, antibodies, cytokines, growth factors, enzyme inhibitors, antibiotics, antiviral agents, anti-inflammatory including steroids, immune boosters, antimetabolites, soluble cytokine receptors, and vitamin D or agents that increase the level of circulating vitamin D, toll-like receptor agonists, CpG oligodeoxynucleotides, surface charged poly(lactide-co-glycolide) microparticles, poly(lactide) microparticles, any of the above encapsulated into liposomes, archaeosome adjuvants, mucosal adjuvants, polyphosphazenes.
  • Additional therapeutically active agents also include copolymers, which bind to a HLA molecule associated with the disease such as another amino acid copolymer composition of the disclosure.
  • the HLA molecule may be an HLA-DQ molecule, an HLA- DP molecule or an HLA-DR molecule.
  • the enzyme inhibitor may be a protease inhibitor or a cyclooxygenase inhibitor. Examples of the therapeutically active agents to be administered in conjunction with the amino acid copolymer composition are recited in Section IV, "Pharmaceutical Composition" section, though the administration of these agents are not limited to co-administration as a single composition.
  • the additional therapeutic agents may be administered before, concomitantly with, or after the administration of the amino acid copolymer composition, at such time that the effect of the additional therapeutic agents and the effect of the amino acid copolymer composition overlap at some time point.
  • antigen/epitope non-specific treatments and therapies directly targeted at controlling T lymphocytes or their functions may be administered in conjunction with an amino acid copolymer composition of the disclosure.
  • the therapeutic agents useful for such treatment include Muromonab-CD3 (OKT3), antilymphocyte globulin (ALG), antithymocyte globulin (ATG), or interleukin-2 receptor monoclonal antibody ("mAb") daclizumab or basiliximab.
  • Other agents include soluble CTLA-4, an anti-CD154 mAb; anti-CDl la; a humanized mAb which inhibits VLA-4; anti-CD2, 3, or 4 antibodies; and anti-CD 152 antibodies (J.B. Matthews et al., Amer. J. Transplantation, 2003, 3 : 794-80).
  • AD Alzheimer's disease
  • FTDP-17 parkinsonism linked to chromosome 17
  • ALS progressive supranuclear palsy
  • PD PD
  • amino acid copolymer therapeutic or amino acid copolymer-specific antibody therapeutic in combination with one or more additional therapy.
  • the route of administration can be oral, intraperitoneal, intradermal, transdermal, subcutaneous, by intravenous or intramuscular injection, by inhalation, topical, intralesional, or by infusion; liposome- mediated delivery; intrathecal, gingival pocket, rectal, intravaginal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art as one skilled in the art may easily perceive.
  • Administration can be systemic or local. In the event more than one amino acid copolymer composition of the disclosure is being administered to a subject during the same or overlapping time period, such additional therapeutic agent may be administered by the same or a route different rote of administration.
  • the composition is formulated for any of the foregoing routes of administration, such as intradermal, transdermal, intramuscular or subcutaneous.
  • an embodiment of the disclosure is to administer a suitable dose of a therapeutic amino acid copolymer composition of the disclosure that will be the lowest effective dose to produce a therapeutic effect, for example, mitigating symptoms.
  • the therapeutic amino acid copolymer compositions are preferably administered at a dose per subject, which corresponds to a dose per month of at least about 0.5 mg, at least about 1 mg, at least about 2 mg, at least about 5 mg, at least about 10 mg, or at least about 20 mg as appropriate minimal starting dosages, or about x mg, wherein x is an integer between 0.5 and 20.
  • the compositions are administered once a month, twice a month, three times a month, once a week, or once daily.
  • the compositions are administered once a month. In one embodiment of the methods described herein, a dose of about 0.01 to about 10 mg/kg can be administered. In some embodiments, the effective dosage of the amino acid copolymer composition of the present disclosure is about 50 to about 400 micrograms of the composition per kilogram of the subject per day. In some embodiments, each individual dosage in the treatment regimen is from about 0.5 to 20, or more preferably from about 1 to lOmg/dose. In some embodiments, the preferred dosing regimen is 1-10 mg once a month. In some embodiments, the preferred dosing regimen is 1-5 mg, 2-6 mg, 3-7 mg, 4-8 mg, or 5-10 mg once a month.
  • the preferred dosing regiment is 0.5-5 mg twice a month. In some embodiments, the preferred dosing regimen is 0.25-2.5 mg once a week. In some embodiments, the preferred dosing regimen is 0.5-5 mg twice a month. In some embodiments, the preferred dosing regimen is 0.03-0.3 mg daily.
  • the dose of the amino acid copolymer composition of the disclosure will vary depending on the subject, the condition being treated and upon the particular route of administration used. It is routine in the art to adjust the dosage to suit the individual subjects. Additionally, the effective amount may be based upon, among other things, the size of the polypeptides or amino acid copolymers, the biodegradability of the polypeptides or amino acid copolymers, the bioactivity of the polypeptides or amino acid copolymers and the bioavailability of the polypeptides or amino acid copolymers. If the amino acid copolymer compositions do not degrade quickly, such as is expected when the compositions comprise unnatural amino acids or comprise
  • peptidomimetics is bioavailable and highly active, a smaller amount will be required to be effective.
  • the actual dosage suitable for a subject can easily be determined as a routine practice by one skilled in the art, for example a physician or a veterinarian given a general starting point.
  • the physician or veterinarian could start doses of an amino acid copolymer composition of the disclosure employed in the pharmaceutical composition at a level lower than that required in order to achieve the desired therapeutic effect, and increase the dosage with time until the desired effect is achieved.
  • the dosage of the amino acid copolymer composition may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disorder or disease state is observed, or if the disorder or disease state has been ablated, or if an unacceptable side effects are seen with the starting dosage.
  • a therapeutically effective amount of an amino acid copolymer composition of the disclosure is administered to the subject in a treatment regimen comprising intervals administering more than one dose at intervals over some period of time.
  • Treatment regimens with longer dosing intervals (time between administrations), consequently often with lower total exposure of polypeptides or amino acid copolymers, are expected to induce lower titers of antibodies against the polypeptides or amino acid copolymers themselves, while still inducing desired protective effects.
  • Such reduction of neutralizing antibodies are desirable because it is considered likely to help the amino acid copolymer compositions of the disclosure to retain their effectiveness without being neutralized, and it is associated with reduced risk of anaphylactic shocks, providing safer treatments of diseases.
  • an amino acid copolymer composition of the disclosure is administered to be subject at least three times during a treatment regimen, such that there are at least two time intervals between administrations.
  • any of the methods of the disclosure may be practiced using a sustained release formulation comprising an amino acid copolymer composition of the disclosure.
  • a sustained release formulation comprising an amino acid copolymer composition of the disclosure.
  • such method comprises applying a sustained-release transdermal patch or implanting a sustained-release capsule or a coated implantable medical device so that a therapeutically effective dose of the copolymer of the present disclosure is delivered at defined time intervals to a subject of such a method.
  • the amino acid copolymer composition of the subject disclosure may be delivered via a capsule, which allows regulated-release of the compositions over a period of time.
  • Controlled or sustained-release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
  • particulate compositions coated with polymers e.g., poloxamers or poloxamines.
  • a source of an amino acid copolymer composition is stereotactically provided within or proximate to the area where pathology is observed or suspected.
  • An improvement in the symptoms of a subject afflicted with a disease as a result of administration of the amino acid copolymer composition may be noted by a decrease in frequency of recurrences of episodes of the disease symptoms, by decrease in severity of symptoms, and by elimination of recurrent episodes for a period of time after the start of administration.
  • a therapeutically effective dosage preferably reduces symptoms and frequency of recurrences by at least about 20%, for example, by at least about 40%, by at least about 60%, and by at least about 80%, or by about 100% elimination of one or more symptoms, or elimination of recurrences of the autoimmune disease, relative to untreated subjects.
  • the period of time can be at least about one month, at least about six months, or at least about one year.
  • An aspect of the present disclosure is a method of treating a subject afflicted with a disease or condition that would benefit for an antigen-specific immune response (e.g. protein conformational disorders, pathogenic infections, etc.), comprising the steps of administering an antibody prepared using an amino acid copolymer composition of the disclosure.
  • an antigen-specific immune response e.g. protein conformational disorders, pathogenic infections, etc.
  • One aspect of the present disclosure is a method of treatment using antibodies against a composition of the disclosure related to a disease, in particular, a protein conformational disease.
  • the antibodies useful for such method of treatment are antibodies generated against an amino acid copolymer composition of the disclosure, wherein the base peptide sequence is a sequence of a protein known to be associated with a protein conformational disorder. More particularly, such protein is known to form an aggregate or fibril. In particular, antibodies thus generated are specific to the pathological conformation of such protein.
  • the list of relevant diseases is recited above in this specification.
  • One aspect of the present disclosure is a method of treatment using antibodies against a composition of the disclosure related to a disease, in particular, a pathogenic infection.
  • the antibodies useful for such method of treatment are antibodies generated against an amino acid copolymer composition of the disclosure, wherein the base peptide sequence is a sequence of a protein known to be associated with a pathogenic infection.
  • the pathogen is an intracellular pathogen.
  • antibodies thus generated are specific to the pathogenic protein.
  • the antibodies for the use in the method of treatment are modified antibodies having an engineered Fc region, wherein the engineered Fc region confers favorable pharmacodynamic profiles.
  • the Fc region enhances clearance of antibody-antigen complex.
  • the Fc region is not immunogenic to the subject.
  • An aspect of the present disclosure is a method of treating a subject afflicted with a protein conformational disorder, comprising the steps of administering an antibody prepared using an amino acid copolymer composition of the disclosure.
  • the protein conformational disorder is Parkinson's disease.
  • the protein conformational disorder is ALS.
  • the protein conformational disorder is Alzheimer's disease.
  • An aspect of the present disclosure is a method of treating a subject afflicted with a pathogenic infection, comprising the steps of administering an antibody prepared using an amino acid copolymer composition as described above.
  • the pathogen is a bacterium or a virus.
  • the pathogen is of the genus selected from Chlamydia, Rickettsia, Coxiella, Mycobacterium, Francisella, Listeria, Salmonella, Brucella, Legionella, Nocardia, Rhodococcus, Yersinia, and Neisseria.
  • an antibody or antibodies identified by the method to generate antibodies against antigens associated with a disease or condition is cloned.
  • the nucleic acids encoding such antibodies are cloned into an appropriate expression vector and delivered to a cellular site where such antibodies are desirable, at which site the antibodies are expressed.
  • Another aspect of the present disclosure is a method of treating a subject afflicted with a disease or condition, comprising the steps of contacting under sterile conditions the blood of the subject to a membrane or a resin having conjugated with antibodies specific to a protein associated with the disease or condition and prepared using an amino acid copolymer composition of the disclosure, such antibodies described above, wherein the protein associated with the disease or condition binds to such antibodies and is removed from the blood, and returning the blood to the subject.
  • the disease or condition is a protein conformational disorder.
  • the disease or condition is a pathogenic infection.
  • An embodiment of the disclosure is a method of prophylactic treatment of a subject at risk for developing a disease or condition by contacting under sterile conditions the blood of the subject to a membrane or a resin having conjugated with antibodies specific to a protein associated with the disease or condition and prepared using an amino acid copolymer composition of the disclosure, such antibodies described above, wherein the protein associated with the disease or condition binds to such antibodies and is removed from the blood, and returning the blood to the subject, whereby preventing the onset of such disease or condition.
  • the disease or condition is a protein conformational disorder.
  • the disease or condition is a pathogenic infection.
  • An aspect of the disclosure is a composition comprising a scaffold to which antibodies are attached, which antibodies are generated against an amino acid copolymer composition of the disclosure, wherein the base peptide sequence is derived from a sequence of a protein known to be associated with a disease or condition.
  • the base peptide sequence is derived from a sequence of a protein known to be associated with a protein conformational disorder.
  • the base peptide sequence is derived from a sequence of a protein known to be associated with a pathogenic infection.
  • the scaffold is a membrane compatible with haemodialysis.
  • the antibodies are conjugated to such membrane.
  • the antibodies are conjugated to a resin, such as CN-Br agarose resin (for example CN-Br Sepharose® (Pharmacia), to create an immunoaffinity resin.
  • an aspect of the disclosure is a composition comprising antibodies against a composition of the disclosure related to a disease, in particular, a protein conformational disease, or a target (e.g., an antigen).
  • an antibody of the disclosure is an antibody, or combination of antibodies, that binds to at least one protein associated with a protein conformational disorder.
  • an antibody of the disclosure is an antibody, or combination of antibodies, that binds to at least one protein associated with Parkinson's disease.
  • an antibody of the disclosure is an antibody, or combination of antibodies, that binds to at least one protein associated with ALS.
  • an antibody of the disclosure is an antibody, or combination of antibodies, that binds to at least one protein associated with Alzheimer's disease.
  • compositions of the disclosure are used to raise, induce or generate antibodies, such as by administering to a human or non human subject.
  • a composition of the disclosure can be used to isolate antibodies.
  • a composition of the disclosure may be affixed to a column or used in another screening system to isolate antibodies, such as antibodies generated following administration of that same composition to a subject.
  • compositions of the disclosure have numerous other uses. For example,
  • compositions of the disclosure are useful for generating antibodies, such as for use as reagents, and the instant disclosure also comprises a method of generating antibody reagents for use in, for example, research studies in vitro or in vivo.
