Directed sequence polymer compositions and antibodies thereof for the treatment of protein conformational disorders

Abstract

The instant invention comprises a process for the solid phase synthesis of directed epitope peptide mixtures useful in the treatment and diagnosis of protein conformational disorders, such process defined by a set of rules regarding the identity and the frequency of occurrence of amino acids that substitute a base or native amino acid of a known epitope. The resulting composition is a mixture of related peptides for therapeutic use. The invention also pertains to the process of generating antibodies using the directed epitope peptide mixtures as the antigens, and antibodies generated by such process, useful in the treatment and diagnostics of the said protein conformational disorder.

1 - 34 . (canceled) 
     
     
         35 . A process for generating antibodies comprising the steps of:
 (i) preparing a composition comprising directed-sequence polymers (DSPs), wherein the DSP composition has a complexity of greater than 5×10 2  different DSPs, comprising the steps of:
 (1) selecting a base peptide sequence, wherein the base peptide sequence is derived from an epitope of an antigen associated with a protein conformational disorder, 
 (2) synthesizing by solid phase peptide synthesis a first cassette of the DSPs, the cassette having a sequence of amino acid positions corresponding to each amino acid of the base peptide sequence,
 wherein, for at least one amino acid position of the first cassette of the DSPs, an amino acid is added, said amino acid randomly selected from a mixture of amino acids consisting of the original amino acid found at the corresponding amino acid position of the base peptide sequence, optionally alanine (A), and, optionally, at least one conserved substitution, 
 wherein the amino acids in the mixture are present in a fixed molar input ratio relative to each other, determined prior to starting synthesis, 
 wherein the relative molar amount of A is more than 10% and less than 90% of the total amino acid concentration of the DSPs; 
 
 (3) optionally extending the length of the DSPs by
 (a) repeating step (2) for 1 to 15 cycles and elongating the DSPs under the same condition including the input ratio of amino acids in the mixture; 
 (b) repeating step (2) for 1 to 15 cycles and elongating the DSPs, for each cycle, using a different input ratio of amino acids in the mixture; 
 (c) repeating steps (1) and (2) for 1 to 15 cycles and elongating the DSPs using cassettes based on more than one base peptide; 
 (d) assembling 1 to 15 cassettes synthesized in a single cycle of step (2); or 
 (e) assembling 1 to 15 cassettes, the first cassette synthesized under one condition of step (2), and second and more cassettes synthesized under one or more different conditions of step (2); 
 
   wherein the number of cycles selected in step (3) is selected so that the final length of the DSPs is 10 to 300 amino acid residues;   (ii) administering said DSP composition to an animal; and   (iii)(a) isolating antibodies immunoreactive with said DSP composition from said animal, or   (iii)(b) isolating cells that produce antibodies immunoreactive with said DSP composition from said animal, and then isolating antibodies immunoreactive with said DSP composition from said isolated cells.   
     
     
         36 . The process according to  claim 35 , wherein the antibodies are immunoreactive with a protein comprising the base peptide, with or without post-translational modification, and wherein said protein is a full-length protein associated with the protein conformational disorder or a fragment of such full-length protein, and wherein said protein is in a pathological or non-pathological conformation. 
     
     
         37 - 56 . (canceled) 
     
     
         57 . A process for manufacturing a composition comprising directed-sequence polymers (DSPs), wherein the DSP composition has a complexity of greater than 5×10 2  different DSPs, comprising the steps of:
 (1) selecting a base peptide sequence, wherein the base peptide sequence is derived from an epitope of an antigen associated with a protein conformational disorder, 
 (2) synthesizing by solid phase peptide synthesis a first cassette of the DSPs, the cassette having a sequence of amino acid positions corresponding to each amino acid of the base peptide sequence,
 wherein, for at least one amino acid position of the first cassette of the DSPs, an amino acid is added, said amino acid randomly selected from a mixture of amino acids consisting of the original amino acid found at the corresponding amino acid position of the base peptide sequence, optionally alanine (A), and, optionally, at least one conserved substitution, 
 wherein the amino acids in the mixture are present in a fixed molar input ratio relative to each other, determined prior to starting synthesis, 
 wherein the relative molar amount of A is more than 10% and less than 90% of the total amino acid concentration of the DSPs; 
 
 (3) optionally extending the length of the DSPs by
 (a) repeating step (2) for 1 to 15 cycles and elongating the DSPs under the same condition including the input ratio of amino acids in the mixture; 
 (b) repeating step (2) for 1 to 15 cycles and elongating the DSPs, for each cycle, using a different input ratio of amino acids in the mixture; 
 (c) repeating steps (1) and (2) for 1 to 15 cycles and elongating the DSPs using cassettes based on more than one base peptide; 
 (d) assembling 1 to 15 cassettes synthesized in a single cycle of step (2); or 
 (e) assembling 1 to 15 cassettes, the first cassette synthesized under one condition of step (2), and second and more cassettes synthesized under one or more different conditions of step (2); 
 
 
       wherein the number of cycles selected in step (3) is selected so that the final length of the DSPs is 10 to 300 amino acid residues. 
     
     
         58 . The process according to  claim 57 , wherein the antigen is associated with a protein conformational disorder affecting the central and/or peripheral nervous system. 
     
     
         59 . The process according to  claim 57 , wherein the protein conformational disorder is Parkinson's disease. 
     
     
         60 . The process according to  claim 57 , wherein the protein conformational disease is dialysis-related amyloidosis. 
     
     
         61 . The process according to  claim 57 , wherein the first base peptide sequence is selected from the group consisting of SEQ ID NO: 3 through 13. 
     
     
         62 . The process according to  claim 57 , wherein A is more than about 10% and less than about 70% of the total amino acid concentration of the DSPs. 
     
     
         63 . The process according to  claim 57 , wherein A is more than about 15% and less than about 50% of the total amino acid concentration of the DSPs. 
     
     
         64 . The process according to  claim 57 , wherein the conserved substitution is defined according to the amino acid similarity table shown in  FIG. 4 . 
     
     
         65 . The process according to  claim 57 , wherein the conserved substitution is determined based on empirical data of known naturally occurring variants of the epitope. 
     
     
         66 . The process according to  claim 57  wherein, for at least one amino acid position of the first cassette of the DSPs, an amino acid is added, said amino acid randomly selected from a mixture of amino acids comprising an original amino acid found at the corresponding amino acid position of the base peptide sequence, alanine (A), and, optionally, at least one amino acid serving as a conserved substitution.