Chemistry: analytical and immunological testing – Cancer
Reexamination Certificate
2000-08-30
2004-10-26
Caputa, Anthony C. (Department: 1642)
Chemistry: analytical and immunological testing
Cancer
C436S063000, C424S130100, C424S134100, C424S135100, C424S178100, C424S182100, C424S183100, C424S001370, C424S001530, C424S001650, C424S001690, C424S009100, C424S009200, C530S365000, C530S366000, C530S402000
Reexamination Certificate
active
06808930
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to agents which are useful in the diagnosis and treatment of malignancies such as cancer therapy, to processes for their production and to pharmaceutical compositions containing them.
Apoptotic cell death is characterised by loss of cytoplasmic material, nuclear changes with marginalisation of chromatin and by the formation of apoptotic bodies (L. M. Schwartz et al., Immunol. Today 1993, 14:582-590, D. J. McConkey et al. Mol. Aspects Med. (1996) 17:1-115, J. F. Kerr et al., Cancer (1994) 73:2013-26). The reduction in cell viability is accompanied by DNA fragmentation that proceeds in steps with initial formation of high molecular weight (HMW) DNA fragments (50-300 kbp) and the subsequent appearance of oligonucleosome length DNA fragments consisting of oligomers of approximately 200 bp. (M. J. Arends et al., Am. J. Pathol. (1990) 136;593-608, B. Zhivotovosky et al., FEBS Lett. (1994) 352;150-4). Cytoplasmic proteases and Ca
2+
-dependant signaling pathways are activated prior to DNA fragmentation, and are regarded as a prerequisite for the nuclear changes (B. Zhivotovsky et al. Exp. Cell Res. (1995) 221:404-412 m S. Kurar et al., TIBS, (1995) 20:198-202). Agonists like Fas-ligand and TNF first bind to cell surface receptors and then activate transmembrane signaling events that cause cytoplasmic and nuclear changes (L. G. Zheng et al., Nature (1995) 377: 348-351, W. P. Declercq et al, Cytokine (1995, 7:701-9, T. S. Griffith, Science, (1995) 270:1189-1192). Endonuclease activation and DNA fragmentation require that signals from the cytoplasm reach the nucleus. The mechanisms of nuclear uptake and signaling across the nuclear membrane in apoptotic cells remain poorly understood. The transport of macro-molecules from the cytoplasm into the nucleus is highly regulated. Nuclear pore complexes (NPCs) are the sites of exchange of macromolecules between cytoplasm and nucleoplasm (D. A. Jans et al., Physiol. Rev (1996) 76: 651-685, D. Gorlich et al., Science (1996) 271:1513-1518, and Y. Yoneda, J. Biochem. (1997) 121:811-817). The NPCs allow passive diffusion of molecules smaller than 30 kDa but larger proteins like ovalbumin are delayed and bovine serum albumin (66 kDa) does not enter the nucleoplasm. Entry of large molecules or complexes into the nucleus requires active transport and is commonly carrier mediated. The specificity for the carrier may be determined by the so called nuclear targetting or nuclear localization sequences (NLS) that characterize proteins with the ability to enter the nucleus. For example, binding of glucocorticoids to their receptor releases HSP 90 that binds to unoccupied receptors and reveals a NLS in the glucocorticoid receptor sequence that leads to the transport of the glucocorticoid ligand-receptor complex into the nucleus (J. Yang et al., Mol. Cell. Biol. (1994) 14: 5088-98 issn 0270-7306).
A protein complex obtainable from milk that induces apoptosis in tumour cells and immature cells but spares other cells has been described previously (Proc. Natl. Acad. Sci, USA, 92, p8064-8068). The active fraction was initially isolated from human casein by ion-exchange chromatography and was shown by N-terminal amino acid sequencing and mass spectrometry to contain an oligomeric form of &agr;-lactalbumin (described as a multimeric form or “MAL”). Monomeric &agr;-lactalbumin is the major protein component in human milk whey, where it occurs at concentrations around 2 mg/ml (W. E. Heine et al., J. Nutr. (1991) 121: 277-83), but monomeric &agr;-lactalbumin isolated from human whey did not induce apoptosis. Further analysis has provided evidence that the apoptosis-inducing fractions contains oligomeric forms of &agr;-lactalbumin with structural properties distinct from monomeric &agr;-lactalbumin as it occurs in whey. The apoptosis inducing fraction is referred to hereinafter as MAL. It is possible that the mechanism by which the oligomer induces apopotosis may relate to the Ca
2+
binding properties of MAL since apoptosis required extracellular calcium.