  • compositions of the disclosure can be used to immunize a host, such as a mouse or rabbit, and serum from the animal is harvested.
  • monoclonal antibodies may be generated using known techniques following challenge with a composition of the disclosure.
  • Certain antibodies generated or selected by their specific binding to an amino acid copolymer composition of the disclosure are useful to identify specific conformation of a protein in its pathological and non-pathological state. Such antibodies, when conjugated to scaffolds, are further useful for isolating and purifying the target proteins and polypeptides.
  • Such antibodies are also useful in preclinical investigations of candidate pharmaceutical agents, wherein such agent may disrupt or disturb the binding of such antibodies to the target proteins.
  • the antibodies can also be used to detect certain pathological antibodies and to measure the effects of such candidate pharmaceutical agents.
  • Compositions of the disclosure may also be used to isolate antibodies of interest.
  • the instant disclosure also comprises a method of creating antibody reagents for use as diagnostic tools.
  • An embodiment of the disclosure provides, a method of diagnosing a disease or condition, comprising: (i) contacting a biological sample from a subject with an antibody of the disclosure (e.g., a monoclonal or polyclonal antibody raised by immunizing a host with a composition of the disclosure); (ii) contacting a control sample with the antibody; and (iii) measuring specific binding of the antibody to an antigen in the sample; wherein specific binding of the antibodies to the antigen is indicative of the subject being afflicted with the disorder.
  • an antibody of the disclosure e.g., a monoclonal or polyclonal antibody raised by immunizing a host with a composition of the disclosure
  • a control sample e.g., a control sample with the antibody
  • measuring specific binding of the antibody to an antigen in the sample e.g., a control sample with the antibody
  • a number of methods for measuring antibody-protein binding are known in the art, including ELISA, Western blotting, and spot-blot
  • the control sample may be a standard sample, a sample from a second subject known to be free of the pathology that is being investigated, or a sample from the same subject at a different time point to determine the chronological changes of the disease conditions.
  • tests are performed simultaneously using the antibody of the disclosure and a positive control antibody that confirms that the biological sample contains sufficient material.
  • the positive control antibody may recognize any protein that is reasonably expected to be present in all samples (i.e. from both healthy and diseased patients), and may recognize a housekeeping enzyme (for example).
  • the binding of the antibody of the disclosure is quantified; in other embodiments, the binding is evaluated qualitatively.
  • the disease or condition is a protein conformational disorder.
  • the disease or condition is a pathogenic infection.
  • such disorder to be detected is one of the disorders enumerated elsewhere in this application.
  • the diagnostic test may be performed in vivo, identifying the affected locations within the body.
  • the antibody may be labeled in such a manner that it can be detected within a patient's body, e.g. with MRI.
  • This label may be an iron-containing compound, such as ferrous and ferric-containing compounds, e.g. ferric-oxides. Specific examples include Fe 2 0 3 and Fe 3 C"4.
  • Antibodies labeled with iron-containing compounds may also be used for in vitro diagnosis, e.g. when MRI is performed on a biological sample.
  • antibodies generated using compositions of the disclosure can be used for imaging analysis, such as to examine biodistribution of a target in vitro or in vivo.
  • compositions of the disclosure are useful for eliciting the release of a Th2-associated cytokine or chemokine from cells of the immune system in vitro or in vivo.
  • the immune cells are monocytes.
  • the Th2 associated cytokine is IL-4, IL-5, IL-6, IL-10, or IL-13.
  • the Th2 associated chemokine is CCL17 or CCL22.
  • the compositions of the disclosure are useful for eliciting the release of CCL22 from immune cells. Indeed, exemplary compositions described herein (e.g. DP-C016.B, DP-C016.C, etc.) have been shown to induce CCL22 release from monocytic cells. Accordingly, the disclosure provides such methods.
  • compositions of the disclosure are useful for eliciting preferential proliferation of CD4+ T-cells T in vitro or in vivo.
  • the compositions of the disclosure elicit preferential CD4+ T-cell proliferation amongst peripheral blood monocytes contacted with the compositions.
  • the compositions of the disclosure elicit preferential CD4+ T-cell proliferation amongst splenocytes contacted with the compositions.
  • exemplary compositions described herein e.g. DP-CO 16.B, DP- C016.C, etc.
  • the disclosure provides such methods.
  • compositions of the disclosure may be used to study a disease or condition.
  • compositions of the disclosure may be used to identify other proteins involved in a disease or condition.
  • Compositions of the disclosure may be used to evaluate disease pathology and progression in cells or animal models of the condition.
  • Compositions of the disclosure may be used to generate reagents suitable for further studying a disease or condition in vitro or in vivo.
  • Compositions of the disclosure may be used to induce/promote CD4+ T-cell proliferation in vitro or in vivo, such as in animal models of disease.
  • compositions of the disclosure can be administered alone or with an adjuvant or other agent - and including when provided as a pharmaceutical composition
  • compositions of the disclosure e.g., various formulations, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem.
  • Methods of introduction can be enteral or parenteral, including but not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous,
  • parenteral introduction includes intramuscular, subcutaneous, intravenous, intravascular, and intrapericardial administration. In certain embodiments, introduction is intracranially or intraventricularly or into the cerebrospinal fluid.
  • the present disclosure provides systemic delivery of one or more doses of
  • composition of the disclosure includes, for example, subcutaneous, intravenous, or intramuscular. In certain embodiments, delivery comprises subcutaneous delivery.
  • administration may comprise contacting a culture of cells with a composition of the disclosure, e.g., by adding composition to the culture media.
  • compositions and route of administration is chosen depending on the particular use of the technology. For example, a different composition and/or route of administration may be appropriate when using the compositions of the disclosure for research purposes, such as in vitro or in an animal model, versus when using for diagnostic or therapeutic purposes in human patients or animal subjects. Similarly, the route of administration may vary
  • the target e.g., the antigen upon which the ARs are based.
  • One of skill in the art can select the appropriate route of administration depending on the particular application of the technology.
  • compositions of the disclosure are useful in the treatment of a disease or condition amenable to treatment with an immunotherapy (e.g., where it would be beneficial to both increase the immune response, preferably T-cell proliferation and/or to generate an antigen-specific immune response.
  • Suitable diseases or conditions include, for example, protein conformational disorders, pathogenic infections, other conditions amenable to treatment via immunotherapy, etc.).
  • compositions of the disclosure are suitable for use in animal models of disease and/or to cells in culture.
  • compositions of the disclosure are provided as pharmaceutical compositions.
  • compositions of the disclosure may be formulated as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and/or excipients.
  • compositions and pharmaceutical compositions of the disclosure may be formulated for administration in any convenient way for, for example, use in human or veterinary medicine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of the compositions of the disclosure include those suitable for oral, nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration.
  • the amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.
  • methods of preparing these formulations or compositions include combining the therapeutic agent and a carrier and, optionally, one or more accessory ingredients.
  • the formulations can be prepared with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for parenteral administration may comprise one or more compositions of the disclosure in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents, which delay absorption, such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars,
  • compositions of the disclosure are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to human beings.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • compositions of the disclosure for use in the methods of the present disclosure can be determined by standard clinical techniques and may vary depending on the particular indication or use. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • compositions of the disclosure are sterile and/or non-pyrogenic.
  • the compositions are sterile and/or substantially pyrogen free.
  • the formulations of the disclosure are pyrogen-free formulations, which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever-inducing, thermostable substances
  • FDA Food & Drug Administration
  • EU endotoxin units
  • the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
  • compositions and methods described herein specifically contemplates any combination of the features of compositions of the present disclosure (alone or in combination) with the features described for the various pharmaceutical compositions and route of administration described in this section.
  • Exemplary pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible.
  • the carrier is suitable for oral, rectal, transmucosal (including by inhalation), parenteral, intravenous, intramuscular, intraperitoneal, intradermal, transdermal, topical, or subcutaneous administration.
  • One exemplary pharmaceutically acceptable carrier is physiological saline.
  • compositions of the present disclosure are well-known and generally described in, for example, Remington 's Pharmaceutical Science (18 th Ed., ed. Gennaro, Mack Publishing Co., Easton, PA, 1990).
  • Various pharmaceutically acceptable excipients are well- known in the art and can be found in, for example, Handbook of Pharmaceutical Excipients (4 th ed., Ed. Rowe et al. Pharmaceutical Press, Washington, D.C.).
  • the composition can be formulated as a solution, microemulsion, liposome, capsule, tablet, or other suitable forms.
  • the active component, which comprises the copolymer may be coated in a material to protect it from inactivation by the environment prior to reaching the target site of action.
  • the pharmaceutical compositions of the present disclosure are preferably sterile and non- pyrogenic at the time of delivery, and are preferably stable under the conditions of
  • the composition further comprises components to enhance stability, permeability, and/or bioavailability, such as particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.
  • the pharmaceutical compositions also include additional therapeutically active agents.
  • additional ingredient can be one or more of: an additional amino acid copolymer composition that binds to a different target, an additional amino acid copolymer composition where the ARs are derived from a different portion of the same target, an antibody which activates inflammatory molecules, or cytokines.
  • Further additional ingredient can be activating cytokines and chemokines (as described in Shaw, Jennifer, Infection and Immunity, 69:4667-4672, 2001) taken from the group consisting of ⁇ , Mipla, Mip-2, Mip3a, IP- 10, MCP-1, TCA-3, IL-1, IL-18, IL-6, ⁇ , MIF, IL-12, CCL19, and CCL21.
  • the pharmaceutical composition does not include these additional agents, but any one or more of them can be administered or provided in a separate
  • vitamin D that is or becomes biologically active within the body of the subject receiving such form of vitamin D may also be used as an additional ingredient.
  • the two main forms of vitamin D are: vitamin D3 or cholecalciferol, which is formed in the skin after exposure to sunlight or ultraviolet light, and ergocalciferol or vitamin D2 which is obtained by irradiation of plants or plant materials or foods. The differences are situated in the side chain.
  • Vitamin D3 may be obtained from natural sources such as fatty fish such as herring and mackerel. In the body, two other forms of vitamin D3 can be found.
  • Vitamin D3 is hydroxylated in the liver into 25-hydroxyvitamin D3 (25(OH)D), and subsequently in the kidney into 1,25-dihydroxyvitamin D3 (l,25(OH)2D), which is the active metabolite that stimulates the calcium absorption from the gut (Feldman et al. , 2005).
  • l,25(OH)2D is sufficiently available, 24,25-dihydroxyvitamin D (24,25(OH)2D) is formed in the kidney, which is further catabolized.
  • the pharmaceutical composition does not include these additional agents, but any one or more of them can be administered or provided in a separate composition as part of a method of the disclosure (e.g., coadministration at the same time or at differing times).
  • the composition has intrinsic adjuvant properties and is capable of raising an immune response without an adjuvant. In some embodiments, only a minimal or greatly reduced amount of adjuvant is required. In either case, in certain embodiments, pharmaceutical compositions of the disclosure optionally contain an additional adjuvant. In some embodiments, the adjuvant minimizes a broad inflammatory response (e.g., does not promote a broad inflammatory response when administered). In some embodiments, the adjuvant promotes a broad inflammatory response. Alternatively, in certain embodiments, any of the methods described herein may optionally comprise administering an additional adjuvant before, concurrently with, or after administration of a compositions or
  • the adjuvant minimizes a broad inflammatory response. In some embodiments, the adjuvant promotes a broad inflammatory response. In some embodiments, an amino acid copolymer composition of the disclosure is formulated and/or administered with alum as an adjuvant. In some
  • an amino acid copolymer composition of the disclosure is formulated and/or administered with aluminum hydroxide or aluminum phosphate.
  • the composition, whether formulated and/or administered with or without an adjuvant is formulated for intradermal, transdermal, intramuscular or subcutaneous injection.
  • the composition, whether formulated and/or administered with or without an adjuvant is formulated for intramuscular delivery.
  • the composition, whether formulated and/or administered with or without adjuvant is formulated for transdermal delivery.
  • Another aspect of the disclosure is a pharmaceutical composition
  • a pharmaceutical composition comprising one or more antibodies generated and produced using the process described herein elsewhere.
  • An antibody or antibodies that react to a protein conformational disease can be used to neutralize pathological proteins that such antibodies specifically bind, or to facilitate clearing from the body of a patient afflicted with such disease.
  • compositions suitable for antibodies are generally known in the art, including buffers and excipients, and preservative agents such as protease inhibitors that are suitable for pharmaceutical use.
  • the pharmaceutical compositions of the present disclosure are preferably sterile and non-pyrogenic at the time of delivery.
  • the compositions may be formulated for administration by injection, e.g., by bolus injection or continuous infusion in a parenteral, intravenous, intraperitoneal, intramuscular, or subcutaneous manner.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.
  • a suitable vehicle e.g., sterile pyrogen free water
  • antibodies are single chain variable fragments, to facilitate transport into the tissues due to its smaller size compared to naturally occurring antibodies.
  • Such antibodies may further be associated with a carrier or agent to cross the blood brain barrier, for example, an anti-transferring antibody. See, for example, Friden et al., Anti- transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier, PNAS June 1, 1991 vol. 88 no. 11 4771-4775.
  • the disclosure provides methods of identifying suitable amino acid copolymer compositions for use in one or more methods of the disclosure.
  • suitable amino acid copolymer compositions are identified based on their ability to induce proliferation of CD4+ T-cells, such as in assays using peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs are human PBMCs.
