U.S. patent application number 11/482834 was filed with the patent office on 2007-05-03 for vectors derived from antibodies for transferring substances into cells.
This patent application is currently assigned to INSTITUT PASTEUR. Invention is credited to Alexandre Avrameas, Stratis Avrameas, Bruno Blondel, Gerard Buttin, Therese Couderc, Susan Michelson, Marie-Francoise Saron, Therese Ternynck, Donato Zipeto.
Application Number | 20070098741 11/482834 |
Document ID | / |
Family ID | 9510010 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070098741 |
Kind Code |
A1 |
Ternynck; Therese ; et
al. |
May 3, 2007 |
Vectors derived from antibodies for transferring substances into
cells
Abstract
The present invention provides novel polypeptides which can
effectively penetrate into cells thereby transporting a substance
of interest into the cells.
Inventors: |
Ternynck; Therese; (Paris,
FR) ; Avrameas; Alexandre; (Vitry Sur Seine, FR)
; Buttin; Gerard; (Paris, FR) ; Avrameas;
Stratis; (Paris, FR) ; Saron; Marie-Francoise;
(Paris, FR) ; Blondel; Bruno; (Bures Sur Yvette,
FR) ; Couderc; Therese; (Paris, FR) ;
Michelson; Susan; (Noisy, FR) ; Zipeto; Donato;
(Paris, FR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
INSTITUT PASTEUR
Paris
FR
Universite Pierre et Marie Curie
Paris Cedex 05
FR
|
Family ID: |
9510010 |
Appl. No.: |
11/482834 |
Filed: |
July 10, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10460471 |
Jun 13, 2003 |
|
|
|
11482834 |
Jul 10, 2006 |
|
|
|
09497997 |
Feb 4, 2000 |
6635248 |
|
|
10460471 |
Jun 13, 2003 |
|
|
|
PCT/FR98/01740 |
Aug 4, 1998 |
|
|
|
09497997 |
Feb 4, 2000 |
|
|
|
Current U.S.
Class: |
424/204.1 ;
424/135.1 |
Current CPC
Class: |
C12N 2710/16611
20130101; A61K 38/00 20130101; A61P 31/12 20180101; C07K 16/1072
20130101; A61P 31/18 20180101; C07K 16/44 20130101; C07K 2317/565
20130101; C07K 16/28 20130101; C12N 2710/16111 20130101; C12N
2770/32611 20130101; C07K 2317/80 20130101; A61K 47/6883 20170801;
A61K 2039/505 20130101; A61P 31/22 20180101; A61K 47/62 20170801;
C07K 16/18 20130101; Y02A 50/30 20180101; C07K 2319/00
20130101 |
Class at
Publication: |
424/204.1 ;
424/135.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 39/12 20060101 A61K039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 4, 1997 |
FR |
97 09972 |
Claims
1. A polypeptide, characterized in that: it is constituted by a
unique or repeated peptide motif; and it comprises an amino acid
sequence endowing it with the capacity to penetrate into cells and,
if necessary, to transport thereto a substance of interest.
2. A polypeptide characterized in that: it is constituted by a
unique or repeated peptide motif; it comprises an amino acid
sequence constituted by one or more different antibody fragment(s);
and it is capable of penetrating into cells.
3. A polypeptide according to claim 1 or claim 2, characterized in
that it comprises all or a portion of a hypervariable region of an
antibody.
4. A polypeptide according to claim 1 or claim 2, characterized in
that it comprises a fragment of a heavy antibody chain.
5. A polypeptide according to claim 4, characterized in that it
comprises all or a portion of the CDR3 region of an antibody.
6. A polypeptide according to claim 4 or claim 5, characterized in
that it comprises all or a portion of the CDR2 region of an
antibody.
7. A polypeptide according to claim 5 or claim 6, characterized in
that it comprises all or a portion of the CDR3 region and all or a
portion of the CDR2 region of an antibody.
8. A polypeptide according to claim 7, characterized in that it
essentially consists of a fusion between the CDR3 region of an
antibody and the CDR2 region of an antibody.
9. A polypeptide according to any one of the preceding claims,
characterized in that it comprises at most 100 amino acids.
10. A polypeptide according to claim 9, characterized in that it
comprises 3 to 60 amino acids, preferably 3 to 30 amino acids.
11. A polypeptide according to any one of the preceding claims,
characterized in that the antibody fragment is a fragment of an
antibody capable of penetrating into cells.
12. A polypeptide according to claim 11, characterized in that the
antibody fragment is a fragment of a polyreactive antibody.
13. A polypeptide according to claim 12, characterized in that the
antibody fragment is a fragment of an anti-DNA antibody.
14. A polypeptide according to claim 1 or claim 2, characterized in
that it comprises a region with a sequence selected from SEQ ID No.
1, 2, 3 and 8, or any functional homologue.
15. A polypeptide according to any one of the preceding claims,
characterized in that it further comprises a region composed of
basic amino acids, in particular lysine.
16. A polypeptide according to claim 1, characterized in that the
amino acid sequence is capable of being obtained by screening a
peptide library for cell penetration.
17. A polypeptide characterized in that it comprises a polylysine
region and a region derived from a penetrating polyreactive
antibody, and in that it is capable of penetrating into cells.
18. A polypeptide according to any one of claims 1 to 17, with the
capacity of reacting in vitro with anionic macromolecules such as
double or single strand RNA, DNA, or with cationic macromolecules
such as histones.
19. A polypeptides according to any one of claims 1 to 17, with the
capacity of reacting in vitro with heparin and heparin
sulphate.
20. A polypeptide according to claim 1 or claim 2, characterized in
that it is also capable of causing a substance to penetrate into a
cell.
21. Use of a polypeptide according to any one of the preceding
claims, for preparing a composition intended to transfer substances
into cells.
22. Use of a polypeptide according to any one of claims 1 to 20 for
the preparation of an antiviral composition.
23. A polypeptide according to any one of claims 1 to 20,
characterized in that it is coupled to a substance.
24. A vector for transferring a substance into a cell,
characterized in that it comprises a polypeptide according to any
one of claims 1 to 20 to which said substance is coupled.
25. A vector according to claim 24, characterized in that the
coupling is a covalent coupling.
26. A vector according to claim 25, characterized in that coupling
is effected by a covalent maleimide, succinimide, peptide,
disulphide, amine, acid, biotin-streptavidin or p-benzoquinone
covalent type bond.
27. A vector according to claim 24, characterized in that said
substance is a nucleic acid.
28. A vector according to claim 24, characterized in that said
substance is a protein.
29. A vector according to claim 24, characterized in that said
substance is a drug.
30. A vector according to claim 24, characterized in that said
substance is an antigen.
31. A eukaryotic cell containing a polypeptide according to any one
of claims 1 to 20.
32. A eukaryotic cell containing a vector according to claim
21.
33. A method for transferring a substance into a cell in vitro, ex
vivo or in vivo comprising: coupling said substance to a
polypeptide as defined in claim 1, 2 or 17; and incubating the cell
with the product of said coupling.
34. A method according to claim 33, characterized in that the cell
is incubated with the coupling product in the presence of
glycerol.
35. A pharmaceutical composition comprising a vector according to
claim 24 in which the substance is an active principle of a drug,
in association with a physiologically acceptable vehicle.
36. A vaccine comprising a vector according to claim 24 in which
the substance is an antigen, in association with a physiologically
acceptable vehicle.
37. An antiviral composition comprising a polypeptide according to
any one of claims 1 to 20 or an antibody or an antibody fragment
according to claim 13.
38. Use of an antibody or antibody fragment according to claim 13
for the preparation of an antiviral composition.
39. Use according to claim 38, characterized in that the antibodies
are monoclonal antibodies.
40. Use according to claim 22 or claim 38, in combination with an
antiviral agent.
41. Use according to claim 22 or claim 38, characterized in that
the virus is the human acquired immunodeficiency virus (HIV), a
polio virus, a herpes virus or a cytomegalovirus.
42. A method for modifying a cell with the aim of improving the
resistance of that cell to a virus, comprising bringing said cell
into contact with one or more polypeptides according to any one of
claims 1 to 20, or polyreactive antibodies or antibody fragments
having the capacity to bind DNA.
43. A composition comprising cells incubated ex vivo in the
presence of one or more polypeptides according to any one of claims
1 to 20 or antibodies or antibody fragments as defined in claim
13.
44. A composition according to claim 43, characterized in that the
cells are human peripheral blood mononuclear cells.
Description
[0001] The present invention relates to active transfer of haptens,
proteins, peptides, nucleic acids and other molecules into cells.
More particularly, the present invention relates to novel
polypeptides which can effectively penetrate into cells, in
particular eukaryotic cells, and transport thereto a substance of
interest which is capable of constituting novel antiviral
compositions. This invention is of major importance as it has
application in a variety of fields, in particular that of gene
therapy and vaccines.
[0002] Gene therapy remains dependent on a considerable number of
parameters, among them the development of vectors which are capable
of transferring active principles endowed with predetermined
specific properties to the cytoplasm of cells of the host organism
under consideration in the absence of genetic alterations
associated with the use of such vectors, and with no degradation of
the biological activity of the transferred active principles.
Current knowledge is that in spite of the effort achieved in
developing vectors of viral or non viral origins, not all of these
conditions have been satisfactorily fulfilled.
[0003] Further, the possibility of transporting substances
efficiently into cells is also important for all biotechnological
applications. Thus transferring substances into cells in vitro or
ex vivo can be used either to produce proteins or peptides, or to
regulate gene expression, or to analyse the properties of a given
substance in that cell. In vivo, the transfer of a substance to a
cell can also act to create models for studying diseases in animals
or for studying the effect of a given compound on an organism.
[0004] The present invention thus aims to provide a novel type of
vector which is both effective and is more innocuous than viral
vectors in current use.
[0005] International patent application WO 97/02840 describes the
use of antibodies or their F(ab')2 and Fab' fragments which can
penetrate into the interior of living cells, as immunovectors for
intracytoplasmic and intranuclear transfer of biologically active
substances. While such vectors are highly effective, their use can
produce problems in some applications. The use of antibodies or
F(ab')2 antibody fragments involves the production of high titers
of these molecules with qualities which are compatible with
therapeutic use. Further, the use of molecules with the size and
complexity of antibodies can constitute a further disadvantage, in
particular as regards use. U.S. Pat. No. 5 635 383 illustrates a
further type of complex vector based on polylysine for transferring
nucleic acids into cells.
[0006] The present application relates to novel polypeptides with
advantageous properties both for transferring of substances into
cells and as antiviral agents. The primary structure of these
polypeptides is much simpler than antibodies and they are of
reduced size. Further, preparation is easy and their potential
applications are highly varied.
[0007] More particularly, the present invention stems from the
discovery by the inventors that it is possible to identify, from
whole antibodies, limited regions carrying a cellular penetration
activity. The invention also stems from the discovery that it is
possible to isolate, from whole antibodies, in particular from a
single chain of these antibodies, peptides or polypeptides endowed
with cell penetration activity. The present invention constitutes
the first demonstration that a fragment of a single chain of an
antibody can effectively penetrate into cells. The present
invention also constitutes the first demonstration that such a
fragment is also capable, advantageously, of transporting a
substance of interest into said cell, and can preferably have an
antiviral activity.
[0008] The present invention thus provides novel molecules which
are particularly adapted to transfer biologically active substances
into eukaryotic cells, particularly mammalian cells.
[0009] In a first aspect, the invention provides a polypeptide
characterized in that: [0010] it is constituted by a unique or
repeated peptide motif; and [0011] it comprises an amino acid
sequence endowing it with the capacity to penetrate into cells and,
if necessary, to transport thereto a substance of interest.
[0012] In this regard, the invention concerns a polypeptide
characterized in that: [0013] it is constituted by a unique or
repeated peptide motif; and [0014] it comprises an amino acid
sequence constitute by one or more different antibody fragment(s);
and [0015] it is capable of penetrating into cells.
[0016] In one implementation of the invention, the polypeptides
thus comprise one or more fragment(s) of an antibody which may or
may not be different. In their simplest form, antibodies (molecules
from the immunoglobulin superfamily) are constituted by four chains
which are associated together (for example IgG) two heavy chains H,
and two light chains L (FIG. 1) These four chains are associated
together post-synthesis to form a molecule with a molecular weight
of about 150,000 kD. The antigenic specificity of antibodies is
provided by variable domains involving a number of regions of a
heavy chain and a number of regions of a light chain (FIG. 1).
[0017] Polypeptides can also be constituted by sequences
originating from other immunoglobulin representatives such as
IgM.
[0018] Each heavy chain of an antibody is composed of about 450
amino acids, and comprises different domains termed the constant
domain (C), variable domain (V) and joining domains (D and J).
Particular motifs are found in the variable domains, termed CDR
(Complementarity Determining Region) which can readily be localised
by sequence alignment (C. Janeway and P. Travers, 1996,
Immunobiology, Academic Press, "The Structure of a Typical Antibody
Molecule"). For an analysis of the sequences of the variable
regions, reference should also be made to the article by T. T. Wu
and E. Kabat (J. Exp. Med., 1970, Vol. 132, p. 211-250). CDR motifs
themselves comprise hypervariable regions.
