Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1999-10-13
2003-06-10
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S389100, C530S389800, C530S391100, C530S391500, C530S391700, C530S402000, C530S408000, C424S001110, C424S001650, C424S001690, C424S009300, C424S009322, C424S009351, C424S193100, C424S194100, C436S063000, C436S504000, C436S512000, C435S007900, C435S188000, C435S810000
Reexamination Certificate
active
06576746
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods for introducing thiol-containing linkers onto disease targeting agents that contain disulfide bonds, the radiolabeled targeting agents and drug conjugates produced using these methods, and use of the radiolabeled or drug-bearing targeting agents for diagnosis and treatment.
2. Description of Related Art
Free thiols offer a unique chemical handle for the attachment of numerous species to specific targeting agents, because of the specificity of the thiol group for reactive groups such as haloacetates, maleimides, and activated sulfonyl groups, and for reduced metal species such as reduced pertechenetate and perrhenate, and certain other thiophilic metals such as zinc, copper, mercury, cadmium, platinum, palladium, lead and bismuth. However, a complicating issue with many proteins, polypeptides and peptides is the presence of disulfide bonds that are critical to their structural integrity. The inherent reactivity of the thiol group can lead to breakage of such disulfide bonds, with possible formation of mixed disulfides, and an inability of the proteins, polypeptides and peptides to bind to their target antigen or receptor.
Antibody fragments, as well as sub-Fab′ fragments, single-chain antibodies, diabodies, polypeptides and peptides, offer advantages for in-vivo targeting of radioimaging and radio-therapeutic isotopes and drugs because the smaller fragments will target and clear faster than an intact IgG or larger protein. For example, a radiolabeled antibody fragment delivers a dose of a therapeutic or imaging isotope to a target more quickly than intact IgG and the faster clearance will minimize the radiometric dose to the non-target tissues. Rapid targeting is especially important for isotopes with short half-lives such as Tc-99m (t½=6 hr) or Re-188 (t½=17 hr). Tc and Re cations bind strongly to thiol-containing ligands but conjugation of these ligands to antibodies or antibody fragments can present some difficulties.
A divalent antibody fragment such as a F(ab′)
2
fragment should have increased total targeting compared to a Fab′ fragment because the divalent binding region will increase the affinity of the protein for the antigen. F(ab′)
2
fragments are made up of two Fab′ fragments joined by one or more disulfide bonds, which are sensitive to reduction by free thiols both during and after the conjugation of a thiol-containing moiety. Thus, it is necessary to conjugate a thiol-containing ligand to the protein either using a protected thiol which is subsequently deprotected, or using a low thiol concentration and hydrophilic thiols to minimize the interaction of the free thiols on the ligand with the disulfides on the antibody.
Another problem that can occur during conjugation to a non-specific site on a targeting agent is that the conjugate may be bound to or near the antigen-binding region of an antibody or the receptor-binding region of a peptide/polypeptide, which can reduce or eliminate the binding affinity of the antibody or peptide for the antigen or receptor. The conjugation of haptens to periodate-oxidized carbohydrate sites (aldehydes and ketones) is one method of site-specifically forming conjugates. The carbohydrate regions can be genetically engineered into specific sites on proteins or peptides so, for example, it is possible to place a carbohydrate at a site on a F(ab′)
2
fragment that will not interfere with the binding of the antibody fragment to the antigen.
The presence of carbohydrate residues on the light chains of certain IgGs has been established. Such residues remain on F(ab′)
2S
and F(ab)
2S
after pepsin or papain digestion, respectively. As such, they represent a masked potential site-specific chemical handle for haptenic attachment. In addition, certain murine antibodies have been reengineered to produce humanized complementarity determining region versions of the same antibodies, while simultaneously engineering glycosylation sites at positions remote to the antibody's antigen-binding site. This enables the insertion of carbohydrate at desired positions within the protein, including insertion of carbohydrate in the CH
1
domain and the variable region on either light or heavy chains.
It is also necessary to have a conjugate which is sufficiently stable in-vitro, and in-vivo so that the biodistribution of the radiolabel reflects the biodistribution of the antibody fragment. If the linkage of the conjugate to the antibody or the attachment of the radioisotope to the conjugate is unstable then there may be a substantial reduction of radioisotope that reaches the target. The radioisotope that separates from the protein may contribute to the background activity, which would further obscure targeting.
A continuing need exists to prepare thiol-containing disulfide-linked targeting vectors which can be, readily radiolabeled with thiophilic metal ions for use in radioimaging and radiotherapy, or substituted with drugs for targeted chemotherapy. Such an invention must successfully address the multiple problems discussed above.
SUMMARY OF THE INVENTION
One object of the present invention is to provide conjugates of disulfide-containing targeting proteins, polypeptides and peptides, e.g., divalent antibody fragments and (SV)
2S
, with thiol-containing ligands without cleaving the disulfide bonds of the targeting proteins.
Another object of the invention is to use the substituted thiol group attached to the disulfide-containing proteins or peptides as a specific chemical handle to further attach certain radioisotopes or chemotherapy agents.
Another object of the invention is to provide radiolabeled proteins that are stable in vitro and in vivo.
Yet another object is to provide methods for the use of stably substituted disulfide-containing proteins, polypeptides and peptides for radiodiagnosis, radiotherapy and chemotherapy of disease.
These and other objects are achieved by providing a method of producing a diagnostic or therapeutic conjugate of a protein, polypeptide or peptide containing at least one disulfide bond which is necessary to maintain its biological activity, and bearing at least one thiol-containing moiety linked thereto through a hydrazone or hydrazine linkage, comprising contacting the protein, polypeptide or peptide with a thiol-reactive diagnostic or therapeutic agent, either preformed or generated in situ, to form a stable diagnostic or therapeutic conjugate of the protein, polypeptide or peptide without substantial cleavage of the disulfide bond.
In the foregoing method, the thiol-containing moiety linked to the protein, polypeptide or peptide through a hydrazone or hydrazine linkage is joined by reacting the disulfide bond-containing protein, polypeptide or peptide which also contains an aldehyde or ketone group with a thiol-hydrazine of the formula HS-Q-NHNH
2
, wherein Q is a linking moiety selected from the group consisting of alkyl groups, aryl groups, cycloalkyl groups, peptides, and combinations thereof; and optionally reducing the resultant hydrazone to a hydrazine.
The diagnostic or therapeutic agent in the conjugate can be a thiol-binding cationic. radioisotope or a drug derivative comprising a thiol-binding linker.
In one preferred embodiment, the protein is a glycosylated divalent antibody fragment whose partially oxidized carbohydrate portion is joined through the hydrazone or hydrazine linkage to the thiol-containing moiety.
Preformed, stable kits for effecting radiolabeling according to the foregoing method also are provided.
DETAILED DESCRIPTION
The present inventors have developed a method for conjugating a thiol-containing peptide linker or ligand to a disulfide-containing protein or peptide, e.g., a F(ab′)
2
, through a carbonyl function, e.g., a periodate oxidized carbohydrate portion of the protein, without reducing disulfide bonds that maintain structure and/or conformation related to activity, e.g., reduction of a F(ab′)
2
fragment to Fab&
Griffiths Gary L.
McBride William J.
Foley:Lardner
Immunomedics Inc.
Low Christopher S. F.
Mohamed Abdel A.
LandOfFree
Site-specific labeling of disulfide-containing targeting... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Site-specific labeling of disulfide-containing targeting..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Site-specific labeling of disulfide-containing targeting... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3108037