  • suitable amino acid copolymer compositions are identified based on their ability to stimulate Th2 chemokine (e.g., CCL22, CCL17, etc.) secretion from monocytes.
  • the Th2 chemokine is CCL22.
  • the composition may be capable of stimulate secretion/release of additional chemokines.
  • the monocytes are mouse monocytes. In some embodiments, the monocytes are human monocytes.
  • a method of screening for suitable amino acid copolymer compositions comprises testing candidate compositions in one or both of the assays described above (i.e., a CD4+ T cell proliferation assay and a Th2 chemokine (e.g., CCL22, CCL17, etc.) release assay).
  • a CD4+ T cell proliferation assay i.e., a CD4+ T cell proliferation assay and a Th2 chemokine (e.g., CCL22, CCL17, etc.) release assay).
  • a Th2 chemokine e.g., CCL22, CCL17, etc.
  • the ability of an amino acid copolymer composition to stimulate Th2 chemokine (e.g., CCL22, CCL17, etc.) release is predictive of its CD4+ T-cell proliferative ability.
  • a Th2 chemokine (e.g., CCL22, CCL17, etc.) release assay alone is sufficient to identify suitable compositions.
  • the method comprises providing a candidate amino acid copolymer composition and evaluating the composition in one or both of an assay to evaluate CD4+ T-cell proliferation or Th2 chemokine (e.g., CCL22, CCL17, etc.) release from monocytes.
  • the effect of the candidate composition may be evaluated versus one or more relevant controls, such as a single polypeptide or a mixture of polypeptides lacking RCR region(s).
  • a candidate amino acid composition that tests positive, relative to suitable controls, in one or both assays is identified as a suitable amino acid copolymer composition (e.g., suitable for use in vitro or in vivo to promote CD4+ T-cell proliferation or Th2 chemokine (e.g., CCL22, CCL17, etc.) release from monocytes; suitable for use as an immunotherapeutic; suitable to study an immune stimulatory response in vitro and/or in vivo; etc.).
  • a suitable amino acid copolymer composition e.g., suitable for use in vitro or in vivo to promote CD4+ T-cell proliferation or Th2 chemokine (e.g., CCL22, CCL17, etc.) release from monocytes; suitable for use as an immunotherapeutic; suitable to study an immune stimulatory response in vitro and/or in vivo; etc.
  • Figure 2 shows a schematic for the design of an amino acid copolymer composition (a composition based on the DP-CO 16.
  • B template arrangement, sequence, and input amino acid input distribution comprising a high complexity (500 or greater peptides) mixture of polypeptides based on antigenic sequences derived from Tau.
  • B is designed to target multiple phosphorylation patterns among 9 potential phosphorylation sites in the C- terminus of Tau and can be used to generate specific antibodies against many variants of early phosphorylated Tau protein in oligomers and paired helical filaments (PFIFs).
  • PFIFs paired helical filaments
  • the substantially full-length polypeptides of DP-CO 16.B in this schematic are 60 amino acids long and the amino acid positions, relative to full-length polypeptides based on the depicted template arrangement, of the polypeptides of the composition are indicated in Row 1 of the top and bottom panels (the row labeled peptide #).
  • the DP-C016.B polypeptides e.g., the peptides in the mixture of polypeptides comprising the DP-CO 16.
  • RCRs random copolymer regions
  • the amino acid at each position is selected from Alanine, Lysine or Glutamic Acid
  • the RCRs comprise A/K/E motifs (N-terminal RCR a (corresponding to positions 1-8 in Figure 2) and RCRb (corresponding to positions 28-36 in Figure 2)) and two antigenic regions (ARs) based on sequences derived from human Tau-F (AR a (corresponding to positions 9-27 in Figure 2) and ARb (corresponding to positions 37-60 in Figure 2)).
  • the template arrangement of DP-C016.B comprises: RCRa-ARa-RCRb-ARb, although other template arrangements are described herein.
  • RCRa and RCRb differ in length, and ARa and ARb correspond to or comprise different base peptide sequences, although each derived from human Tau-F.
  • complexity across the polypeptides in the composition comes from both the RCRs and the ARs although, in other examples, complexity may come only from the RCRs or from the RCRs and one, but not both, ARs.
  • the RCRs and ARs are contiguous. However, they may be interconnected by one or more linkers, such as by one or more intervening amino acid residues. Moreover, the polypeptides in the composition may include one or more additional amino acid residues at the N- or C-terminus, including natural or non-natural amino acids.
  • Row 2 of the top and bottom panels indicates the positions in human Tau-F (SEQ ID No. 17) that correspond to residues in the AR, which are or may be phosphorylated in this composition.
  • Row 3 of the top and bottom panels indicates the two random copolymer regions (RCRs) corresponding to positions 1-8 (RCRa) and positions 28- 36 (RCRb) (designated by the residue A - although the actual sequence in an individual peptide will vary according to the input % of A/K/E; where for this example, the input % is provided in Rows 5-7), and the base peptide sequences for the two antigenic regions derived from human Tau-F (ARa (corresponding to positions 9-27 of Figure 2) and ARb
  • the sequence of the AR given in Row 3 corresponds to the base peptide sequence and reflects original amino acid residues derived from the corresponding portion of the target, human Tau-F.
  • Row 4 of the top and bottom panels indicates the input percentage of phosphorylation at the residues in the first AR at positions corresponding to positions 13, 15, 19, 22 and 23 (corresponding to Y394, S396, S400, T403, and S404 of human Tau-F), and in the second AR at positions corresponding to positions 41, 46, 52 and 54 (corresponding to S422, T427, S433 and S435 of human Tau-F).
  • phosphorylated tyrosine is provided at an input percentage of 45% at the position
  • Rows 5-7 of the top and bottom panels indicate the different proportions of alanine, lysine, and glutamic acid that are provided at each position for generating the individual polypeptides of the mixture. Additional amino acids such as conserved substitutions may be provided at any of the positions in the antigenic region.
  • the input percentage for each of the two RCRs is 50% A, 40% K and 10% E, for each position of each RCR. As described herein, other ratios, distributions, and input percentages for the RCRs are also contemplated. Similarly, RCRs of differing sizes (e.g., shorter or longer than depicted) are contemplated, as described herein.
  • Rows 8 and 9 of the top and bottom panels indicate the percentage of positive and negatively charged residues available at each position.
  • the estimated percentages of alanine, positively charged residues and negatively charged residues across the entire length of the polypeptides across the composition are indicated in the rightmost column of the bottom panel.
  • the relative molar input percentages of alanine, lysine, and glutamic acid were 50%, 40%, and 10%, respectively. Therefore, each polypeptide has an A, K, or E at each position of its random copolymer regions, and the distribution in the polypeptides in the composition is influenced by these input percentages.
  • the AR at positions corresponding to positions 9-27 is based on a base peptide sequence which corresponds to positions 390-408 of human Tau-F (SEQ ID No. 17) and the AR at positions corresponding to positions 37-60 is based on a base peptide sequence which corresponds to positions 418-441 of human Tau-F (SEQ ID No. 17).
  • most positions of the ARs e.g.
  • positions 10-12, 14, 16-18, 20, 21, 24-27, 37-40, 42- 44, 47, 49-51, 55, 57-60) specify a relative molar input percentage of 10% alanine for generating complexity at those positions across the mixture of polypeptides, with the original amino acid (relative to the native human Tau-F sequence) making up the remaining 90% of the input molar percentage of amino acid at each position and for generating complexity at those positions across the mixture. Therefore, in this example, at these positions, each polypeptide has either the original amino acid indicated in Row 3 or alanine in the ARs.
  • additional complexity could be provided by, for example, also permitting variation at a position based on a conserved substitution, such as an orthologous substitution, a residue that is present in a different species, a phosphorylated or nitrated residue, a residue that is observed based on polymorphism or natural variation across humans, or a substitution with a similar amino acid residue that is considered interchangeable or a conservative substitution.
  • a conserved substitution such as an orthologous substitution, a residue that is present in a different species, a phosphorylated or nitrated residue, a residue that is observed based on polymorphism or natural variation across humans, or a substitution with a similar amino acid residue that is considered interchangeable or a conservative substitution.
  • positions 9, 45, 48, 53, and 56 no amino acids other than the residue present at the corresponding position of the base peptide are provided.
  • each polypeptide has the phosphorylated amino acid, the non-phosphorylated amino acid, or alanine.
  • the relative molar input percentage of phospho-serine is 100%. Additional amino acids such as conserved substitutions may be provided at any of the positions. This provides a description of this particular composition. However, other compositions based on, for example, differing template arrangements, input percentages in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated and provided.
  • a polypeptide mixture according to the schematic shown in Figure 2 was successfully synthesized using solid-phase peptide synthesis (SPPS). Briefly, polypeptides based on this template arrangement and input percentages were synthesized from C-terminus to N-terminus using Fmoc-based SPPS. Automated peptide synthesis is performed on a continuous flow peptide synthesizer. The first amino acid at the C-terminal position is a Norleucine (Nle) covalently coupled to the resin support (not shown in Figure 2). At each position and for each polypeptide generated, Fmoc is cleaved and an amino acid is randomly selected from the amino acid solution described in Figure 2 for each position.
  • SPPS solid-phase peptide synthesis
  • the amino acid solution for coupling contains 10% alanine (A) and 90% leucine (L), and thus, for each polypeptide, alanine or leucine is incorporated at a position corresponding to position 60.
  • the final step involves cleavage of the linear polypeptide from the solid resin support.
  • DP-C016.B was recognized by an anti-Tau paired helical filament pS396 monoclonal antibody (data not shown). This demonstrates that the polypeptides of the mixture are in the correct conformation to induce antibodies against paired helical filaments.
  • Figure 3 shows a schematic for the design of an amino acid copolymer composition (a composition based on the DP-C016.C template arrangement, sequence, and input amino acid input distribution) comprising a high complexity mixture of polypeptides based on antigenic sequences derived from Tau.
  • DP-C016.C like DP-C016.B, is designed to target multiple phosphorylation patterns among 14 potential phosphorylation sites in the C-terminus of Tau and can be used to generate specific antibodies against many variants of early phosphorylated Tau protein in oligomers and paired helical filaments (PHFs).
  • Full-length polypeptides of DP-C016.C in this schematic are about 60 amino acids long and the amino acid positions, relative to full-length polypeptides based on the depicted template arrangement, of the polypeptides of the composition are indicated in Row 1 of the top and bottom panels (the row labeled peptide #).
  • DP-C016.C polypeptides are each based on a template arrangement having 2 random copolymer regions (RCRs) wherein the amino acid at each position is selected from Alanine, Lysine, Glutamic Acid or Phenylalanine, i.e., the RCRs comprise A/K/E/F motifs (N-terminal RCRa (corresponding to positions 1-8 in Figure 3) and RCRb (corresponding to positions 28-36 in Figure 3)) and two antigenic regions (ARs) based on sequences derived from human Tau-F (ARa (corresponding to positions 9-27 in Figure 3) and ARb (corresponding to positions position 37-60 in Figure 3)).
  • RCRs random copolymer regions
  • the template arrangement of DP-C016.C comprises: RCRa-ARa-RCRb-ARb, although other template arrangements are described herein.
  • RCRa and RCRb differ in length, and ARa and ARb correspond to or comprise different base peptide sequences, although each derived from human Tau-F.
  • complexity across the polypeptides in the composition comes from both the RCRs and the ARs although, in other examples, complexity may come only from the RCRs or from the RCRs and one, but not both, ARs.
  • the RCRs and ARs are contiguous. However, they may be interconnected by a linker, such as by one or more intervening amino acid residues. Moreover, the polypeptides in the composition may include one or more additional amino acid residues at the N- or C- terminus, including natural or non-natural amino acids.
  • Row 2 of the top and bottom panels indicates the positions in human Tau-F (SEQ ID No. 17) that correspond to residues in the AR which are or may be phosphorylated in this composition.
  • Row 3 of the top and bottom panels indicates the two random copolymer regions (RCRs) corresponding to positions 1-8 (RCRa) and positions 28- 36 (RCRb) (designated by the residue A - although the actual sequence in an individual polypeptide will vary according to the input % of A/K/E/F; where for this example, the input % is provided in Rows 5-8), and the base peptide sequences for the two antigenic regions derived from human Tau-F (ARa (positions 9-27) and ARb (positions 37-60)).
  • Row 4 of the top and bottom panels indicates the input percentage of phosphorylation at the residues in the first AR at positions corresponding to positions 13, 15, 19, 22 and 23 (corresponding to Y394, S396, S400, T403, and S404 of human Tau-F), and in the second AR at positions corresponding to positions 41, 46, 52 and 54 (corresponding to S422, T427, S433 and S435 of human Tau-F).
  • phosphorylated tyrosine is provided at an input percentage of 45% at the position
  • Rows 5-8 of the top and bottom panels indicate the different proportions of alanine, lysine, glutamic acid, and phenylalanine that are provided at each position for generating the individual polypeptides of the mixture. Additional amino acids such as conserved
  • substitutions may be provided at any of the positions in the antigenic region.
  • the input percentage for each of the two RCRs is 50% A, 40% K, 5% E, and 5% F, for each position of each RCR.
  • other ratios, distributions, and input percentages for the RCRs are also contemplated.
  • RCRs of differing sizes are contemplated, as described herein.
  • Rows 9 and 10 of the top and bottom panels indicate the percentage of positive and negatively charged residues available at each position.
