Peptides which can be used as vectors for the intracellular addressing of active molecules

The invention relates to a peptide of sequence (I) or (Ia): ##STR1## where X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.13, X.sub.14, X.sub.15, X.sub.16 each represent an .alpha.-amino acid, X.sub.6 representing tryptophan. This peptide comprises between 6 and 10 hydrophobic amino acids. The invention also relates to the use of the said peptide for introducing, into living cells, a molecule which is active on the cellular functions.

FIELD OF THE INVENTION 
The present invention relates to a class of peptides capable of crossing 
the cellular membranes and of reaching the various compartments of the 
cell. 
BACKGROUND OF THE INVENTION 
The problem of the entry, into living cells, of various substances having 
pharmacological properties, and of their access to the various 
intracellular compartments, in particular the cytoplasmic compartment and 
the nuclear compartment, is of great importance both for research and for 
therapeutic use. 
A limited number of means for introducing substances such as polypeptides 
and oligonucleotides into the intracellular compartments is currently 
known. Among the various techniques currently proposed, there may be 
mentioned: 
1. The transfection of genes, and derived techniques which make it possible 
to enhance in vivo or in vitro the transfection rates, such as 
precipitation with calcium phosphate, the use of cationic lipids, 
electroporation, trituration (scrape loading), the use of viral vectors, 
and the like. 
2. The binding to cellular membrane receptors; these receptors are 
subsequently endocytozed, and release the bound molecules into the 
cytoplasmic compartment. In this category, there may be mentioned the 
folate receptor, the diphtheria toxin or transcription factors such as the 
TAT protein of the HIV retrovirus. The mechanism of transport involving 
these receptors is still poorly known, but requires in all cases an 
endocytosis stage. 
3. The homeodomain-type peptides. Previous work carried out by the team of 
inventors on the homeodomain of the transcription factor Antennapedia 
(AntpHD) have made it possible to show that the homeodomain peptides cross 
the plasma membranes by an energy-independent process which is therefore 
distinct from endocytosis. The 3rd helix of the homeodomain peptide has 
the same properties [JOLIOT et al., Proc. Natl. Acad. Sci., USA, 88, p. 
1864-1868 (1991); DEROSSI et al., J. Biol. Chem. 269, 14, p. 10444-10450, 
(1994)]. 
These properties have been used to internalize, in cells, polypeptides and 
oligonucleotides linked to the homeodomain or to helix 3, [PEREZ et al., 
J. Cell. Science, 102, p. 712-722, (1992)] by genetic fusion or 
biochemical bonding. This entry is quantitative, and the vector and its 
load are found in 100% of the cells; in addition, the internalization is 
independent of the relevant cell type. 
The smallest fragment of the homeodomain capable of crossing the membranes 
and of serving as vector for other peptides or for oligonucleotides is a 
peptide of 16 amino acids, corresponding to helix 3. This peptide, which 
comprises amino acids 43 to 58 of the homeodomain, is named below. By way 
of example, the sequence of the peptide 43-58 of the homeodomain Antp is 
the following: 
Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Met-Lys-Trp-Lys-Lys 
This sequence is represented in the sequence listing in the annex under the 
number SEQ ID NO:1. 
The mechanism by which this peptide could penetrate into living cells has 
been the subject of various studies, and it was assumed until now that its 
alpha-helix structure was essential for the internalization. The team of 
inventors has previously shown that certain substitutions or deletions in 
the peptide sequence, which modified the structure of the peptide, 
interfered with the activity of the said peptide. For example, a peptide 
in which the 2 Trp's (48 and 56) are replaced by two Phe's, or a peptide 
comprising amino acids 41 to 55 of the homeodomain are not internalized 
(DEROSSI et al., 1994, publication cited above). Another team [BRUGIDOU et 
al., Biophys. Biochem. Res. Com., 214:2, pp 685-693, (1995)] observed the 
internalization of peptides constituting structural analogues of the 
peptide 43-58; for that, they constructed, from a peptide 43-58 (differing 
from the peptide 43-58 of the homeodomain Antp in that the 2 isoleucines 
at positions 45 and 47 are replaced by valines), variants of the 
retro-inverse type. The retro-inverse variants, which make it possible to 
mimic the three-dimensional structure of natural peptides, consist of 
amino acids of the D series (instead of amino acids of the L series in the 
natural peptides) linked according to a sequence which is the reverse of 
that of the peptide to be reproduced. 
