U.S. patent application number 09/853080 was filed with the patent office on 2002-06-06 for antigenic polypeptide sequences of factor viii, and fragments and/or epitopes of these sequences.
Invention is credited to Di Giambattista, Mario, Laub, Ruth.
Application Number | 20020068303 09/853080 |
Document ID | / |
Family ID | 25314986 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068303 |
Kind Code |
A1 |
Laub, Ruth ; et al. |
June 6, 2002 |
Antigenic polypeptide sequences of factor VIII, and fragments
and/or epitopes of these sequences
Abstract
The present invention is related to the antigenic polypeptide
sequence of Factor VIII.
Inventors: |
Laub, Ruth; (Brussels,
BE) ; Di Giambattista, Mario; (Braine-Le-Comte,
BE) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
25314986 |
Appl. No.: |
09/853080 |
Filed: |
May 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09853080 |
May 9, 2001 |
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08765837 |
Sep 7, 1999 |
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08765837 |
Sep 7, 1999 |
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PCT/BE95/00068 |
Jul 14, 1995 |
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Current U.S.
Class: |
435/7.1 ;
530/325; 530/326; 530/383; 530/387.1; 530/388.25 |
Current CPC
Class: |
G01N 2333/755 20130101;
C07K 2317/21 20130101; C07K 16/36 20130101; A61K 38/00 20130101;
A61K 39/0008 20130101; A61K 2039/505 20130101; C07K 2317/76
20130101; G01N 33/6878 20130101; A61K 39/00 20130101; C07K 14/755
20130101; A61P 37/00 20180101; Y10S 530/806 20130101; A61P 7/00
20180101 |
Class at
Publication: |
435/7.1 ;
530/383; 530/325; 530/326; 530/387.1; 530/388.25 |
International
Class: |
C07K 005/00; C07K
007/00; C07K 016/00; C07K 017/00; A61K 038/00; A61K 038/04; C07K
001/00; C07K 014/00; A61K 035/14; G01N 033/53; C12P 021/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 14, 1994 |
BE |
09400666 |
Claims
What is claimed is:
1. An isolated or purified antigenic polypeptide comprising the
amino acid sequence of factor VIII or a fragment thereof.
2. An isolated or purifed antigenic polypeptide comprising a factor
VIII polypeptide which lacks the following fragments: alanine
322-serine 750, leucine 1655-arginine 1689, lysine 1694-proline
1782 and possibly the fragment aspartic acid 2170-tyrosine
2332.
3. The polypeptide according to claim 1 or claim 2, wherein said
polypeptide exhibits an immunoaffinity for the receptors of T
and/or B lymphocytes.
4. An isolated or purified antigenic fragment of factor VIII,
wherein said fragment is selected from the group consisting of the
polypeptide sequences A1, A2, A3 or C of factor VIII or a portion
thereof.
5. The antigenic fragment of claim 4 wherein said fragment is
selected from the group consisting of the sequence fragment
arginine 1649 to arginine 2031 inclusive, the sequence fragment
threonine 1739 to aspartic acid 1831 inclusive, the sequence
fragment arginine 1803 to arginine 1917 inclusive and any of the
preceding fragments wherein at least one amino acid has been
deleted.
6. An isolated or purified polypeptide comprising an epitope of
factor VIII, wherein said epitope is selected from the group
consisting of: (a) the epitope arginine 1648 to tyrosine 1664
inclusive, defined by the following sequence: SEQ ID NO: 1: Arg Asp
Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr, (b)
the epitope of (a) wherein one or more amino acids of the
tetrapeptide Arg-Asp-Ile-Thr or one or two of the last amino acids
of the peptide Asp-Tyr; (c) the epitope aspartic acid 1681 to
arginine 1696 inclusive, defined by the following sequence: SEQ ID
NO: 2: Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr
Arg, (d) the epitope of (c) wherein one or more amino acids of the
epitope Asp-Glu-Asp-Glu are deleted; (e) the epitope threonine 1739
to tyrosine 1748 inclusive, defined by the following sequence: SEQ
ID NO: 3: Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr; (f) the epitope
asparagine 1777 to phenylalanine 1785 inclusive, defined by the
following sequence: SEQ ID NO: 4: Asn Gln Ala Ser Arg Pro Tyr Ser
Phe; (g) the epitope of (f) wherein one or two amino acids of the
terminal dipeptide Ser-Phe or the tetrapeptide Pro-Tyr-Ser-Phe are
deleted; (h) the epitope glutamic acid 1794 to tyrosine 1815
inclusive, defined by the following sequence: SEQ ID NO: 5: Glu Asp
Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr
Lys Thr Tyr; (i) the epitope of (h) wherein one or more amino acids
from the first tripeptide Glu-Asp-Gln or the first nonapeptide
Glu-Asp-Gln-Arg-Gln-Gly-Ala-Glu-Pro are deleted; (j) the epitope
methionine 1823 to aspartic acid 1831, defined by the following
sequence: SEQ ID NO: 6: Met Ala Pro Thr Lys Asp Glu Phe Asp; (k)
the epitope glutamic acid 1885 to phenylalanine 1891 inclusive,
defined by the following sequence: SEQ ID NO: 7: Glu Thr Lys Ser
Trp Tyr Phe; (l) the epitope glutamic acid 1885 to alanine 1901
inclusive, defined by the following sequence: SEQ ID NO: 8: Glu Thr
Lys Ser Trp Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala; (m) the
epitope of (l) wherein one or more amino acids from the
heptapeptide Gly-Thr-Lys-Ser-Trp-Phe-Thr or from the tripeptide
Cys-Arg-Ala are deleted; (n) the epitope aspartic acid 1909 to
arginine 1917 inclusive, defined by the following sequence: SEQ ID
NO: 9: Asp Pro Thr Phe Lys Glu Asn Tyr Arg; (o) the epitope
comprised between serine 2018 and histidine 2031 inclusive, defined
by the following sequence: SEQ ID NO: 10: Ser Asn Lys Cys Gln Thr
Pro Leu Gly Met Ala Ser Gly His; and (p) any of the preceding
epitopes (a) to (o) wherein at least one amino acid has been
deleted.
7. The antigenic fragment of claim 4, wherein said fragment is
selected from the group consisting of alanine 108 to methionine 355
inclusive, alanine 108 to alanine 227 inclusive and any of the
preceding fragments wherein at least one amino acid has been
deleted.