  • the estimated percentages of alanine, positively charged residues and negatively charged residues across the entire length of the polypeptides across the composition are indicated in the rightmost column of the bottom panel.
  • the relative molar input percentages of alanine, lysine, glutamic acid, and phenylalanine were 50%, 40%, 5%), and 5% respectively. Therefore, each polypeptide has an A, K, E, or F at each position of its random copolymer regions, and the distribution in the polypeptides in the composition is influenced by these input percentages.
  • the ARa at positions corresponding to positions 9-27 is based on a base peptide sequence which corresponds to positions 390-408 of human Tau-F (SEQ ID No. 17) and the ARb at positions corresponding to positions 37-60 is based on a base peptide sequence which corresponds to positions 418-441 of human Tau-F (SEQ ID No. 17).
  • most positions of the ARs e.g.
  • positions 10-12, 14, 16-18, 20, 21, 24-27, 37-40, 42- 44, 47, 49-51, 55, 57-60) specify a relative molar input percentage of 10% alanine for generating complexity at those positions across the mixture of polypeptide, with the original amino acid (relative to the native human Tau-F sequence) making up the remaining 90% of the input molar percentage of amino acid at each position and for generating complexity at those positions across the mixture. Therefore, in this example, at these positions, each polypeptide has either the original amino acid indicated in Row 3 or alanine in the ARs.
  • additional complexity could be provided by, for example, also permitting variation at a position based on a conserved substitution, such as an orthologous substitution, a residue that is present in a different species, a residue that is observed based on polymorphism or natural variation across humans, or substitution with a similar residue.
  • a conserved substitution such as an orthologous substitution
  • no amino acids other than the residue present at the corresponding position of the base peptide are provided.
  • positions corresponding to Y394, S400, T403, T427, S433, and S435 relative molar input percentages of 10% alanine, 45% non-phosphorylated amino acid, and 45% phosphorylated amino acid are provided for the generation of the mixture.
  • each polypeptide has the phosphorylated amino acid, the non-phosphorylated amino acid, or alanine.
  • the relative molar input percentage of phosphor-serine is 100%. Additional amino acids such as conserved substitutions may be provided at any of the positions.
  • the first amino acid at the C-terminal position is a Norleucine (Nle) covalently coupled to the resin support (not shown in Figure 3). This provides a description of this particular composition. However, other compositions based on, for example, differing template arrangements, input percentages in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated and provided.
  • DP-C016.C differs from DP-C016.B based on the RCRs (e.g., the RCRs in DP-
  • C016.C include an input percentage of phenylalanine).
  • DP-C016.B and DP- C016.C span the same antigenic regions.
  • a polypeptide mixture according to the schematic shown in Figure 3 was successfully synthesized using solid-phase peptide synthesis. Briefly, polypeptides based on this template arrangement and input percentages were synthesized from C-terminus to N-terminus using Fmoc-based SPPS, as described above. To improve the coupling efficiency and generate a higher yield in each step, longer coupling times or the addition of redundant coupling reactions may be used at one or more positions.
  • DP- C016.C was recognized by an anti-Tau paired helical filament pS396 monoclonal antibody (data not shown). This demonstrates that the polypeptides of the mixture are in the correct conformation to induce antibodies against paired helical filaments.
  • exemplary amino acid copolymer compositions (DP- C016.B and DP-C016.C) was evaluated by measuring their ability to induce CCL22 secretion in macrophage-derived RAW264.7 cells. Biological activity of these amino acid copolymer compositions was compared to that of (i) DP-CO 16.
  • A which is a complex mixture of peptides with one antigenic region corresponding to residues 388-441 of SEQ ID No.
  • RAW264.7 cells were incubated in 96-well round-bottom plates (3E+05 cells/well) in the presence of increasing concentrations (0; 0.0128; 0.064; 0.32; 1.6; 8 and 40 ⁇ ) of a polypeptide mixture (DP-CO 16. A, DP-CO 16.B, or DP-CO 16.C), single control peptides (Non-Phospho Peptide cnt or Phospho Peptide Cnt), or COPAXONE ® (0; 1.25 ; 2.5 ; 5 ; 10 ; 20 ; and 40 ⁇ ).
  • LPS at 1 ⁇ g/mL was used for the CCL22 assay. Each condition was tested in triplicate per run. Plates were duplicated for testing of cytotoxicity and of CCL22 production.
  • Mouse CCL22/MDC Immunoassay Briefly, after a 2 hour incubation in CCL22 coated plates, wells were washed then sequentially incubated for 2 hours with HRP-coupled anti- MDC polyclonal antibody and for 30 minutes in the dark with substrate solution.
  • An analytical batch included 8 calibration samples run in duplicate to generate the standard curve and study samples tested once. Negative control (supernatants of
  • Remaining supernatant was stored at -80°C for potential re-testing if needed (out of range values for example).
  • FIG. 4 shows CCL22 production by RAW monocytic cells stimulated with
  • compositions of the disclosure represented by DP-C016.B and DP-C016.C are comparable to or better than COPAXONE ® in terms of CCL22 release by the tested monocytic cell line.
  • Control compositions comprising single phosphorylated or non-phosphorylated peptides spanning the same region of Tau covered by DP-CO 16 did not stimulate CCL22 production in the RAW monocytic cells.
  • DP-C016.A which has an antigenic region spanning the same region of Tau covered by DP-C016.B and DP-C016.C but lacks any random copolymer regions (i.e. DP- CO 16.
  • A has no K/A or K/A/E or K/A/E/F RCRs sequences
  • DP-CO 16.B and DP-CO 16. C which have relatively short 8-9 amino acid RCRs linked to ARs, promote CCL22 release.
  • DP- C016.B and DP-C016.C also induce anti-hyper-phosphorylated Tau specific antibodies (data not shown).
  • DP-C016.B and DP-C016.C were evaluated using a CFSE proliferation assay. Their proliferative activity was compared to that of (i) DP-CO 16.
  • A which is a peptide mixture with an antigenic region corresponding to residues 388-441 of SEQ ID No. 17 but which lacks any RCRs,
  • two single control peptides Non-Phospho Peptide cnt or Phospho Peptide Cnt
  • PBMCs peripheral blood mononuclear cells
  • CPD citrate-phosphate-dextrose
  • PBMCs were stained with 5 ⁇ of
  • CFSE Carboxyfluorescein Diacetate Succinimidyl Ester
  • COPAXONE ® or immobilized anti-CD3 (10 ⁇ g/mL) plus soluble anti-CD28 (5 ⁇ g/mL) antibodies as a positive control and incubated for 6 days at 37°C, 5% C02 in the dark.
  • proliferating cells were characterized by flow cytometry after staining with anti-CD3, anti-CD4 and anti-CD8.
  • Figures 5 A and 5B show the results of the proliferation assays.
  • the proliferation index combines % of proliferating cells with mean fluorescence intensity (MFI) ((% CD4+ T- cells/MFI) x 100).
  • MFI mean fluorescence intensity
  • DP-CO 16.B has proliferative properties similar to COPAXONE ® based on % proliferative CD4+ T-cell s and shift in mean fluorescence intensity (MFI) and induces potent CD4+ T-cell proliferation.
  • MFI mean fluorescence intensity
  • DP-CO 16.B showed higher median levels of CD4+ T-cell proliferation than COPAXONE ® . 5.8% CD4+ T-cells divide up to once a day when cultured with DP-C016.B at 2.5 ⁇ over 6 days.
  • DP-C016.C also induced significant CD4+ T-cell proliferation.
  • Very limited proliferation of CD8+ T-cells is observed with either DP- C016.B or DP-C016.C.
  • DP-C016.A which has an antigenic region spanning the same region of Tau covered by DP-C016.B and DP-C016.C but lacks any RCRs (i.e. DP- CO 16.
  • A has no K/A or K/A/E or K/A/E/F RCR sequences
  • the single control peptides also do not significantly induce CD4+ T-cell proliferation.
  • DP-CO 16.B and DP-CO 16. C which have short 8-9 amino acid long random copolymer regions linked to antigenic regions, induce CD4+ T-cell proliferation, while also inducing anti-hyper-phosphorylated Tau specific antibodies (data not shown).
  • Example 5. DP-C016.B and DP-C016.C induce sustained immunogenicity in mice
  • A, DP- C016.B, or DP-C016.C was evaluated in vitro. Further, the ability of DP-C016.A, DP- C016.B, or DP-C016.C to raise immunogen-specific antibodies in mice injected with the compositions was evaluated.
  • IF A incomplete Freund's adjuvant
  • IF A incomplete Freund's adjuvant
  • Each ml of IF A contained 0.85 ml of paraffin oil and 0.15 ml of mannide monooleate.
  • the mice were injected once a week for a total of 4 subcutaneous injections without the use of anesthetics. The injection was carried out in the form of an emulsion in IF A, using glass syringes with metal connectors. The compounds were injected at 2 concentrations, one concentration per group. A control group injected with IF A alone was also tested. In addition, there were three un-injected mice. One week after the fourth injection, all the mice were sacrificed. The naive mice were also sacrificed and their sera were used as negative control in the anti-DP-C016.A Ab assay.
  • Splenocyte proliferation assay On the day of sacrifice, splenocyte suspensions were prepared and cultured in 96-well plates for three days with medium alone or 6 concentrations of each of the corresponding copolymer composition (100, 30, 10, 3, 1 and 0.3 ⁇ g/mL), or ammonium bicarbonate 0.2 mM, which corresponded to the bicarbonate concentration in the highest compound concentration tested, or phytohaemagglutinin (PHA) (at 5 or 20 ⁇ g/mL).
  • DP-CO 16.B was used for the recall response with splenocytes from IFA-treated mice. Each culture condition was performed in quadruplicate (4 wells on a 96-well plate) except for the PHA tested in duplicates.
  • 3H-thymidine was added and cells were cultured for an additional -18 hours. Prior to thymidine incorporation, 50 iL of the cell culture supernatant was collected for possible future analysis.
  • Anti-immunogen antibody determination Sera from the 35 injected mice and from the 3 un-injected mice were collected. The 30 sera from mice injected with the compounds were assayed for the determination of anti-immunogen antibodies using a titer assay approach.
  • the anti-immunogen antibody assay plate consisted of test sample(s), and negative control (sera collected from non-injected mice or from mice injected with IF A), titered down (8 dilutions) with dilution buffer. All the plates were coated with the respective composition used for the injection. The presence of anti-immunogen antibodies was detected using both anti-IgGl and anti-IgG2a antibodies, in separate reactions.
  • Figure 6A shows the results of the splenocyte proliferation assay in mice immunized with 0.25 mg/kg.
  • A, DP- C016.B, or DP-C016.C were collected and re-stimulated in vitro with various concentrations of the corresponding composition, the T-cell response induced by DP-CO 16.
  • B and DP- C016.C was greater than that of DP-C016.A. Therefore, DP-C016.B and DP-C016.C were able to induce specific T-cells in the spleen of mice immunized with the compositions while DP-CO 16.
  • Figure 6B shows the results of antibody ELISA against the respective immunogen.
  • Serum samples from mice injected with the compositions were assayed for the presence of anti-immunogen antibodies, and significantly higher titers of the anti-immunogen IgGl antibodies were present in the sera of mice injected with DP-C016.B and DP-C016.C compared to the sera of mice injected with DP-C016.A.
  • the presence of IgGl antibodies is indicative of a Th2 response.
  • DP-CO 16.B and DP-CO 16. C which comprise relatively short 8-9 amino acid long random copolymer regions (RCRs), were significantly more immunogenic than DP- CO 16.
  • A a composition that lacks random copolymer regions.
  • the greater immunogenicity of DP-CO 16.B and DP-CO 16. C indicates that the compositions have potential value in generating a robust immune response in elderly patients whose immune systems generally tend to be weaker.
  • DP-C003.E an amino acid copolymer composition (a composition based on the DP-
  • Figure 22 shows a schematic for the design of DP-C003.E.
  • DP-C003.E is designed to target multiple phosphorylation and nitration patterns covering Aspl21, Asnl22, Tyrl25, Tyrl33, and Tyr 136 of human a-synuclein and can be used to generate specific antibodies.
  • the full- length polypeptides of DP-C003.E are about 61 amino acids long.
  • the template arrangement of DP-C003.E comprises: ARa-RCRa-AR a -RCRb-AR c .
  • the DP-C003.E polypeptides are each based on a template arrangement having two 5 amino acid-long random copolymer regions (RCRs) wherein the amino acid at each position is selected from Alanine or Lysine i.e., the RCRs comprises K/A motifs and three antigenic regions that are all based on the same base peptide sequence corresponding to residues 121-137 of SEQ ID No. 20.
  • each peptide has an A or K at each position of its RCRs with the distribution according to these percentages.
  • the AR at positions corresponding to 1-17, the AR at position corresponding to 23-39, and the AR at positions corresponding to 41-57, of Figure 22 are based on a base peptide sequence which corresponds to positions 121-137 of human a-synuclein (human a-synuclein set forth in SEQ ID No. 20.
  • DP-C003.F and DP-C003.G comprising high complexity mixtures of polypeptides based on antigenic sequences derived from ⁇ -synuclein, were designed and synthesized according to the methods of the disclosure.
  • the DP-C003.F and DP-C003.G polypeptides are each based on a template arrangement having two 5 amino acid-long random copolymer regions (RCRs) wherein the amino acid at each position is selected from Alanine or Lysine i.e., the RCRs comprises K/A motifs and three antigenic regions that are all based on the same base peptide sequence corresponding to residues 121-137 of SEQ ID No. 20.