The inventors have now sought to define the minimum characteristics of the 
amino acid sequences capable of serving as vector for the internalization 
and addressing of polypeptides and oligonucleotides, and have, for this 
purpose, synthesized several peptides by specifically modifying certain 
residues. 
SUMMARY OF THE INVENTION 
The subject of the present invention is a peptide corresponding to one of 
the following sequences (I) or (Ia): 
##STR2## 
where X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5, X.sub.6, X.sub.7, 
X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12, X.sub.13, X.sub.14, 
X.sub.15, X.sub.16 each represent an .alpha.-amino acid, which peptide is 
characterized in that it comprises between 6 and 10 hydrophobic amino 
acids, and in that X.sub.6 represents tryptophan, with the exception of 
the following peptides: 
the peptide whose sequence is represented in the sequence listing in the 
annex under the number SEQ ID NO:1; 
the peptides in which X.sub.3 and X.sub.5 each represent a valine residue. 
The hydrophobic amino acids are alanine, valine, leucine, isoleucine, 
proline, phenylalanine, tryptophan and methionine. 
The rest of the amino acids which enter into the constitution of the 
peptides in accordance with the invention are non-hydrophobic amino acids 
which may be polar amino acids (glycine, serine, threonine, cysteine, 
tyrosine, asparagine, glutamine), acidic amino acids (aspartic or glutamic 
acid) or basic amino acids (lysine, arginine or histidine), or a 
combination of amino acids of these three categories. 
According to a preferred embodiment of a peptide in accordance with the 
invention, it comprises 6 hydrophobic amino acids and 10 non-hydrophobic 
amino acids. 
The linkage of polar or charged non-hydrophobic amino acids, and of 
hydrophobic amino acids confers on the peptides in accordance with the 
invention an amphiphilic character which, according to the experiments 
carried out by the inventors, appears to be essential for their 
properties. These experiments have, in addition, made it possible to 
highlight other characteristics essential for intracellular translocation, 
and to show that: 
intracellular translocation does not require a specific receptor, and can 
therefore affect all cellular types; 
the alpha-helix structure does not play a part in intracellular 
translocation, (but undoubtedly plays a role in nuclear addressing); 
the amphiphilic properties of the peptide, as well as the presence of a Trp 
residue appear, on the other hand, to be important for translocation. 
Preferably, a peptide in accordance with the invention consists of 
sequences comprising from 1 to 6 non-hydrophobic amino acids and from 1 to 
6 hydrophobic amino acids, alternately distributed along the peptide 
chain. 
According to another embodiment of a peptide in accordance with the 
invention, X.sub.1, X.sub.2, X.sub.4, X.sub.9, X.sub.15 and X.sub.16 are 
non-hydrophobic amino acids and X.sub.3, X.sub.7 and X.sub.14 are 
hydrophobic amino acids. According to a preferred feature of this 
embodiment, X.sub.14 represents tryptophan or isoleucine. 
The intracellular penetration properties of the peptides in accordance with 
the invention are comparable to those of helix 3 of a homeodomain peptide, 
and allow their use as internalization and intracellular addressing vector 
for introducing, into living cells, molecules which are active on the 
cellular functions, in particular other peptides or nucleotide sequences. 
To obtain an addressing, which is more specifically cytoplasmic, of the 
active molecule which it is desired to introduce, a peptide in accordance 
with the invention in which at least one of the amino acids in position 
X.sub.3, X.sub.7 and X.sub.14 is a proline, will be preferably used. 
For the implementation of the present invention, the polypeptide or 
oligonucleotide to be transported is bound to a peptide in accordance with 
the invention. 
