Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1994-01-13
2003-01-21
Chan, Christina (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387300, C530S402000, C530S409000, C530S866000, C435S972000, C436S547000, C436S512000
Reexamination Certificate
active
06509451
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to cross-linked antibodies, to compositions containing them, to processes for their preparation, and to their use in medicine and for diagnosis.
BACKGROUND TO THE INVENTION
Antibody conjugates, in which antibodies are covalently attached to reporter or effector groups, have been used in diagnosis, and, to a more limited extent, in therapy. The antibody is generally selected on the basis of its affinity and specificity for a particular target antigen, such that, in use, it should be able to deliver the reporter of effector group to a desired site and maintain it there for a length of time.
Bispecific heterodimeric antibodies have been previously described in which Fab′ fragments have been joined via a thioether linkage [Glennie M. J. et al J. Immunol. 139, 2367, (1987)]. Antibodies in which the fluorescein derivative crabescein has been linked across a disulphide bond have also been described [Packard, B. P. et al, Biochemistry 25, 3548 (1986)].
We have now found that by cross-linking at least two chains of an antibody in a cross-linkage away from the antigen binding domains the binding capacity of the modified antibody may be advantageously enhanced relative to the unmodified antibody. In vivo, modified antibodies of this type also have good blood clearance and, in the presence of a tumour, give advantageously high tumours; blood and tumour: bone ratios. Such antibodies, when labelled with a reporter or effector group thus offer potential advantages over conventional labelled antibodies.
SUMMARY OF THE INVENTION
Thus, according to one aspect of the invention we provide a cross-linked antibody conjugate comprising a labelled antibody molecule having at least one non-disulphide interchain bridge, said bridge being attached to each chain at one or more bridging sites located outside of the antigen binding domains of the antibody.
In the antibody conjugates according to the invention, the interchain bridge may link any heavy or light chain in the antibody molecule to one or more other heavy and/or light chains in the same molecule. Preferably, however, the bridge will link two chains, particularly two heavy chains. More than one interchain bridge group may be present, although conjugates with one such bridge are preferred.
The term non-disulphide interchain bridge is intended to mean that S-S bridges of the type normally found in antibodies are excluded.
The bridging site in each antibody chain may generally be at the side-chain of an amino acid residue forming part of the chain but not directly participating in the antigen binding domain. Suitable amino acids include those with a side-chain containing an amino, sulphydryl, carboxyl, phenolic or other aromatic or heteroaromatic functional group through which the interchain bridge may be linked. Particular amino acid residues of these types include lysine, cysteine, glutamic acid, aspartic acid and tyrosine residues. Alternatively, the bridging site may be at a carbohydrate residue attached to the antibody chain, particularly an oxidised carbohydrate residue containing at least one aldehyde group through which the intercahin bridge may be linked.
Particularly preferred bridging sites are the sulphydryl groups of cysteine residues, for example those normally functioning in interchain disulphide bridges. Preferred sites of this type are sulphydryl groups of cysteine residues present in heavy chains in the hinge region of the antibody.
In another preference, the bridging site may be at the side-chain of an amino acid residue not normally present in the immunoglobulin, but which has been introduced through the use of recombinant DNA techniques as described hereinafter. Such sites include the sulphydryl and amino groups of cysteine and lysine residues respectively.
The interchain bridges in conjugates according to the invention may in general be of any desired length or composition. Suitable bridges include residues of homo- or heterofunctional cross-linking reagents, particularly homo- or heterobifunctional cross-linking reagents. Thus, the bridges may be such that they link two or more bridging sites. Particular bridges include optionally substituted polyvalent, especially bivalent, radicals of aliphatic, aromatic or araliphatic compounds.
The interchain bridge may for example have the structure [—X
1
—]
m
—Y
1
—Y
2
—[—X
2
—]
n
(where X
1
and X
2
is each the residue of a reactive functional group, Y
1
and Y
2
together form the remainder of the bridge and m and n, which may be the same or different is each an integer 1 or more).
In bridges of the above particular types Y
1
and Y
2
together may be straight or branched C
1-20
alkylene (e.g. C
1-10
alkylene such as C
4-10
alkylene, e.g. butylene, pentylene, hexylene, or heptylene), C
2-20
alkenylene or C
2-20
alkynylene chains, optionally interrupted by one or more —O— or —S— atoms or C
5-8
cycloalkylene (e.g. cyclopentylene or cyclohexylene), C
6-12
aromatic (e.g. phenylene or substituted phenylene), C
5-10
heteroaromatic (e.g. furanyl, pyridyl), —N(R
1
)— (where R
1
is a hydrogen atom or a C
1-6
alkyl group), —CON(R
1
)— or —N(R
1
)CO— groups.
In general, residues of reactive functional groups represented by X
1
or X
2
include residues of any groups capable of reacting with any thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group. Thus, for example, X
1
or X
2
may be —CH
2
—, —S—, —NH—, —NHN═, —N(CH
3
)N═, —NHCONHN═, —NHCSNHN═, —N(Ph)N═ (where Ph is phenyl), —NC(O)—, —NC(S)—, —CO—,
—Het
1
C(Het
2
)CH
2
— (where Het
1
and Het
2
, which may be the same or different, is each a nitrogen containing heterocyclic group, e.g. a pyridyl group, or Het
1
is a nitrogen containing heterocyclic group and Het
2
is a hydrogen atom), or
(where Alk is a C
1-4
alkyl, e.g. methyl group).
It will be appreciated that when the bridge is branched a reactive functional group may be provided in each branch, thus allowing attachment of the bridge to each chain through more than one bridging site.
The term labelled antibody molecule in conjugates according to the invention is intended to mean an antibody molecule to which a reporter or effector group is attached.
Reporter group is to be understood to mean any group or compound which is easily detectable by analytical means in vitro and/or in vivo and which confers this property to the conjugate. Effector group is to be understood to mean any group or compound which is capable of eliciting a change in, or a response from, a biological system and which also confers this property to the conjugates of the invention.
Suitable reporter or effector molecules include radionuclides, e.g.
125
I and
131
I; chelated metals; fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy; pharmacological agents, including cytotoxic compounds and toxins, e.g. ricin and fragments thereof; enzymes; and hormones.
Chelated metals include chelates of di- or tripositive metals having a coordination number from 2 up to 8 inclusive. Particular examples of such metals include technetium (Tc), rhenium (Re), cobalt (Co), copper (Cu), gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and Scandium (Sc). In general the metal is preferably a radionuclide. Particular radionuclides include
99m
Tc,
186
Re,
188
Re,
58
Co,
60
Co,
67
Cu,
195
Au,
199
Au,
110
Ag,
111
Ag,
203
Pb,
206
Bi,
207
Bi,
111
In,
67
Ga,
68
Ga,
88
Y,
90
Y,
160
Tb,
153
Gd and
47
Sc.
The chelated metal may be for example one of the above types of metal chelated with any suitable polydentate chelating agent, for example acyclic or cyclic polyamines, polyethers, (e.g. crown ethers and derivatives thereof); polyamides; porphyrins; and carbocyclic derivatives.
In general, the type of chelating agent will depend on the metal in use. One particularly useful group of chelating agents in conjugates according to the invention, however, are acyclic and cyclic polyamines, es
Celltech Limited
Chan Christina
Mathews, Collins Shepherd & McKay, P.A.
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