8. An isolated or purified polypeptide comprising an epitope of
factor VIII, wherein said epitope is selected from the group
consisting of: (a) the epitope alanine 108 to valine 128 inclusive,
defined by the following sequence: SEQ ID NO: 11: Ala Ser Glu Gly
Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys
Val; (b) the epitope of (a) wherein the terminal amino acids
alanine and/or valine are deleted; (c) the epitope glutamic acid
181 to leucine 192 inclusive, defined by the following sequence:
SEQ ID NO: 12: Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu; (d)
the epitope of (a) wherein one or two amino acids of the terminal
dipeptide Thr-Leu are deleted; (e) the epitope aspartic acid 203 to
alanine 227 inclusive, defined by the following sequence: SEQ ID
NO: 13: Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met
Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala; (f) the epitope of (e)
wherein one or more amino acids of the nonapeptide
Asp-Arg-Asp-Ala-Ala-Ser-Ala-Arg-Ala are deleted; (g) the epitope
aspartic acid 327 to methionine 355 inclusive, defined by the
following sequence: SEQ ID NO: 14: Asp Ser Cys Pro Glu Glu Pro Gln
Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr
Asp Ser Glu Met; (h) the epitope of (g) wherein one or more amino
acids of the dipeptide Asp-Ser or the octapeptide
Asp-Asp-Leu-Thr-Asp-Ser- -Glu-Met are deleted; and (i) any of the
preceding epitopes (a) to (h) wherein at least one amino acid has
been deleted.
9. The antigenic fragment of claim 4, wherein said fragment is
selected from the group consisting of aspartic acid 403 to aspartic
acid 725 inclusive, histidine 693 to aspartic acid 725 inclusive
and any of the preceding fragments wherein at least one amino acid
has been deleted.
10. An isolated or purified polypeptide comprising an epitope of
factor VIII, wherein said epitope is selected from the group
consisting of: (a) the epitope aspartic acid 403 to lysine 425
inclusive, defined by the following sequence: SEQ ID NO: 15: Asp
Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln Arg Ile Gly
Arg Lys Tyr Lys Lys; (b) the epitope of (a) wherein one or more
amino acids of the tetrapeptide Asp-Asp-Arg-Ser are deleted; (c)
the epitope valine 517 to arginine 527 inclusive, defined by the
following sequence: SEQ ID NO: 16: Val Glu Asp Gly Pro Thr Lys Ser
Asp Pro Arg; (d) the epitope of (c) wherein one or the two amino
acids of the dipeptide Pro-Arg are deleted; (e) the epitope
tyrosine 555 to glutamine 565 inclusive defined by the following
sequence: SEQ ID NO: 17: Tyr Lys Glu Ser Val Asp Gly Arg Gly Asn
Gln; (f) the epitope histidine 693 to glycine 701 inclusive,
defined by the following sequence: SEQ ID NO: 18 His Asn Ser Asp
Phe Arg Asn Arg Gly; (g) the epitope serine 710 to aspartic acid
725 inclusive, defined by the following sequence: SEQ ID NO: 19 Ser
Cys Asp Lys Asn Thr Gly Asp Tyr Try Gly Asp Ser Tyr Glu Asp; (h)
the epitope leucine 730 to serine 741 inclusive, defined by the
following sequence: SEQ ID NO: 20: Leu Leu Ser Lys Asn Asn Ala Ile
Glu Pro Arg Ser; (i) the epitope of (h) wherein the terminal amino
acid serine and/or the first amino acid leucine are deleted; (j)
the epitope serine 817 to serine 830 inclusive, defined by the
following sequence: SEQ ID NO: 21: Ser Asp Asp Pro Ser Gly Ala Ile
Asp Ser Asn Asn Ser; and (k) any of the preceding epitopes (a) to
(j) wherein at least one amino acid has been deleted.
11. The antigenic fragment of claim 4, wherein said fragment is
selected from the group consisting of lysine 2085 to isoleucine
2251 inclusive, leucine 2273 to tyrosine 2332 inclusive, lysine
2085 to glycine 2121 inclusive, serine 2182 to leucine 2251
inclusive and any of the preceding fragments wherein at least one
amino acid has been deleted.
12. An isolated or purified polypeptide comprising an epitope of
factor VIII, wherein said epitope is selected from the group
consisting of: (a) the epitope isoleucine 2081 to serine 2095
inclusive, defined by the following sequence: SEQ ID NO: 22: Ile
His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser; (b) the
epitope of (a) wherein one or more amino acids of the tetrapeptide
Ile-His-Gly-Ile are deleted; (c) the epitope tyrosine 2105 to
glycine 2121 inclusive, defined by the following sequence: SEQ ID
NO: 23: Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser
Thr Gly; (d) the epitope of (c) wherein one or more amino acids of
the tripeptide Tyr-Ser-Leu are deleted; (e) the epitope asparagine
acid 2128 to asparagine acid 2138 inclusive, defined by the
following sequence: SEQ ID NO: 24: Asn Val Asp Ser Ser Gly Ile Lys
His Asn; (f) the epitope histidine 2152 to arginine 2163 inclusive,
defined by the following sequence: SEQ ID NO: 25: His Pro Thr His
Tyr Ser Ile Arg Ser Thr Leu Arg; (g) the epitope serine 2181 to
asparagine acid 2198 inclusive, defined by the following sequence:
SEQ ID NO: 26: Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser
Tyr Phe Thr Asn; (h) the epitope of (g) wherein one or more amino
acids of the first dipeptide Ser-Tyr or one or more amino acids
from the terminal tripeptide Phe-Thr-Asn are deleted; (i) the
epitope serine 2204 to glutamine 2222 inclusive, defined by the
following sequence: SEQ ID NO: 27: Ser Pro Ser Lys Ala Arg Leu His
Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln; (j) the epitope
glutamine 2235 to leucine 2251 inclusive, defined by the following
sequence: SEQ ID NO: 28: Gln Lys Thr Met Lys Val Thr Gly Val Thr
Thr Gln Gly Val Lys Ser Leu; (k) the epitope of (j) wherein one or
two amino acids of the terminal dipeptide Ser-Leu or one or more
amino acids of the tetrapeptide Val-Lys-Ser-Leu are deleted; (1)
the epitope glycine 2242 to leucine 2251 inclusive, defined by the
following sequence: SEQ ID NO: 29: Gly Val Thr Thr Gln Gly Val Lys
Ser Leu; (m) the epitope of (l) wherein one or two amino acids of
the terminal dipeptide Ser-Leu are deleted; (n) the epitope
isoleucine 2262 to glutamine 2270 inclusive, defined by the
following sequence: SEQ ID NO: 30: Ile Ser Ser Ser Gln Asp Gly His
Gln; (o) the epitope leucine 2273 to serine 2289 inclusive, defined
by the following sequence: SEQ ID NO: 31: Leu Phe Phe Gln Asn Gly
Lys Val Lys Val Phe Gln Gly Asn Gln Asp Ser; (p) the epitope
proline 2292 to tyrosine 2305 inclusive, defined by the following
sequence: SEQ ID NO: 32: Pro Val Val Asn Ser Leu Asp Pro Pro Leu
Leu Thr Arg Tyr; (q) the epitope of (p) wherein one or more amino
acids of the terminal tripeptide Thr-Arg-Tyr are deleted; (r) the
epitope glutamic acid 2322 to tyrosine 2332 inclusive, defined by
the following sequence: SEQ ID NO: 33: Glu Val Leu Gly Cys Glu Ala
Gln Asp Leu Tyr; and (s) any of the preceding epitopes (a) to (r)
wherein at least one amino acid has been deleted.
