Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-06-19
2001-08-07
Kemmerer, Elizabeth (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S023400, C435S320100, C435S325000, C435S410000, C435S348000, C435S252300, C435S254110, C435S069300, C514S002600, C514S012200, C424S130100
Reexamination Certificate
active
06271368
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to nucleic acid molecules encoding preproproteins having after maturation the biological activity of the mistletoe lectin dimer, to vectors comprising these nucleic acid molecules, to hosts transformed with said vectors and to polypeptides and/or polypeptide dimers which are encoded by these nucleic acid molecules. The polypeptides and/or polypeptide dimers of the invention are widely therapeutically applicable. Thus, the present invention further relates to immunotoxins as well as to pharmaceutical compositions that contain the polypeptides and/or the polypeptide dimers of the invention. Additionally, the invention relates to diagnostic compositions comprising the nucleic acid molecules of the invention, the polypeptides and/or the polypeptide dimers of the invention and/or primers which hybridize specifically to the nucleic acid molecules of the invention. Finally, the invention relates to plant protective agents comprising the polypeptides of the invention and/or the polypeptide dimers of the invention.
BACKGROUND OF THE INVENTION
Mistletoe extracts have been therapeutically used for centuries. Since the beginning of this century, mistletoe preparations have been used in cancer therapy with varying success [Bocci, 1993; Gabius et al., 1993; Gabius & Gabius, 1994; Ganguly & Das, 1994]. Hajto et al. [1989, 1990] could show that the therapeutic effects are mediated in particular by socalled mistletoe lectins (viscumins,
Viscum album
agglutinins, VAA). Besides a cytotoxic effect today the art in particular discusses (unspecific) immunostimulation, the positive effects of which are used for the accompanying therapy and after-care of tumor patients. An increase in quality of life is possibly mediated in such patients by the secretion of endogeneous endorphins [Heiny and Beuth, 1994]. Numerous in vitro [Hajto et al., 1990; Männel et al., 1991; Beuth et al., 1993] and in vivo [Hajto, 1986; Hajto et al., 1989; Beuth et al., 1991; Beuth et al., 1992] studies as well as clinical studies [Beuth et al., 1992] report an increased release of inflammatory cytokines (TNF-&agr;, IL-1, IL-6) as well as an activation of cellular components of the immunological system (T
H
cells, NK cells).
Today a 60 kDa mistletoe lectin protein is considered the active principle of the mistletoe extracts which can be biochemically obtained from extracts [Franz et al., 1977; Gabius et al., 1992]. The ML protein consists of two covalently S—S linked subunits, the A chain of which being responsible for the enzymatic inactivation of ribosomes [Endo et al., 1988] and its B chain being responsible for carbohydrate binding. Biological activity today is mainly attributed to the lectin activity of the B chain [Hajto et al., 1990].
However, little is known about the structure/function relationships of the mistletoe lectin (ML). The contribution which the single-chains and their different biochemical and enzymatic activities make to the mode of action and/or the therapeutic effects observed is not yet clear. The analysis of the structure/function relationships is rendered difficult by contamination of the preparations with other plant ingredients of the mistletoe [Stein & Berg, 1994]. It is discussed that the activity of extract preparations may depend on the different compositions of the preparations, which in turn may depend on the kind of the host tree (e.g., apple, pine, poplar) [Hülsen et al., 1986]. Both viscotoxins [Garcia-Olmedo et al., 1983; Mendez et al., 1990] and other viscumins (such as ML-2, ML-3) are said to have similar effects [Eifler et al., 1994]. Even ML preparations which have been highly purified biochemically (affinity chromatography) are substantially heterogeneous (FIG.
8
).
This heterogeneity relates to the biochemically assessable activities of the chains, to the evoked in vitro and in vivo effects and to the protein structures as such. Structural variants are discussed for the glycosylations of the ML A and B chains as well as for sequence variations. Gabius et al. [1992] and Dietrich et al. [1992] show a sequence variability of the A1 and A2 chains of ML-1.
For a more detailed analysis of the therapeutic effects of the mistletoe lectin it is desirable that it is available as structurally homogeneous substance in pure form. Furthermore, it is important for the scientists to be able to prepare mistletoe lectin or its components in large amounts in pure form so that it/they can be employed on a large scale as active ingredient of pharmaceutical compositions. These aims could not be nearly attained by the processes so far known in the art. Isolation from plant material according to the present state of the art will always yield a heterogeneous mixture of substances. The heterogeneity of plant mistletoe lectin preparations inter alia results from the posttranslational processing of the ML-1 to the isoforms ML-2 and ML-3 so that mistletoe lectin preparations have a varying content of ML-1, ML-2 and ML-3 depending on the method of isolation or the duration of fermentation [Jäggy et al., 1995]. Any of the above-mentioned isoforms additionally is largely microheterogeneous which is illustrated in
FIG. 8
for ML-1 by isoelectric chromatofocusing.
OBJECTS AND SUMMARY OF THE INVENTION
The problem underlying the present invention is therefore to provide mistletoe lectin in pure form and in amounts allowing its use on a large scale. The problem is solved by the embodiments characterized in the claims.
The invention thus relates to a nucleic acid molecule encoding (a) a preproprotein having after maturation the biological function of the mistletoe lectin dimer and having the nucleotide sequence depicted in
FIG. 4
c
; (b) a fragment of the preproprotein according to (a), with the fragment being a biologically active component of the mistletoe lectin dimer; which (c) distinguishes itself from the nucleic acid molecule according to (a) or (b) by degeneration of the genetic code; or (d) hybridizes to the nucleic acid molecule according to (a), (b) or (c) under stringent conditions and encodes a polypeptide having the biological function and/or activity indicated in (a) or (b).
By providing the gene sequences of mistletoe lectin for the first time, recombinant, highly purified individual chains (rMLA, rMLB) that can be reassociated in vitro and thus give a rML holoprotein that is homogeneous in terms of its protein chemistry, its enzymatic activity and its structure could be obtained starting from said sequence. The reassociated recombinant protein is not variable nor microheterogeneous, particularly with respect to its primary structure and the posttranslational modifications (glycosylation, phosphorylation) and hence is particularly useful for therapy both as holoprotein, as partial chain and in form of subfragments.
According to the present invention a “fragment” of a mistletoe lectin preproprotein is understood to be any fragment, not only a naturally occurring fragment, being a biologically active component of the mistletoe lectin dimer. For reasons of clarity it is pointed out that the person skilled in the art obviously understands such a biologically active component of the mistletoe lectin dimer also to be those components that are components of the singlechains of the dimer. Therefore, also the single-chains or fragments thereof that form part of the sequence depicted in
FIG. 4
c
are covered by the present invention. In the present invention, the term “naturally” in conjunction with “component of the mistletoe lectin” is understood such that the so characterized fragment is either a chain of the mistletoe lectin dimer or a subfragment of the chain that naturally occurs in said chain. These fragments are preferably biologically active.
In the present invention, the term “biologically active” is understood such that these fragments have at least one biological function of the chains or of the
Baur Axel
Eck Jürgen
Lentzen Hans
Zinke Holger
Frommer & Lawrence & Haug LLP
Kemmerer Elizabeth
Kowalski Thomas J.
Madus Ag Köln
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