The products of fusion of a peptide in accordance with the invention with 
another peptide sequence, or with an oligonucleotide sequence may be 
obtained by different means known per se. In the case of a polypeptide, 
conventional genetic engineering or peptide synthesis techniques may be 
used for example. In the case of an oligonucleotide, there may be used, 
for example, the technique described by LEMAITRE et al. [Proc. Natl. Acad. 
Sci. USA, 84, 648-652, (1987)], or that of MATSUEDA et al. [Chemistry 
Letters, p. 951-952, (1978) and Int. J. of Peptide and Protein Research, 
p. 107-112 (1986)].

DETAILED DESCRIPTION OF THE INVENTION 
The present invention will be understood more clearly with the aid of the 
additional description which follows, which refers to an example showing 
the properties of various peptides in accordance with the invention. 
It should be understood, however, that this example is given solely by way 
of illustration of the subject of the invention and does not in any manner 
constitute a limitation thereto. 
EXAMPLE 
10 peptides, whose sequences are represented in the sequence listing in the 
annex under the numbers SEQ ID NO:1 to SEQ ID NO:10 were synthesized. All 
these peptides were, in addition, provided at their N-terminal end with an 
aminopentanoic arm and a biotin allowing their internalization to be 
monitored; the peptides thus modified are represented in Tables I and II 
below. 
Table I 
__________________________________________________________________________ 
43-58 
Biot-Apa-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys 
SEQ ID NO:1 
41-55 
Biot-Apa-Thr-Glu-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys 
SEQ ID NO:2 
58-43 
Biot-Apa-Lys-Lys-Trp-Lys-Met-Arg-Arg-Asn-Gln-Phe-Trp-Ile-Lys-Ile-Gln-Arg 
SEQ ID NO:3 
43-58 D 
Biot-Apa-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys 
SEQ ID NO:4 
Pro50 
Biot-Apa-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Pro-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys 
SEQ ID NO:5 
3Pro 
Biot-Apa-Arg-Gln-Pro-Lys-Ile-Trp-Phe-Pro-Asn-Arg-Arg-Lys-Pro-Trp-Lys-Lys 
SEQ ID NO:6 
__________________________________________________________________________ 
Table II 
__________________________________________________________________________ 
Met-Arg 
Biot-Apa-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Met-Arg-Arg-Lys-Trp-Lys-Lys 
SEQ ID NO:7 
7 Arg 
Biot-Apa-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Arg-Trp-Arg-Arg 
SEQ ID NO:8 
W/R 
Biot-Apa-Arg-Arg-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Trp-Arg-Arg-Trp-Arg-Arg 
SEQ ID NO:9 
LR/ 
Biot-Apa-Arg-Leu-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Leu-Arg-Arg-Leu-Arg-Arg 
SEQ ID NO:10 
__________________________________________________________________________ 
Legend to Table I 
43-58: Peptide helix 3rd 
41-55: Control: Non-internalized peptide (see DEROSSI et al., 1994) 
58-43: Reverse sequence 
43-58 D: Peptide 3rd helix (43-58) consisting of amino acids of the D 
series 
Pro50: Peptide 43-58 with a Proline in 50 (instead of Gln) 
3Pro: Peptide 43-58 with 3 Prolines at positions 45, 50 and 55, in place of 
the Ile, Gln and Lys residues respectively. 
Legend to Table II 
Met-Arg: Peptide 43-58 with Met in 52 (instead of Arg) and Arg in 54 
(instead of Met) 
7Arg: All the Lysines of the peptide 43-58, except that in 46 are replaced 
with Arg's 
W/R: Peptide 43-58 highly modified, consists essentially of a succession of 
Trp and Arg 
L/R: Peptide W/R with Leu's in place of Trp's. 
The entry of these various peptides into cells in culture was studied under 
the same conditions and using the same procedures as those described by 
DEROSSI et al. (1994, publication cited above). 
The entry of the peptides into the nerve cells or fibroblasts in culture 
was examined at 4 and 37.degree. C. 