13. A purified or isolated conformational epitope, which contains
at least two different epitopes according to any one of claims 7, 9
and 11 and any of the preceding epitopes wherein at least one amino
acid has been deleted.
14. A pool of more than three fragments or epitopes according to
any of the preceding claims 5 to 12.
15. A complex comprising a carrier protein or a carrier peptide
linked to an antigenic fragment or epitope from factor VIII.
16. An inhibitor of factor VIII, which exhibits an immunoaffinity
with an element selected from the group consisting of factor VIII,
an antigenic fragment thereof, an epitope therefrom, and a complex
comprising any of the foregoing polypeptides.
17. The inhibitor according to claim 16, wherein said inhibitor is
an anti-factor VIII antibody or antibody fragment.
18. An anti-inhibitor directed against the inhibitor of factor VIII
according to claim 16 or 17.
19. An anti-inhibitor according to claim 18, wherein said
anti-inhibitor is an anti-anti-factor VIII idiotype antibody or
antibody fragment.
20. A pharmaceutical composition comprising an adequate
pharmaceutical carrier and at least one element selected from the
group consisting of factor VIII, an antigenic fragment thereof, an
epitope therefrom, a complex comprising any of the foregoing
polypeptides, an inhibitor of any of the foregoing polypeptides and
an anti-inhibitor of any of the foregoing polypeptides.
21. A diagnostic and/or purification device, which comprises at
least one elment selected from the group consisting of factor VIII,
an antigenic fragment thereof, an epitope therefrom, a complex
comprising any of the foregoing polypeptides, an inhibitor of any
of the foregoing polypeptides and an anti-inhibitor of any of the
foregoing polypeptides.
22. The device according to claim 21, wherein said device is a
diagnostic kit.
23. The device according to claim 21, wherein said device is a
chromatography column or filter.
24. A method for a therapeutic treatment and/or prevention of an
immune disorder in mammal, wherein the pharmaceutical composition
according to claim 20 is administered to a mammal presently or
potentially having said immune disorder, in an amount effective to
treat and/or prevent said immune disorder.
25. A method for a therapeutic treatment and/or prevention of an
immune disorder in a mammal, wherein a physiological fluid such as
serum obtained from said mammal is put into the chromatography
column of claim 23 in order to allow binding of the inhibitors of
factor VIII present in said serum to an element selected from the
group consisting of factor VIII, an antigenic fragment thereof, an
epitope therefrom, and a complex comprising any of the foregoing
polypeptides, wherein the physiological liquid is eluted from said
chromatography column and the physiological liquid from which the
inhibitors of factor VIII have been removed is reinjected to the
patient.
26. The therapeutic treatment and/or prevention method according to
claim 24 or 25, wherein the immune disorder is induced by an
element selected from the group consisting of inhibitors of factor
VIII, inhibitors of the binding of factor VIII to the von
Willebrand factor, inhibitors of the binding of factor VIII to the
factor IX, inhibitors of the binding of factor VIII to the factor X
and inhibitors of the binding of factor VIII to membrane
phospholipids.
27. A process for identifying and obtaining inhibitors and/or
anti-inhibitors comprising: attaching an element selected from the
group consisting of factor VIII, an antigenic fragment thereof, an
epitope therefrom, a complex comprising any of the foregoing
polypeptides, an inhibitor of any of the foregoing polypeptides and
an anti-inhibitor of any of the foregoing polypeptides to a solid
support of a chromatography column; passing a physiological fluid
from a patient containing inhibitors of factor VIII through said
chromatography column; eluting said column; and collecting the
fractions containing inhibitors of factor VIII which have exhibited
an immunoaffinity with said element.
28. The process according to claim 27, further comprising the steps
of: attaching the collected inhibitors of factor VIII to the solid
support of a chromatography column; passing a physiological fluid
from a patient containing anti-inhibitors of factor VIII through
said chromatography column; eluting said column; and collecting the
fractions containing anti-inhibitors of factor VIII which have
exhibited an immunoaffinity with said inhibitors of factor VIII.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation-in-Part of U.S.
application Ser. No. 08/765,837, filed Sep. 7, 1999, which was a
U.S. National Phase application of PCT/BE95/00068, filed Jul. 14,
1995, which claims priority to Belgian Application BE 9400666,
filed Jul. 14, 1994, the disclosures of which are incorporated
herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to the antigenic polypeptide
sequences of factor VIII, to fragments and epitopes of these
sequences and to the major parts of these epitopes, to the
inhibitors which are directed against these sequences, its
fragments, its epitopes and/or major parts of these epitopes, and
to anti-inhibitors which are directed against the said
inhibitors.
[0003] The present invention also relates to a pharmaceutical
composition and to a diagnostic device comprising at least one of
the above mentioned molecules.
BACKGROUND OF THE INVENTION
[0004] FVIII is a large multi-domain protein of 2,332 amino acids
made up of three structural domains, A, B and C which are arranged
in the order A1:a1:A2:a2:B:a3:A3:C1:C2. The A domains possess more
than 40% homology and are also homologous to ceruloplasmin (for
recent review, see Pratt (2000) and Saenko (1999)). 30% homology
also exists between the A domains of factor V and FVIII. The C
domain occurs twice and is reported to be able to bind
glyco-conjugates and phospholipids having a net negative charge. It
exhibits homology with lectins which are able to bind to negatively
charged phospholipids. The platelet attachment site has been
located in this region (C2 domain) (Foster et al., (1990)).
[0005] These antigenic determinants consist of fragments 351-365
(A1 domain-heavy chain), 713-740 (A2 domain), 1670-1684 (A3
domain-light chain) (NH.sub.2 end of the light chain) or else
2303-2332 (C2 domain-light chain) (Foster C, (1990)), fragments
701-750, 1663-1689, 330-472, 1694-1782 (EP-0 202 853), 322-740 and
2170-2322.
[0006] The U.S. Pat. No. 5,744,446 describes an hybrid human/animal
Factor VIII having a sequence of amino acids selected from the
group of the A2 domain fragments 373-540, 373-508, 445-508,
484-508, 404-508, 489-508 and 484-489, with corresponding sequences
of porcine or murine Factor VIII, said hybrid being used for the
treatment of Factor VIII deficiencies.
[0007] The antibodies which recognize these various sites
interfere, with the activation of FVIII, the binding of vWf, FIXa,
FXa, APC or phospholipids. The specific antibody response to FVIII
vary considerably among individuals, and epitopes for inhibitor
antibodies have to be determined for all FVIII domains (see for
recent review Scandella, 2000; Lollar, 2000).
