A-β immunogenic peptide carrier conjugates and methods of producing same

• US 9,089,510 B2
• Filed: 07/07/2011
• Issued: 07/28/2015
• Est. Priority Date: 12/17/2003
• Status: Expired due to Fees

• Current Assignee: Janssen Sciences Ireland UC (Johnson & Johnson)
• Original Assignee: Janssen Sciences Ireland UC (Johnson & Johnson), Wyeth LLC (Pfizer Inc.)
• Inventors: Arumugham, Rasappa G., Prasad, A. Krishna, Hagen, Michael
• Primary Examiner(s): GUDIBANDE, SATYANARAYAN R
• Application Number:
  US13/178,428
• Publication Number:
  US 20110287042A1
• Time in Patent Office: 1,482 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 2039/6031 Proteins
• CPC Class Codes: A61K 2039/6081 Albumin; Keyhole limpet haemocyanin [KLH]
• CPC Class Codes: A61K 2039/62 characterised by the link between antigen and carrier
• CPC Class Codes: A61K 39/0007 Nervous system antigens; Prions
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61K 47/646 the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
• CPC Class Codes: A61P 25/28 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
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

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.

Claims

1. A method of producing an immunogenic conjugate, the method comprising the steps of:

(a) derivatizing one or more functional groups of a carrier protein to generate an activated functional group on the carrier protein;

(b) reacting a peptide immunogen comprising an Aβ

fragment with the carrier protein of step (a) under conditions to form a conjugate, wherein the Aβ

fragment comprises residues 16-23 of Aβ

(SEQ ID NO;

21) and the peptide immunogen is covalently attached to the activated functional group on the carrier protein; and

(c) further reacting the conjugate of step (b) with N-acetylcysteamine to inactivate any remaining activated functional group on the carrier protein to generate the immunogenic conjugate, wherein a capping molecule, formed by reaction of the conjugate with the N-acetylcysteamine, is covalently attached to the remaining activated functional group on the carrier protein,

wherein the immunogenic conjugate has the formula;

2. The method of claim 1, further comprising a step of introducing a reactive group into the peptide immunogen before step (b).

3. The method of claim 2, wherein the step of introducing the reactive group into the peptide immunogen comprises adding an amino acid residue having the reactive group.

4. The method of claim 3, 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 —

NH₂.

5. The method of claim 4, 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 peptide immunogen during peptide synthesis.

6. The method of claim 5, wherein the cysteine residue is added to the carboxy-terminus of the peptide immunogen.

7. The method of claim 5, wherein the cysteine residue is added to the amino-terminus of the peptide immunogen.

8. The method of claim 5, wherein the peptide immunogen further comprises a peptide linker disposed between the Aβ

fragment and the cysteine residue.

9. The method of claim 2, wherein the step of 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.

10. The method of claim 9, wherein the thiolating reagent is N-acetylhomocysteine thiolactone.

11. The method of claim 9, wherein the amino acid residue is localized at the amino-terminus of the peptide immunogen or at carboxy-terminus of the peptide immunogen.

12. The method of claim 1, wherein the immunogenic conjugate comprises from 5 to 25 peptide immunogens covalently attached to the carrier protein.

13. The method of claim 12, wherein the immunogenic conjugate comprises from 12 to 20 peptide immunogens covalently attached to the carrier protein.

14. The method of claim 13, wherein the immunogenic conjugate comprises from 12 to 15 peptide immunogens covalently attached to the carrier protein.

15. The method of claim 6, wherein the peptide immunogen is KLVFFAED-C(SEQ ID NO:

45).

16. A method of producing an immunogenic conjugate, the method comprising:

(a) derivatizing one or more lysine residues of a CRM₁₉₇ carrier protein to generate an activated functional group on the carrier protein;

(b) reacting a peptide immunogen comprising residues 16-23 of Aβ

(SEQ ID NO;

21) and a C-terminal cysteine residue with the carrier protein of step (a) under conditions to form a conjugate; and

(c) further reacting the conjugate of step (b) with N-acetylcysteamine to inactivate any remaining activated functional group on the carrier protein to generate the immunogenic conjugate,

wherein the immunogenic conjugate has the formula;

17. The method of claim 16, wherein n is an integer greater than or equal to 5, but less than or equal to 25.

18. The method of claim 17, wherein n is an integer greater than or equal to 12, but less than or equal to 15.

19. The method of claim 18, wherein the peptide immunogen is KLVFFAED-C(SEQ ID NO:

45).

20. The method of claim 16, wherein the immunogenic conjugate has the formula:

21. The method of claim 20, wherein n plus p equals from 20 to 22.