Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~32~ ~7
HOECHST AKTIENGESELLSCHAFT HOE 87/F 113 Dr. KL/AW
Specification
Bifunctional proteins
Interleukin-2, called IL-2 hereinafter, acts as T-celL
growth factor. IL-2 potentiates the activity of killer
cells such as NK (natural killer) cells, cytotoxic T-
1Q cells and LAK (lymphokine-activated killer) ceLls.
~y contrast~ granulocyte macrophage colony stimula~ing
factor~ called GM-CSF hereinafter~ stimulates the forma-
~ion of granulocytes and macrophages from hemopoietic
precursor cells. Combination of the two bioLogical
activities is of interest for human treatment with and
without administra~ion of cytostatics~ However, the
~tabilities of IL-2 and GM-CSF differ, which may result
in problems on direct administration of the two com-
0 ponents and t-hus i-n a decrease in the therapeutic
success.
The problem of the difference in stability can be solved
according to the invention by linking these two proteins
to a bifunctional protein.
Fusion proteins of the general formula
Met - X - Y - Z or Met - ~ - Y - X
(Ia) (Ib)
"
have aLready been proposed for the preparation, by
genetic manipulation, of optionally modified GM SSF in
~hich X essentially denotes the amino acid sequence of
approximately the first 100 amino acids of, preferably
human,lL-2, Y denotes a direct bond if the amino acid or
amino acid sequence adjacent to the desired protein
allows the desired pro~ein to be cLeaved off, or
. . :
., . :
: . . .
13~2~ ~7
-- 2 ~
otherwise denotes a bridging member which is composed of
one or more genetically encodable amino acids and which
allo~s the cLeavage off, and Z is a sequence wh;ch is
composed of genetically encodable amino acids and which
represents the desired GM~CSF protein. It is also pos-
sible dur;ng this to make use - more or less - up to
the end of the DNA sequence coding for IL-2~ and thus
generate b;ologically active IL-2 - modified where appro-
pr;ate - as a "by-product" (not prior~published European
Patent Application with the publication number (EP-A)
0,228,018 and South African Patent ~6/~557).
In contras~ to the earlier proposal, the invention re-
lates not to the use of the proteins as intermediate but
to the use in ~ethods for the therapeutic treatment of
the human body and to medicaments ~hich contain fusion
proteins of this type or ~hich are composed of fusion
proteins of this type. A further aspect of the invention
relates to the use of these fusion proteins for the
preparation of a medicament for the treatment of mal;g-
nant neoplasms.
The fus;on protein used according to the invention is
thus composed of two b;ologically active components,
namely of an IL-2 constituent~ ~hich can be modified
in a manner known per s~, on the one hand, and of a GM-
CSF constituPnt, which can likewise be mod;fied, on the
other hand and, where appropria~e, of a bridging member
corresponding to the defini~ion Y in the formulae g;ven
above. The arrangement of the t~o components preferably
corresponds to the formula Ia. The principle according
to the invention can also be used for the preparation of
other novel bifunctional proteins.
The figure shows the construction of the plasmid pB30
which codes for a bifunctional protein according to the
invention.
' ' ~' ' :
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-- 3 --
Mod;f;cat;ons of the IL-2 ~olecule have been disclosed,
reference being made here only to EP-A 0,091~53~,
0,109,748, 0,118,617, 0,136,489 and 0~163,249 by way of
example.
s
Furthermore, the not prior-published EP-A 0,Z19,839 pro-
poses an IL-2 derivati~e in which the first seven N-
terminal amino acids are deleted.
Modi~ications of ~he GM-CSF molecule have been proposed
in EP-A 0,228,018.
Further alterations to ~he two active constituents of the
molecule can be carried out in a manner kno~n per se,
~ention being made here only of specific mutagenesis by
~ay of example.
The bridging ~ember Y advantageously has the formula II
- Asp - (aa)X - Pro - (II) d
in which x denotes an integer up to about Z0, and aa
denotes any desired genetically encodable amino acid
with the exception of cyste;ne.
It is advantageous in the formula II for the IL-2 con-
stituent to be arranged at the left-hand end, and con
sequently the GM-CSF constituent to be arranged at the
right-hand end.
Particularly preferred embodiments of Y have the amino
acid se~uence
-Asp-Pro-Met-Ile-Thr-Thr-Tyr-Ala-Asp-Asp-Pro or
-Asp-Pro-Met-Ile-Thr-Thr-Tyr-Leu-Glu-Glu-Leu-Thr-
Ile-Asp-Asp-Pro-
it again being preferable for the IL-2 constituent to be
arranged at the left-hand end and the GM-CSF constituent
to be arranged at the right-hand snd~
~ . ~
~ 3 2 ~ 7
~ 4
The bifunctional proteins accord;ng to the invent;on can
be expressed ;n a manner kno~n per se. It is possible in
bac~erial express;on systems for the route of direct
expression to be followed. Suitable for this purpose are
3ll known host-vector systems with hosts such as bacter;a
of the spec;es Streptomyces, B. subtilis, Salmonella
typh;murium or Serrat;a marcescens, espec;ally E. coli.