[0008] Other antibodies, which do not inhibit standard activity
tests in vitro, can exert an influence on the behavior of FVIII
with the other constituents of the coagulation cascade while
attaching themselves to sites in the molecule which are at a
substantial distance from the active sites. These antibodies, can
interfere with the natural state of folding of FVIII by altering
some of its properties.
[0009] Emergence of alloantibodies (inhibitors) that neutralize
infused FVIII activity may seriously complicate FVIII replacement
therapy. Reported inhibitor incidence rates in hemophiliacs vary
considerably. They range around 6-35% (Vermylen et al, 1998).
Candidates for genetic predispositions such as large deletions and
intron 22 inversion have been found associated with a high
incidence of inhibitors and genes that are involved in the immune
response as genes MHC class I and class II (Tuddenham and McVey,
1998). Repeat switching from one FVIII product to another and the
possibility that some FVIII concentrates are more immunogenic may
also explain the appearance of inhibitors (Vermylen et al, 1998).
Different methods of preparing FVIII could exert an influence on
its structure, its physicochemical properties or its natural
micro-environment; Laub et al. (1999); Raut et al. (1998)).
Clinically relevant anti-FVIII autoantibodies are rare in
non-hemophilic patients (annual frequency in the population:
1-{fraction (5/10)}.sup.6) (Morrisson and Ludlam) (1995). They are
associated with a number of autoimmune diseases and are often
characterized by life-threatening hemorrhage. On the other hand,
anti-FVIII antibodies have also been described in healthy subjects
(Algiman et al, 1992; Moreau et al, 2000), without any apparent
effect on the subjects' levels of circulating FVIII.
[0010] Self proteins or derived peptides may elicit an immune
response if presented to CD4 T cells at inflammatory sites by
professional antigen presenting cells. Using pools of overlapping
synthetic peptides spanning the sequences of individual FVIII
domains, Reding et al. (2000) showed reactive CD4.sup.+to FVIII in
healthy subjects and hemophilia patients. Several FVIII domains
were recognized: A3 domain was recognized more strongly and
frequently and each domain forms several epitopes.
[0011] Techniques such as western blotting, immunoprecipitation,
and enzyme-linked immunosorbent assays (ELISAs), using well-defined
FVIII proteolytic fragments, a large recombinant peptide library,
or synthetic peptide arrays, have been used to map different
FVIII-inhibitor binding sites located mainly in the A2 and C2
domains. However, none of these techniques has made it possible to
build a model for identification of inhibitor and non-inhibitor
epitopes. Only a few epitopes have been mapped to discrete
sequences (<20 amino-acid residues). To solve this problem,
Palmer et al (1997) synthesized 96 undecamer peptides (11
amino-acid residues) representing 80% of the complete residue
sequence of FVIII. They succeeded in determining the epitope
specificity of 9 patients' inhibitory antibodies. Other useful
techniques are analysis of FVIII gene mutations and their effects
on the FVIII molecule as well as phage display technology (van den
Brink et al, 2000). All these methodologies, however, are time
consuming, rather costly, and largely dependent on patient
availability. Certain areas of the FVIII molecule may be "hot
spots" containing commonly recognized clusters of inhibitor
epitopes, e.g., regions in the A2 domain, A3 domain, and C2 domain.
The reason for these "hot spots" in generating an inhibitor
response remains poorly understood (Reisner et al, 1995).
[0012] Currently, a predominant notion among hemophilic patients,
clinicians and "fractionators" is that of having available a
purified FVIII which is devoid of all pathogenic plasma
contaminants and secondary effects. Different animal models could
be used as hemophilia dogs, SCID mice, hemophilia mice . . . but
until now, no satisfactory experimental model exists which makes it
possible to forecast the immunogenicity or the immuno-modulatory
effect of the FVIII preparations, or the susceptibility of the
host, before they have been administered clinically.
[0013] Patients who develop an anti-FVIII immune response find
themselves in a serious situation which necessitates the use of
severe, aggressive and excessively expensive measures.
[0014] One of the frequently treatment, is the induction of immune
tolerance by administration of very high doses of FVIII (150 IU/kg
twice a day) in association or not with prothrombin complex
concentrates and is assigned as "Bonn Protocol". Treatment options
are also to by-pass the FVIII inhibitor activity by use of PCC
(preferably an activated PCC [APCC]) or FVIIa. Specific antibodies
as consequence of the infusion of these alternative agents could be
produced, impairing the treatment. As an alternative agent porcine
FVIII may be used to achieve haemostasis in patients with
antibodies that do not substantially crossreact with porcine FVIII
before or during the treatment (Lollar, 2000).
[0015] A potential alternative approach to inhibit the production
of inhibitors is blockade of the T cell/B cell collaboration
mediated by through receptor ligand binding signal events
(Ewenstein et al, 2000). Preliminary clinical trials were performed
using a humanized mouse monoclonal antibody to human T cell CD40
ligand (CD 154).
[0016] A profitable strategy for reducing the level of inhibitors
has consisted in subjecting patients to an extracorporeal
circulation to enable solid-phase absorption of the total IgG.
[0017] The immunoabsorbant could be sepharose-bound staphylococcal
protein A or sepharose-bound polyclonal sheep antibodies to total
human immunoglobulin (Knobf and Derfler, 1999). The foreign
proteins (protein A, sheep anti-human Ig) could leak from the
column and triggered the immune system of the recipient; moreover
problems could raised as sanitization (ICH Topic Q5A, Directive
92/79/EC).
[0018] The infusion of polyvalent intravenous immunoglobulins
(IVIG), where appropriate combined with an immunosuppressive
treatment, has been found to be relatively effective, although the
reason for this effectiveness is still not fully established.
Various hypotheses involving feed-back inhibition of IgG synthesis,
stimulation of IgG clearance or activation of T suppressor cells
have been advanced. An interesting explanation is that these
commercial intravenous immunoglobulins might contain antibodies
which are able to react with the variable parts (idiotypes) of the
anti-FVIII antibodies and neutralize these antibodies (Dietrich et
al. (1992)).
[0019] Unfortunately, none of these approaches has been found to be
satisfactory in terms of safety, efficacy, efficiency and cost.
[0020] The state of the art in epitope structure prediction was
limited given to the fact that non-continuous amino acid residues
seem to constitute most important epitope and that the dynamics of
binding is often not integrated into the epitope prediction
equation making epitope structure prediction a complex
four-dimensional problem (Van Regenmortel, Methods: A companion to
Methods in Enzymology, 9, page 465-472, 1996).
[0021] According to the author, most of the antibodies raised
against intact proteins do not react with any peptide fragment
derived from the parent protein indicating that such antibodies are
directed to discontinuous epitopes (conformational epitopes).
[0022] This author states also that low success rate of antigenic
prediction is due to the fact that predictions concerns only
continuous epitopes and it is unrealistic to reduce the complexity
of epitopes that always possess conformational features to one
dimensional linear peptide model.