[0019] The present application stems from the demonstration that it
is possible to obtain regions which are limited in size and of
simple structure with particularly advantageous properties from the
antibody structure. Thus, starting from a molecule which is complex
(four associated chains) and large (150000 kD), the Applicant has
succeeded in constructing polypeptides with a single chain, with
the capability of penetrating into cells and of transporting
thereto substances of interest. The properties of the polypeptides
of the invention are all the more remarkable since their sequences
corresponding to those of one or more fragments of only one of the
chains of an antibody and thus in order to be active, there is no
need for constant regions originating from a heavy chain and a
light chain. Polypeptides of the invention obtained by chemical
synthesis have the same properties.
[0020] The term "polypeptide" as used in the present invention
defines a molecule comprising a concatenation of amino acids, with
a size in the range 3 to 100 amino acids, for example less than 60
amino acids. Still more preferably, it is a molecule comprising a
concatenation of 3 to 60 amino acids, advantageously 3 to 30.
Particularly preferred polypeptides advantageously comprise more
than about 10 amino acids. The polypeptide of the invention can
also comprise certain structural modifications, of a chemical or
enzymatic nature for example. Thus the polypeptide of the invention
can comprise certain functional groups which, by chemical or
enzymatic reaction, can couple with another substance. The
polypeptides of the invention can also be chemically modified in
order to render them more resistant to proteases or less visible to
the immune system. The polypeptides of the invention can be
obtained by any method which is known to the skilled person, in
particular by chemical synthesis, for example using peptide
synthesisers, or by fragmentation or deletion from larger
polypeptides, natural or otherwise. They can also be prepared using
recombinant DNA techniques, by expression of a corresponding
nucleic acid in a eukaryotic or prokaryotic host cell,. Clearly,
they can result from combinations of these different methods.
[0021] To this end, the polypeptides of the invention can be
produced from libraries of nucleic acids or peptides, such as
synthesised combinatorial libraries.
[0022] The term "unique peptide motif" means that, in contrast to
antibodies or Fab or F(ab')2 type antibody fragments, for example,
the polypeptides of the invention comprise only a single chain of
amino acids. The term "repeated peptide motif" means that the
polypeptides of the invention can comprise different peptide blocks
assembled together, optionally chemically, to form a single
chain.
[0023] The term "penetrate" or "penetrating" as used in the present
invention means a polypeptide which is capable of passing from the
external medium to the intracellular medium, in particular into the
cell cytoplasm. This capacity can be determined in different
manners, in particular using a cell penetration test comprising
initial incubation of the polypeptide to be studied in the presence
of culture cells followed, after fixing and permeabilisation of
these cells, by revealing the presence of said polypeptide inside
said cell. Revealing can be achieved by a further incubation with
labelled antibodies directed against said polypeptide and
detection, in the cytoplasm or in the immediate proximity of the
nucleus or even in the nucleus, of the antigen-antibody type
immunological reaction between the polypeptide and the labelled
antibody. A previously labelled polypeptide of the invention
followed by detection of said labelling in these cellular
compartments can also be used for revealing. Such a cell
penetration test has been described, for example, in International
patent application WO 97/02840.
[0024] As indicated above, the present invention stems from
demonstrating the existence of reduced regions of an antibody
endowed with cell penetration properties and which can also act to
transport substances of interest. More particularly, the inventors
have sought the presence of regions endowed with cell penetration
properties. and which could be used as a vector in place of whole
antibodies in the structure of certain penetrating antibodies such
as those described in WO 97/02840. To this end, the inventors have
first determined the complete sequence of heavy and light chains of
three particular monoclonal antibodies, J20.8, F4.1 and F14.6.
These antibodies are anti-DNA antibodies, polyreactive, which are
produced by hybridoma deposited at the CNCM [National Collection of
Micro-organism Cultures] under numbers I-1605, I-6506 and I-1607
(see patent application cited above). Alignment of these sequences
and their comparative analysis have revealed the following
remarkable elements: [0025] the existence of a region of very high
homology (65-70%) in the CDR2 region of these three antibodies; and
[0026] the presence, in these three antibodies, of CDR3 regions
which are rich in lysine and arginine (basic amino acids).
[0027] With regard to these results, and given that the majority of
peptides capable of transport and nuclear localisation are rich in
lysine and arginine, the Applicants then synthesised series of
polypeptides corresponding to different regions of these
antibodies, and in particular to the CDR2 and CDR3 regions, and
hybrid constructions in which certain of these regions were fused
together (in particular a CDR2-3 peptide carrying CDR2 and CDR3
regions in succession). A biotin residue was also introduced to the
N-terminal side of these polypeptides, to enable them to be
detected easily.
[0028] These polypeptides were then tested for their capacity to
penetrate into cells. The results obtained show that, remarkably,
certain of these polypeptides have the capacity to penetrate
effectively into cells. In particular, the results obtained show
that the group of polypeptides which comprise all or a portion of
the CDR3 region are capable of penetrating into cells.
[0029] More preferably, the polypeptides of the invention are thus
constituted by a unique chain comprising at least one fragment of
the heavy chain of an antibody. Still more preferably, they
comprise at least a fragment of the variable region of the heavy
chain of an antibody.
[0030] In a particular implementation, the invention concerns
polypeptides as defined above comprising all or a portion of the
CDR3 region of an antibody.
[0031] Further, the results obtained have also shown that
polypeptides also containing all or a portion of the CDR2 region
also have the capability of penetrating into cells. To this end,
polypeptides which combine all or a portion of the CDR3 region and
all or a portion of the CDR2 region have entirely remarkable cell
penetration capacities.
[0032] Thus in a further implementation, the polypeptides of the
invention comprise all or a portion of the CDR2 region of an
antibody.
[0033] In a particularly interesting implementation, the
polypeptides of the invention more preferably comprise all or a
portion of the CDR3 region and all or a portion of the CDR2 region.
This type of polypeptide is particularly advantageous as it is
capable of mass penetration into the interior of living cells.
[0034] More particularly, the expression "all or a portion" as used
in the present application means that the polypeptides of the
invention can comprise either the whole of the CDR region concerned
of an antibody, or only a portion thereof, it being understood that
the polypeptide retains a cell penetration capacity (functional
homologue). A portion of the CDR region can consist of a CDR region
which is free of one or more terminal amino acids, in particular
one, two or three terminal amino acids. It may also be a CDR region
where one or more internal residues have been deleted or
substituted by other amino acids, preferably amino acids of the
same nature (for example basic amino acids). Advantageously, less
than 30% of the internal residues of the CDR region are modified,
preferably less than 20% and more preferably less than 15%.
[0035] Preferred polypeptides of the invention are thus
polypeptides comprising all or a portion of a CDR3 region of an
antibody. By way of illustration, CDR3 regions with sequence SEQ ID
No. 1, 2, 3, 8 or the sequences shown in FIG. 2 and FIG. 3 or any
functional homologue can be cited.
[0036] The antibody fragments can themselves constitute the
polypeptide of the invention. They can also be modified by adding
residues to one or both of their extremities. In particular, it may
be advantageous to add amino acids which give the fragment, in
particular the CDR region, a better spatial configuration. It may
also be advantageous to add one or more essentially basic amino
acids, lysine and/or arginine in type, to stabilise the polypeptide
and increase its interaction with the cell membranes. Further, as
indicated above, the polypeptides of the invention may comprise
several regions of an antibody chain, such as a CDR2 region and a
CDR3 region. These regions can in particular be fused together or
spaced by amino acids as described above.
[0037] Particular polypeptides of the invention are polypeptides
comprising a CDR3 region of an antibody or polypeptides essentially
comprising a fusion between the CDR3 region of an antibody and the
CDR2 region of an antibody. Examples of such polypeptides are the
CDR3 polypeptides and the CDR2-3 polypeptide the sequences for
which are given in the Examples.
[0038] Experiments carried out with these polypeptides, in
particular polypeptides comprising the CDR3 region, and more
particularly those comprising the CDR3 region and the CDR2 region,
clearly show that: [0039] 1) incubating PtK2, HeLa or 3T3 cells for
one hour with complete culture medium (10% foetal calf serum)
containing the polypeptide is sufficient for the polypeptide to be
massively transported into the cytoplasm of all the cells and into
the nucleus of a large proportion of these cells, the proportion
being variable depending on the line concerned. [0040] 2) When
cells are incubated for 2 hours in complete culture medium
containing pre-formed peptide-streptavidin complexes coupled to
peroxidase (MW.gtoreq.100000) or peptide-streptavidin coupled to
alkaline phosphatase (MW.gtoreq.180000), the corresponding enzymes
are detected in the cytoplasm of all of the cells of the culture
and weakly to intensely detected in the majority of the nuclei of
these cells. No intracellular coloration is observed when the cells
are incubated in the presence of streptavidin coupled with
peroxidase, streptavidin coupled with alkaline phosphatase or with
streptavidin or the enzymes in their native forms.
[0041] The polypeptides of the invention, in particular of type
CDR3 and CDR2-3, and their peptide-streptavidin-enzyme complexes
are transported in large quantities into a large proportion of
human peripheral cells and particularly into activated T
lymphocytes.
[0042] In general, the polypeptides of the invention can be
constructed using different techniques which are known to the
skilled person (supra), starting from any given antibody, in
particular any given monoclonal antibody.
[0043] Preferably, the polypeptides of the invention are obtained
by chemical synthesis or are constructed from a fragment or several
fragments of one or more penetrating antibody(ies), preferably a
penetrating monoclonal antibody. The existence of antibodies which
can penetrate inside cells and in particular into the nuclei of
human lymphocytes when these cells are incubated in vitro in a
culture medium containing a serum originating from patients with
disseminated lupus erythematosus (DLE) was reported for the first
time by Alarcon-Segovia et al. In 1978 (Nature, 271). Recently,
this type of antibody has been detected in the lupus mouse MRL
lpr/lpr, but also in the NZB mouse with an autoimmune hemolytic
disease syndrome and even in the normal BALB/c mouse. Certain
monoclonal antibodies prepared from the spleen of these mice have
been shown to be capable of penetrating in vitro into the nucleus
of cells maintained in culture (Vlahakos et al., J. Am. Soc.
Nephrol. 2 (1992) 1345; Eyal Raz et al., Eur. J. Immuno. 23 (1993)
383). Further, it has been shown that these antibodies are also
capable, when injected into mice, of penetrating into several types
of cells, and are found in their nuclei (Okudaira et al., Arthritis
and Rheumatism, 30 (1987) 669).
[0044] In general, any antibody can be selected with a view of
determining its penetrating character. This selection can be made,
for example, using a cell penetration test comprising initial
incubation of the antibody under study in the presence of cells, in
particular cells into which it is desired to transport a substance
of interest, followed by fixing and permeabilisation of these
cells, revealing the presence or the absence of this antibody in
the plasmic membrane, the cytoplasm or in the immediate proximity
of the nucleus or even in the nucleus. Revealing can, for example,
be effected by incubation with a second labelled antibody, directed
against the test antibody, followed by detection of the
immunological reaction of the antigen-antibody type between these
two antibodies. Such a test has been described in detail, for
example in French patent FR-9508316.
[0045] Still more preferably, the fragment of antibody used to
construct a polypeptide of the invention is a fragment of a
polyreactive antibody, in particular penetrating and polyreactive.
A polyreactive antibody is an antibody which is capable of
recognising several different antigens. In general, such antibodies
have a particularly high affinity for a particular type of antigen
and are capable of recognising one or more other antigens with a
lower affinity. The polyreactivity of antibodies can be
demonstrated by any conventional immunological technique, such as
the methodology described by Sibille et al. (Eur. J. Immuno. 1997,
27: 1221-1228).
[0046] Advantageously, the polyreactive antibodies used in
constructing the polypeptides of the invention are capable of
reacting with nucleic acids, free or complexed with proteins
(anti-DNA antibodies). This property can be demonstrated using the
ELISA technique or by passing the antibodies over a column or any
other support on which DNA has already been immobilised. The
anti-DNA antibodies are thus retained. on the support and can be
eluted and isolated using conventional techniques. In general, the
avidity for DNA of the anti-DNA antibodies used in the context of
the invention is of the order of 1.times.10.sup.6 M to
2.times.10.sup.7 M. Preferably, these antibodies recognise genomic
DNA, in particular genomic DNA. In a particular implementation, the
antibodies used are polyreactive antibodies which recognise the
genomic DNA of human hematopoietic cells. Still more preferably,
they are antibodies which are capable of reacting with nucleic
acids and recognise, inter alia, proteins such as Tat from the HIV
retrovirus, and/or constituents of the cell surface and of the cell
cytoskeleton.
[0047] To construct a polypeptide of the invention, the selected
antibody is then used as follows: [0048] a) if the sequence of the
variable region of the heavy or light chain of this antibody is not
accessible, it is determined in a first stage. Conventional
sequencing techniques can be used, as illustrated in the examples;
[0049] b) the CDR regions are localised by sequence alignment with
other sequences of antibody chains, or by any other technique;
[0050] c) fragments of this sequence are prepared, or the
polypeptides corresponding to regions of this sequence are
synthesised, and assembled if necessary. To this end, any
conventional technique which is known to the skilled person can be
used (peptide sequencers, using a restriction enzyme, ligases,
etc.); [0051] d) the fragment obtained may be modified by addition,
deletion or substitution of amino acids; [0052] e) the cell
penetration capacity of the polypeptide obtained is then tested
under the conditions described above, also the capacity of
transporting substances such as fluorescein or peroxidase.
[0053] Optionally, steps c), d) and e) or d) and e) are repeated so
as to improve the penetration efficacy or the general properties of
the polypeptides of the invention.
[0054] In a supplemental subsequent step f), the polypeptide
obtained, with the capacity to penetrate into cells, is then used
in a coupling reaction with a given substance to generate a vector
as will be defined below.