The DNA sequence which codes for the desired protein is
incorporated in a known manner into a vec~or which en-
sures satisfactory expression in the chosen express;on
system.
It is exped;ent to choose for this purpose the promoter
and operator from the group trp, lac, tac, PL or PR of
phage ~, hsp, omp or a synthetic promoter, as described
in~ for example, German OffenlegungssGhrift 3D430,683
and in EP-A 0,173,149. The tac promoter-operator seq-
uence is advantageous and is now commercially available
(for example pKK~23-3 expression vector, Pharmacia,
"Molecular Biologicals, Chemicals and Equipment for
Molecular Biology", 1g84, page 63).
On expression of the protein according to the invent;on,
it may prove expedient to moclify individual triplets for
the first few amino acids after the ATG start codon in
order to prevent any base-pairing at the level of the mRNA.
Such modif;cations, such as deletions or additions of in-
dividual a~ino acids~ are familiar to the exper~, and the
invention also relates to them.
For expression in yeasts - preferably S. cerevisiae - it is
expedient ~o use a secretion systemr for examPle heter-
ologous expression via the ~-factor system, which has been
described several times.
It is advantageous for the expression of the bifunc~ional
molecule in yeast if dibasic peptide sequences and
,.: .. ..
: . ~
.:
1~22~
-
glycosylation sites in the bifunctional prote;n have
been destroyed by appropriate exchange of individual
amino acidsO This results in many possible combinations
which may also influence the biological actio~O
The expression of IL-2 in yeast is disclosed in EP-A
0,142,268, and that of GM-CSF in EP-A 0~188~350n
The administration of the bifunctioRal proteins according
to the invention corresponds to that of the two components.
Howeverr because of the greater stability a lower ~osage
is possible in many cases, the dosage being in the lower
part of the range of those hitherto proposed.
The invention is illustrated in detail in the examples
which follow~ Unless indica~ed otherwise, percentage
data and ratios relate to weight.
FxamPle 1
The plasmid p159i6 (EP-A2 0,163~49, Figure 5; (1) in
the present figure) contains a synthet;c gene coding for
IL-2 between an EcoRI and a SalI cleavage site. The DNA
sequence for this gene is represented in the sa;d EP-A2
as "DNA sequence I". A TaqI cleavage site is located in
~he region of triplets 127 and 1280 The IL-2 part-
sequence (2) ;s cut out of this plasmid by cutting with
EcoRI and TaqI~ and is isolated.
,~
The plasmid pHG23 (3) which codes for GM-CSF is disclosed
in EP-A2 0,183,350~ The GM~CSF cDNA is representPd in
Figure Z in this EP-A2. The plasmid pHG23 ;s obtained
when the cDNA sequence is incorporated in the PstI
cleavage site of pBR322, use being made of, on the one
hand, the PstI cleavage site at the 5' end and, on the
other hand, a PstI site in~roduced at the 3' end by GC
tail;ng. The DNA sequence (4) ~hich contains most of the
GM-CSF gene is isolated from this plasmid by cutting
with SfaNI and PstI~
~.
2 2 ~ 5 ~
-- 6 --
The folLo~;ng oligonucleotide (5) is synthesized by the
phosphite method:
128 (133)
Ile Ile Ser Thr Leu Asp Pro Met Ile
CG ATC ATC TCT ACC CTG GAC CCG ATG ATC
TAG TAG AGA TGG GAC CTG GGC TAC TAG
(TaqI)
1 2
Thr Thr Tyr Ala Asp Asp Pro (Ala) (Pro)
ACC ACC TAT GCG GAC GAT CCG GC
TGG TGG ATA CGC CTG CTA GGC CGT GGG
(SfaNI)
The oligonucLeotide (5? extends at:the 5' end the DNA
sequence of IL-2, there being, however, Asp in place of
Thr in position 133. At the 3' end of this o~igonucleo-
tide are located the nucleotides which have been deleted
from the cDNA by cutting w;th SfaNI.