[0023] Similarly, Palmer et al. (1997) using synthetic peptide
arrays to identify novel Factor VIII inhibitor epitopes note that
each patient pattern of anti-factor VIII antibody reactivity
appears to be polyclonal, directed against multiple sites located
within the amino and carboxyl terminus of the protein and seems to
be unique for each plasma investigated (see also above). Moreover,
this author notes that it is difficult to predict the importance
that any given antibody: epitope interaction may have on Factor
VIII coagulation activity based on the results of synthetic peptide
assays alone (due to the incomplete understanding of the
relationship between structure and function of different factor
VIII domains and the possibility that both inhibitor and
non-inhibitory antibodies may be present in a patient's plasma.
[0024] Therefore, the documents of the state of the art do not
suggest to identify antigenic linear peptides upon a macro-molecule
(such as Factor VIII) and that linear epitopes could be used for
the diagnostic and/or the therapy of immune disorders induced by
inhibitors directed against Factor VIII.
[0025] The present invention aims to obtain antigenic polypeptide
sequences of factor VIII, fragments and epitopes of these
sequences, whose purpose is to improve the diagnosis and/or therapy
(including prevention) of immune disorders (in particular those
induced by inhibitors of FVIII and inhibitors of FVIII, especially
inhibitors of the binding of the von Willebrand factor (vWf), to
the FIX and/or to membrane phospholipids (PL)), and which allows a
screening between non-inhibitory and inhibitory anti-FVIII allo- or
auto-antibodies (allo- or auto-immunoglobulins).
[0026] Another aim of the invention is to obtain inhibitors which
exhibit an immunoaffinity with these antigenic polypeptide
sequences, fragments and/or epitopes, as well as to obtain
anti-inhibitors, in particular antibodies or (T)cell receptors,
which are directed against the above-mentioned said inhibitors and
whose purpose is to improve the diagnosis and/or therapy (or
prevention) of immune disorders.
[0027] A further aim of the invention is to obtain said molecules
at high purity, in industrial level, without contaminants (viruses,
prions, . . . ) and according to the GMP practices in the field of
therapy and diagnostics (ICH topic QSA, Directive 92/79/EC,
etc.).
SUMMARY OF THE INVENTION
[0028] Some embodiments of the present invention are described in
the following numbered paragraphs.
[0029] Paragraph 1:
[0030] An antigenic polypeptide sequence, which is the polypeptide
sequence of factor VIII.
[0031] Paragraph 2:
[0032] An antigenic polypeptide sequence, which lacks the following
fragments: alanine 322-serine 750, leucine 1655-arginine 1689,
lysine 1694-proline 1782 and possibly the fragment aspartic acid
2170-tyrosine 2332.
[0033] Paragraph 3:
[0034] The sequence according to paragraph 1 or 2, which is
immunogenic.
[0035] Paragraph 4:
[0036] The sequence according to paragraph 3, which exhibits an
immunoaffinity for the receptors of T and/or B lymphocytes.
[0037] Paragraph 5:
[0038] An antigenic fragment of the sequence according to paragraph
1 or 2, which is selected from the group consisting of the
polypeptide sequences Al, A2, A3 or C of factor VIII.
[0039] Paragraph 6:
[0040] The antigenic fragment of the polypeptide sequence A3
according to paragraph 5, which is selected from the group
consisting of the sequence fragment arginine 1649 to arginine 2031
inclusive, the sequence fragment threonine 1739 to aspartic acid
1831 inclusive and/or the sequence fragment arginine 1803 to
arginine 1917 inclusive.
[0041] Paragraph 7:
[0042] A sequence epitope of the fragment according to paragraph 6,
which is selected from the group consisting of:
[0043] the epitope arginine 1648 to tyrosine 1664 inclusive,
defined by the following sequence:
[0044] SEQ ID NO:1:
[0045] Arg Asp Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile
Asp Tyr,
[0046] possibly deleted from one or more amino acids of the
tetrapeptide Arg-Asp-Ile-Thr or one or two of the last amino acids
of the peptide Asp-Tyr, the epitope aspartic acid 1681 to arginine
1696 inclusive, defined by the following sequence:
[0047] SEQ ID NO:2:
[0048] Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr
Arg,
[0049] possibly deleted from one or more amino acids of the epitope
Asp-Glu-Asp-Glu,
[0050] the epitope threonine 1739 to tyrosine 1748 inclusive,
defined by the following sequence:
[0051] SEQ ID NO:3:
[0052] Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr,
[0053] the epitope asparagine 1777 to phenylalanine 1785 inclusive,
defined by the following sequence:
[0054] SEQ ID NO:4:
[0055] Asn Gln Ala Ser Arg Pro Tyr Ser Phe,
[0056] possibly deleted from one or two amino acids of the terminal
dipeptide Ser-Phe or the tetrapeptide Pro-Tyr-Ser-Phe,
[0057] the epitope glutamic acid 1794 to tyrosine 1815 inclusive,
defined by the following sequence:
[0058] SEQ ID NO:5:
[0059] Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys
Pro Asn Glu Thr Lys Thr Tyr,
[0060] possibly deleted from one or more amino acids from the first
tripeptide Glu-Asp-Gln or the first nonapeptide
Glu-Asp-Gln-Arg-Gln-Gly-A- la-Glu-Pro,
[0061] the epitope methionine 1823 to aspartic acid 1831, defined
by the following sequence:
[0062] SEQ ID NO:6:
[0063] Met Ala Pro Thr Lys Asp Glu Phe Asp,
[0064] the epitope glutamic acid 1885 to phenylalanine 1891
inclusive, defined by the following sequence:
[0065] SEQ ID NO:7:
[0066] Glu Thr Lys Ser Trp Tyr Phe,
[0067] the epitope glutamic acid 1885 to alanine 1901 inclusive,
defined by the following sequence:
[0068] SEQ ID NO:8:
[0069] Glu Thr Lys Ser Trp Phe Thr Glu Asn Met Glu Arg Asn Cys Arg
Ala,
[0070] possibly deleted from one or more amino acids from the
heptapeptide Gly-Thr-Lys-Ser-Trp-Phe-Thr or from the tripeptide
Cys-Arg-Ala,
[0071] the epitope aspartic acid 1909 to arginine 1917 inclusive,
defined by the following sequence:
[0072] SEQ ID NO:9:
[0073] Asp Pro Thr Phe Lys Glu Asn Tyr Arg,
[0074] and the epitope comprised between serine 2018 and histidine
2031 inclusive, defined by the following sequence:
[0075] SEQ ID NO:10:
[0076] Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His.
[0077] Paragraph 8:
[0078] An antigenic fragment of the polypeptide sequence Al
according to paragraph 5, which is alanine 108 to methionine 355
inclusive, preferably alanine 108 to alanine 227 inclusive.