[0055] An alternative to this method lies in the use of one or more
libraries of nucleic acids or peptides as the starting material.
Thus rather than starting from the sequence for an antibody, it is
possible to construct, for example by combinatorial chemistry,
libraries of peptides or nucleic acids coding for peptides
representing functional homologues of the CDR2 or CDR3 antibody
regions.
[0056] The peptides or combinations of peptides or nucleic acids of
these libraries are then prepared, optionally modified and tested
for their activity using steps c) to e) of the above method.
[0057] Preferably in step c) of the above method, the prepared
fragments comprise all or a portion of the CDR3 region of an
antibody.
[0058] In step d), modifications can, for example, consist of
introducing certain supplemental amino acids, either simply for
technical reasons (ease of synthesis, coupling between different
regions, etc) or for structural or physicochemical reasons.
Concerning amino acids for "filling", amino acids which are
relatively neutral on the structural and physicochemical level are
advantageously used. Regarding structural reasons, as indicated
above, adding residues can improve the conformation of the
polypeptide and thus potentialise its activity. As an example,
introducing nuclear localisation factor sequences (NLF) can
increase the intranuclear transfer potentials. Further, it may also
be desirable to increase the basic nature of the polypeptides.
[0059] To this end, to improve the compaction properties of the
polypeptides of the invention, in particular as regards nucleic
acids, polypeptides have been constructed which carry lysine
residues on the N-terminal side. Advantageously, the number of
lysine residues is less than 30, more preferably between 10 and
20.
[0060] The results presented in the examples confirm the
penetration properties of these polypeptides, and their capacity
for effective transport of substances of interest, in particular
nucleic acids.
[0061] Whatever the additions made, the polypeptide of the
invention as prepared, for example, using the above protocol,
preferably comprises at most 100 amino acids. Still more
preferably, it comprises 3 to 60 amino acids, preferably 3 to 40
amino acids.
[0062] In a further aspect, the invention concerns the use of a
polypeptide as defined above to transfer substances into cells, in
vitro, ex vivo or in vivo.
[0063] In a still further aspect, the invention concerns a vector
for transferring a substance into a cell, characterized in that it
comprises a polypeptide as defined above to which said substance is
coupled.
[0064] In one implementation of the invention, the polypeptide
comprises a sequence of amino acids endowing it with the ability to
penetrate into cells enabling it to transport into said cell
substances of biological interest which are associated therewith,
for example haptens or macro-molecules of hundreds to thousands of
kD, such as drugs, proteins or nucleic acids.
[0065] The sequence of amino acids and the substances of
therapeutic interest associated with it can, for example, be
coupled or bonded via covalent or non covalent bonds.
[0066] A polypeptide of the invention is advantageously constituted
by peptides or macro-molecules with the capability of penetrating
into living cells, and more particularly from peptide derivatives
of antibodies or antibody fragments as described in International
patent application WO 97/02840 or from other peptides comprising
one or more hypervariable antibody portions, or synthetic
molecules, not directly related to an antibody type structure,
which can be obtained, for example, by screening a peptide library
for cell penetration.
[0067] A particular polypeptide of the invention is thus composed
of a unique or repeated peptide motif, and comprises a sequence of
amino acids which endow it with the capacity to penetrate into
cells and transport a substance of interest thereto, this sequence
being capable of being obtained by screening a peptide library for
cell penetration. The conditions for screening such libraries have
been described above.
[0068] A further particular polypeptide of the invention is
composed of a unique or repeated peptide motif and comprises a
sequence of amino acids which endow it with the ability to
penetrate into cells and transport thereto a substance of interest,
this sequence being composed of a peptide comprising one or more
hypervariable antibody portions.
[0069] The coupled substance can be any product of interest, in
particular a biological, pharmaceutical or agro-alimentary product.
In particular, it may be a nucleic acid, such as a ribonucleic acid
or a deoxyribonucleic acid. This nucleic acid can also be from a
variety of origins, in particular human, viral, animal, eukaryotic
or prokaryotic, plant, synthetic, etc. This nucleic acid can also
be a variety of sizes, from a simple oligonucleotide to a genome or
a fraction thereof. In particular, it may be a viral genome or a
plasmid. The substance can also be a protein, such as an enzyme,
hormone, cytokine, apolipoprotein, growth factor, etc. A particular
type of substance is represented by antigens. As indicated below,
the polypeptides of the invention can advantageously act as an
adjuvant and stimulate the immune response directed against an
antigen.
[0070] More generally, the substance can be any active principle of
a drug, be it a chemical, biochemical or synthetic product.
[0071] To enable its transfer into a cell, said substance is thus
coupled to a polypeptide of the invention.
[0072] The term "coupled" as used in the invention means any type
of interaction enabling a physical association between the
substance and the polypeptide. Preferably, however, the interaction
is sufficiently stable for the vector not to dissociate before cell
penetration. For this reason, the preferred coupling is covalent
coupling.
[0073] Covalent coupling can be effected by different techniques
which are known to the skilled person. In particular, it can be
effected using maleimide, succinimide, peptide, disulphide and
thioether bonds. Reference should be made in this respect to
"Bioconjugate Techniques" by Greg T. HERMANSON (Academic Press,
1996).
[0074] A particular method consists, for example, of adding a
cystein residue which can be readily used for disulphide,
thioether, amine or acid bonds to one extremity of the polypeptide
of the invention. A further approach consists of chemically
coupling a biotin group, which then enables any substance bonded to
streptavidin to be coupled. Coupling can also be effected using
p-benzoquinone (FR-7537392 and U.S. Pat. No. 4,925,921, for
example).
[0075] In general, any chemical, biochemical, enzymatic or genetic
coupling method which is known in the literature can be used.
[0076] Further, a vector of the invention can comprise a
polypeptide as described above to which a number of identical or
different substances are coupled.
[0077] The examples below clearly demonstrate that the polypeptides
of the invention have the ability not only to penetrate into cells,
but also to transport substances of interest thereto. The examples
demonstrate enzyme type protein transport. It should be understood
that enzymes can be substituted by any other molecule of interest
such as nucleic acids, peptides or drugs, under the same
conditions.
[0078] The examples also demonstrate the capacity of the peptides
of the invention to transfer nucleic acids into cells. For this
particular application, coupling between the peptide and the
nucleic acid is generally non-covalent coupling, based on ionic
interactions, electrostatic interactions or Van der Waals forces.
More particularly, when used to transfer nucleic acids into cells,
a peptide of the invention advantageously comprises a region
constituted by basic amino acids, for example lysine in type,
enabling a complex (polyplex) to be formed with the negatively
charged nucleic acids. Thus in one particular implementation, the
invention concerns the use of a peptide as defined above, carrying
a polylysine region, for transferring nucleic acids (i.e.,
plasmids, cosmids, linear fragments, genes, antigens, antisense,
oligonucleotides, etc) into cells. In one particular aspect, the
invention thus provides a peptide comprising a polylysine region
and a region derived from a penetrating polyreactive antibody, and
capable of penetrating into cells. More particularly, the
polylysine region advantageously comprises 5 to 30 lysine residues,
preferably 5 to 20, which are advantageously not interrupted by
other residues. The region derived from the penetrating antibody
can be defined as above. Such a polypeptide advantageously
comprises less than 100 residues, as explained above.
[0079] Further, the fact that the polypeptides of the invention
enable massive transport of proteins into cells has also prompted
the Applicant to examine the possibility of using them as an
intracellular antigen transport agent, endowing them with an
adjuvant effect and leading to an increase in the immune response
against these antigens. Thus mice received several injections with
the streptavidin-peroxidase conjugate alone or complexed with a
polypeptide of the invention. The results obtained show that the
use of a polypeptide of the invention can increase by on average 4
to 8 times the titer of anti-streptavidin antibodies and
anti-peroxidase antibodies.
[0080] The invention also concerns a method for transferring a
substance into a cell in vitro, ex vivo or in vivo comprising:
[0081] coupling said substance to a polypeptide as defined above;
and [0082] bringing the cell into contact with the product of said
coupling.
[0083] For in vitro or ex vivo use, contact can be effected by
simple incubation of the cells with the coupling product (vector).
For in vivo use, contact is generally effected by administering the
coupling product (vector) to the organism under consideration. As
indicated above, when coupling is covalent in type, the vector can
comprise one or more molecules of interest. Further, for
non-covalent coupling, for example for nucleic acids, the vector is
generally formed by incubating the peptide and the nucleic acids in
a medium enabling them to form a complex. The respective quantities
of the partners are easily adjusted by the skilled person as a
function of the nature of the peptide (length and charge), nucleic
acid and cell type. By way of example, coupling can be carried out
at peptide concentrations of 0.01 to 100 nmoles of peptide per
.mu.g of nucleic acid, preferably 0.01 to 10 nmoles/.mu.g. Further,
in the method of the invention, it may be advantageous to use, in
addition, a stabilising agent or facilitator such as glycerol. Thus
the results shown in the examples show that in the presence of
glycerol, the transfection efficacy of nucleic acids can be
improved by a factor of close to 40. Thus a particular.
implementation of the method of the invention comprises bringing
cells into contact with the coupling product in the presence of a
stabilising agent, in particular glycerol. Advantageously,
transfection is carried out in vitro or ex vivo in the presence of
glycerol, at concentrations or 0.1 to 2 M, for example. It should
be understood that these concentrations can be adjusted by the
skilled person.
[0084] In a still further aspect, the invention provides a cell, in
particular a eukaryotic cell, containing a polypeptide or a vector
as defined above. This cell is advantageously a mammalian cell, in
particular an animal or human cell. In particular, it may be a cell
of the hematopoietic system, such as a progenitor cell or a strain
cell or a lymphocyte cell (T, B). It may also be a cell presenting
the antigen such as macrophages or dendritic cells.
[0085] Further, the inventors have also shown that the polypeptides
of the invention and the polyreactive antibodies are endowed with
their own biological properties. The invention demonstrates that
these polypeptides (peptide derivatives) or antibodies are capable
of inducing a biological effect which is distinct from their
ability to vectorise active substances. Unexpectedly, the present
invention shows in particular that these antibodies and peptide
derivatives are capable by themselves of exerting an antiviral
activity on different cell populations and on different types of
virus.
[0086] The present invention thus also concerns a novel approach to
inhibiting viral replication and/or infection in cells. In
particular, the present invention concerns the use of particular
antibodies or antibody fragments as antiviral agents, in particular
to inhibit viral replication and/or infection in cells. The
invention also describes a novel method for treating cells to
render them more resistant to viral replication and/or infection.
The invention also concerns populations of cells treated by
antibodies or polypeptides which are less sensitive to viral
replication and/or infection. This property can be implemented in
vitro, ex vivo or in vivo, optionally in combination with other
agents, to reduce infection and development of a virus, in
particular infection and/or replication of the human acquired
immunodeficiency virus, polio virus, herpes virus or
cytomegalovirus, for example.
[0087] Thus in a yet still further aspect the invention concerns
the use of one or more polypeptides as defined above in preparing
an antiviral composition. More particularly the invention concerns
the use of one or more antibodies or antibody fragments in
preparing an antiviral composition, characterized in that said
antibodies or antibody fragments are polyreactive and are capable
of binding to a nucleic acid, preferably DNA.
[0088] More particularly, an antiviral composition as defined in
the invention consists of a composition which is capable of
inhibiting infection of a target cell by a virus and/or replication
of a virus in a target cell.
[0089] As indicated above, this aspect of the present invention
stems from the demonstration of the unexpected biological
properties of the polypeptides described above and, more generally,
of certain polyreactive antibodies, i.e., susceptible of
recognising a plurality of antigens, and more specifically having
the capability to bind DNA.
[0090] In a more particular aspect, then, the invention concerns
the use of polyreactive antibodies or antibody fragments, or
anti-DNA and penetrating derivative polypeptides, as an antiviral
agent.
[0091] As indicated above, the polyreactive antibodies or antibody
fragments used are preferably capable of recognising at least one
proteic antigen of cellular and/or viral origin. More particularly,
in addition to DNA, these antibodies or antibody fragments
recognise at least one viral antigen such as a viral protein
envelope antigen. In one particular implementation, the invention
concerns the use of polyreactive antibodies or antibody fragments
which are capable of binding a protein or a peptide of the human
acquired immunodeficiency virus (HIV). This property can be tested
using any conventional immunological technique. Thus HIV proteins
or peptides can be immobilised on any suitable support (plate,
column, beads, etc.), and to incubate this support with the
antibodies. The formation of an antigen-antibody complex can then
be detected using any conventional technique (immunofluorescence,
enzymatic reaction, etc.). In general, the avidity for HIV proteins
or peptides of the anti-DNA antibodies used in the invention is of
the order of 10.sup.6 to 10.sup.7 M.
[0092] Preferably, the antibodies used have the capability of
binding HIV Tat and/or rev proteins, more preferably the Tat
protein or a peptide of that protein. Particular peptides which can
be cited are peptides comprising residues 22-37 and 46-60 of the
HIV Tat protein.
