Polypeptide and protein derivatives and process for their...

Details Polypeptide and protein derivatives and process for their... Polypeptide and protein derivatives and process for their...

1994-05-12

2004-01-06

Ponnaluri, Padmashri (Department: 1639)

Drug, bio-affecting and body treating compositions

Conjugate or complex of monoclonal or polyclonal antibody,...

C424S179100, C530S402000, C530S403000, C530S406000, C530S410000, C530S387100

Reexamination Certificate

active

06673347

ABSTRACT:

TECHNICAL FIELD
This invention relates to novel polypeptide and protein derivatives in which polypeptides and proteins are conjugated by bridging molecules to the same kind of polypeptides or proteins, other kinds of proteins or polypeptides, reporter groups or cytotoxic agents.
BACKGROUND OF THE INVENTION
In the diagnosis of many forms of disease, as well as when following the effects of treatment, it would often be desirable to use labelled proteins that bind to specific target structures in the body. For example, when diagnosing or treating cancer, it would be desirable to be able to detect both primary tumours and metastases using labelled tumour-specific antibodies. Many reports have appeared on the labelling of proteins and antibodies by random chemical attack on their side chains. In such a process, most frequently, the side chains of the tyrosines are iodinated (Mach et al., Cancer Research 43, 5593-5600 [1983]), or the side-chains of the lysines are acylated. In this latter case the acylation is often by groups that chelate metals (e.g. Hnatowich et al., Science 220, 613-615 [1983]). Subsequently, the chelating groups can be used to bind radioactive metals. It has also been suggested but not yet been satisfactorily tested to bind to such molecules paramagnetic ions for nuclear magnetic resonance (NMR) imaging (Brady et al., Magnetic Resonance in Medicine 1, 286 [1984]). The labelling of proteins, especially of antibodies, however, has so far always been effected in a more or less random way.
Random substitutions on biological active proteins, for example random substitutions on antibody molecules, can have a number of drawbacks:
1. If by chance a particularly reactive site were to lie in the active site of the protein a substitution at this site would possibly inactivate the protein, e.g. a particularly valuable monoclonal antibody might be rendered totally useless if by chance a side chain particularly reactive towards substitution were to lie in the antigen-binding site. The substitution would then inactivate the antibody.
2. Even when the active site of the protein (e.g. an antibody) escapes serious damage, a high number of substitutions on the protein—which may be desirable, e.g. in order to have a high intensity in case of radioactive labelling via chelating groups—might change its physico-chemical properties (e.g. solubility).
3. A random, multiple substituted product constitutes a heterogenous mixture of molecules with different properties, with attendant problems of assuring constant properties from batch to batch.
SUMMARY OF THE INVENTION
The present invention relates to novel polypeptide and protein derivatives, to a process for their preparation, to their use and to novel intermediates therefor. The novel polypeptides and proteins of the present invention are, more specifically, polypeptides and proteins which are conjugated via an intermediate grouping containing at least one radical of the formula —C(R)═N— (or —N═C(R)—) or —CH(R)—NH— (or —NH—CH(R)—), wherein R is hydrogen, an aliphatic, cycloaliphatic, aromatic or araliphatic hydrocarbon group which group may be substituted, with themselves or each other, with a different polypeptide or protein or with a reporter group or a cytotoxic agent. These compounds are obtained by condensation of two reactants one of which is an aldehyde (or acetalized aldehyde) or ketone the other being an amino compound thus yielding a Schiff base or azomethine type compound which, if desired or necessary, can be stabilized in a further reaction, viz. by reduction of the —C(R)═N— (or —N═C(R)—) radical to a —CH(R)—NH— (or —NH—CH(R)—) radical.
DESCRIPTION OF THE INVENTION
The present invention in a major aspect makes use of the fact that enzymes can direct bifunctional reagents with suitable reactive groups at specific sites in polypeptides or proteins (e.g. antibodies). These sites are preferably the carboxyl terminus of the polypeptide chain, which is at least in terms of primary structure in most cases far from the active site of proteins. This is especially true for antibody molecules where the carboxyl terminus is furthest away from the antigen-binding site. Therefore problem No. 1 mentioned above can be eliminated by the process of the present invention. The limitation of the substitution to a specific site such as the carboxyl terminus, will also eliminate problems No. 2 and No. 3, above.
However, in a further aspect the present invention makes use of the fact that specific bifunctional reagents with suitable reactive groups preferably or specifically react at non carboxy terminus sites of the molecule, viz. with specific side chains or the amino terminal amino group in a non-enzymatic reaction.
Examples of bifunctional reagents with suitable reactive groups are compounds with an amino group at one end and with a formyl or amino group (preferably in protected form) at the other end, such as o-, m- or p-formylphenylalanine.
Therefore, the polypeptide and protein derivatives of the present invention can be prepared by a condensation reaction between an aldehyde or ketone and an amino compound to yield the desired derivative of the azomethine or Schiff base type and, if desired, subsequent reduction of the —C═N— radical (which is relatively labile in case one of the reaction partners is an amine and the product is a Schiff base) to form a corresponding derivative containing a —CH
2
—NH— radical. The amino compound can be an amine, an O-alkylated hydroxylamine or a hydrazide. In the case of an O-alkylated hydroxylamine reacting with a carbonyl compound (aldehyde or ketone) oximes are obtained containing a —C(R)═N—O— radical. Since such compounds are relatively stable no subsequent reduction, albeit possible, is necessary to form a corresponding derivative containing a —CH(R)—NH—O— radical. In the case of a hydrazide reacting with a carbonyl compound the reaction product will contain a —C(R)═N—NH— radical which again is relatively stable and needs no reduction to form a corresponding derivative containing a —CH(R)—NH—NH— radical.
The basic reaction scheme of which the present makes use is >C═O+H
2
N—→>C═N—→>CH—NH—. In this scheme, one complementary group (carbonyl or amino) is placed at the N- or C-terminus of a protein or polypeptide under mild conditions. To obtain specificity (discrimination between an attached amino group and lysine side chains of the protein or polypeptide) a reactive amino group attached to a protein must be an aromatic one, i.e. must be directly attached to an aromatic group, such as phenyl or it must be directly attached to —O— or to —NH—CO—, i.e. be an O-alkyl-hydroxylamine or a hydrazide, respectively.
If at least one of the reactive groups (carbonyl or amino group) of the reaction partners is aromatic, preferably if both are aromatic, it was found that the condensation reaction is rapid, and highly efficient even at surprisingly low concentrations of reactants. The reactivities involved are sufficiently great to permit the attachment of, e.g. a polymeric chelating group to the specific site, which means that at the cost of a single modification at a specific site on the protein known to be safe for this purpose, it is possible to introduce virtually as many of the desired substituent groups as required for high radioactivity. This feature again permits to overcome problem No. 2, addressed above, since a high number of substitutions spread over the whole protein in order to achieve a high enough intensity of labelling is no longer required.
For the reasons discussed above, it is usually preferable to have the group which is to participate in the condensation reaction to form a Schiff base type compound attached specifically, via enzymatic methods, to the carboxy terminus of the protein or polypeptide.
Under certain circumstances, however, it may be satisfactory and convenient to form Schiff base links via groups introduced elsewhere and groups introduced by other methods. Usually, but not always, such me

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