Immunochemical conjugates: method and composition

• US 4,220,565 A
• Filed: 01/18/1979
• Issued: 09/02/1980
• Est. Priority Date: 01/18/1979
• Status: Expired due to Term

• Current Assignee: The Scripps Research Institute
• Original Assignee: Scripps Clinic & Research Foundation (Scripps Health)
• Inventors: Katz, David H.
• Primary Examiner(s): Danison, Walter C.
• Application Number:
  US06/004,333
• Time in Patent Office: 593 Days
• Field of Search: 260/6, 260/112.7, 528/311, 424/78, 424/88, 424/91, 525/432
• US Class Current: 525/54.1
• CPC Class Codes: A61K 2039/6093 Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/36 from pollen
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: C07K 1/10 using coupling agents C07K1/006 takes precedence
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins

Abstract

Techniques for preparing stable conjugates of protein-D-GL which can then be isolated in pure form are disclosed, using hen egg ovalbumin (OVA) as a prototype protein, and conjugation and purification methods are described which involve (1) application of a coupling method employing m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as the coupling reagent, and (2) introduction of biotin moieties into D-GL molecules to allow application of avidinbiotin system for affinity chromatographic purification of conjugates. These methods are applicable to the preparation of D-GL conjugates of insulin and Ragweed Antigen E and are of general applicability.

Claims

1. An improved method of preparing immunosuppressive conjugates of D-glutamic acid-D-lysine copolymer and an antigen, comprising the steps of:

(a) reacting D-glutamic acid-D-lysine copolymer with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-D-GL or with S-acetylmercaptosuccinic anhydride to form SH-D-GL;

(b) reacting antigen with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-Antigen or with S-acetylmercaptosuccinic anhydride to form SH-Antigen;

(c) reacting MB-D-GL with SH-Antigen or reacting SH-D-GL with MB-Antigen to form said conjugates of D-glutamic acid-D-lysine copolymer with antigen.

2. An improved method for preparing conjugates of D-glutamic acid-D-lysine copolymer and insulin comprising the steps of:

(a) reacting D-glutamic acid-D-lysine copolymer with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-D-GL or with S-acetylmercaptosuccinic anhydride to form SH-D-GL;

(b) reacting insulin with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-In or with S-acetylmercaptosuccinic anhydride to form SH-In; and

(c) reacting MB-D-GL with SH-In or reacting SH-D-GL with MB-In to form said conjugate of D-glutamic acid-D-lysine copolymer conjugate with insulin.

3. An improved method for preparing conjugates of D-glutamic acid-D-lysine copolymer and ragweed antigen E comprising the steps of:

(a) reacting D-glutamic acid-D-lysine copolymer with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-D-GL or with S-acetylmercaptosuccinic anhydride to form SH-D-GL;

(b) reacting ragweed antigen E with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-AgE or with S-acetylmercaptosuccinic anhydride to form SH-AgE; and

(c) reacting MB-D-GL with SH-AgE or reacting SH-D-GL with MB-AgE to form said conjugate of D-glutamic acid-D-lysine copolymer conjugate with ragweed antigen E.

4. An improved method of preparing immunosuppressive conjugates of D-glutamic acid-D-lysine copolymer and a protein, comprising the steps of:

(a) reacting D-glutamic acid-D-lysine copolymer with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-D-GL or with S-acetylmercaptosuccinic anhydride to form SH-D-GL;

(b) reacting protein with either m-maleimidobenzoyl-N-hydroxysuccinimide ester to form MB-protein or with S-acetylmercaptosuccinic anhydride to form SH-protein;

(c) reacting MB-D-GL with SH-protein or reacting SH-D-GL with MB-protein to form said conjugates of D-glutamic acid-D-lysine copolymer with protein.