[0093] In this antiviral application, the antibodies used can be
polyclonal or monoclonal antibodies. Polyclonal antibodies can be
isolated directly from the serum of subjects (immunised or not
immunised, healthy or diseased) by removing blood and isolating
antibodies in the presence of immunoadsorbents (for example protein
A coupled to a sepharose type support). Polyclonal antibodies which
can be used in the invention can in particular be obtained from
healthy or diseased animal serum, in particular from rodents, more
particularly from mice. They may in particular be autoimmune lupic
mice which have high levels of natural antibodies recognising
various antigens. Polyclonal antibodies can also be obtained from
human serum, for example from patients with disseminated lupus
erythematosus, which are also known to present a high natural level
of polyreactive antibodies. Concerning monoclonal antibodies, they
can be prepared using conventional immunological techniques, by
removing splenocytes from immunised or non immunised animals,
healty or pathological, fusion with myeloma cells, then clonal
dilution and selection of hybridomas producing antibodies. These
techniques have been widely documented, in particular by S. L.
Morriso and V. T. Oi, in "Advances in Immunology (1989), 44: 65-92;
J. G. R. Hurrell, "Monoclonal Hybridoma Antibiotics: Techniques and
Applications", CRC Press 1982. These techniques have also been
illustrated in the examples. Polyreactive monoclonal antibodies can
also be artificially synthesised or humanised from animal
monoclonal antibodies.
[0094] The antibodies used can be immunoglobulins of different
types, in particular IgG or IgM. It should be understood that other
types of antibodies can also be used (IgE, IgA, etc.) provided that
they have the required properties.
[0095] Further, as indicated above, the use of not only intact
antibodies but any polypeptide of the invention is also possible,
also any fragment of these antibodies which retains the required
properties. These fragments can also be (Fab')2, (Fab') or ScFv
fragments. These fragments can result from enzymatic antibody
digestion, or can be obtained by recombination or by synthesis. The
preparation of such fragments (for example by enzymatic treatments)
has been widely documented in the literature and can thus be
carried out by the skilled person using simple routine operations
starting from antibody preparations.
[0096] As indicated above, this aspect of the present invention
stems in part from the discovery of the antiviral properties of
such polypeptides, antibodies and fragments, in particular their
ability to inhibit viral replication in a target cell or infection
of a target cell by a virus. The term "infection" means penetration
of the virus or viral genome into the target cell, and the term
"replication" essentially means replication of the viral genome in
said target cell.
[0097] The present invention can be employed to inhibit the cycle
of different viruses, more particularly a RNA virus (retrovirus) or
a DNA virus. Further, it may be a virus with tropism for man or for
different animals, in particular mammals (dogs, cats, rabbits,
cattle, etc.). More preferably, the present invention can inhibit a
virus such as the human immunodeficiency virus (HIV), polio virus,
herpes virus or cytomegalovirus (CMV).
[0098] A particular implementation of the invention comprises the
use of one or more polyreactive antibodies or fragments of such
antibody for preparing a composition intended to inhibit infection
of a target cell by HIV and/or replication of HIV in a target cell,
characterized in that said antibodies or antibody fragments have
the ability to bind DNA. More preferably, the antibodies used also
have the ability to bind a protein or a peptide of the human
acquired immunodeficiency virus. More particularly, the invention
can be used to inhibit infection and/or replication of different
strains of HIV, in particular HIV-1 and HIV-2.
[0099] More particularly, the invention concerns the use of one or
more polypeptides as described above to prepare a composition
intended to inhibit viral replication and/or infection.
[0100] Within the context of the invention, the term "target cell"
means any cell which is naturally susceptible of being infected by
a virus, preferably susceptible of enabling replication of the
virus. In the case of HIV, the target cells are constituted by
cells of the immune system, in particular lymphocytic cells. More
specific examples of HIV target cells are T lymphocytes, in
particular auxiliary T lymphocytes (CD4+). Other HIV target cells
for the invention are more generally constituted by peripheral
mononuclear cells, in particular human (PBMC). Regarding the polio
virus, an example of target cells are epithelial cells. In general,
the present invention can be employed to interfere with the
development of a virus in any type of target cell for the
antibodies used.
[0101] Advantageously, a polypeptide of the invention is derived
from recombinant ScFv fragments, capable of reacting with DNA or
with other anionic or cationic macromolecules, in particular
heparin and heparin sulphate, and obtained from lymphocytes
originating from normal patients or patients with different
diseases in particular disseminated lupus erythrematosus.
[0102] The mechanism(s) for the action of the compounds of the
invention still have to be elucidated. In this respect, a series of
recent results, in particular for the herpes virus, in particular
the human herpes virus for example the type 1 herpes simplex virus
or cytomegalovirus (CMV) appears to indicate that the peptides of
the invention affix to cellular receptors used by the virus
themselves to penetrate into the host cells. Fixing of peptides on
these receptors is followed by internalisation of the
peptide-receptor complexes, which results in a reduction in the
number of cellular receptors remaining available to fix the virus.
This reduction sometimes appears to be especially significant as in
the case of CMV. However, other mechanisms, in particular on the
level of the nucleus, could also be involved in the unexpected
properties of these antibodies and derivatives.
[0103] Advantageously, the antiviral activity of the polypeptides
means a significant reduction in replication or infection.
Advantageously, the inhibition produced by the polypeptides or
antibodies of the invention corresponds to a reduction by a factor
of at least 1.5 with respect to the level of infection and/or
replication in the same cells or cell populations in the absence of
treatment. More preferably, inhibition corresponds to a reduction
by a factor of at least 4. This inhibition can be quantified, for
example by measuring the viral plaques, the levels of viral
antigens present in the cells, cell viability, etc. In a particular
implementation, the inhibition efficacy is evaluated by measuring
the levels of viral antigens such as p24 and/or gp120 antigens, for
HIV.
[0104] In a particular implementation, the invention concerns the
use of antibodies or polypeptides to induce an inhibition of a
factor of at least 2 in viral replication in the target cells.
[0105] In a first implementation, the invention comprises using a
single type of polypeptide or antibody or antibody fragment as
defined above. As illustrated in the examples, by using a single
compound it is in fact possible to inhibit HIV-1 replication in
target cells by a factor of more than 10, in particular of the
order of 100. The use of a single compound can also produce an
inhibition by a factor of more than 2 in the replication of type-1
polio virus in target cells.
[0106] Further, a pronounced anti-herpetic reaction has been
observed for certain antibodies and peptides of the invention. It
is important to indicate that this action is also observed with
HSV-1 thymidine-kinase.sup.- (TK.sup.-) on which acyclovir has no
effect.
[0107] Further, a pronounced anti-CMV action has also been
observed, in particular with the K19-pF4-1 polypeptide. In a series
of experiments, it has been demonstrated that K19-pF4.1 inhibited
the infection of cells by CMV by almost 100%. - It is important to
note that even the synthesis of early antigens was completely
inhibited (detection both by immunofluorescence and radiolabelling,
followed by immunoprecipitation).
[0108] In a further implementation, the invention comprises the use
of a plurality of polypeptides, antibodies and/or antibody
fragments as defined above. As illustrated in the examples, certain
of these compounds, in combination, can exert a synergistic
inhibition effect on viral replication in target cells. Thus,
unexpectedly, certain antibodies, alone, have a moderate inhibiting
activity but in combination induce an inhibition of HIV-1
replication in target cells by a factor of more than 10, in
particular of the order of 100.
[0109] In a further implementation, the invention also comprises
the use of one or more polypeptides, antibodies and/or antibody
fragments as defined above in combination with one or more
antiviral agents. Examples of such antiviral agents are AZT, DDI
and antiproteases. In this regard, the present application also
concerns a product comprising: [0110] one or more polypeptides,
antibodies and/or antibody fragments as defined above; and [0111]
an antiviral agent; for simultaneous, separate or intermittent
use.
[0112] The present invention can be used to inhibit viral
replication and/or infection in vitro, ex vivo or in vivo.
[0113] For in vitro or ex vivo use, the target cells, or a cell
composition comprising target cells, are generally incubated in the
presence of compounds as defined above. The doses of the
polypeptides, antibodies or antibody fragments used are generally
in the range about 1 to 500 .mu.g per 10.sup.6 cells, preferably
about 1 to 100 .mu.g/10.sup.6 cells. These doses can of course be
adapted by the skilled person without difficulty. Incubation is
carried out in any suitable cell culture medium, and under the
normal temperature conditions (for example between ambient
temperature and about 37.degree. C.). The media used are any mammal
cell culture media known to the skilled person, such as RPMI, DMEM,
MEM, etc. Incubation can be carried out using any suitable
apparatus such as a dish, flask, ampoule, pouch, tube, syringe,
etc., preferably under sterile conditions. Advantageously,
incubation is carried out for a period in the range about 1 hour to
about 5 days, depending on the use and aim. As an example, cells
can be incubated for a period in the range from about 1 hour to
about 12 hours. The incubated cells can then be administered to a
subject (autologous), and the subject can also receive one or more
administrations of antibodies or antibody fragments.
[0114] For in vivo use, the compounds can be administered by
different routes, such as systemic, intramuscular, or
sub-cutaneously, for example. Preferred routes are systemic (in
particular i.v.) and sub-cutaneous. The doses used can also be
adapted by the skilled person as a function of the stage of the
subject, the desired aim and the number and/or frequency of
administrations.
[0115] Preferably, the invention is employed to inhibit viral
infection or replication in target cells ex vivo. To this end,
target cells are removed from a subject (PBMC, for example),
incubated ex vivo with compounds as defined above (for example for
1 to 6 hours, at 37.degree. C., in a sterile pouch), then
re-administered to the subject. The subject can also receive one or
more administrations of the compounds, optionally in combination
with one or more other antiviral agents. The compounds or
compositions as described above are particularly suitable for
preventive use, i.e., to inhibit viral replication or infection in
healthy or seropositive subjects but who have not developed the
symptoms of the disease. The invention can also be used as a
maintenance treatment, used alone or in combination with other
antiviral agents, as explained above.
[0116] The invention thus also concerns a method for improving the
efficacy of antiviral agents comprising the combined use of
polypeptide, antibody or antibody fragments as defined above.
[0117] The invention also concerns a method for modifying a cell
with the aim of reducing infection of this cell by a virus and/or
replication of a virus in this cell (i.e., to improve viral
resistance of this cell), comprising bringing said cell into
contact with one or more polypeptides, antibodies or antibody
fragments as defined above. The invention also concerns any
population of cells incubated in the presence of polypeptides,
antibodies or antibody fragments as defined above. More
particularly, such populations can be PBMC cells or other cells of
the immune system, optionally packaged in a sterile container.
Preferably, such a cellular composition generally comprises
10.sup.5 to 10.sup.8 cells. These cellular compositions can be used
to study the viral cycle, to search for inhibiting compositions or
associations of inhibiting compounds, or optionally to reduce the
risks and effects of a viral infection in vivo after
administration.
[0118] The invention also concerns a pharmaceutical composition
comprising, in association with a physiologically acceptable
vehicle, a vector as defined above in which the substance is an
active principle of a drug.
[0119] The invention still further concerns a vaccine comprising,
in association with a physiologically acceptable vehicle, a vector
as defined above in which the substance is an antigen.
[0120] The present invention will now be described in more detail
using the following non limiting examples which are provided by way
of illustration.
KEY TO FIGURES
[0121] FIG. 1: Diagram of the structure of an antibody.
[0122] FIG. 2: Peptide sequence of the variable regions of the
heavy chain of penetrating monoclonal antibody. The CDR regions are
shown. "-" means identical amino acids.
[0123] FIG. 3: Sequence for CDR2 and CDR3 regions of the VH domains
in cells penetrating anti-DNA antibody.
[0124] FIG. 4: ELISA demonstration of the in vivo adjuvant effect
of the polypeptides of the invention.
[0125] FIG. 5: DNA transfection activity of the CDR2-3-PL19 peptide
in CCL39 cells.
[0126] FIG. 6: Polyfection of 3T3 cells by the K19-P3 peptide at
different peptide/plasmid ratios.
[0127] FIG. 7: Polyfection of 3T3 cells by the K19-P3 peptide as a
function of incubation time of the cells with the complex.
[0128] FIG. 8: Polyfection of 3T3 cells in the absence (FIG. 8a) or
presence (FIG. 8b) of a facilitator agent: glycerol.
[0129] FIG. 9:Polyfection of CCL39 cells in the absence (FIG. 9a)
or presence (FIG. 9b) of a facilitator agent: glycerol.
[0130] FIG. 10: Infectious titers and determination of the p24
antigen of HIV1 produced in a culture medium for human circulating
mononuclear cells (PBMC) treated with different preparations of
antibody and infected with HIV1 Lai. Average values for three
independent experiments.
[0131] FIG. 11: Infectious titers and determination of the p24
antigen of HIV1 produced in human PBMC culture medium treated with
different preparations of polypeptides or antibody and infected
with strain HIV1 BX 08.
[0132] FIGS. 11A and 11B: Measurement of infectious titers on day
13 in two series of independent experiments (experiments III and
V).
[0133] FIG. 11C: Quantification of p24 antigen in supernatants of
cultures inoculated with 10.sup.-2 dilution on day 9. (experiment
V).
[0134] FIG. 12: Inhibition of replication of polio virus by
polypeptides by measuring the viral titer reduction factor on day
5.
[0135] Solid bars: wild-type polio virus strain.
[0136] Hatched bars: attenuated polio virus strain.
[0137] FIG. 13: Demonstration of inhibition of CMV protein
synthesis after treatment with K19-pF4.1 (PL) polypeptide, P3
peptide, or no peptide (NT), before viral infection (13a, 13b),
during viral infection (13c) or after viral infection (13d). It was
revealed using an anti-pp150 antibody (for late proteins),
anti-pp65 (for early proteins) and anti-IE (for very early
proteins).
