Title:  Method of affinity cross-linking biologically active immunogenic peptides to antibodies

United States Patent:  6,238,667

Appl. No.:  070907

A method of affinity cross-linking a peptide to an antibody by photo-chemically activating an azido compound in a peptide including said azido compound; adding an antibody to the photochemically activated peptide; and allowing the photochemically activated peptide and the antibody to react. The azido compound has an affinity for a hydrophobic structure in the variable domain of the antibody which binds to nucleotides or nucleosides, binding the peptide into a native binding pocket of the immunoglobulin (Ig) structure of an antibody. The site of cross-linking is located away from the antigen binding site in the Fv domain avoiding the compromise of antigen recognition. A composition of a peptide cross-linked to an antibody is also disclosed.

SUMMARY OF THE INVENTION

The present invention provides a method of affinity cross-linking a peptide to an antibody comprising the steps of

(a) photo-chemically activating an azido compound in a peptide comprising said azido compound;

(b) adding an antibody to said photochemically activated peptide; and

(c) allowing said photochemically activated peptide and said antibody to react, wherein said azido compound has an affinity for a hydrophobic structure in the variable domain of said antibody which binds to nucleotides or nucleosides, binding said peptide into a native binding pocket of the immunoglobulin (Ig) structure of an antibody, and wherein the site of cross-linking is located away from the antigen binding site in the Fv domain avoiding the compromise of antigen recognition.

In a preferred embodiment the photoreactive azido compound is created by oxidizing azido-adenosine with periodite to produce an azido-dialdehyde compound, wherein said dialdehyde is reacted via a Schiff-base reaction with primary amines of peptides of said antibody and photolyzed with UV light into an affinity site of said antibody.

In an alternative embodiment the azido compound is 5-azido tryptophan or 6-azido tryptophan, and wherein said azido compound is added to the C-terminal or N-terminal position of the peptide by standard peptide synthesis technology.

The invention also provides a composition and a pharmaceutical composition comprising a photochemically activated peptide having an N or C terminal azido compound, wherein said N or C terminal azido compound is crosslinked to an antibody and said azido compound has an affinity for a hydrophobic structure in the variable domain of said antibody which binds to nucleotides or nucleosides, binding said peptide into a native binding pocket of the immunoglobulin (Ig) structure of an antibody, and wherein the site of cross-linking is located away from the antigen binding site in the Fv domain avoiding the compromise of antigen recognition.

The invention of inserting biologically and immunologically active peptides into the variable domain of antibodies includes peptides which present T-cell and B-cell epitopes, comprise selfbinding, stimulate lymphocytes and allow transport across biological membranes.

The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed description and figures, wherein only the preferred embodiments of the invention are shown and described, simply by way of illustration of the best mode of carrying out the invention. As is readily recognized the invention is capable of modifications within the skill of the relevant art without departing from the spirit and scope of the invention.

Claim 1 of 17 Claims

What is claimed is:

1. A method of affinity cross-linking a peptide to an antibody so that the peptide becomes attached to the antibody at a location where the peptide does not compromise the antigen recognition of the antibody, the method comprising the steps of

(a) providing an antibody, the antibody having a variable domain, the variable domain including a hydrophobic structure, the hydrophobic structure defining a binding pocket having a tryptophan-binding site, and wherein the hydrophobic structure is located away from the antigen binding site that is in the Fv domain of the antibody,

(b) providing a peptide that has an azido tryptophan residue, the azido tryptophan residue having an affinity for the hydmphobic-:structure of the variable domain of the antibody,

(c) photo-chemically activating the azido tryptophan residue of the peptide, and

(d) allowing the peptide and the antibody to interact whereby the photo-chemically activated azido tryptophan residue binds to the hydrophobic structure of the variable domain and reacts with the tryptophan-binding site whereby the peptide becomes cross-linked to the antibody,

whereby, because the location of the hydrophobic structure is away from the antigen binding site that is in the Fv domain of the antibody, the cross-linked peptide does not compromise the antigen recognition of the antibody.

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