  • RCRs 5 amino acid-long random copolymer regions
  • each peptide has an A or K at each position of its RCRs with the distribution according to these percentages.
  • DP-C003.E had a relative molar input percentage of alanine of 10% along each of the positions in the ARs, with the exception of any native alanine residues or serine residues (positions 1-3, 5-8, 10-17, 23-25, 27-30, 32-39, 45-47, 49-52, 54-61), DP-C003.7 and DP- COOS.8, respectively, had relative molar input percentages of alanine of 30% and 50%, at each of these positions of the AR.
  • DP-C003.F and DP-C003.G differed from DP- C003.E in the composition of the ARs, with DP-C003.F and CP-C003.G having a substantially higher input percentage of alanine across the ARs, and thus, a substantially higher total input percent of alanine across the ARs and RCRs.
  • DPC003.E stimulated CCL22 release from RAW monocytic cells more effectively than either DPC003.F or DPC003.G.
  • FIG. 7 shows a schematic for the design of another amino acid copolymer composition (a composition based on the DP-C003.A template arrangement, sequence, and input amino acid input distribution) comprising a high complexity of polypeptides based on antigenic sequences derived from a-synuclein.
  • DP-C003.A is designed to target multiple phosphorylation and nitration patterns covering Tyrl25, Tyrl33, Tyrl36 and Serl29 of human a-synuclein and can be used to generate specific antibodies.
  • the full-length polypeptides of DP-C003.A based on this template arrangement are about 53 amino acids long and the amino acid positions, relative to full-length polypeptides based on the depicted template arrangement, of the composition are indicated in Row 1 (the row labeled peptide #).
  • the DP-C003.A polypeptides e.g.
  • the polypeptides in the mixture of polypeptides comprising the DP-C003.A composition are based on a template arrangement having a maximum of 2 random copolymer regions (RCRs) wherein the amino acid at each position is selected from lysine, alanine or glutamic acid, i.e., the RCRs comprise K/A/E motifs (N-terminal RCR a (corresponding to positions 1-9 in Figure 7) and RCRb
  • the template arrangement of DP-C003.A comprises: RCR a -AR a - RCRb-ARb, although other template arrangements are described herein.
  • complexity across the polypeptides in the composition comes from both the RCRs and ARs although, in other examples, complexity may come only from the RCRs or from the RCRs and one, but not both, ARs. In this example, the RCRs and ARs are contiguous.
  • polypeptides in the composition may include on or more additional amino acid residues at the N- or C-terminus, including naturally occurring or non-natural amino acids.
  • Row 2 of the top and bottom panels indicates the positions in human ⁇ -synuclein (SEQ ID No. 35) that correspond to residues that may be phosphorylated or nitrated, and for which phosphorylation or nitration is among the diversity available in the polypeptides in the mixture.
  • Rows 3, 4, and 5 indicate the possible amino acids, which are or may be incorporated at each position in the peptide.
  • the two copolymer regions (RCRs) correspond to positions 1-9 (RCRa) and positions 27-35 (RCRb).
  • the two antigenic regions (ARs) correspond to positions 10-26 (ARa) and to positions 36-52 (ARb).
  • the sequence of the AR given in Row 3 corresponds to the base peptide sequence and reflects original amino acid residues derived from the corresponding portion of the target, human a-synuclein (SEQ ID No. 35).
  • Rows 6, 7, and 8 indicate the molar input percentages of the corresponding, color-coded, amino acid above.
  • the relative molar amino acid input percentages of glycine (G) and alanine (A) are 90% and 10%, respectively. Additional amino acids such as conserved substitutions may be provided at any of the positions in the antigenic region.
  • the two RCRs in this example are 9 amino acids in length and contain either lysine (K), alanine (A), or glutamic acid (E) at each position.
  • RCRs of differing sizes are contemplated, as described herein.
  • the relative molar input percentages of lysine, alanine, or glutamic acid were 40%, 50%, or 10% relatively.
  • the relative molar input percentage of lysine, alanine, or glutamic acid was selected from within the foregoing ranges, independently for each position.
  • the AR at positions corresponding to 10-26 of Figure 7 is based on a base peptide sequence which corresponds to positions 123-137 of human a-synuclein (human a-synuclein set forth in SEQ ID No. 35; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 36).
  • the AR at positions corresponding to 36-52 of Figure 7 is based on a base peptide sequence which corresponds to positions 121-137 of human a-synuclein (SEQ ID No. 35; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 37).
  • most positions of the ARs (e.g. positions 10-12, 15-17, 19-21, 23-24, 26, 36-38, 41-43, 45-47, 49-50, and 52) specify a relative molar input percentage of 10% alanine for generating complexity at those positions across the mixture of polypeptides, with the original amino acid (relative to the human ⁇ -synuclein sequence) making up the remaining 90% of the input molar percentage of amino acid at each position and for generating complexity at those positions across the mixture. Therefore, in this example, at these positions, each polypeptide has either the original amino acid indicated in Row 3 or alanine in the ARs.
  • additional complexity could be provided by, for example, also permitting variation at a position based on a conserved substitution, such as an orthologous substitution, a residue that is present in a different species, a residue that is observed based on polymorphism or natural variation across humans or substitution with a similar residue.
  • a conserved substitution such as an orthologous substitution
  • no amino acids other than the residue present at the corresponding position of the base peptide are provided.
  • relative molar input percentages of 10% alanine, 45% non-phosphorylated amino acid, and 45% nitrated amino acid are provided for the generation of the mixture.
  • each polypeptide has the nitrated amino acid, the non-nitrated amino acid, or alanine.
  • the relative molar input percentage of phosphor-serine is 100%.
  • Norleucine is found at the C-terminal most amino acid at position 53 of Figure 7. Additional amino acids such as conserved substitutions may be provided at any of the positions. This provides a description of this particular composition. However, other compositions based on, for example, differing template arrangements, input percentages in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated and provided.
  • a polypeptide mixture according to the schematic shown in Figure 7 was successfully synthesized using solid-phase peptide synthesis. Briefly, the polypeptides based on this template arrangement and input percentages were synthesized from C-terminus to N-terminus using Fmoc-based SPPS, as described above. To improve the coupling efficiency and generate a higher yield in each step, longer coupling times or the addition of redundant coupling reactions may be used at one or more positions.
  • the disclosure provides compositions manufactured based on this template arrangement, sequence and input amino acid distribution.
  • exemplary copolymer compositions based on antigenic sequences derived from a-synuclein include DP-C003.B ( Figure 8), DP-C003.C ( Figure 9), and DP-C003.D ( Figure 10). Each are briefly described herein.
  • DP-C003.B an amino acid copolymer composition (a composition based on the DP- C003.B template arrangement, sequence, and input amino acid input distribution) comprising a high complexity mixture of peptides based on antigenic sequences derived from a- synuclein, is designed and synthesized according to the methods of the disclosure.
  • the full- length peptides of DP-C003.B are about 57 amino acids long.
  • DP-C003.B is designed to target multiple phosphorylation and nitration patterns covering Aspl21, Asnl22, Tyrl25, Tyrl33, and Tyr 136 of human a-synuclein and can be used to generate specific antibodies.
  • the template arrangement of DP-C003.B comprises: ARa-RCRa-ARb-RCRb-ARc.
  • B peptides e.g., the peptides in the mixture of peptides comprising the DP-C003.B composition
  • each peptide has a K, A, or E at each position of its RCRs with the distribution according to these percentages, and wherein the actual input percentage is independently selected for each position.
  • the AR at positions corresponding to 1-17 and the AR at positions corresponding to 41-57 of Figure 8 is based on a base peptide sequence which corresponds to positions 121- 137 of human a-synuclein (human a-synuclein set forth in SEQ ID No. 35; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 37).
  • the AR at positions corresponding to 21-37 of Figure 8 is based on a base peptide sequence which most closely corresponds to positions 123-137 of human ⁇ -synuclein (SEQ ID No. 35;
  • DP-C003.C an amino acid copolymer composition (a composition based on the DP- C003.C template arrangement, sequence, and input amino acid input distribution) comprising a high complexity mixture of peptides based on antigenic sequences derived from a- synuclein, is designed and synthesized according to the methods of the disclosure.
  • the full- length polypeptides of DP-C003.C are about 63 amino acids long.
  • DP-C003.C is designed to target multiple phosphorylation and nitration patterns covering Tyrl25, Serl29, Tyrl33, and Tyr 136 of human ⁇ -synuclein and can be used to generate specific antibodies.
  • the template arrangement of DP-C003.C comprises: RCRa-ARa- RCRb - ARb -RCRc- ARc-RCRd .
  • the DP-C003.C polypeptides (e.g., the polypeptides in the mixture of polypeptides comprising the DP-C003.C composition) each have four 3 amino acid-long random copolymer regions (RCRs) wherein the amino acid at each position is selected from lysine, alanine, or glutamic acid i.e., the RCRs comprises K/A/E motifs and three antigenic regions that are all based on the same base peptide sequence corresponding to residues 121-137 of SEQ ID No. 35.
  • each peptide has a K, A, or E at each position of its RCRs with the distribution according to these percentages and wherein the actual input percentage is independently selected for each position.
  • the AR at positions corresponding to 4-20 and the AR at positions corresponding to 44-60 of Figure 9 is based on a base peptide sequence which corresponds to positions 121- 137 of human ⁇ -synuclein (human a-synuclein set forth in SEQ ID No. 35; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 37).
  • the AR at positions corresponding to 24-40 of Figure 9 is based on a base peptide sequence which most closely corresponds to positions 123-137 of human a-synuclein (SEQ ID No. 35; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 36).
  • DP-C003.D an amino acid copolymer composition (a composition based on the DP- C003.D template arrangement, sequence, and input amino acid input distribution) comprising a high complexity mixture of peptides based on antigenic sequences derived from a- synuclein, is designed and synthesized according to the methods of the disclosure.
  • the full- length polypeptides of DP-C003.D are about 63 amino acids long.
  • DP-C003.D is designed to target multiple phosphorylation and nitration patterns covering Tyrl25, Serl29, Tyrl33, and Tyr 136 of human ⁇ -synuclein and can be used to generate specific antibodies.
  • the template arrangement of DP-C003.D comprises: RCRa-ARa- RCRb-ARb-RCRc-ARc-RCRd.
  • the DP-C003.D polypeptides e.g., the polypeptides in the mixture of peptides comprising the DP-C003.D composition
  • each peptide has a R, A, or E at each position of its RCRs with the distribution according to these percentages and wherein the actual input percentage is independently selected for each position.
  • the AR at positions corresponding to 4-20 and the AR at positions corresponding to 44-60 of Figure 10 is based on a base peptide sequence which corresponds to positions 121- 137 of human ⁇ -synuclein (human a-synuclein set forth in SEQ ID No. 35; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 37).
  • the AR at positions corresponding to 24-40 of Figure 9 is based on a base peptide sequence which most closely corresponds to positions 123-137 of human ⁇ -synuclein (SEQ ID No. 35;
  • compositions with antigenic specificity to ⁇ -synuclein could comprise RCRs (such as two or three RCRs) that are 3 amino acids, or 4 amino acids, or 5 amino acids, or 6 amino acids, or 7 amino acids in length (or even 8 or 9 as described herein).
  • the length of each RCR may be selected independently from length of the other RCRs.
  • All of the foregoing compositions may have a Norleucine or another non-naturally occurring amino acid at either the C-terminus or the N-terminus.
  • Figure 11 shows a schematic for the design of an amino acid copolymer composition (a composition based on the DP-CO 16. E template arrangement, sequence, and input amino acid input distribution) comprising a high complexity of polypeptides based on antigenic sequences derived from Tau.
  • DP-CO 16.E is designed to target multiple phosphorylation patterns among 11 potential phosphorylation residues found in the C-terminus of human Tau- F and can be used to generate specific antibodies against many variants of
  • hyperphosphorylated Tau protein found in neuropathological hallmarks such as
  • NFTs neurofibrillary tangles
  • PFIFs paired helical filaments
  • the full-length polypeptides of DP-C016.E in this schematic are about 51 amino acids long and the amino acid positions, relative to full-length polypeptides based on the depicted template arrangement, of the composition are indicated in Row 1 (the row labeled peptide #).
  • E polypeptides e.g. the polypeptides in the mixture of
  • E composition are based on a template arrangement having a maximum of 2 random copolymer regions (RCRs) wherein the amino acid at each position is selected from lysine, alanine or glutamic acid, i.e., the RCRs comprise K/A/E motifs (N-terminal RCR a (corresponding to positions 1-3 in Figure 11) and RCRb
  • the template arrangement of DP- C016.F comprises: RCRa-ARa- RCRb-ARb, although other template arrangements are described herein.
  • ARa corresponds to or comprises a region of PHF1 epitope
  • ARb corresponds to or comprises a region comprising the pS422 and the pS433 epitopes of Tau-F.
  • complexity across the polypeptides in the composition comes from both the RCRs and ARs although, in other examples, complexity may come only from the RCRs or from the RCRs and one, but not both, ARs.
  • the RCRs and ARs are contiguous. However, they may be interconnected by a linker, such as by one or more intervening amino acid residues.
  • the polypeptides in the composition may include on or more additional amino acid residues at the N- or C-terminus, including naturally occurring or non-natural amino acids.