METHOD AND APPARATUS
Mice and Cell Lines
[0138] BALB/c (NZB.times.NZW) F1 mice were kept in the animal house
at the Institut Pasteur. Cells from different species and from
different tissues were used: PtK2 cells (kidney fibroblasts),
GMA-32 cells (hamster lung), 3T3 cells (mouse embryo fibroblasts),
CCL39 cells (hamster fibroblasts), HeLa cells (human cervical
carcinoma), VERO cells (monkey kidney), HEp-2 cells (human larynx
carcinoma), JURKAT cells and CEM cells (human T lymphoblasts) all
available from the ATCC Collection. These different cell types were
cultivated in RPMI medium or in DMEM medium containing 10% of
inactivated foetal calf serum and supplemented with L-glutamine,
sodium pyruvate and non-essential amino acids and antibiotics
(complete culture medium) at 37.degree. C. in a moistened
atmosphere containing 5% Co.sub.2.
Monoclonal Antibodies
[0139] The preparation and isolation of monoclonal antibodies
J20.8, F4.1 and F14.6 have been described in French patent
application FR-9508316. These antibodies are polyreactive anti-DNA
antibodies and also recognise different antigens such as peptides
22-37 and 46-60 from the Tat protein. These antibodies are murine
IgG2a cells. The anti-Tat antibody used is a monoreactive murine
IgGl monoclonal antibody recognising the Tat protein of HIV-1.
[0140] These antibodies were purified on a protein A sepharose
column (Ey et al., Immunochemistry 15 (1978) 429). The
polyreactivity of these purified antibodies as regards double
stranded DNA and other antigens was tested using ELISA employing
the methodologies described in the literature (Guilbert et al., J.
Immunol. 128 (1982) 2779).
Peptide Synthesis
[0141] The peptides were synthesised using techniques which are
known to the skilled person. Thus the peptides were produced by
solid phase synthesis on Fmoc resin. Trifluoroacetic acid (TFA) was
used for cleavage and the peptides were purified on a
semi-preparative HPLC-RPCS column (Eurosil Bioselect 5 .mu., 300 A
(1;6.times.25 cm) and eluted at 1.1 ml/min with a 0.1% TFA solution
and an acetonitrile gradient (10-70%). The lyophilised peptides
were dissolved in 0.15 mM NaCl and sterilised with a 0.22 .mu.m
filter. To determine the peptide concentration, aliquots were
hydrolysed at 110.degree. C. in the presence of 6N HCl-2% phenol
then analysed using a Beckman 6300 amino acid analyser.
Viral Strains
[0142] The experiments described below were carried out using the
following viral strains: HIV-1 BX08 strain (primary isolate,
sub-type B); HIV-1 Lai strain; polio virus type 1 (wild type PV1
Mahoney strain and attenuated Sabin PV1); cytomegalovirus Ad169
strain and Herpes Virus Simplex type I.
EXAMPLES
1. Sequencing of Monoclonal Antibodies
[0143] The nucleotide sequence of the VH and VL regions of
monoclonal antibodies J20.8, F4.1 and F14.6 were determined. To
this end, total RNA was extracted from hybridoma cells using the
guanidine thiocyanate technique (Schwartz et al., Biol. Cell. 73
(1991) 7) then separated by formaldehyde/agarose gel
electrophoresis. The messenger RNAs obtained was then transformed
into complementary DNA using a reverse transcriptase kit (Life
Technologies, Eragny, France) and used as a primer in amplification
reactions (PCR) using Taq DNA polymerase (Boehringer, Mannheim,
Germany) following the manufacturer's instructions. The
oligonucleotide primers used to generate the complementary DNA
were:
[0144] firstly, a primer corresponding to the conserved sequences
of IgG2a immunoglobulins: TABLE-US-00001 (SEQ ID N.sup.o 11)
5'-GTTCTGACTAGTGGGCACTCTGGGCT
[0145] and secondly, four primers for the VH region: TABLE-US-00002
(SEQ ID N.sup.o 12) 5'-GAGGTTCAGCTCGAGCAGTCTGGGGC (SEQ ID N.sup.o
13) 5'-GAGGTGAAGCTCGAGGAATCTGGAGG (SEQ ID N.sup.o 14)
5'-GAAGTGCAGCTCGAGGAGTCTGGGG (SEQ ID N.sup.o 15)
5'-GAGGTTCAGCTCGAGCAGTCTGGAGC
[0146] The PCR amplification products were then purified using a
Geneclean kit (Bio 101, Vista, Calif.). Chemical sequencing was
carried out by Genome Express (Grenoble, France). The nucleotide
sequences were analysed using GENBANK and EMBL databases held at
the Institut Pasteur (Information Science Unit) using GCG sequence
analysis software (GCG) (Devereux J., "The GCG Sequence Analysis
Software Package", 1989), and the corresponding amino acid
sequences were deduced.
[0147] The sequence for the VH regions of these antibodies is shown
in FIG. 2. Alignment of these sequences enables the CDR regions
(CDR1, CDR2 and CDR3) present in these sequences to be localised.
This alignment also demonstrates the existence of a substantial
structural homology between the CDR2 regions and the common
structural characteristics between the CDR3 regions, in particular
the presence of basic residues (arginine and lysine). The sequences
for the CDR2 and CDR3 regions of other antibodies are shown in FIG.
3.
2. Construction of Penetrating Polypeptides
[0148] Starting from the sequences shown in FIG. 1, different
polypeptides comprising all or a portion of the CDR3 region and/or
the CDR2 region of the antibodies were prepared. Synthesis was
carried out by peptide synthesisers (see Method and Apparatus
section). The following polypeptides were synthesised, where m is 1
or 0:
[0149] CDR3: TABLE-US-00003 SEQ ID n.sup.o 1:
(Thr).sub.m-(Arg).sub.m-(Gln).sub.m-Lys-Tyr-Asn-Lys-Arg-Ala-
(M-D-Y-W-G-Q-G-T).sub.m
[0150] A variation of this sequence is, for example the sequence
TRQKYNKRA(MDYWGQGT).sub.m. A further variation (functional
homologue) is, for example, the sequence
Ala-Arg-Gln-Lys-Tyr-Asn-Lys-Arg-Ala-Met-Asp-Tyr (SEQ ID No. 8).
TABLE-US-00004 SEQ ID n.sup.o 2:
(Thr).sub.m-(Arg).sub.m-(Gln).sub.m-Lys-Tyr-Gly-Lys-Arg-Gly-
(M-D-Y-W-G-Q-G-T).sub.m
[0151] A variation of this sequence is, for example the sequence
TRQKYNKKRG(MDYWGQGT).sub.m. TABLE-US-00005 SEQ ID n.sup.o 3:
(Thr).sub.m-(Arg).sub.m-(Gln).sub.m-Ala-Arg-Ala-Thr-Trp-Asp-Trp
(F-A-Y-W-G-Q-G-T).sub.m
[0152] A variation of this sequence is, for example the sequence
TRGARATWDW(FAYWGQGT).sub.m.
[0153] In sequences 1 to 3 above,
MDYWGQGT=Met-Asp-Tyr-Trp-Gly-Gln-Gly-Thr and
FAYWGQGT=Phe-Ala-Tyr-Trp-Gly-Gln-Gly-Thr. Further, the formula
(a-b-c).sub.m means that a single, some or all of the residues
mentioned in brackets are present or are not present. CDR2:
TABLE-US-00006 SEQ ID n.sup.o 4:
(Val).sub.m-(Ala).sub.m-Tyr-Ile-Ser-Arg-Gly-Gly-Val-Ser-Thr-
Tyr-Tyr-Ser-Asp-Thr-Val-Lys-Gly-(Arg).sub.m-(Phe).sub.m-
(Thr).sub.m (CDR2(1)).
[0154] A variation of this sequence is, for example, the sequence
VAYISRGGVSTYYSDTVKGRF or VAYISRGGVSTYYSDTVKGRFT. TABLE-US-00007 SEQ
ID n.sup.o 5:
(Val).sub.m-(Ala).sub.m-Tyr-Ile-Ser-Arg-Gly-Gly-Gly-Ile-Phe-
Tyr-Tyr-Glu-Asp-Ser-Ile-Lys-Gly-(Arg).sub.m-(Phe).sub.m
(CDR2(2)).
[0155] A variation of this sequence is, for example, the sequence
VAYISRGGIFYYQDSIKGRF. TABLE-US-00008 SEQ ID n.sup.o 6:
(Val).sub.m-(Ala).sub.m-Ala-Ile-Ser-Arg-Gly-Gly-Gly-Tyr-Ser-
Tyr-Tyr-Leu-Asp-Ser-Val-Lys-Gly-(Arg).sub.m-(Phe).sub.m-
(Thr).sub.m-(Ile).sub.m (CDR2(3)).
A variation of this sequence is, for example, the sequence
VAAISRGGGYSYYLDSVKGRFTI.
[0156] CDR2-3 TABLE-US-00009 SEQ ID n.sup.o 7 (p3 or Pf4.1
peptide): Val-Ala-Tyr-Ile-Ser-Arg-Gly-Gly-Val-Ser-Thr-Tyr-
Tyr-Ser-Asp-Thr-Val-Lys-Gly-Arg-Phe-Thr-Arg-Gln-
Lys-Tyr-Asn-Lys-Arg-Ala.
A variation of this sequence is, for example, the sequence
VAYISRGGVSTYYSDTVKGRFTRQKYNKRAVAY.
[0157] Functionalised CDR2-3: TABLE-US-00010 (corresponding to the
sequence SEQ ID n 7)
Biotinyl-Val-Ala-Tyr-Ile-Ser-Arg-Gly-Gly-Val-Ser-
Thr-Tyr-Tyr-Ser-Asp-Thr-Val-Lys-Gly-Arg-Phe-Thr-
Arg-Gln-Lys-Tyr-Asn-Lys-Arg-Ala- (SEQ ID n.sup.o 9)
Cys-Val-Ala-Tyr-Ile-Ser-Arg-Gly-Gly-Val-Ser-Thr-
Tyr-Tyr-Ser-Asp-Thr-Val-Lys-Gly-Arg-Phe-Thr-Arg-
Gln-Lys-Tyr-Asn-Lys-Arg-Ala-.
[0158] An active group (SH) was introduced into sequence SEQ ID No.
9 via cystein to enabling coupling to another substance.
[0159] Functionalised CDR2: TABLE-US-00011
Biotinyl-VAYISRGGVSTYYSDTVKGRFT (Biotinyl-Val-Ala-
Tyr-Ile-Ser-Arg-Gly-Gly-Val-Ser-Thr-Tyr-Tyr-Ser-
Asp-Thr-Val-Lys-Gly-Arg-Phe-Thr), corresponding to sequence SEQ ID
n.sup.o 4.
[0160] Functionalised CDR3: TABLE-US-00012
Biotinyl-Ala-Arg-Gln-Lys-Tyr-Asn-Lys-Arg-Ala-Net- Asp-Tyr
(corresponding to sequence SEQ ID n.sup.o 8).
3. Study of the Penetration of Polypeptides into Cells
[0161] Cultured PtK2 fibroblasts seeded the day before in an amount
of 5.times.10.sup.4 cells per well onto glass sheets, are incubated
at 37.degree. C., 1-18 hours in complete RPMI 1640 culture medium
(or DMEM) (10% foetal calf serum, 2 mM L-glutamine and 1 mM of
sodium pyruvate) containing a biotinylated polypeptide of the
invention (5-20 .mu.g/ml). The cells were then washed with PBS and
fixed with 2% of p-formaldehyde at 4.degree. C. for 10 minutes then
washed with PBS.
[0162] The cells were then incubated with a solution of
streptavidin conjugated with 5 .mu.g/ml of peroxidase in PBS for 30
minutes, then washed with PBS and incubated in the peroxidase
cytochemical substrate (diaminobenzidine+H.sub.2O.sub.2). After
washing, the cells were examined microscopically.
[0163] The results obtained show that after 1 hour of culture, the
polypeptides comprising all or a portion of a CDR3 were visible by
peroxidase coloration in the cytoplasm of all of the cells and in
the nucleus of a large number of cells. The results also show that
the polypeptide CDR2-3 (in particular the pF4.1 polypeptide with
sequence SEQ ID No. 7) penetrated massively and rapidly into the
cells and most reached the nucleus of said cells. These results
thus show that it is possible to generate polypeptides with a high
cell penetration capacity from a CDR3 type fragment.
4. Cell Penetration of Polypeptide-streptavidin Vectors Coupled to
Enzymes
[0164] This example illustrates how the polypeptides of the
invention can be coupled to an active substance and used to
transport said substance into cells.
[0165] The vectors were prepared by incubating 1.4 .mu.g of
biotinylated CDR2-3 polypeptide (pF4.1) with 10 .mu.g of
streptavidin conjugated with peroxidase or with alkaline
phosphatase in a volume of 10 .mu.l for 15 minutes at laboratory
temperature. The mixture was then diluted in 0.5 ml of complete
culture medium before being deposited on the cells in culture.
After 2 hours of culture, the cells were washed with PBS, fixed
with p-paraformaldehyde, washed then incubated in the peroxidase
cytochemical substrate (diaminobenzidine+H.sub.2CO.sub.2) or that
of alkaline phosphatase (Naphthol AsMx+Fast Red tetrazolium
salt).