The entry of the peptides of Table I was tested by confocal microscopy, gel 
electrophoresis and ELISA. 
The entry of the peptides of Table II was tested only by confocal 
microscopy; indeed, the absence of lysine in these peptides makes the 
attachments very difficult and therefore requires a very rapid observation 
which is incompatible with transfers onto filters or ELISA tests (multiple 
washes). 
1) Internalization at 37.degree. C. and at 4.degree. C. of the peptides 
43-58, 58-43, D43-58, Pro50 and 3Pro 
E15 embryonic cortex cells were incubated (1.1.times.10.sup.6 cells/ml) for 
2 h at 37.degree. C. or at 4.degree. C. with the peptides 43-58, 58-43, 
D43-58, Pro50 and 3Pro, at the concentration of 44 .mu.M for a quantity 
1X, or without peptide (well C). 
The presence of the peptides in the incubation medium and in the cells 
after washing them with trypsin was analysed on a 12-22% SDS-PAGE 
electrophoresis gel and by electrotransfer. 
The results are illustrated in FIGS. 1A, 1B (37.degree. C.) and 2 
(4.degree. C.). 
Legend to FIG. 1: 
A: Autoradiogram of the cellular extracts after revealing by 
bioluminescence (LUMINOL.RTM., AMERSHAM). 
B: Autoradiogram of the incubation media after revealing by 
bioluminescence. 
Legend to FIG. 2: 
The well SP corresponds to the loading onto the gel of 1 .mu.l of substance 
P. 
All the peptides of Table I, with the exception of 41-55, are internalized 
at 4 and 37.degree. C. and recovered in the cells. The peptide 43-58 is 
degraded (about 50%) at 37.degree. C. but not at 4.degree. C. 
2) Cellular localization of the peptides 43-58, 51-55, 58-43, D43-58, Pro50 
and 3Pro at 37.degree. C. and 4.degree. C. 
The E15 cortex and striatum cells are cultured on glass coverslips at a 
density of 25,000 cells/cm.sup.2 for 2 days. The peptides are all added at 
a final concentration of 20 .mu.M. After 2 h of incubation at 37.degree. 
C., the cells are washed, fixed and the presence of biotin is revealed by 
fluorescent streptavidin. The sections observed by confocal microscopy are 
presented in FIGS. 3 (37.degree. C.) and 4 (4.degree. C.). The cells which 
have integrated the peptide appear fluorescent, on a black background. 
______________________________________ 
Legend to FIG. 3: 
1 : peptide 43-58, 2 : peptide 58-43, 
3 : peptide D43-58, 4 : peptide Pro50, 
5 : peptide 3Pro, 6 : peptide 41-55. 
Legend to FIG. 4: 
1 : peptide 43-58, 2 : peptide 58-43, 
3 : peptide D43-58, 4 : peptide Pro50, 
5 : peptide 3Pro, 6 : peptide 41-55. 
______________________________________ 
The presence of prolines (Pro50 or 3Pro) does not prevent internalization 
but appears to interfere with nuclear addressing. The entry of the 
peptides tested is also observed in fibroblasts (not shown). 
3) Colorimetric assay of the internalizations of the peptides 43-58, 51-55, 
58-43, D43-58, Pro50 and 3Pro at 37.degree. C. and 4.degree. C. 
E15 (B) or E16 (A) cortex and striatum cells were incubated 
(1.1.times.10.sup.6 cells/ml) with peptides at a concentration of 17 .mu.M 
(A) or 44 .mu.M (B) for quantities 1X, or without peptides (9). The cells 
were washed with 0.5 M NaCl (A) or with trypsin (B). They were cultured on 
ELISA plate, fixed and biotinylated peptides were revealed using a 
coloured substrate (PNPP) of alkaline phosphatase coupled to streptavidin. 
The results are illustrated in FIGS. 5A and 5B. 
Legend to FIGS. 5A and 5B: 
A: experiment at 37.degree. C.; 
B: experiment at 4.degree. C. 
