A-BETA IMMUNOGENIC PEPTIDE CARRIER CONJUGATES AND METHODS OF PRODUCING SAME
The present invention is directed to methods of producing conjugates of Aβ peptide immunogens with protein/polypeptide carrier molecules, which are useful as immunogens, wherein peptide immunogens are conjugated to protein carriers via activated functional groups on amino acid residues of the carrier or of the optionally attached linker molecule, and wherein any unconjugated reactive functional groups on amino acid residues are inactivated via capping, thus retaining the immunological functionality of the carrier molecule, but reducing the propensity for undesirable reactions that could render the conjugate less safe or effective. Furthermore, the invention also relates to such immunogenic products and immunogenic compositions containing such immunogenic products made by such methods.
1 - 379 . (canceled)
380 . A method for inducing an immune response in a mammalian subject, the method comprising administering to the subject an effective amount of a immunogenic composition comprising an immunogenic conjugate of a peptide immunogen with a carrier protein, the immunogenic conjugate generated by:
(a) introducing a reactive group into an amino acid residue of the peptide immunogen, wherein the peptide immunogen is an Aβ peptide or a fragment or analog thereof;
(b) derivatizing one or more functional groups of the carrier protein to generate an activated functional group on the carrier protein;
(c) reacting the peptide immunogen of step (a) with the carrier protein of step
(b) under conditions to form a conjugate, wherein the reactive group of the peptide immunogen is covalently attached to the activated functional group on the carrier protein; and
(d) further reacting the conjugate of step (c) with a capping reagent to inactivate any remaining activated functional group on the carrier protein to generate the immunogenic conjugate.
381 . The method of claim 380 , wherein the carrier protein is selected from the group consisting of human serum albumin, keyhole limpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin, influenza hemagglutinin, PADRE polypeptide, malaria circumsporozite (CS) protein, hepatitis B surface antigen (HBSAg19-28), Heat Shock Protein (HSP) 65, Mycobacterium tuberculosis , cholera toxin, cholera toxin mutants with reduced toxicity, diphtheria toxin, CRM 197 protein that is cross-reactive with diphtheria toxin, recombinant Streptococcal C5a peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes ORF 1664, Streptococcus pyogenes ORF2452, Streptococcus pneumoniae pneumolysin, pneumolysin mutants with reduced toxicity, Chlamydia pneumoniae ORF T367 , Chlamydia pneumoniae ORF T858, Tetanus toxoid, HIV gp120 T1, components recognizing microbial surface adhesive matrix molecules (MSCRAMMS), growth factors, hormones, cytokines and chemokines.
382 . The method of claim 381 , wherein the carrier protein is CRM 197 .
383 . The method of claim 380 , wherein the peptide immunogen is an Aβ fragment.
384 . The method of claim 383 , wherein the Aβ fragment is selected from the group consisting of Aβ1-3, 1-4, 1-5, 1-6, 1-7, 1-9, 1-10, 1-11, 1-12, 1-16, 1-28 3-6, 3-7, 13-28, 15-24, 16-22, 16-23, 17-23, 17-24, 18-24, 18-25, 17-28, 25-35, 33-42, 35-40, and 35-42.
385 . The method of claim 380 , wherein the functional group of the carrier protein is derivatized using a cross-linking reagent.
386 . The method of claim 385 , wherein the functional group is derivatized with a haloacetylating agent.
387 . The method of claim 380 , wherein the capping reagent that is used to inactivate any activated functional group on the carrier protein is selected from the reagent group consisting of cysteamine, N-acetylcysteamine, ethanolamine, sodium hydroxide, sodium carbonate, ammonium bicarbonate and ammonia.
388 . The method of claim 380 , wherein introducing the reactive group into the peptide immunogen comprises adding an amino acid residue having the reactive group.
389 . The method of claim 388 , wherein the amino acid residue is a cysteine residue in which the reactive group comprises —SH, or the amino acid residue is an arginine residue and the reactive group comprises a guanidyl group, or the amino acid residue is a glutamate or aspartate residue and the reactive group comprises —COOH, or the amino acid residue is a lysine residue and the reactive group comprises —NH 2 .
390 . The method of claim 384 , wherein the reactive group introduced into the peptide immunogen comprises a —SH of a cysteine residue, and wherein the cysteine residue is introduced into the Aβ peptide immunogen by addition during peptide synthesis.
391 . The method of claim 390 , wherein the cysteine residue is localized at the carboxy-terminus of the Aβ peptide immunogen.
392 . The method of claim 380 , wherein introducing the reactive group into the peptide immunogen comprises generating a pendant thiol group on an amino acid residue susceptible to such modification via a thiolating reagent.
393 . The method of claim 392 , wherein the thiolating reagent comprises N-acetylhomocysteine thiolactone.
394 . The method of claim 388 , wherein the amino acid residue is localized at the amino-terminus of the Aβ peptide immunogen or at the carboxy-terminus of the Aβ peptide immunogen.
395 . The method of claim 392 , wherein the amino acid residue is localized at the amino-terminus of the Aβ peptide immunogen or at the carboxy-terminus of the Aβ peptide immunogen.
396 . The method of claim 380 , wherein the carrier protein further comprises one or more polypeptide linkers covalently attached to the carrier protein, and wherein the one or more functional groups comprise a substituent of the one or more polypeptide linkers.
397 . The method of claim 380 , further comprising administering to the subject one or more adjuvants selected from the group consisting of GM-CSF, 529SE, IL-12, aluminum phosphate, aluminum hydroxide, Mycobacterium tuberculosis, Bordetella pertussis , bacterial lipopolysaccharides, aminoalkyl glucosamine phosphate compounds, MPL™ (3-O-deacylated monophosphoryl lipid A), a polypeptide, Quil A, STIMULON™ QS-21, a pertussis toxin (PT), an E. coli heat-labile toxin (LT), IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon-α, interferon-β, interferon-γ, G-CSF, TNF-α and TNF-β.