[0166] The results obtained show that the corresponding enzymes
were detected in the cytoplasm of all of the cultured cells and
weakly to intensely in the majority of the cell nuclei. In
contrast, no intracellular coloration was observed when the cells
were incubated in the presence of streptavidin coupled with
peroxidase, streptavidin coupled with alkaline phosphatase or with
streptavidin or the enzymes in their native forms.
5. Construction and Activity of a Polypeptide Comprising
Supplemental Lysine Residues
[0167] A CDR2-3-PL19 (polylysine) (also termed K19-P3 or K19-pF4.1)
was synthesised and purified (ALTERGEN). The sequence of the
polypeptide is as follows: TABLE-US-00013 SEQ ID n.sup.o 10
(NH2-(K19)-VAYISRGGVSTYYSDTVKGRFTRQKYNKRA-COOH).
[0168] CCL39 cells (hamster fibroblasts) (5.times.10.sup.4 cells)
were placed in 24 well culture plates for 18 hours before
transfection in MEM+10% FCS (foetal calf serum) culture medium.
Transfections were carried out in MEM+10% FCS with no other
auxiliary agent (CHLOROQUINE). The peptide-PL and free polylysine
PL (corresponding to 19 lysines) were complexed with the pCMVLUC
plasmid (respectively 24 .mu.g and 70 .mu.g per 6 .mu.g of plasmid)
for 30 minutes. The complex was then added dropwise to the CCL39.
After incubating for 5 hours, the medium was replaced with fresh
medium. Luciferase expression was assayed 24 hours later. The cells
were washed twice with PBS. After washing, the cells were lysed
with 100 .mu.l of lyse buffer (PROMEGA) for 10-15 minutes. The
cells were then centrifuged for 7 minutes at 4.degree. C. to remove
cellular debris. 20 .mu.l of this lysate was mixed with 100 .mu.l
of luciferase buffer (PROMEGA). The relative luciferase units (RLU)
were recorded on a LUMAT LB9501 (BERTHOLD). The protein
concentration was determined using a BIORAD PROTEIN ASSAY-1 kit and
the amount of luciferase in each sample was normalised per mg of
protein, each transfection being carried out three times.
[0169] The results obtained are shown in FIG. 5. They show that in
complete medium and with no auxiliary agent, the peptide-PL
transfects with an efficacy of 2.times.10.sup.6 RLU/mg of proteins,
i.e., about 1000 times more than polylysine alone and more. than a
peptide recently described (Wadhwa et al., Bioconjugate Chem. 1997,
8:81-88), but where the activity was dependent on the presence of
100 .mu.M of chloroquine.
[0170] Transfecting cells with the CDRK19-P3 polypeptide is thus
particularly advantageous since it can be carried out in a complete
culture medium and in the absence of auxiliary agent. Current
transfection systems using polylysine all require the addition of
an auxiliary agent, usually chloroquine which is toxic for the
cells. This chloroquine prevents degradation in the lysosomes of
conjugate-polylysine complexes internalised by the conventional
endocytosis route.
[0171] In contrast, the present invention does not require the use
of such auxiliary agents.
6. Polyfection of 3T3 Cells with the K19-P3 Peptide
[0172] This example illustrates the transfer properties of nucleic
acids of the peptides of the invention in 3T3 cells.
[0173] 3T3 cells (8.times.10.sup.4 cells) were distributed into 24
well plates the day prior to transfection. Polyplexes between the
pCMV LUC plasmid and the K19-P3 peptide or control peptides CW-K19
and K19 were prepared by incubating 3 .mu.g of plasmid in 50 .mu.l
of 0.15 M NaCl for 20 minutes at 20.degree. C., with different
quantities of peptide. More particularly, the polyplexes were
produced in stoichiometries of 0.05 to 1.4 nmoles of peptides per
.mu.g of DNA.
[0174] Shortly before transfection, the cells were washed, then
incubated for different periods with 0.5 ml of complete culture
medium. The polyplexes were added to the cells for 1 h15, 2h30,
5h30 and 24h at 37.degree. C. in a moist atmosphere (92% air, 8%
CO.sub.2). The medium was eliminated and the cells incubated again
for 24 hours at 37.degree. C. in 1 ml of fresh medium. Each
experiment was carried out at least three times. The luciferase
activity was determined as for Example 5.
[0175] The results obtained are shown in FIGS. 6 and 7.
[0176] FIG. 6 shows an increase in luciferase expression correlated
with an increase in the concentration of peptide, the maximum
activity being observed at a concentration of 0.6 nmoles of
peptide/.mu.g of DNA. This concentration corresponded to a charge
ratio of R=4.4. At higher concentrations, expression remained
constant.
[0177] FIG. 7 also shows that, at a charge ratio R of 4.4, exposure
of cells for 24 hours to the polyplex did not induce any toxicity.
After incubating for 5h30, the luciferase activity which was
measured was 2-3.times.10.sup.7 RLU/mg of protein for the K19-P3
peptide. Transfection after incubating for 24 h was increased by a
factor of about 1.3 with respect to incubating for 5h30.
7. Polyfection in the Presence of Glycerol
[0178] This example shows that transfection efficacy can be
improved using a composition comprising a peptide of the invention
and a stabiliser such as glycerol.
[0179] In this example, transfections were carried out on 3T3 cells
and CCL39 cells, as described in Example 6, in a complete medium
containing or not containing glycerol (0.23 M) in a peptide/DNA
charge ratio of 2.2 (incubation for 5h30 at 37.degree. C.).
[0180] Further, by way of comparison, transfection was carried out
in the presence of polyethylene immine (PEI), 25 kDa (Aldrich, St
Louis, Mo.) in a charge ratio of 2.2 in the presence of glycerol
and 5 in the absence of glycerol.
[0181] The results obtained are shown in FIGS. 8 and 9.
[0182] These results show that glycerol (0.23 M) induces an
increase in polyfection (determined by measuring the luciferase
activity) by a factor of more than 5, possibly up to 40. Thus in
CCL39 cells (FIG. 9), an increase by a factor of 38 was
demonstrated, illustrating the importance of a composition of the
invention comprising glycerol, in particular for transfecting cells
which are difficult to transfect. Further, similar results were
obtained by varying the concentration of the glycerol in the
medium, up to a value of 1.15 M.
[0183] The results shown in FIGS. 8 and 9 also show that the
transfection efficacy of the peptides of the invention, which are
non toxic and of a simple, defined structure, are higher than 25
kDa PEI in the presence of glycerol, both for 3T3 cells and for
CCL39 cells.
8. Use of a Polypeptide as an Immunoadjuvant
[0184] The vector was formed by incubating 14 .mu.g of biotinylated
CDR2-3 polypeptide+40 .mu.g of streptavidin conjugated with
peroxidase (Sigma) for 15 minutes then diluting in 0.1 ml of PBS
before being injected into each mouse. Injection was via the pads.
The control mice received 40 .mu.g of streptavidin conjugated with
peroxidase in 0.1 ml of PBS.
[0185] The mice were bled every week. A repeat injection was
carried out under the same conditions one month after the first
injection. An ELISA test showed that the mice which had received
the CDR2-3-streptavidin conjugated with peroxidase complex
responded with anti-streptavidin and anti-peroxidase IgG
antibodies, but with very few IgM, with substantially higher values
than those which had received streptavidin conjugated with
peroxidase alone and from the 14.sup.th day (FIG. 4). The repeat
injection caused an increase in the antibody titer in the two
groups, but the values were always higher in the group which had
received the complex. On the basis of these results, it thus
appears that under the test conditions, the polypeptides of the
invention are capable of increasing the titer of antibodies
directed against a given antigen by a factor of at least 4 to
8.
[0186] The same experiments can be reproduced using not a protein
as the antigen but a nucleic acid coding for said antigen. Further,
these experiments can also be repeated under the same conditions
with a polypeptide comprising supplemental basic residues, in
particular a polylysine.
9. HIV Inhibition
[0187] This example illustrates the antiviral properties of
polyreactive antibodies of the invention on HIV-1 Lai strain.
Cells
[0188] The target HIV cells used were human peripheral blood
mononuclear cells (PBMC). These cells were obtained from healthy
subjects using any technique which is known to the skilled person
(Ficoll gradients, leukapheresis, etc.). The PBMC cells were
activated by phytohemagglutinin for about 3 days and kept in
culture at 37.degree. C. in a CO.sub.2 atmosphere in the presence
of interleukin-2.
Assay of p24 Antigen
[0189] The p24 protein was assayed in cell culture supernatants
using an ELISA test employing a commercially available kit
(Diagnostic Pasteur).
[0190] Human peripheral blood mononuclear cells (PBMC), after
activation in phytohemagglutinin, were incubated at different
concentrations with the three antibodies J20-8, F14-6 and F4-1 for
4 hours at 37.degree. C. After eliminating the antibodies, the
cells were infected with successive dilutions of HIV-1 Lai (1 hour
at 37.degree. C.), washed, incubated with fresh culture medium in
the presence or not in the presence of antibody and the supernatant
was examined every 3 or 4 days for the presence of the P24 antigen
of HIV-1 to evaluate the level of viral replication in treated or
untreated cells. The results of these titrations of HIV-1 Lai on
PBMC cells treated or not treated with the antibodies are shown in
FIG. 10. These results show that the antibody F4-1 is capable of
reducing the titer of the virus by about log 2 with respect to
untreated cells or cells treated with monoreactive anti-Tat
antibody. Further, these results also show that the antibody F14-6,
which does not inhibit HIV-1 when it is tested alone, caused a
reduction in the infectious titer in synergy with the antibody
J20-8 which alone had no inhibiting activity.
[0191] It is highly probable that the action of the antibody of the
invention is exerted once the antibody has penetrated into the
cells, as illustrated by the fact that pre-incubation of cells with
the antibody can induce a strong inhibition of HIV-1
infectiousness. Further, preliminary experiments indicate that
treatment with antibody does not modify virus-cell recognition via
the CD4 molecule, which suggests a specific and intranuclear effect
of the antibodies of the invention on HIV infectiousness and
replication.
10. Inhibition of HIV
[0192] This Example again illustrates the properties of the
compositions of the invention, in particular polypeptides, on a
further HIV isolate.
[0193] Human peripheral blood mononuclear cells (PBMC) were
activated in the presence of phytohemagglutinin for 3 days, then
re-suspended in a culture medium in the presence of interleukin-2.
The cells were then pre-incubated for 4 hours at 37.degree. C. with
25 .mu.g or 50 .mu.g per 2.times.10.sup.6 of the following
polypeptides or antibodies: [0194] P3 peptide: this peptide
corresponds to the peptide pF4.1 in SEQ ID No. 7. [0195] P3PL
peptide: this peptide corresponds to peptide K19-pF4.1 of SEQ ID
No. 10. [0196] F4-1 antibody.
[0197] By way of comparison, the cells were also incubated in the
presence of AZT.
[0198] After incubation, the cells were washed in fresh culture
medium then infected with 0.5 ml of a viral HIV-1 BX 08 strain
dilution, (10.sup.-3 to 10.sup.-5 or 10.sup.-1 to 10.sup.-4
depending on the experiment) into a stock of 2.times.10.sup.6 cells
for 1 hour at 37.degree. C. The cells were then washed 3 times in
fresh medium and re-suspended in medium containing the above
peptides or antibodies (12.5 .mu.g or 25 .mu.g) and distributed
over 48 well plates in an amount of 4 wells per dose of virus and
peptide/antibody. The culture medium, containing
peptides/antibodies, was changed every 3 or 4 days. Then viral
production (consequence of infection and replication) was estimated
by assaying the p24 antigen in the culture supernatants, under the
conditions described in the Method and Apparatus section.
[0199] The results of several series of experiments are shown in
FIGS. 11A, 11B and 11C.
[0200] As for Example 9, these results illustrate the capacity of
polyreactive anti-DNA antibody or polypeptides to reduce
replication of HIV in target cells, at different doses.
11. Inhibition of Polio Virus
[0201] This example illustrates the antiviral properties of the
products of the invention for polio virus.
[0202] In order to test the inhibiting power of the peptides
K19pF4.1, K19pJ20.8, K19pF14.6 on polio virus replication, two
strains of type 1 polio virus (PV1) were used: the PVA/Mahoney wild
type strain and the PV1/Sabin attenuated strain. The inhibiting
power of the peptides was evaluated by measuring the titer
reduction factor of a viral suspension of polio virus in the
presence of peptide. The viral suspension titer was determined in
terms of the cytopathogenic 50 dose (CPD50) per ml on Hep-2c human
epithelial cells using a dilution limit microtechnique (Melnick et
al., 1979, Melnick, J. L., Wenner, H. A., and Philips, C. A.,
(1979). Enteroviruses, in "Diagnostic procedure for viral,
rickettsial and chlamydial infections" (E. H. Lennette and N. J.
Schmidt, Eds), pp. 471-534, American Public Health Association,
Washington D.C.). The titer reduction factor thus corresponds to
the ratio between the titer for the viral suspension in the absence
of peptide and that in the presence of the test peptide.
Apparatus
[0203] Peptides Peptides derived from anti-DNA monoclonal antibody:
K19pF4.1 (SEQ ID No. 10); TABLE-US-00014 K19pFJ20.8
(K).sub.19-V-A-Y-I-S-R-G-G-G-I-F-Y-Y-Q-D-S-I-K-G-R-F-T-
R-E-K-Y-G-K-R-G-M-D-Y; K19PF14.6
(K).sub.19-A-I-S-R-G-G-G-Y-S-Y-Y-L-D-T-V-K-R-T-A-R-A-T-
W-D-W-F-A-Y.