[0079] Paragraph 9:
[0080] A sequence epitope of the fragment according to paragraph 8,
which is selected from the group consisting of:
[0081] the epitope alanine 108 to valine 128 inclusive, defined by
the following sequence:
[0082] SEQ ID NO:11:
[0083] Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu
Lys Glu Asp Asp Lys Val,
[0084] possibly deleted from the terminal amino acids alanine
and/or valine,
[0085] the epitope glutamic acid 181 to leucine 192 inclusive,
defined by the following sequence:
[0086] SEQ ID NO:12:
[0087] possibly deleted from one or two amino acids of the terminal
dipeptide Thr-Leu,
[0088] the epitope aspartic acid 203 to alanine 227 inclusive,
defined by the following sequence:
[0089] SEQ ID NO:13:
[0090] Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met
Gln Asp Arg Asp Ala Ala Ser
[0091] Ala Arg Ala,
[0092] possibly deleted from one or more amino acids of the
nonapeptide Asp-Arg-Asp-Ala-Ala-Ser-Ala-Arg-Ala,
[0093] and the epitope aspartic acid 327 to methionine 355
inclusive, defined by the following sequence:
[0094] SEQ ID NO:14:
[0095] Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu
Glu Ala Glu
[0096] Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met,
[0097] possibly deleted from one or more amino acids of the
dipeptide Asp-Ser or the octapeptide
Asp-Asp-Leu-Thr-Asp-Ser-Glu-Met,
[0098] Paragraph 10:
[0099] An antigenic fragment of the antigenic polypeptide sequence
A2 according to paragraph 5, which is aspartic acid 403 to aspartic
acid 725 inclusive, preferably histidine 693 to aspartic acid 725
inclusive.
[0100] Paragraph 11:
[0101] A sequence epitope of the fragment according to paragraph
10, which is selected from the group consisting of:
[0102] the epitope aspartic acid 403 to lysine 425 inclusive,
defined by the following sequence:
[0103] SEQ ID NO:15:
[0104] Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln
Arg Ile Gly Arg Lys
[0105] Tyr Lys Lys,
[0106] possibly deleted from one or more amino acids of the
tetrapeptide Asp-Asp-Arg-Ser,
[0107] the epitope valine 517 to arginine 527 inclusive, defined by
the following sequence:
[0108] SEQ ID NO:16:
[0109] Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg,
[0110] possibly deleted from one or the two amino acids of the
dipeptide Pro-Arg,
[0111] the epitope tyrosine 555 to glutamine 565 inclusive defined
by the following sequence:
[0112] SEQ ID NO:17:
[0113] Tyr Lys Glu Ser Val Asp Gly Arg Gly Asn Gln,
[0114] the epitope histidine 693 to glycine 701 inclusive, defined
by the following sequence:
[0115] SEQ ID NO:18
[0116] His Asn Ser Asp Phe Arg Asn Arg Gly,
[0117] the epitope serine 710 to aspartic acid 725 inclusive,
defined by the following sequence:
[0118] SEQ ID NO:19
[0119] Ser Cys Asp Lys Asn Thr Gly Asp Tyr Try Gly Asp Ser Tyr Glu
Asp,
[0120] the epitope leucine 730 to serine 741 inclusive, defined by
the following sequence:
[0121] SEQ ID NO:20:
[0122] Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser,
[0123] possibly deleted from the terminal amino acid serine and/or
the first amino acid leucine,
[0124] the epitope serine 817 to serine 830 inclusive, defined by
the following sequence:
[0125] SEQ ID NO:21:
[0126] Ser Asp Asp Pro Ser Gly Ala Ile Asp Ser Asn Asn Ser.
[0127] Paragraph 12:
[0128] An antigenic fragment of the antigenic polypeptide sequence
C according to paragraph 5, which is lysine 2085 to isoleucine 2251
inclusive, or leucine 2273 to tyrosine 2332 inclusive, preferably
lysine 2085 to glycine 2121 inclusive or serine 2182 to leucine
2251 inclusive.
[0129] Paragraph 13:
[0130] A sequence epitope of the fragment according to paragraph
12, which is selected from the group consisting of:
[0131] the epitope isoleucine 2081 to serine 2095 inclusive,
defined by the following sequence
[0132] SEQ ID NO:22:
[0133] Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser
Ser,
[0134] possibly deleted from one or more amino acids of the
tetrapeptide Ile-His-Gly-Ile,
[0135] the epitope tyrosine 2105 to glycine 2121 inclusive, defined
by the following sequence:
[0136] SEQ ID NO:23:
[0137] Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser
Thr Gly,
[0138] possibly deleted from one or more amino acids of the
tripeptide Tyr-Ser-Leu,
[0139] the epitope asparagine acid 2128 to asparagine acid 2138
inclusive, defined by the following sequence:
[0140] SEQ ID NO:24:
[0141] Asn Val Asp Ser Ser Gly Ile Lys His Asn,
[0142] the epitope histidine 2152 to arginine 2163 inclusive,
defined by the following sequence:
[0143] SEQ ID NO:25:
[0144] His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg,
[0145] the epitope serine 2181 to asparagine acid 2198 inclusive,
defined by the following sequence:
[0146] SEQ ID NO:26:
[0147] Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe
Thr Asn,
[0148] possibly deleted from one or more amino acids of the first
dipeptide Ser-Tyr or one or more amino acids from the terminal
tripeptide Phe-Thr-Asn,
[0149] the epitope serine 2204 to glutamine 2222 inclusive, defined
by the following sequence:
[0150] SEQ ID NO:27:
[0151] Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala
Trp Arg Pro Gln,
[0152] the epitope glutamine 2235 to leucine 2251 inclusive,
defined by the following sequence:
[0153] SEQ ID NO:28:
[0154] Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
Ser Leu,
[0155] possibly deleted from one or two amino acids of the terminal
dipeptide Ser-Leu or one or more amino acids of the tetrapeptide
Val-Lys-Ser-Leu,
[0156] the epitope glycine 2242 to leucine 2251 inclusive, defined
by the following sequence:
[0157] SEQ ID NO:29:
[0158] Gly Val Thr Thr Gln Gly Val Lys Ser Leu,
[0159] possibly deleted from one or two amino acids of the terminal
dipeptide Ser-Leu, the epitope isoleucine 2262 to glutamine 2270
inclusive, defined by the following sequence:
[0160] SEQ ID NO:30:
[0161] Ile Ser Ser Ser Gln Asp Gly His Gln,
[0162] the epitope leucine 2273 to serine 2289 inclusive, defined
by the following sequence:
[0163] SEQ ID NO:31:
[0164] Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln
Asp Ser,
[0165] the epitope proline 2292 to tyrosine 2305 inclusive, defined
by the following sequence:
[0166] SEQ ID NO:32:
[0167] Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr,
[0168] possibly deleted from one or more amino acids of the
terminal tripeptide Thr-Arg-Tyr,
[0169] the epitope glutamic acid 2322 to tyrosine 2332 inclusive,
defined by the following sequence:
[0170] SEQ ID NO:33:
[0171] Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr.