1: peptide 43-58 1X 
2: peptide 43-58 4X 
3: peptide 58-43 1X 
4: peptide D43-58 
5: peptide Pro50 
6: peptide 3Pro 
7: peptide 41-55 1X 
8: peptide 41-55 4X, sample not present in A 
9: no peptide. 
4) Internalization at 37.degree. C. of the peptides Met-Arg, W/R and L/R 
E15 cortex and striatum cells are cultured on glass coverslips at a density 
of 25,000 cells/cm.sup.2 for 2 days. The peptides are added at a final 
concentration of 20 .mu.M for peptide 43-58 and of 2 .mu.M for the others 
and the cells are incubated for 2 h at 37.degree. C. The cells are washed, 
fixed and the biotin of the peptides is revealed by streptavidin-FITC. The 
results are illustrated in FIG. 6. 
Legend to FIG. 6: 
1: peptide 7Arg 
2: peptide Met-Arg 
3: peptide W/R 
4: peptide L/R. 
Internalization and cytoplasmic and nuclear addressing of Met-Arg, of 7Arg 
and of W/R are observed. However, the peptide L/R is found in a fraction 
which is apparently of the vesicular type, probably of endocytic 
character. 
CONCLUSION 
The results obtained with the peptides of Table I make it possible to 
conclude that: 
internalization does not require a receptor since the peptides 58-43 and 
43-58D are internalized. However, the first has a sequence which is 
different from that of the initial peptide (even if its general 
amphiphilic helical structure is conserved) , and the peptide consisting 
of amino acids of the D series should not interact with a natural receptor 
composed of amino acids of the L series; 
the alpha-helix structure is not necessary since the introduction of a 
proline and a fortiori of 3 destroys the helicity. However, it is noted 
that the addition of prolines may interfere with the nuclear addressing. 
As regards the peptides of Table II, it may be concluded from the results 
obtained that: 
the amphiphilicity is necessary for internalization, but it is not 
sufficient since L/R does not have the properties of W/R 
the presence and the position of Trp residues are important. 
Since the inventors have also demonstrated the internalization of the 
homeodomain of the Engrailed homeoprotein, which does not have the Trp 
residue in 56 (which is replaced by an Ile residue) but has conserved that 
in 48, it can be concluded therefrom that only Trp 48 is important for 
translocation. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 10 
- (2) INFORMATION FOR SEQ ID NO: 1: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#1: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Ar - #g Met Lys Trp Lys Lys 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 15 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#2: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Thr Glu Arg Gln Ile Lys Ile Trp Phe Gln As - #n Arg Arg Met Lys 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#3: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Lys Lys Trp Lys Met Arg Arg Asn Gln Phe Tr - #p Ile Lys Ile Gln Arg 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 4: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
- (ix) FEATURE: 
(A) NAME/KEY: Peptide 
(B) LOCATION:1..16 
(D) OTHER INFORMATION:/pro - #duct= "amino acids of the D 
series" 
#4: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Ar - #g Met Lys Trp Lys Lys 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 5: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#5: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Gln Ile Lys Ile Trp Phe Pro Asn Arg Ar - #g Met Lys Trp Lys Lys 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 6: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#6: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Gln Pro Lys Ile Trp Phe Pro Asn Arg Ar - #g Lys Pro Trp Lys Lys 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 7: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#7: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Gln Ile Lys Ile Trp Phe Gln Asn Met Ar - #g Arg Lys Trp Lys Lys 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 8: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#8: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Ar - #g Met Arg Trp Arg Arg 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 9: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 16 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#9: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Arg Trp Arg Arg Trp Trp Arg Arg Trp Tr - #p Arg Arg Trp Arg Arg 
# 15 
- (2) INFORMATION FOR SEQ ID NO: 10: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 15 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: peptide 
#10: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 
- Arg Leu Arg Arg Leu Leu Arg Arg Leu Leu Ar - #g Arg Leu Arg Arg 
# 15 
__________________________________________________________________________