MAL may be derived from other sources of &agr;-lactalbumin such as bovine, sheep or goats milk or human whey.
It has now been found that MAL is taken up by susceptable cells (i.e. tumour cells) and accumulated in cell nuclei. This high uptake by the nucleus, combined with its oligomeric protein structure, means that MAL would provide a useful carrier for other moieties for example, cytotoxins or chemotherapeutic agents whose effect would supplement the a effect of MAL in killing tumour cells, or diagnostic reagents such as dyes or radio- or other labels which would allow identification of tumour cells, whilst at the same time, allowing MAL to exert a killing effect on those cells.
SUMMARY OF THE INVENTION
The present invention provides an agent comprising a protein complex comprising an oligomeric form of &agr;-lactalbumin (MAL) and a further reagent which is combined with MAL such that it is carried into the nucleoplasm of cells which are susceptible to MAL.
The said further reagent may be coupled by conjugation or by covalent bonding for example by way of a linking or spacer group as would be understood in the art. Enzymatic reactions can mediate or facilitate the coupling.
Recombinant production techniques allows also the possiblity that MAL could be produced in the form of a fusion protein with the said further reagent.
Examples of said further reagents include cytoxins such as known chemotherapeutic reagents used for the treatment of cancer, microbial toxins such as diptheria toxin and monoclonal antibodies. Alternatively, the said further reagent comprises a labelling agent such as biotin or radioactive labels such as
125
I. For example, a labelling group can be introduced into a protein using an enzymatic reaction or by having a labelled building stone (such as radioactive isotopes e.g.
14
C,
35
S, ) within the protein.
125
I-labelling can be performed enzymatically by coupling
125
I to the protein with the help of lactoperoxidase. Biotinylation of the protein is performed by letteing D-biotinoyl-&egr;-aminocaproic acid-N-hydroxysuccinimide ester react with the protein by forming a stable amide bond to free amino groups in the protein.
Protein may also be labelled by adding radioactive amino acid during the production of a recombinantly produced protein.
Depending upon the nature of the said further reagent, the complex of the invention can be used in the diagnosis and/or treatment of cancer. For this purpose, the complex is suitably formulated as a pharmaceutical composition and these form a further aspect of the invention.
The complex can be administered in the form of an oral mucosal dosage unit, an injectable composition, or a topical composition. In any case the protein is-normally administered together with the commonly known carriers, fillers and/or expedients, which are pharmaceutically acceptable.
In case the protein is administered in the form of a solution or cream for topical use the solution contains an emulsifying agent for the protein complex together with a diluent or cream base. Such formulations can be applied directly to the tumour, or can be inhaled in the form of a mist into the upper respiratory airways.
In oral use the protein is normally administered together with a carrier, which may be a solid, semi-solid or liquid diluent or a capsule. Usually the amount of active compound is between 0.1 to 99% by weight of the preparation, preferably between 0.5 to 20% by weight in preparations for injection and between 2 and 50% by weight in preparations for oral administration.
In pharmaceutical preparations containing complex in the form of dosage units for oral administration the compound may be mixed with a solid, pulverulent carrier, as e.g. with lactose, saccharose, sorbitol, mannitol, starch, such as potato starch, corn starch, amylopectin, cellulose derivatives or gelatine, as well as with an antifriction agent, such as magnesium stearate, calcium stearate, polyethylene glycol waxes or the like, and be pressed into tablets.
Hakansson Per Anders
Svanborg Catharina
Burns Doane Swecker & Mathis L.L.P.
Caputa Anthony C.
Holleran Anne L.
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