  • Row 2 of the top and bottom panels indicates the positions in human Tau-F (SEQ ID No.
  • Rows 3, 4, and 5 indicate the possible amino acids which are or may be incorporated at each position in the peptide.
  • the two copolymer regions (RCRs) correspond to positions 1-3 (RCRa) and positions 23-25 (RCRb).
  • the two antigenic regions (ARs) correspond to positions 4-22 (AR a ) and to positions 26-50 (ARb).
  • the sequence of the AR given in Row 3 corresponds to the base peptide sequence and reflects original amino acid residues derived from the corresponding portion of the target, human Tau-F.
  • Rows 6, 7, and 8 (the rows labeled 1 st Row %, 2 nd Row%, and 3 rd Row %,
  • the two RCRs in this example are 3 amino acids in length and contain either lysine (K), alanine (A), or glutamic acid (E) at each position.
  • RCRs of differing sizes are contemplated, as described herein.
  • the relative molar input percentages of lysine, alanine, or glutamic acid were 30%-80%, 15%-55%, or 5%-15% relatively.
  • the relative molar input percentage of lysine, alanine, or glutamic acid was selected from within the foregoing ranges, independently for each position.
  • the AR at positions corresponding to 4-22 of Figure 11 is based on a base peptide sequence which corresponds to positions 390-408 of human Tau-F (human Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 9).
  • the AR at positions corresponding to 26-50 of Figure 11 is based on a base peptide sequence which corresponds to positions 414-438 of human Tau-F (human Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 31).
  • most positions of the ARs e.g.
  • positions 5-7, 9, 11-13, 15-16, 19-22, 27, 29-33, 35-37, 40, 42-44, 48, and 50 specify a relative molar input percentage of 10% alanine for generating complexity at those positions across the mixture of polypeptides, with the original amino acid (relative to the human Tau-F sequence) making up the remaining 90% of the input molar percentage of amino acid at each position and for generating complexity at those positions across the mixture. Therefore, in this example, at these positions, each polypeptide has either the original amino acid indicated in Row 3 or alanine in the ARs.
  • additional complexity could be provided by, for example, also permitting variation at a position based on a conserved substitution, such as an orthologous substitution, a residue that is present in a different species, a residue that is observed based on polymorphism or natural variation across humans or substitution with a similar residue.
  • a conserved substitution such as an orthologous substitution
  • a residue that is present in a different species a residue that is observed based on polymorphism or natural variation across humans or substitution with a similar residue.
  • no amino acids other than the residue present at the corresponding position of the base peptide are provided.
  • phosphorylated amino acid the non-phosphorylated amino acid, or alanine.
  • the relative molar input percentage of phosphor-serine is 100%.
  • Norleucine is found at the C-terminal most amino acid at position 51 of Figure 1 1. Additional amino acids such as conserved substitutions may be provided at any of the positions. This provides a description of this particular composition. However, other compositions based on, for example, differing template arrangements, input percentages in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated and provided.
  • a polypeptide mixture according to the schematic shown in Figure 1 1 was successfully synthesized using solid-phase peptide synthesis. Briefly, the polypeptides based on this template arrangement and input percentages were synthesized from C-terminus to N- terminus using Fmoc-based SPPS, as described above. To improve the coupling efficiency and generate a higher yield in each step, longer coupling times or the addition of redundant coupling reactions may be used at one or more positions.
  • the disclosure provides compositions manufactured based on this template arrangement, sequence and input amino acid distribution.
  • Figure 12 shows a schematic for the design of an amino acid copolymer composition (a composition based on the DP-C016.F template arrangement, sequence, and input amino acid input distribution) comprising a high complexity of polypeptides based on antigenic sequences derived from Tau.
  • the disclosure provides compositions manufactured based on this template arrangement, sequence and input amino acid distribution.
  • DP-C016.F is designed to target multiple phosphorylation patterns among 14 potential phosphorylation residues found in the C-terminus of human Tau-F and can be used to generate specific antibodies against many variants of hyperphosphorylated Tau protein found in neuropathological hallmarks such as neurofibrillary tangles (NFTs), Neuropil threads, plaques and paired helical filaments (PFIFs).
  • NFTs neurofibrillary tangles
  • PFIFs paired helical filaments
  • F polypeptides e.g. the polypeptides in the mixture of polypeptides comprising the DP-CO 16.
  • RCRs random copolymer regions
  • the amino acid at each position is selected from lysine, alanine or glutamic acid
  • the RCRs comprise K/A/E motifs (N-terminal RCR a (corresponding to positions 1-3 in Figure 12), RCRb (corresponding to positions 23-25 in Figure 12), and RCRc (corresponding to positions 42-44 in Figure 12)) and three antigenic regions (ARs) based on sequences derived from human Tau-F (AR a (corresponding to positions 4-22 in Figure 12) ARb (corresponding to positions 26-41 in Figure 12), and AR C (corresponding to positions 45-59 in Figure 12)).
  • the template arrangement of DP-C016.F comprises: RCRa-AR a - RCRb-ARb- RCR C -AR C , although other template arrangements are described herein.
  • ARa corresponds to or comprises a region of a PHF1 epitope
  • ARb correspond to or comprises a region comprising the pS422 epitope of Tau-F
  • ARc corresponds to or comprises a region comprising the pS433 epitope of Tau-F.
  • complexity across the polypeptides in the composition comes from both the RCRs and ARs although, in other examples, complexity may come only from the RCRs or from the RCRs and one, but not both, ARs.
  • the RCRs and ARs are contiguous. However, they may be interconnected by a linker, such as by one or more intervening amino acid residues.
  • the polypeptides in the composition may include one or more additional amino acid residues at the N- or C- terminus, including naturally occurring or non-natural amino acids. In this example, additional C-terminal amino acids are included.
  • Row 2 of the top and bottom panels indicates the positions in human Tau-F (SEQ ID No. 17) that correspond to residues in the AR, which are or may be phosphorylated in this composition.
  • Rows 3, 4, and 5 indicate the possible amino acids which are or may be incorporated at each position in the peptide.
  • the three copolymer regions (RCRs) correspond to positions 1-3 (RCRa), positions 23-25 (RCRb), and regions 42- 44 (RCRc).
  • the three antigenic regions (ARs) correspond to positions 4-22 (ARa), positions 26-41 (ARb), and positions 45-59 (ARc), in each case as depicted for full-length sequences in Figure 12.
  • the sequence of the AR given in Row 3 corresponds to the base peptide sequence and reflects original amino acid residues derived from the corresponding portion of the target, human Tau-F.
  • Rows 6, 7, and 8 (the rows labeled 1 st Row %, 2 nd Row%, and 3 rd Row %,
  • the three RCRs in this example are 3 amino acids in length and contain either lysine (K), alanine (A), or glutamic acid (E) at each position. RCRs of differing sizes (e.g. shorter or longer than depicted) are contemplated, as described herein.
  • the relative molar input percentages of lysine, alanine, or glutamic acid were 30%-80%, 15%-55%, or 5%-15%, relatively. In some embodiments, the relative molar input percentage of lysine is 35%-75%, 40-75%), or 50-70%).
  • the relative molar input percentage of lysine, alanine or glutamic acid was selected from within the foregoing ranges, independently for each position.
  • the AR at positions corresponding to 4-22 of Figure 12 is based on a base peptide sequence which corresponds to positions 390-408 of human Tau-F (human Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 9).
  • the AR at positions corresponding to 26-41 of Figure 12 is based on a base peptide sequence which corresponds to positions 414-429 of human Tau-F (human Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 32).
  • the AR at positions corresponding to positions 45-59 of Figure 12 is based on the base peptide sequence which corresponds to positions 424-438 of human Tau-F (human Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 33).
  • base peptide sequence which corresponds to positions 424-438 of human Tau-F (human Tau-F sequence set forth in SEQ ID No. 17; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 33).
  • most positions of the ARs e.g.
  • positions 5-7, 9, 11-13, 15-16, 19-22, 27, 35-37, 40, 45-46, 49, 51-53, 57, and 59) specify a relative molar input percentage of 10%> alanine for generating complexity at those positions across the mixture of polypeptides, with the original amino acid (relative to the human Tau-F sequence) making up the remaining 90%> of the input molar percentage of amino acid at each position and for generating complexity at those positions across the mixture. Therefore, in this example, at these positions, each polypeptide has either the original amino acid indicated in Row 3 or alanine in the ARs.
  • additional complexity could be provided by, for example, also permitting variation at a position based on a conserved substitution, such as an orthologous substitution, a residue that is present in a different species, a residue that is observed based on polymorphism or natural variation across humans or substitution with a similar residue.
  • a conserved substitution such as an orthologous substitution
  • a residue that is present in a different species a residue that is observed based on polymorphism or natural variation across humans or substitution with a similar residue.
  • no amino acids other than the residue present at the corresponding position of the base peptide are provided.
  • each polypeptide has the phosphorylated amino acid, the non- phosphorylated amino acid, or alanine.
  • the relative molar input percentage of phosphor-serine is 100%.
  • Norleucine is found at the C-terminal most amino acid at position 63 of Figure 12.
  • alanine used in the ARs, which is, in certain embodiments, an important feature. Additional amino acids such as conserved substitutions may be provided at any of the positions. This provides a description of this particular composition.
  • compositions based on, for example, differing template arrangements, input percentages in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated and provided.
  • alternate phosphorylation sites and phosphorylation patterns in the C-terminal region of tau may be targeted ( Figure 13).
  • DP-C016.E differs from DP-C016.F ( Figure 12) based on the both the RCRs and ARs.
  • E template arrangement contains two RCRs as compared to the three RCRs in DP-CO 16.
  • the one antigenic region (AR a ) (which corresponds to positions 390-408 of human Tau-F (SEQ ID No. 17)) is identical between DP-CO 16.E and DP-CO 16.F.
  • ARb and AR C of DP-CO 16.F correspond to positions 414-429 and 424-438 of human Tau-F (SEQ ID No.
  • DP-C016.E combines these regions into one antigenic region (ARb) which corresponds to positions 414- 438 of human Tau-F (SEQ ID No. 17). These differences contribute to DP-C016.E having an estimated net charge of 1.1 at pH7 and DP-CO 16.F having an estimated net charge of 3.1 at pH7 (data not shown).
  • ARb antigenic region
  • DP-C016.E having an estimated net charge of 1.1 at pH7 and DP-CO 16.F having an estimated net charge of 3.1 at pH7 (data not shown).
  • the net charge and nature of the ARs contributes to overall solubility and lack of solubility prevents evaluation of immunogenicity of the complex polypeptide mixture. Accordingly, where the ARs are of a character that contributes to poor solubility and/or alpha-helical structure, increasing charge helps improve solubility so that the immunogenic properties may be fully evaluated.
  • the estimated net charge is a proxy for the predicted charge (e.g., estimated charge) at pH7
  • composition of the disclosure is based on the template sequence where: (i) the net charge of the ARs, is calculated based on the base peptide sequence without the inclusion of potential variation at each amino acid position of the AR, (ii) the net charge of the RCRs, is calculated for a hypothetical polypeptide in which for every 1 position in which a positively charged amino acid is assumed to occur, alanine is assumed to occur in two positions (e.g., if template arrangement and template sequence for a polypeptide mixture includes 1 RCR of 3 amino acid residues in length, estimated net charge is calculated based on the assumption that the RCR contains 1 positively charged residue and two alanines); and (iii) and additional amino acids outside of the ARs and RCRs and present in the design are included (e.g., a 4 residue N- or C- terminal tail in the design is accounted for based on its sequence and charge). For the purpose of this calculation, negatively charged amino acids are not considered for the RCR.
  • Polypeptide mixtures according to the schematics shown in Figures 11 and 12 were successfully synthesized using solid-phase peptide synthesis. Briefly, the polypeptides based on this template arrangement and input percentages were synthesized from C-terminus to N- terminus using Fmoc-based SPPS, as described above. To improve the coupling efficiency and generate a higher yield in each step, longer coupling times or the addition of redundant coupling reactions may be used at one or more positions.
  • the disclosure provides compositions manufactured based on these template arrangements, sequences and input amino acid distributions.
  • Example 9 Effect of DP-C016.E and DP-C016.F on CD4+ T-cell proliferation
  • DP-CO 16.E and DP-CO 16.F were evaluated using a CFSE proliferation assay. Their proliferative activity was compared to that of (i) DP-CO 16. B, which is a polypeptide mixture based on the template arrangement, amino acid sequence, and amino acid input percentages described in Figure 2, (ii) DP-CO 16.D, which is a polypeptide mixture with a template arrangement comprising first antigenic region corresponding to residues 387-407 of SEQ ID No. 17 and second antigenic region corresponding to 417-432 of SEQ ID No. 17, and two 5 amino acid long RCRs, and (iii) COPAXONE®.
  • PBMCs peripheral blood mononuclear cells
  • CPD citrate-phosphate-dextrose
  • PBMCs were stained with 5 ⁇ of
  • CFSE Carboxyfluorescein Diacetate Succinimidyl Ester
  • proliferating cells were characterized by flow cytometry after staining with anti-CD3, anti-CD4 and anti-CD8.
  • Figure 14 shows the results of the proliferation assays.
  • the stimulation index combines % of proliferating cells with mean fluorescence intensity (MFI) ((% CD4+ T- cells/MFI) x 100).