[0172] Paragraph 14:
[0173] A conformational epitope, which contains at least two
different epitopes according to any one of the preceding paragraphs
8, 10 and 12.
[0174] Paragraph 15:
[0175] A pool of more than three fragments or epitopes according to
any of the preceding paragraphs 6 to 14.
[0176] Paragraph 16:
[0177] A recombinant factor VIII having an amino acid sequence
deleted from one or more of the fragments or the epitopes according
to any one of the paragraphs 6 to 14.
[0178] Paragraph 17:
[0179] A complex comprising a carrier protein or a carrier peptide
linked to an element which is selected from the group consisting of
the fragment and/or the epitope according to any one of the
paragraphs 6 to 14.
[0180] Paragraph 18:
[0181] An inhibitor of factor VIII, which exhibits an
immunoaffinity with the sequence, the fragment, the epitope, the
pool and/or the complex according to any one of the preceding
paragraphs.
[0182] Paragraph 19:
[0183] The inhibitor according to paragraph 18, which is an
anti-factor VIII antibody or antibody fragment.
[0184] Paragraph 20:
[0185] An anti-inhibitor, which is directed against the inhibitor
of factor VIII according to paragraph 18 or 19.
[0186] Paragraph 21:
[0187] The anti-inhibitor according to paragraph 20, which is an
anti-anti-factor VIII idiotype antibody or antibody fragment.
[0188] Paragraph 22:
[0189] A pharmaceutical composition, which comprises an adequate
pharmaceutical carrier and at least one element selected from the
group consisting of the sequence, the fragment, the epitope, the
pool, the complex, the recombinant factor VIII or the inhibitor
and/or the anti-inhibitor according to any one of the preceding
paragraphs.
[0190] Paragraph 23:
[0191] A diagnostic and/or purification device, which comprises at
least one element which is selected from the group consisting of
the sequence, the fragment, the epitope, the pool, the complex, the
inhibitor and/or the anti-inhibitor according to any one of the
preceding paragraphs.
[0192] Paragraph 24:
[0193] The device according to paragraph 23, which is a diagnostic
kit.
[0194] Paragraph 25:
[0195] The device according to paragraph 23, which is a
chromatography column or filter.
[0196] Paragraph 26:
[0197] A method for a therapeutic treatment and/or prevention of an
immune disorder in mammal, wherein the pharmaceutical composition
according to paragraph 22 is administered to the mammal patient
presently or potentially having said immune disorder, in an amount
effective to treat and/or prevent said immune disorder.
[0198] Paragraph 27:
[0199] A method for a therapeutic treatment and/or prevention of an
immune disorder in a mammal patient, wherein a physiological fluid
such as serum obtained from said mammal patient is put into the
chromatography column of paragraph 25 in order to allow a binding
with the inhibitors of factor VIII present in said serum with the
sequence, the fragment, the epitope, the pool and/or the complex
according to any of the preceding paragraphs 1 to 15, wherein the
physiological liquid is eluted from said chromatography column and
the physiological liquid from which the inhibitors of factor VIII
have been removed is reinjected to the patient.
[0200] Paragraph 28:
[0201] The therapeutic treatment and/or prevention method according
to paragraph 26 or 27, wherein the immune disorder is induced by an
element selected from the group consisting of inhibitors of factor
VIII, inhibitors of the binding of factor VIII to the von
Willebrand factor, to the factor IX, the factor X and/or to
membrane phospholipids.
[0202] Paragraph 29:
[0203] A process for identifying and obtaining inhibitors and/or
anti-inhibitors according to paragraph 18 or 19, comprising the
steps of:
[0204] selecting an element from the group consisting of the
sequence, the fragment, the epitope, the pool and/or the complex
according to any one of the preceding paragraphs 1 to 15 or 17,
attached to a solid support of a chromatography column,
[0205] passing a physiological fluid from a patient containing
inhibitors of factor VIII through said chromatography column,
[0206] eluting said column, and
[0207] collecting the fractions containing inhibitors of factor
VIII which have exhibited an immunoaffinity with said element.
[0208] Paragraph 30:
[0209] The process according to paragraph 29, further comprising
the steps of:
[0210] attaching the collected inhibitors of factor VIII upon the
solid support of a chromatography column,
[0211] passing a physiological fluid from a patient containing
anti-inhibitors of factor VIII through said chromatography
column,
[0212] eluting said column, and
[0213] collecting the fractions containing anti-inhibitors of
factor VIII which have exhibited an immunoaffinity with said
inhibitors of factor VIII.
BRIEF DESCRIPTION OF THE DRAWINGS
[0214] FIG. 1. This figure depicts the hydrophilicity, flexibility
and accessibility graph of the A3 sequence of Factor VIII,
renumbered 1 to 371 amino acids (surface value for each amino
acid).
[0215] FIG. 2. This FIG. (2a) represents the elution profile
related to the purification of human anti-SEQ. ID NO: 32 antibodies
by affinity chromatography in peptide-Sepharose column. Cohn
fraction II+III solution (50 mL) was loaded onto the column (1 mL
gel) at a flow rate of 1 mL/min. The separation of specific
antibodies was performed as described in the Examples. The arrow
indicates the position of specific human anti-SEQ. ID NO: 32. IgG
purified from Cohn fraction II+III. The clotting activity of FVIII
(2b) was measured as described in Examples in the presence of
increasing amount of anti-SEQ. ID NO: 32. The of FVIII
activity=(FVIII activity in the presence of antibody/FVIII activity
in the absence of antibody) * 100.
[0216] FIG. 3. This figure represents the human anti-peptide
antibody immunoreactions with FVIII polypeptides after Western
Blotting (panel A from left to right: human antibodies HAP1 through
HAP4, specific for different FVIII epitope sequences found in the
FVIII HC--see also Table 2 and panel B: human antibodies specific
for the P5 peptide and the FVIII LC sequences, P7, P8, and P9--see
also Table 2). The RAP9 lane shows the reactivity of FVIII
polypeptieds towards purified rabbit antibodies specific for the
peptide sequence Arg.sup.1797-Tyr.sup.1815 (see also Table 2).
[0217] FIG. 4. This figure represents the ELISA reactivity of the
four inhibitor plasmas with different peptide sequences. Inhibitors
present in four patient plasmas were analyzed by ELISA test using
as coated antigens the different selected FVIII epitopes synthetic
peptides, as indicated by the ordinate.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0218] The present invention relates to the antigenic polypeptide
sequences of factor VIII and/or fragments of these sequences, as
described by Verhar et al. (1984), the disclosure of which is
incorporated herein by reference in its entirety.