[0204] Peptide used as negative control: K19PT corresponding to an
ovalbumin peptide with 20 amino acids carrying 19 N-terminal
lysines.
Virus
[0205] Wild type polio virus type 1 strain: PV1/Mahoney;
[0206] Attenuated polio virus type 1 strain: PV1/Sabin.
Cells
[0207] Hep-2c human epithelial cells originating from an epidermoid
carcinoma of the larynx.
Protocol
[0208] On day 1, the cells were placed in culture. To this end, a
suspension of 2.5.times.10.sup.5 cells/ml in a MEM medium, 10%
foetal calf serum (FCS), 0.5% gentamycin was prepared, and 5 plates
of 96 wells were seeded with 200 .mu.l/well (i.e., 5.times.10.sup.4
cells/well). The cells were incubated at 37.degree. C. in the
presence of 5% CO.sub.2.
[0209] On day 0, the cells were pre-incubated with the peptides for
2 hours. To this end, the peptides indicated above were diluted to
50 .mu.g/ml in MEM medium, 10% FCS, 0.5% gentamycin. The wells were
emptied by aspiration, then 100 .mu.l/well of medium+peptide (one
plate per peptide) was added. As a control a plate was prepared
with 100 .mu.l/well or medium with no peptide for titrating viral
suspension in the absence of peptide. The mixture was incubated for
2 hours at 37.degree. C. in the presence of 5% of CO.sub.2.
[0210] In parallel, dilutions of viral suspensions were prepared,
of 10 in 10 up to 10.sup.-4, then dilutions of 4 in 4 up to
10.sup.-8.8 in MEM medium, with no FCS, 0.5% gentamycin (50
.mu.l/well and 4 wells/dilution).
[0211] For use during infection, the peptides were diluted in MEM
medium, 3% FCS, 0.5% gentamycin so as to obtain a final
concentration of 25 .mu.g/ml of peptide (knowing that for the test,
150 .mu.l of medium containing the peptide was added to 50 .mu.l of
viral dilution).
[0212] The infection step was carried out by emptying the wells (by
aspiration) then adding the following elements: [0213] 150
.mu.l/well of diluted peptide or medium for the untreated plate;
[0214] 50 .mu.l/well of 10.sup.-5.8 to 10.sup.-8.8 viral dilutions,
4 wells/dilution. Incubation was at 37.degree. C. for 5 days in the
presence of 5% of CO.sub.2.
[0215] 5 days after infection, the CPD50/ml titers were determined
as described above.
[0216] The results obtained are shown in the Table below and in
FIG. 12. These results show the capacity of the polypeptides of the
invention, used alone, to inhibit replication of different strains
of polio virus. TABLE-US-00015 TABLE TITER LOG REDUCTION VIRUS
PEPTIDE (TITER/ML) FACTOR PV1/Mahoney none 9.65 K19pF4-1 8.75 8
K19pJ20-8 8.9 6 K19PF14-6 9.05 4 K19PT 8.75 8 PV1/Sabin none 9.5
K19pF4-1 8 32 K19pJ20-8 8.6 8 K19PF14-6 8.6 8 K19PT 8.6 8
[0217] In particular, these results show that the K19-pF4.1 peptide
has a very pronounced inhibiting effect on type 1 polio virus
replication, by a factor of more than 30. This experiment
illustrates the multiple applications of the present invention for
inhibiting different types of virus.
12. Inhibition of Cytomegalovirus (CMV) Replication
[0218] This example illustrates the antiviral properties of
compounds of the invention on cytomegalovirus.
Apparatus
Cells
[0219] Human diploid fibroblasts and primary human astrocytomas
(U373MG) were used. They were cultivated in a Dulbecco medium with
a supplemental 2 mM of glutamine and 10% of foetal calf serum.
Peptides
[0220] The compound used was the K19pF4-1 peptide derivative.
Virus
[0221] The CMV Ad169 strain (ATCC VR538) was used. CMV titration
was carried out by counting the plaques formed under carboxymethyl
cellulose (0.6%).
Procedure
[0222] The cells were treated with trypsine, washed, distributed in
the wells of culture plates in an amount of 10.sup.5 per well and
cultivated for 6 to 24 hours. The polypeptides, in sterile
solution, were added to the cultures in a final concentration of 25
to 50 .mu.g/ml. The cells were infected in an infection
multiplicity of 1 pfu/cell.
[0223] Three operating procedures were followed: [0224] a) the
polypeptides were added 4 hours before viral infection; [0225] b)
the polypeptides were added at the same time as the virus; [0226]
c) the polypeptides were added after absorption of the virus on the
cells (2 hours at 37.degree. C.)
[0227] The antiviral effect was evaluated: [0228] 1) visually by
observing the morphological appearance of the cells under an
optical microscope; [0229] 2) by titrating the viral multiplication
by counting the plaques formed 5 days after infection; and [0230]
3) by Western blot analysis of the appearance of very early, early
and late viral infection proteins. Results
[0231] The results obtained showed that: [0232] 1) the cells
treated with K19-pF4-1, before or at the same time as the viral
infection, displayed no morphological modifications (cytopathogenic
effects: CPE). In contrast, the CPE of cells treated with K19-pF4.1
at the end of viral adsorption was large 24 to 48 hours after
infection. Treated or untreated cells after viral adsorption with
K19-pF4.1 or free polylysine of 19 residues (K19) demonstrated
large CPEs. [0233] 2) Virus replication was inhibited by 99.5% in
cells treated with K19-pF4.1 before, and at the same time, as viral
infection (see Table below). [0234] 3) In cells treated with
K19-F4.1 before viral infection, the appearance of neither early
proteins nor late proteins of the CMV was noted (FIGS. 13a and
13b). In cells treated with K19-pF4.1 at the same time as viral
infection, no appearance of late proteins was noted, but the
appearance, albeit reduced in quantity, of very early proteins was
noted (FIG. 13c) K19-pF4.1 had no action if it was added to cells
after viral adsorption (FIG. 13d).
[0235] In conclusion, the results show that the K19-pF4.1
polypeptide significantly inhibits CMV replication when the cells
are treated with this polypeptide before viral infection. When it
is added to cells at the same time as CMV, the peptide is also
effective, but can be to a lesser extent (appearance of early
antigens). TABLE-US-00016 TABLE NUMBER OF PLAQUES FORMED % Mean SD
reduct'n Day 1 No rx 50 50 0 33.333 28.9 K19-pF4.1 100 125 75 100.0
25.0 Day 4 No rx 18000 50750 31500 33416.7 16458.9 K19-pF4.1 0 250
250 166.7 144.3 99.5
[0236] CMV titrations 5 days after infection with 25 .mu.g/ml of
cells pretreated with K19-pF4.1 or with no treatment (no Rx).
13. Inhibition of Herpes Virus
[0237] This example illustrates the antiviral properties of the
compounds of the invention on the herpes simplex virus.
[0238] The antiviral activity was determined by measuring the
cytopathogenic effect (CPE) induced by a HSV-1 TK.sup.+virus (i.e.,
expressing thymidine kinase) and by a HSV-1 TK.sup.- virus (i.e.,
not expressing thymidine kinase, and thus insensitive to AZT).
[0239] The following peptides and antibodies were used: K19,
K19-pF4.1, K19-pJ20.8, K19-pF14.6 and F14.6.
[0240] Acyclovir.RTM. was used as the reference anti HSV-1 TK.sup.+
compound (2 mg, MW 225, Wellcome). The Acyclovir was dissolved n 2
ml of DMEM 1X medium supplemented with antibiotics, then 50 .mu.l
of 1N NaOH was added. The volume was adjusted to 4.44 ml with DMEM
1X medium, AB, and the pH was adjusted to 7-7.4 using 1N HCl. The
neutralised solutions were sterilised on 0.45 .mu.m filters, then
preserved in aliquots at -20.degree. C. The solution (10.sup.-2 M)
was diluted to 1/10 to 1/10.sup.4 just before use (from 10.sup.-3
M) and added in an amount of 50 .mu.l 1 hour after infection.
[0241] The viral stocks (HSV-1 TK.sup.+, seventh passage over Vero
cells, and HSV-1 TK.sup.-, fourth passage) were diluted to 1/100 in
culture medium prior to distribution on plates (50 .mu.l/well).
[0242] The CPE inhibition test is a semi-quantitative test which
measures the % survival of the cells. The following protocol was
followed: Vero cells were trypsinised (trypsin-versene, Eurobio),
distributed in flat bottomed 96-well plates (Falcon) in an amount
of 2.times.10.sup.4 cells per well, in 50 .mu.l of DMEM medium
(Bioproducts) supplemented with antibiotics, L-glutamine and 5% FCS
(Eurobio), then incubated for 24 hours. The peptide/antibodies,
sterilised on a 0.22 .mu.M filter, were added in an amount of 50 to
400 .mu.g/ml (i.e., 20 to 2.5 .mu.g/well) using the following
schedule: [0243] H-24: cells into culture; [0244] H-0: infection;
[0245] H-2, H-1, H0, H+1: add peptides; [0246] H+48: CPE test.
[0247] 48 hours after infection, the medium was removed and the
wells were washed 3 times with 200 .mu.l of sterile PBS buffer
containing Ca2+ and Mg.sup.2+, so as to eliminate dead cells.
Neutral Red (RAL, 0.3%) was added (100 .mu.l/well) and the cells
were incubated for 2 more hours. The cells were then washed 3 times
as described above to eliminate colorant which had not been
incorporated into the cells. The cells were then exploded by adding
100 .mu.l of 1% SDS in distilled water. After 1 hour at 4.degree.
C. to completely leach out the Neutral Red, the crystals were
dissolved with a pipette. Air bubbles were destroyed by a current
of hot air (hair dryer) and the plates were read, using a
microplate reader at 570 nm using a 630 nm reference filter. The
optical density blank was air.
[0248] The following controls were used: [0249] Reference cells:
non infected and non treated, incubated with Neutral Red; [0250]
Reference virus: infected cells with different dilutions of a
factor of 2 of virus, from 100% CPE (dilution 1/1) to 12.5% CPE
(1/8 dilution); [0251] Antiviral reference: cells treated with
acyclovir and infected; and [0252] Toxicity reference: non infected
cells, treated with different peptides/antibodies.
[0253] Viral activity was determined as follows: AI = 100 .times. (
OD .times. .times. cell + virus + peptide ) - OD ( ref .times.
.times. virus .times. .times. 100 .times. % .times. .times. CPE OD
.times. .times. ref .times. .times. cells - OD .times. .times. ref
.times. .times. virus .times. .times. 100 .times. % .times. .times.
CPE . ##EQU1##
[0254] The results obtained are shown in the following tables. The
results clearly show an inhibition of the CPE effect induced by a
HSV-1 TK.sup.+ and TK.sup.- virus. The observed effect is
particularly pronounced for the K19-pJ20.8 peptide, which is very
active against the two viral strains, in particular with incubation
prior to or simultaneously with viral infection.
[0255] The results presented below thus demonstrate the important
antiviral properties of the antibodies/peptides of the invention,
used alone or in combination, on different types of virus such as
HIV, polio virus, CMV and HSV-1. These results illustrate the
applications of the products of the invention to minimise the
effects of viral infection in vitro, ex vivo or in vivo.
TABLE-US-00017 Control tables REFERENCE VIRUS Dilution HSV1
TK.sup.+ HSV1 TK.sup.- 0/0 (control cell) 1.292 .+-. 0.146 1.292
.+-. 0.146 1/1 0.163 .+-. 0.060 0.297 .+-. 0.068 1/2 0.206 .+-.
0.062 0.216 .+-. 0.072 1/4 0.217 .+-. 0.041 0.252 .+-. 0.058 1/8
0.227 .+-. 0.039 0.289 .+-. 0.029 ANTIVIRAL REFERENCE (ACYCLOVIR
.RTM.) Conc. (M)/HSV HSV1 TK.sup.+ HSV1 TK.sup.- 10.sup.-3/0 0.909
.+-. 0.016 0.789 .+-. 0.160 10.sup.-4/0 1.027 .+-. 0.042 0.834 .+-.
0.086 10.sup.-5/0 0.975 .+-. 0.093 0.869 .+-. 0.053 10.sup.-6/0
0.960 .+-. 0.025 0.816 .+-. 0.001 10.sup.-3/HSV 0.626 .+-. 0.050
0.289 .+-. 0.055 62% 0% 10.sup.-4/HSV 0.587 .+-. 0.149 0.173 .+-.