  • MFI mean fluorescence intensity
  • DP-CO 16.F has proliferative properties similar to DP-CO 16.B based on % proliferative CD4+ T-cells and shift in mean fluorescence intensity (MFI) and induces potent CD4+ T-cell proliferation. 2.2% CD4+ T-cells divide up to once a day when cultured with DP-CO 16.F at 2.5 ⁇ over 6 days.
  • DP-CO 16.D did not significantly induce CD4+ T-cell proliferation in this experiment, although we note that it was insoluble, and thus, no conclusions can be drawn regarding DP-CO 16. D based on this experiment.
  • DP- CO 16. D has an estimated net negative charge of -3.8 at pH7.
  • DP-CO 16.E also had poor solubility.
  • DP-CO 16.F and DP-CO 16. B induce CD4+ T-cell proliferation, while also inducing anti-hyper-phosphorylated Tau specific antibodies (data not shown).
  • Example 10 DP-C016.F induces a strong and specific immune response in mice
  • the injection was carried out in the form of an emulsion in IF A, using glass syringes with metal connectors.
  • the compounds were injected at one concentration per group.
  • a control group injected with IFA alone was also tested.
  • DP-C016.C and DP-C016.F are examples of compounds of the disclosure.
  • One week after the fourth injection (Day 29) all the mice were sacrificed. The naive mice were also sacrificed and their sera were used as negative control in the anti-DP-C016 and Anti-PHFl Ab assay.
  • Anti-immunogen antibody determination Sera from the 15 injected mice and from the 3 un-injected mice were collected. The sera from mice injected with the compounds were assayed for the determination of anti-immunogen antibodies using a titer assay approach.
  • the anti-immunogen antibody assay plate included test sample(s), and negative control (sera collected from non-injected mice or from mice injected with IFA), titered down (8 dilutions) with dilution buffer. All the plates were coated with the respective composition used for the injection. The presence of anti-immunogen antibodies was detected using both anti-IgGl and anti-IgG2a antibodies, in separate reactions.
  • Figure 15A shows the results of antibody ELISA against DP-CO 16 or PHF1.
  • Serum samples from mice injected with the compositions were assayed for the presence of anti-DP-C016 or anti-PHFl antibodies, and significantly higher titers of the anti- DP-CO 16 or anti-PHFl IgGl antibodies were present in the sera of mice injected with DP-C016.C or DP- CO 16. F compared to the sera of mice injected with IFA alone. The presence of IgGl antibodies is indicative of a Th2 response.
  • DP-CO 16.F and DP-CO 16. C induced a similar immunogenic profile.
  • the significant immunogenicity indicates that such compositions have potential value in generating a robust immune response, including in elderly patients whose immune systems generally tend to be weaker.
  • Figure 15B shows the results of the antibody ELISA against a short linear peptide covering pS422 and recombinant Tau.
  • One of the key biomarkers for hyperphosphorylation of Tau is the phosphorylation of position pS422 (corresponding to position 422 of SEQ ID No. 17).
  • recombinant Tau represents the major microtubule associated protein, which is part of a normal mature neuron.
  • Figure 15B shows that high titers of anti-pS422 antibodies are present, while very low titers of anti-recombinant Tau antibodies are present. This indicates that the high complexity polypeptides of DP-CO 16. F and, to a lesser extent, DP-CO 16. C, induce a robust, but specific antibody response against pathologic Tau.
  • Figure 15C shows the results of the antibody ELISA against PHF-Tau.
  • hyperphosphorylated Tau leads to conformational changes and, ultimately, to the formation of pathogenic species such as paired helical filaments (PHFs).
  • Figure 15C shows that high titers of anti-PFIF Tau antibodies are induced by DP-C016.F. The significant
  • Figure 15D shows the specificity of the antibodies induced by DP-C016.C and DP-
  • C016.F against human Alzheimer's disease tissue As described above, C57B1/6 mice were injected once a week for four weeks with the DP-C016.C and DP-C016.F compositions emulsified in IFA. On day 29, terminal bleeding was conducted and sera were collected. The antibodies induced by DP-C016.C and DP-C016.F were isolated from sera samples. These antibodies were applied to post-mortem brain tissue from Alzheimer's disease patients.
  • Antibodies bound to antigens were visualized using immunofluorescence antibody assay (IFA).
  • IFA immunofluorescence antibody assay
  • Figure 16 shows a schematic for the design of an amino acid copolymer composition (a composition based on the DP-C024.1 template arrangement, sequence, and input amino acid input distribution) comprising a high complexity of polypeptides based on antigenic sequences derived from HPV L2.
  • DP-C024.1 is designed to target the minor capsid protein L2, which localizes along the inner surface of the virion, and plays a role in capsid stabilization through interaction with the major capsid protein LI.
  • the DP-C024.1 polypeptides can be used to generate specific antibodies against many variants of human papilloma virus, which provide protection against a broad range of HPV variants. Without being bound by theory, antibodies to L2 may be particularly useful in a prophylactic vaccine, such as in subject who has not already been infected with HPV.
  • the full-length polypeptides of DP-C024.1 based on the template arrangement, sequence, and amino acid input distribution shown in this schematic are about 69 amino acids long and the amino acid positions, relative to full-length polypeptides based on the depicted template arrangement, of the composition are indicated in Row 1 (the row labeled peptide #).
  • the DP-C024.1 polypeptides e.g.
  • the polypeptides in the mixture of polypeptides comprising the DP-C024.1 composition are based on a template arrangement having a maximum of 3 random copolymer regions (RCRs) wherein the amino acid at each position is selected from lysine, alanine or glutamic acid, i.e., the RCRs comprise K/A/E motifs (N-terminal RCRa (corresponding to positions 1-4 in Figure 16), RCRb (corresponding to positions 33-36 in Figure 16), and RCRc (corresponding to positions 65-68 in Figure 16), and two antigenic regions (ARs) based on sequences derived from FIPV L2 (ARa (corresponding to positions 5- 32 in Figure 16) and ARb (corresponding to positions 37-64 in Figure 16).
  • RCRs random copolymer regions
  • the template arrangement of DP-C024.1 comprises: RCRa-ARa- RCRb-ARb-RCRc, although other template arrangements are described herein.
  • both AR a and ARb correspond to or comprise a region of an L2 protein of FIPV (e.g., a base peptide sequence based on a portion a L2 protein).
  • complexity across the polypeptides in the composition comes from both the RCRs and ARs although, in other examples, complexity may come only from the RCRs or from the RCRs and one, but not both, ARs.
  • the RCRs and ARs are contiguous. However, they may be interconnected by a linker, such as by one or more intervening amino acid residues.
  • the polypeptides in the composition may include on or more additional amino acid residues at the N- or C-terminus, including naturally occurring or non-natural amino acids.
  • Row 2 of the top and bottom panels indicates the positions that correspond to residues, which may undergo post-translational modifications to form cysteine bridges (positions 21 and 27), or residues, which represent boundaries of the L2 protein epitope in this example (positions 13 and 40).
  • Rows 3, 4, 5, and 6 indicate the possible amino acids, which are or may be incorporated at each position in the peptide.
  • the three copolymer regions (RCRs) correspond to positions 1-4 (RCRa), positions 33-36 (RCRb), and positions 65-68 (RCRc).
  • the two antigenic regions (ARs) correspond to positions 5-32 (ARa) and to positions 37-64 (ARb).
  • the sequence of the AR given in Row 3 corresponds to the base peptide sequence and reflects original amino acid residues derived from the corresponding portion of the target, an HPV L2.
  • Rows 7, 8, 9, and 10 indicate the molar input percentages of the corresponding, color-coded, amino acid above. For example, at position 5, the relative molar amino acid input percentages of serine (S) and alanine (A) are 90% and 10%, respectively. Additional amino acids such as conserved substitutions may be provided at any of the positions in the antigenic region.
  • the three RCRs in this example are 4 amino acids in length and contain either lysine (K), alanine (A), or glutamic acid (E) at each position. RCRs of differing sizes (e.g. shorter or longer than depicted) are contemplated, as described herein.
  • the relative molar input percentages of lysine, alanine, or glutamic acid were 40%, 50%, or 10%, relatively.
  • the relative molar input percentage of lysine, alanine, or glutamic acid was selected from within the foregoing ranges, independently for each position
  • the AR at positions corresponding to 5-32 of Figure 16 and the AR corresponding to positions 37-64 of Figure 16 are based on a base peptide sequence which substantially corresponds to positions 13-40 of an FIPV L2 (exemplary longer L2 protein set forth in SEQ ID No. 30; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 34).
  • base peptide sequence which substantially corresponds to positions 13-40 of an FIPV L2 (exemplary longer L2 protein set forth in SEQ ID No. 30; corresponding base peptide sequence used in this example set forth in SEQ ID NO: 34).
  • most positions of the ARs e.g.
  • positions 5, 7-10, 12, 14, 17-18, 20, 22-28, 31, 37, 39-42, 44, 46, 49-50, 52, 54-60, and 63 specify a relative molar input percentage of 10% alanine for generating complexity at those positions across the mixture of polypeptides, with the original amino acid (relative to the HPV L2 sequence) making up the remaining 90% of the input molar percentage of amino acid at each position and for generating complexity at those positions across the mixture. Therefore, in this example, at these positions, each polypeptide has either the original amino acid indicated in Row 3 or alanine in the ARs.
  • additional complexity is provided by, for example, also permitting variation at a position based on a conserved substitution, such as an orthologous substitution, a residue that is present in a different strain, a residue that is observed based on polymorphism or natural variation, or substitution with a similar residue.
  • a conserved substitution such as an orthologous substitution
  • a residue that is present in a different strain a residue that is observed based on polymorphism or natural variation, or substitution with a similar residue.
  • no amino acids other than the residue present at the corresponding position of the base peptide are provided.
  • Norleucine is found at the C-terminal most amino acid at position 69 of Figure 16.
  • compositions based on, for example, differing template arrangements, input percentages in the RCRs or ARs, and/or base peptide sequence or antigen are contemplated and provided.
  • a polypeptide mixture according to the schematic shown in Figure 16 can be successfully synthesized using solid-phase peptide synthesis. Briefly, the polypeptides based on this template arrangement and input percentages may be synthesized from C-terminus to N-terminus using Fmoc-based SPPS, as described above. To improve the coupling efficiency and generate a higher yield in each step, longer coupling times or the addition of redundant coupling reactions at one or more positions may be used.
  • the disclosure provides
  • Figure 17 shows a representative example of a MALDI-TOF-MS spectra comparing 2 different preparations of an amino acid copolymer composition, DP-CO 16.
  • F comprising a high complexity of polypeptides based on antigenic sequences derived from Tau and the template arrangement and amino acid inputs for DP-C016.F set forth in Example 8.
  • MALDI-TOF-MS Sample Preparation An amino acid copolymer composition was synthesized using solid phase peptide synthesis according to the method previously described in Example 8. Matrix solutions for MALDI-TOF-MS were prepared using the amino acid copolymer composition. Each sample contained about lOmg/ml of 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapinic acid) in an aqueous solvent solution comprising 0.1% acqueous trifluoroacetic acid (TFA) and acetronitrile. The amino acid copolymer composition was mixed with the matrix solution to create cocrystallized samples for analysis.
  • TFA trifluoroacetic acid
  • the cocrystallized samples were deposited onto the stainless steel autosampler pins of a MALDI-TOF-MS instrument and allowed to air dry.
  • the sample was irradiated at 337 nm with a nitrogen laser resulting in the analytes being protonated and desorbed into the gas phase.
  • the mass/charge (m/z) value was determined in a TOF mass analyzer.
  • the resulting composition synthesized had a purity of about 53% (e.g., calculated at about 52.61%).
  • Purity refers to the percentage of polypeptides in the composition based on the template arrangement that are substantially full-length polypeptides.
  • “about 53% purity” refers to a composition in which about 53% of the polypeptides in the composition that are based on the template arrangement are substantially full-length polypeptides.
  • DP-C016.F was prepared as described in Example 8. DPC016.F was then loaded onto Polylactic Acid (PLA) particles, particles which protect foreign antigens (Ags) from degradation and dilution. PLA particles are efficiently captured by phagocytes and improve Ag processing and presentation to T-cells.
  • Figure 18 shows a representative example of a MALDI-TOF-MS spectra of DP-CO 16.F loaded onto PLA particles.
  • PLA Polylactic Acid
  • MALDI-TOF-MS Sample Preparation An amino acid copolymer composition was synthesized using solid phase peptide synthesis according to the method previously described in Example 8. The DP-C016.F was then loaded onto PLA particles. Matrix solutions for MALDI-TOF-MS were prepared using the amino acid copolymer composition loaded onto PLA particles. Each sample contained about lOmg/ml of 3,5-dimethoxy-4-hydroxy cinnamic acid (sinapinic acid) in an aqueous solvent solution comprising 0.1% acqueous trifluoroacetic acid (TFA) and acetronitrile. The amino acid copolymer composition was mixed with the matrix solution to create cocrystallized samples for analysis.
  • TFA trifluoroacetic acid
  • the cocrystallized samples were deposited onto the stainless steel autosampler pins of a MALDI-TOF-MS instrument and allowed to air dry.
  • the sample was irradiated at 337 nm with a nitrogen laser resulting in the analytes being protonated and desorbed into the gas phase.
  • the mass/charge (m/z) value was determined in a TOF mass analyzer.