The preparation of the expression plasmid pE~1000 (6) is
proposed in the (not prior-pubLished) EP-A 0,227,938
(Figure 1). This plasmid is a derivative of the plasmid
ptac 11 tAmann et al., Gene 25 t1983) 167 - 178), in
~hich a synthetic sequence which contains a SalI cleavage
site has been incorporated in the recognition site for
coRI~ The eKpression plasm;d pKK 177.3 is obta;ned in
this way. Insertion of the lac repressor ~Farabaughv
Nature 274 (1978) 765 - 769) results in the plasmid
pJF118. The latter is opened at the unique restriction
cleavage site for AvaI, and is shortened by about 1000
bp in a known manner by exonuclease treatment and ;s
Ligated. The ~lasmid pE~100D ~6) is obtained. Opening
of this plasmid in the polylinker using the enzymes EcoRI
and PstI results in the linear;zed expression plasmid (7).
Th;s linearized plasm;d DNA (7~ is now ligated with the
DNA fragment (2) which codes ~or th~ IL-2 sequence, with
_ 7 _ ~ 3 22~ ~t~
the synthetic oligonucleotide (5) and ~ith the cDNA
fragment (4). The result is the plasm;d pB30 (8) whish
is transformed ;nto the E. coli strain Mc10~1. The
plasmid DNA from individual clones is isolated and
character;zed by restriction analysis.
Example 2
If the following synthetic oligonucleotide
128 (133)
Ile Ile Ser Thr Leu Asp Pro Met Ile Thr Thr Tyr
CG ATC ATC TCT ACC CTG GAC CCG A~6 ATC ACC ACC TAT
TAG TAG AGA TGG ~AC CTG GGC TAC TAG TGG TGG ATA
(TaqI)
1 2
Leu Glu Glu Leu Thr Ile Asp Asp Pro (Ala) (Pro)
CTA GAA GAG CTC ACG ATC GAC GAT CCG GC
GAT CTT CTC GAG TGC TAG CTG CTA GGC CGT GGG
(SfaNI)
is used in place cf oligonucleotide (5) in the example 1,
the result is the plasmid pB310
ExampLe 3
~ompetent cells of the E. coli strain W3110 are trans-
formed with the plasmid pB30 or p~31. An overnight
culture of the strain is diluted in the ratio of about
1:100 with L~ medium (J. H. M;ller~ Experim~nts in Molec.
Gen., Cold Spring Harbor Lab~, 1972), which contains 5a
~glml ampicillin, and the growth ;s followed by measure-
~ent of the OD. At OD = 0~5 the culture is adjusted to
a concentration of 2 mM in isopropyl-~-D-thiogalactopyra-
noside (IPTG) and, after 150 - 180 m;nutes, the bacteria
are spun down. These bacteria are treated in a buffer
m;~ture ~7M urea, 0.1% SDS, 0.1M sodium phosphate, pH
7.0) for about 5 minutes, and samples are applied to an
SDS polyacrylamide gel electrophoresis pLate. This
confirms 4he expression of the b;fun~tional protein.
- 8 - ~322~57
The stated cond;tions apply to shake cultures; for larger
fermentations it is expedient to modify the OD values and
nutrient media and vary the IPTG concentrations appro-
priately.
s
Example 4
E~ coli ~3110 cells which contain the plasmid pB30 or
p931 are, after inductionr spun down, resuspended in
sodium phosphate buffer (pH 7) and again spun down. Th8
bacteria are taken up in the same buffer and then dis-
rupted ~French Press, (R)Dynomi71~. The disrupted cells
are spun do~n. The supernatant and sediment are analyzed
by SDS polyacrylam;de gel electrophorese as described in
Example 3. Staining of the protein bands reveals that the
bifunctional protein ;s located in the sediment from the
disruption. The sediment is washed several times with
chaotropic buffers and finally ~ith water, resulting in
further enrichment of the desired protein. The protein
concentration is then determined in the aqueous protein
suspension. The suspension is now adjusted to a concen-
tration of 5 M in guanidinium hydrochloride and 2 mM in
dithiothreitol (DTT jD The mixture is stirred under
nitrogen for about 30 m;nutes and then diluted with 50 mM
tris buffer (pH 8.5) so that the protein concentration is
100 ~g/ml. It is now dialyzed against this tris buffer
and, after two changes of the buffer, dialyzed against
water. The protein treated in this way is sterile fil-
tered and its biological activity is checked~ It s~ full bio-
logical action both in the interleukin-2-
dependent CTLL 2 cell proliferation assay and in thehuman bone marrow assay. Mixed colonies of granulocytes
and ~acrophages are observed in these.
The bifunctional protein can be further purified by
interleukin-2-specific affinity chromatography~ The
protein is st;ll active in both assays. In contrast, an
. coli extract of the untransformed strain ~3110 whish
has been treated as described shows no act;vity.
- - ::
: ~ : : : . ~;,
~ ~2~
Other conditions are expedient for the industrial pre-
paration of the product, for exa~ple for the folding of
the protein and its purification~ Suitable purification
processes - wh;ch are known per se - are ion e~change,
adsorption, gel fil~ration and preparative HPLC chroma-
tography.
.