0.001 49% 0% 10.sup.-5/HSV 0.223 .+-. 0.017 0.169 .+-. 0.019 7% 0%
10.sup.-6/HSV 0.235 .+-. 0.032 0.239 .+-. 0.111 9% 0%
[0256] TABLE-US-00018 Test tables Peptide .mu./well 20 10 5 HSV1
TK.sup.+ H - 2 K19 0 0 0 K19pF4-1 7 2 1 K19PJ20-8 78 44 1 K19pF14-6
TOX.sup.(*.sup.) TOX 1 F14-6 0 0 0 .sup.(*.sup.)Toxic HSV1 TK.sup.+
H - 1 K19 9 6 8 K19pF4-1 28 30 13 K19PJ20-8 89 74 27 K19pF14-6 TOX
TOX 8 F14-6 0 0 0 HSV1 TK.sup.+ H0 K19 32 14 13 K19pF4-1 28 30 13
K19PJ20-8 86 71 37 K19pF14-6 TOX TOX 69 F14-6 0 0 0 HSV1 TK.sup.+ H
+ 1 K19 0 0 6 K19pF4-1 10 12 10 K19PJ20-8 0 1 0 K19pF14-6 TOX TOX
37 F14-6 0 0 0 HSV1 TK.sup.- H - 2 K19 0 26 27 K19pF4-0 25 5 21
K19PJ20-8 0 46 55 K19pF14-6 TOX TOX 100 F14-6 0 21 24 HSV1 TK.sup.-
H - 1 K19 34 4 0 K19pF4-1 47 7 25 K19PJ20-8 100 49 29 K19pF14-6 TOX
TOX 72 F14-6 0 0 0 HSV1 TK.sup.- H0 K19 40 2 0 K19pF4-1 42 25 15
K19PJ20-8 86 56 38 K19pF14-6 TOX 92 81 F14-6 0 0 0 HSV1 TK.sup.- H
+ 1 K19 2 0 0 K19pF4-1 1 13 1 K19PJ20-8 11 3 1 K19pF14-6 TOX TOX 26
F14-6 0 0 0
[0257] This set of results clearly demonstrates that the
polypeptides of the invention, comprising an antibody region,
preferably comprising all or a portion of a CDR3, are capable (i)
of effectively penetrating into cells; (ii) of transporting
substances thereto, in particular large size substances; (iii), of
acting as an adjuvant in vivo by stimulating the immune response
against a given antigen; and (iv) of exerting an antiviral
activity. Further, the polypeptides of the invention even appear to
be able to transport substances to the cell nuclei, which is of
obvious interest when the substances are nucleic acids or molecules
acting on nucleic acids. Further, the polypeptides of the invention
appear to use a cell penetration mechanism which is different from
the majority of vectors used up to the present time. In particular,
the polypeptides of the invention appear to escape the lysosomes,
which constitutes an additional advantage in that in general,
substantial degradation occurs in those cellular compartments.
Sequence CWU 1
1
36 1 17 PRT Artificial Sequence SYNTHETIC PEPTIDE 1 Thr Arg Gln Lys
Tyr Asn Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly 1 5 10 15 Thr 2 17
PRT Artificial Sequence SYNTHETIC PEPTIDE 2 Thr Arg Gln Lys Tyr Gly
Lys Arg Gly Met Asp Tyr Trp Gly Gln Gly 1 5 10 15 Thr 3 18 PRT
Artificial Sequence SYNTHETIC PEPTIDE 3 Thr Arg Gln Ala Arg Ala Thr
Trp Asp Trp Phe Ala Tyr Trp Gly Gln 1 5 10 15 Gly Thr 4 22 PRT
Artificial Sequence SYNTHETIC PEPTIDE 4 Val Ala Tyr Ile Ser Arg Gly
Gly Val Ser Thr Tyr Tyr Ser Asp Thr 1 5 10 15 Val Lys Gly Arg Phe
Thr 20 5 21 PRT Artificial Sequence SYNTHETIC PEPTIDE 5 Val Ala Tyr
Ile Ser Arg Gly Gly Gly Ile Phe Tyr Tyr Glu Asp Ser 1 5 10 15 Ile
Lys Gly Arg Phe 20 6 23 PRT Artificial Sequence SYNTHETIC PEPTIDE 6
Val Ala Ala Ile Ser Arg Gly Gly Gly Tyr Ser Tyr Tyr Leu Asp Ser 1 5
10 15 Val Lys Gly Arg Phe Thr Ile 20 7 30 PRT Artificial Sequence
SYNTHETIC PEPTIDE 7 Val Ala Tyr Ile Ser Arg Gly Gly Val Ser Thr Tyr
Tyr Ser Asp Thr 1 5 10 15 Val Lys Gly Arg Phe Thr Arg Gln Lys Tyr
Asn Lys Arg Ala 20 25 30 8 12 PRT Artificial Sequence SYNTHETIC
PEPTIDE 8 Ala Arg Gln Lys Tyr Asn Lys Arg Ala Met Asp Tyr 1 5 10 9
31 PRT Artificial Sequence SYNTHETIC PEPTIDE 9 Cys Val Ala Tyr Ile
Ser Arg Gly Gly Val Ser Thr Tyr Tyr Ser Asp 1 5 10 15 Thr Val Lys
Gly Arg Phe Thr Arg Gln Lys Tyr Asn Lys Arg Ala 20 25 30 10 49 PRT
Artificial Sequence SYNTHETIC PEPTIDE 10 Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Val
Ala Tyr Ile Ser Arg Gly Gly Val Ser Thr Tyr Tyr 20 25 30 Ser Asp
Thr Val Lys Gly Arg Phe Thr Arg Gln Lys Tyr Asn Lys Arg 35 40 45
Ala 11 26 DNA Artificial Sequence SYNTHETIC DNA 11 gttctgacta
gtgggcactc tgggct 26 12 26 DNA Artificial Sequence SYNTHETIC DNA 12
gaggttcagc tcgagcagtc tggggc 26 13 26 DNA Artificial Sequence
SYNTHETIC DNA 13 gaggtgaagc tcgaggaatc tggagg 26 14 25 DNA
Artificial Sequence SYNTHETIC DNA 14 gaagtgcagc tcgaggagtc tgggg 25
15 26 DNA Artificial Sequence SYNTHETIC DNA 15 gaggttcagc
tcgagcagtc tggagc 26 16 17 PRT Artificial Sequence SYNTHETIC
PEPTIDE 16 Thr Arg Gln Lys Tyr Asn Lys Arg Ala Met Asp Tyr Trp Gly
Gln Gly 1 5 10 15 Thr 17 18 PRT Artificial Sequence SYNTHETIC
PEPTIDE 17 Thr Arg Gln Lys Tyr Asn Lys Lys Arg Gly Met Asp Tyr Trp
Gly Gln 1 5 10 15 Gly Thr 18 18 PRT Artificial Sequence SYNTHETIC
PEPTIDE 18 Thr Arg Gly Ala Arg Ala Thr Trp Asp Trp Phe Ala Tyr Trp
Gly Gln 1 5 10 15 Gly Thr 19 21 PRT Artificial Sequence SYNTHETIC
PEPTIDE 19 Val Ala Tyr Ile Ser Arg Gly Gly Val Ser Thr Tyr Tyr Ser
Asp Thr 1 5 10 15 Val Lys Gly Arg Phe 20 20 22 PRT Artificial
Sequence SYNTHETIC PEPTIDE 20 Val Ala Tyr Ile Ser Arg Gly Gly Val
Ser Thr Tyr Tyr Ser Asp Thr 1 5 10 15 Val Lys Gly Arg Phe Thr 20 21
20 PRT Artificial Sequence SYNTHETIC PEPTIDE 21 Val Ala Tyr Ile Ser
Arg Gly Gly Ile Phe Tyr Tyr Gln Asp Ser Ile 1 5 10 15 Lys Gly Arg
Phe 20 22 23 PRT Artificial Sequence SYNTHETIC PEPTIDE 22 Val Ala
Ala Ile Ser Arg Gly Gly Gly Tyr Ser Tyr Tyr Leu Asp Ser 1 5 10 15
Val Lys Gly Arg Phe Thr Ile 20 23 33 PRT Artificial Sequence
SYNTHETIC PEPTIDE 23 Val Ala Tyr Ile Ser Arg Gly Gly Val Ser Thr
Tyr Tyr Ser Asp Thr 1 5 10 15 Val Lys Gly Arg Phe Thr Arg Gln Lys
Tyr Asn Lys Arg Ala Val Ala 20 25 30 Tyr 24 47 PRT Artificial
Sequence SYNTHETIC PEPTIDE 24 Lys Lys Lys Lys Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Ala Ile Ser Arg
Gly Gly Gly Tyr Ser Tyr Tyr Leu Asp 20 25 30 Thr Val Lys Arg Thr
Ala Arg Ala Thr Trp Asp Trp Phe Ala Tyr 35 40 45 25 105 PRT
Artificial Sequence SYNTHETIC PEPTIDE 25 Gly Gly Ser Leu Lys Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 1 5 10 15 Ser Tyr Ala Met
Ser Trp Val Arg Gln Thr Pro Ala Lys Arg Leu Glu 20 25 30 Trp Val
Ala Tyr Ile Ser Arg Gly Gly Gly Ile Phe Tyr Tyr Gln Asp 35 40 45
Ser Ile Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr 50
55 60 Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met
Tyr 65 70 75 80 Tyr Cys Thr Arg Glu Lys Tyr Gly Lys Arg Gly Met Asp
Tyr Trp Gly 85 90 95 Gln Gly Thr Ser Val Thr Val Ser Ser 100 105 26
107 PRT Artificial Sequence SYNTHETIC PEPTIDE 26 Glu Thr Gly Gly
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr 1 5 10 15 Phe Ser
Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Ala Lys Arg 20 25 30
Leu Glu Trp Val Ala Tyr Ile Ser Arg Gly Gly Val Ser Thr Tyr Tyr 35
40 45 Ser Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys 50 55 60 Asn Thr Leu Ser Leu Gln Met Ser Ser Leu Arg Ser Glu
Asp Thr Ala 65 70 75 80 Met Tyr Tyr Cys Ala Arg Gln Lys Tyr Asn Lys
Arg Ala Met Asp Tyr 85 90 95 Trp Gly Gln Gly Thr Ser Val Thr Val
Ser Ser 100 105 27 110 PRT Artificial Sequence SYNTHETIC PEPTIDE 27
Ala Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser 1 5
10 15 Gly Phe Thr Phe Ser Asn Tyr Gly Met Ser Trp Val Arg Gln Thr
Pro 20 25 30 Glu Lys Arg Leu Glu Trp Val Ala Ala Ile Ser Arg Gly
Gly Gly Tyr 35 40 45 Ser Tyr Tyr Leu Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp 50 55 60 Asn Ala Arg Asn Thr Leu Tyr Leu Gln
Met Ser Ser Leu Arg Ser Glu 65 70 75 80 Glu Thr Ala Met Tyr Tyr Cys
Ala Arg Thr Ala Arg Ala Thr Trp Asp 85 90 95 Trp Phe Ala Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ala 100 105 110 28 109 PRT
Artificial Sequence SYNTHETIC PEPTIDE 28 Glu Leu Val Arg Gly Ala
Ser Val Lys Val Ser Cys Thr Thr Ser Gly 1 5 10 15 Phe Thr Asn Ile
Lys Asp Asp Tyr Ile His Trp Val Arg Gln Arg Pro 20 25 30 Glu Gln
Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Ala Asn Gly Lys 35 40 45
Thr Lys Tyr Ala Pro Lys Phe Gln Asp Lys Ala Thr Ile Thr Ala Asp 50
55 60 Thr Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser
Glu 65 70 75 80 Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Leu Thr Arg
Trp Tyr Phe 85 90 95 Asp Val Trp Gly Ala Gly Thr Thr Val Thr Leu
Ser Ala 100 105 29 109 PRT Artificial Sequence SYNTHETIC PEPTIDE 29
Gly Leu Val Lys Pro Gly Ala Ser Val Lys Val Ser Cys Asn Val Ser 1 5
10 15 Gly Tyr Ser Phe Thr Gly Tyr Phe Met Asn Trp Val Arg Gln Ser
His 20 25 30 Gly Lys Ser Leu Glu Trp Val Gly Arg Ile Asn Pro Leu
Asn Gly Asp 35 40 45 Thr Phe Tyr Asn Gln Lys Phe Lys Gly Lys Ala
Thr Leu Thr Val Asp 50 55 60 Lys Ser Ser Thr Leu Ala His Met Glu
Leu Arg Leu Arg Lys Ser Glu 65 70 75 80 Asn Ser Ala Val Tyr Tyr Cys
Ala Arg Gly Leu Thr Arg Trp Tyr Phe 85 90 95 Met Val Trp Gly Ala
Gly Thr Thr Val Thr Leu Ser Ala 100 105 30 17 PRT Artificial
Sequence SYNTHETIC PEPTIDE 30 Tyr Ile Ser Arg Gly Gly Gly Ile Phe
Tyr Tyr Gln Asp Ser Ile Lys 1 5 10 15 Gly 31 17 PRT Artificial
Sequence SYNTHETIC PEPTIDE 31 Tyr Ile Ser Arg Gly Gly Val Ser Thr
Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly 32 17 PRT Artificial
Sequence SYNTHETIC PEPTIDE 32 Ala Ile Ser Arg Gly Gly Gly Tyr Ser
Tyr Tyr Leu Asp Ser Val Lys 1 5 10 15 Gly 33 10 PRT Artificial
Sequence SYNTHETIC PEPTIDE 33 Glu Lys Tyr Gly Lys Arg Gly Met Asp
Tyr 1 5 10 34 10 PRT Artificial Sequence SYNTHETIC PEPTIDE 34 Gln
Lys Tyr Asn Lys Arg Ala Met Asp Tyr 1 5 10 35 11 PRT Artificial
Sequence SYNTHETIC PEPTIDE 35 Thr Ala Arg Ala Thr Trp Asp Trp Phe
Ala Tyr 1 5 10 36 52 PRT Artificial Sequence SYNTHETIC PEPTIDE 36
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5
10 15 Lys Lys Lys Val Ala Tyr Ile Ser Arg Gly Gly Gly Ile Phe Tyr
Tyr 20 25 30 Gln Asp Ser Ile Lys Gly Arg Phe Thr Arg Glu Lys Tyr
Gly Lys Arg 35 40 45 Gly Met Asp Tyr 50
* * * * *