  • MALDI-TOF-MS Results The resulting full MALDI-TOF spectrum of DP-CO 16.F recovered from 4 months of loading onto PLA particles is shown in Figure 18.
  • Full-length polypeptides based on this template arrangement are about 63 amino acids and have a mass of about 6900 Daltons, and represents 83% purity.
  • Example 14 DP-C016.F is immunogenic in CD4+ and CD8+ T-cell proliferation assays DP-CO 16. F was tested for its ability to induce human T-cell proliferation in vitro.
  • CD4+ and CD8+ T-cell proliferation assays were conducted using healthy donor PBMC as described above in Example 9.
  • Figure 19A and 19B shows the relative fraction of the proliferating CD4+ and CD8+ T-cells, respectively.
  • DP-CO 16.F 7.5 ⁇ induced CD4+ T-cell proliferation in the majority of healthy donors' PBMCs, significantly better than the tetanus toxoid peptide TET 830 (TT) and as well as the flu HA peptide 317-319 (HA) (Fig. 19A).
  • DP-C016.F did not significantly induce CD8+ T- cell proliferation as compared to TT, indicating the potential for a CD4+ specific response.
  • DP-CO 16.F was loaded onto PLA particles and was tested for its ability to induce a CD4+ T-cell response.
  • the final concentrations of PLA particles were 860, 260, and 90 ⁇ g/mL in the cell culture wells containing 2.5, 0.75, and 0.25 ⁇ of DP-CO 16.F,
  • IFA Incomplete Freund's Adjuvant
  • mice were injected once a week for a total of 4 subcutaneous injections (day 0, 7, 14, and 21) without the use of anesthetics.
  • the injection was carried out in the form of an emulsion in IFA or loaded onto PLA particles, using glass syringes with metal connectors.
  • the compounds were injected at one concentration per group.
  • the groups treated with DP-CO 16.
  • F with PLA particles (0.3mg/kg) and with DP-C016.F with PLA particles (1.2 mg/kg) were further treated on day 49, 77, 105, and 133.
  • the DP-C016.F with PLA particles (0.3mg/kg) and DP-C016.F with PLA particles (1.2 mg/kg) treated groups were bled again.
  • the anti-immunogen antibody assay plate included test samples titered down (8 dilutions) with dilution buffer. All the plates were coated with the one of the following antigens: PFIF1, pS422, recTau and PFIF-tau. The presence of anti-immunogen antibodies was detected using an antibody ELISA.
  • Hind Limb Clasping Score is an assay which allows for the physical assessment of disease severity in a mouse model.
  • the J PL3 mouse model is known to develop hind limb dysfunction with age and increased disease severity.
  • To evaluate the hind limb clasping the mice were suspended by the base of the tail on Day 168. Each mouse was rated for hind limb clasping on a scale of 0 to 2; a score of 0 being no clasping, 1 moderate clasping and 2 severe clasping.
  • Figure 20A shows the results of the antibody ELISA against PHF-tau, which are paired helical filaments isolated from AD patients' autopsies, and recombinant Tau (recTau). Paired helical filaments (PHF) are one of the key pathological hallmarks in
  • recombinant Tau represents the major microtubule associated protein, which is part of a normal mature neuron.
  • Figure 20A shows that high titers of anti-PHF-tau antibodies are present. This indicates that the high complexity polypeptides of DP-C016.F emulsified in IFA 1.2 mg/kg induces a robust antibody response against pathologic Tau. The response to recTau is much lower in non- transgenic mice (data not shown).
  • Figure 20B shows the results of the antibody ELISA against short linear peptides covering either PHF1 or pS422.
  • the PHF-1 site includes pS396 and pS404 of Tau-F, and are commonly phosphorylated in Alzheimer's disease and other tauopathies.
  • position pS422 (corresponding to position 422 of SEQ ID No. 17) represents a key biomarkers for hyperphosphorylated Tau and this position is targeted by DP-C016.F.
  • Figure 20B shows that high titers of anti-PFIFl antibodies are present. There is also an increase in pS422 antibody titer when DP-CO 16.F is emulsified in IF A (1.2 mg/kg).
  • Figure 20C shows the antibody response on day 168 as compared to day 28.
  • the graph shows the results of the antibody ELISA against short linear peptide covering PFIF1, short peptide covering pS422, PFIF-tau, and recombinant Tau (recTau), respectively.
  • Mice treated with DP-CO 16.F loaded onto PLA particles (0.3 mg/kg) showed a sustained anti-tau response over 168 days, with the last treatment occurring on day 133.
  • Figure 20D shows the effect of DP-CO 16.
  • J PL3 mice were treated as described above. The results show a significant inverse correlation between anti-PFIFl antibody titer and hind limb clasping as compared to controls.
  • Example 16 DP-C016.F emulsified in IFA or loaded onto PLA particles increases soluble tau, decreases hyperphosphorylated tau, and decreases tau content in various brain regions of JNPL3 mice
  • mice were administered DP-CO 16. F with either Incomplete Freund's Adjuvant (IFA) or polylactic acid (PLA) particles, and the effect of on tau phosphorylation, total tau contents, and disease state was evaluated.
  • IFA Incomplete Freund's Adjuvant
  • PLA polylactic acid
  • PLA formulation DP-CO 16.F was loaded onto PLA particles. Formulation was optimized to achieve a compromise between yield of adsorption (% of loaded
  • the injections were carried out in the form of an emulsion in IFA or loaded onto PLA particles.
  • the mice were measured for hind limb clasping, antibody response, and were euthanized for histological analysis of tau in the cortex, hindbrain and hippocampus. Soluble, insoluble, and aggregated tau contents were measured. Anti-immunogen antibody determination.
  • the anti-immunogen antibody assay plate included test sample(s) titered down (8 dilutions) with dilution buffer. All the plates were coated with the one of the following antigens: DP-C016.F, recTau and PHF-tau. The presence of anti-immunogen antibodies was detected using both anti-IgGl and anti-IgG2a antibodies, in separate reactions.
  • Hind Limb Clasping Score is an assay which allows for the physical assessment of disease severity in a mouse model.
  • the J PL3 mouse model is known to develop hind limb dysfunction with age and increased disease severity.
  • To evaluate the hind limb clasping the mice were suspended by the base of the tail on Day 119. Each mouse was rated for hind limb clasping on a scale of 0 to 2; a score of 0 being no clasping, 1 moderate clasping and 2 severe clasping.
  • mice were inj ected with DP-CO 16.F emulsified in IF A (0.8 mg/kg), DP-
  • FIG. 21C shows the effect of DP- C016.F emulsified in IF A (0.8 mg/kg); DP-C016.F with PLA particles (0.8 mg/kg); or PLA particles alone on total soluble tau in the cortex, hindbrain and hippocampus of JNPL3 mice.
  • Abnormal accumulation of pathological tau species in the brain of J PL3 transgenic mice has been associated with tauopathic disease. Soluble tau (prefibrillar forms of tau) are potentially more toxic than insoluble tau (tangles), which may serve as a protective mechanism.
  • the DP-C016.F emulsified in IFA (0.8 mg/kg) treatment group also showed the largest decrease in soluble phosphoTau marked by PHF1 and DA31 antibodies in the hindbrain.
  • Figure 21H shows the effect of DP-C016.F emulsified in IFA (0.8 mg/kg); DP- CO 16. F with PLA particles (0.8 mg/kg); or PLA particles alone on hind limb clasping, and its correlation with total soluble tau in the cortex of JNPL3 mice. The results show that total tau content drops in DP-CO 16. F treated mice with severe clasping, and that total tau content increases with clasping severity in vehicle treated mice.
  • DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGW (SEQ ID NO: 1) ;
  • MATLEKLMKAFESLKSF (SEQ ID NO: 5) ;
  • HGAEIVYKSPWSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 6) ;
  • HGAEIVYKSPWSGDTSPRHL (SEQ ID NO: 7);
  • DMVDSPQLATLADEVSASLAKQGL (SEQ ID NO: 10) ;
  • ILARNLVPMV (SEQ ID NO: 21) ;
  • NLVPMVATV (SEQ ID NO: 24);
  • RIQRGPGRAFVTIGK (SEQ ID NO: 25) ;
  • TGSIDMVDSPQLATLADEVSASLAK (SEQ ID NO: 31) ;
  • TGSIDMVDSPQLATLA SEQ ID NO: 32
  • GSEAYEMPSEEGYQDYE (SEQ ID NO: 36) ;
  • Beta-2-microglobulin - human (P61769-1) MSRSVALAVL ALLSLSGLEA IQRTPKIQVY SRHPAENGKS NFLNCYVSGF HPSDIEVDLL KNGERIEKVE HSDLSFSKDW SFYLLYYTEF TPTEKDEYAC R HVTLSQP KIVKWDRDM
  • MOUSE ALPHA SYNUCLEIN (NP_001035916 )
  • Tau-F Microtubule-associated protein tau isoform 2 [Homo sapiens] NP 005901.2
  • TAU HUMAN (UNIPROTKB/SWISS-PROT P10636.5)

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Abstract

L'invention décrit un mélange de polypeptides comprenant une, ou plusieurs régions antigéniques, et une ou plusieurs régions de copolymères aléatoires. Les composés de copolymères d'acides aminés sont capables d'agir en tant qu'agents thérapeutiques spécifiques d'antigènes, et peuvent être utilisé comme vaccins. De telles composés sont synthétisées selon un ensemble de règles concernant l'identité et la fréquence d'occurrence d'acides aminés à chaque position le long des polypeptides. Les composés ainsi obtenues sont utilisées pour de nombreuses applications thérapeutiques, de recherche et de diagnostique.
PCT/US2017/040598 2016-07-01 2017-07-03 Composés d'amino acides de copolymères et leurs applications. WO2018006092A1 (fr)

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AU2017290907A AU2017290907A1 (en) 2016-07-01 2017-07-03 Amino acid copolymer compositions and uses thereof
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
JP2022500584A (ja) * 2018-09-05 2022-01-04 シーメンス エナジー グローバル ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディートゲゼルシャフトSiemens Energy Global Gmbh & Co. Kg 回転防止特徴を有する非接触シール

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WO2003029276A2 (fr) * 2001-10-03 2003-04-10 President And Fellows Of Harvard College Copolymeres destines a la suppression de maladies auto-immunes, et methodes d'utilisation
WO2005120542A2 (fr) * 2004-05-07 2005-12-22 Peptimmune, Inc. Methodes de traitement de maladie a l'aide de copolymeres aleatoires
WO2006031727A2 (fr) * 2004-09-13 2006-03-23 President And Fellows Of Harvard College Peptides destines au traitement de maladies auto-immunes
WO2007078443A2 (fr) * 2005-11-17 2007-07-12 Peptimmune, Inc. Methodes visant a traiter une maladie a l'aide de copolymeres aleatoires
WO2009075854A2 (fr) * 2007-12-10 2009-06-18 Peptimmune Inc. Quantification efficace de mélanges de peptides complexes dans des échantillons de tissus et procédés thérapeutiques améliorés
WO2009128948A1 (fr) * 2008-04-17 2009-10-22 Peptimmune, Inc. Conception et synthèse de compositions de polymères séquencés dirigées et leurs anticorps pour le traitement de troubles conformationnels des protéines
WO2011011706A2 (fr) * 2009-07-24 2011-01-27 The Johns Hopkins University Méthodes et compositions pour le traitement ou la prévention de maladies auto-immunes utilisant des agents immunomodulateurs
WO2012150495A1 (fr) * 2011-05-05 2012-11-08 National Institute Of Immunology Peptides synthétiques et copolymères aléatoires pour le traitement de troubles auto-immuns

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Publication number Priority date Publication date Assignee Title
WO2003029276A2 (fr) * 2001-10-03 2003-04-10 President And Fellows Of Harvard College Copolymeres destines a la suppression de maladies auto-immunes, et methodes d'utilisation
WO2005120542A2 (fr) * 2004-05-07 2005-12-22 Peptimmune, Inc. Methodes de traitement de maladie a l'aide de copolymeres aleatoires
WO2006031727A2 (fr) * 2004-09-13 2006-03-23 President And Fellows Of Harvard College Peptides destines au traitement de maladies auto-immunes
WO2007078443A2 (fr) * 2005-11-17 2007-07-12 Peptimmune, Inc. Methodes visant a traiter une maladie a l'aide de copolymeres aleatoires
WO2009075854A2 (fr) * 2007-12-10 2009-06-18 Peptimmune Inc. Quantification efficace de mélanges de peptides complexes dans des échantillons de tissus et procédés thérapeutiques améliorés
WO2009128948A1 (fr) * 2008-04-17 2009-10-22 Peptimmune, Inc. Conception et synthèse de compositions de polymères séquencés dirigées et leurs anticorps pour le traitement de troubles conformationnels des protéines
WO2011011706A2 (fr) * 2009-07-24 2011-01-27 The Johns Hopkins University Méthodes et compositions pour le traitement ou la prévention de maladies auto-immunes utilisant des agents immunomodulateurs
WO2012150495A1 (fr) * 2011-05-05 2012-11-08 National Institute Of Immunology Peptides synthétiques et copolymères aléatoires pour le traitement de troubles auto-immuns

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
US11827695B2 (en) 2017-02-17 2023-11-28 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
JP2022500584A (ja) * 2018-09-05 2022-01-04 シーメンス エナジー グローバル ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディートゲゼルシャフトSiemens Energy Global Gmbh & Co. Kg 回転防止特徴を有する非接触シール

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