[0219] The "polypeptide sequence of factor VIII" is understood to
be the natural human or animal sequence, which may be glycosylated
and which has been obtained by purification from pools of plasma,
in particular cryoprecipitate, by synthesis and/or by genetic
manipulation (sequence from which portions which are not involved
in the mechanism of blood coagulation may have been deleted) of
factor VIII.
[0220] The present invention relates, in particular, to the
antigenic polypeptide sequences of factor VIII which lacks the
fragments comprised between alanine 322-serine 750, leucine
1655-arginine 1689 and lysine 1694-proline 1782, and possibly also
the fragments comprised between aspartic acid 2170 and tyrosine
2332.
[0221] The present invention relates, in particular, to the
antigenic polypeptide sequences A1, A2, A3 and C (C1 and C2) of
factor VIII.
[0222] A first embodiment of the invention relates to the antigenic
polypeptide sequence A3 of factor VIII, and to fragments and/or
epitopes of this sequence. The said sequence contains the fragments
glutamic acid 1649 to histidine 2031 inclusive, arginine 1652 to
arginine 1917 inclusive or arginine 1803 to arginine 1917
inclusive, of the polypeptide sequence of factor VIII as published
by Verhar et al. (1984) and Toole et al. (1984).
[0223] Preferably, the fragments of the said sequence are arginine
1648 to arginine 1696 inclusive, threonine 1739 to aspartic acid
1831 inclusive or glutamic acid 1885 to arginine 1917
inclusive.
[0224] The fragments, epitopes and major parts thereof are
preferably polypeptidic sequences made of at least 7 amino acids of
the FVIII polypeptidic sequence.
[0225] The invention also relates to the sequence epitopes of these
fragments, in particular:
[0226] the epitope arginine 1648 to tyrosine 1664 inclusive,
defined by the following sequence:
[0227] SEQ ID NO.:1:
[0228] Arg Asp Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile
Asp Tyr,
[0229] and possibly deleted from one or more amino acids of the
tetrapeptide Arg-Asp-Ile-Thr (P7), or one or two of the last amino
acids of the dipeptide Asp-Tyr
[0230] the epitope aspartic acid 1681 to arginine 1696 (P8)
inclusive, defined by the following sequence:
[0231] SEQ ID NO.:2:
[0232] Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr
Arg
[0233] possibly deleted from one or more amino acids of the epitope
Asp-Glu-Asp-Glu,
[0234] the epitope threonine 1739 to tyrosine 1748 inclusive,
defined by the following sequence:
[0235] SEQ ID NO.:3:
[0236] Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr
[0237] the epitope asparagine 1777 to phenylalanine 1785 inclusive,
defined by the following sequence:
[0238] SEQ ID NO.:4:
[0239] Asn Gln Ala Ser Arg Pro Tyr Ser Phe
[0240] possibly deleted from one or more amino acids of the
terminal dipeptide Ser-Phe or tetrapeptide Pro-Tyr-Ser-Phe
[0241] the epitope glutamic acid 1794 to tyrosine 1815 inclusive,
defined by the following sequence:
[0242] SEQ ID NO.:5:
[0243] Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg Lys Asn Phe Val Lys
Pro Asn Glu Thr Lys Thr Tyr,
[0244] possibly deleted from one or more amino acids of the first
tripeptide Glu-Asp-Gln (P9) or the first nonapeptide
Glu-Asp-Gln-Arg-Gln-Gly-Ala-Glu-Pro
[0245] the epitope methionine 1823 to aspartic acid 1831 inclusive,
defined by the following sequence:
[0246] SEQ ID NO.:6:
[0247] Met Ala Pro Thr Lys Asp Glu Phe Asp
[0248] the epitope glutamic acid 1885 to phenylalanine 1891
inclusive, defined by the following sequence:
[0249] SEQ ID NO.:7:
[0250] Glu Thr Lys Ser Trp Tyr Phe
[0251] the epitope glutamic acid 1885 to alanine 1901 inclusive,
defined by the following sequence:
[0252] SEQ ID NO.:8:
[0253] Glu Thr Lys Ser Trp Phe Thr Glu Asn Met Glu Arg Asn Cys Arg
Ala
[0254] possibly deleted from one or more amino acids from the
heptapeptide Glu-Thr-Lys-Ser-Trp-Phe-Thr or from the tripeptide
Cys-Arg-Ala.
[0255] the epitope aspartic acid 1909 to arginine 1917 inclusive,
defined by the following sequence:
[0256] SEQ ID NO.:9:
[0257] Asp Pro Thr Phe Lys Glu Asn Tyr Arg
[0258] the epitope comprised between serine 2018 and histidine 2031
inclusive, defined by the following sequence:
[0259] SEQ ID NO.:10:
[0260] Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His
[0261] Advantageously, the said sequences, specific fragments and
epitopes exhibit an antigenic characteristic which is illustrated
by Table 1.
[0262] Another preferred embodiment of the invention relates to
antigenic polypeptide sequence Al of factor VIII, fragments and/or
epitopes of this sequence.
[0263] Preferably, the fragments of the said sequence are alanine
108 to methionine 355 inclusive, preferably alanine 108 to alanine
227 inclusive.
[0264] The invention also relates to the sequence epitopes of these
fragments, in particular:
[0265] the epitope alanine 108 to valine 128 inclusive, defined by
the following sequence:
[0266] SEQ ID NO.:11:
[0267] Ala Ser Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu
Lys Glu Asp Asp Lys Val
[0268] possibly deleted from the terminal amino acids alanine and
valine (P1)
[0269] the epitope glutamic acid 181 to leucine 192 inclusive,
defined by the following sequence:
[0270] SEQ ID NO.:12:
[0271] Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu
[0272] possibly deleted from one or two amino acids of the terminal
dipeptide Thr-Leu
[0273] the epitope aspartic acid 203 to alanine 227 inclusive,
defined by the following sequence:
[0274] SEQ ID NO.:13:
[0275] Asp Glu Gly Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met
Gln Asp Arg Asp Ala Ala Ser Ala Arg Ala
[0276] possibly deleted from one or more amino acids of the
nonapeptide Asp-Arg-Asp-Ala-Ala-Ser-Ala-Arg-Ala
[0277] the epitope aspartic acid 327 to methionine 355 inclusive,
defined by the following sequence:
[0278] SEQ ID NO.:14:
[0279] Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg Met Lys Asn Asn Glu
Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp Ser Glu Met
[0280] possibly deleted from one or more amino acids from the
terminal dipeptide Asp-Ser or the octapeptide
Asp-Asp-Leu-Thr-Asp-Ser-Glu-Met (P2).
[0281] Another preferred embodiment of the invention relates to the
antigenic polypeptide sequence A2 of factor VIII, fragments and/or
epitopes of this sequence.
[0282] Preferably, the fragments of the said sequence are aspartic
acid 403 to serine 840 inclusive, preferably histidine 693 to
aspartic acid 725 inclusive.
[0283] The invention also relates to the sequence epitopes of these
fragments, in particular:
[0284] the epitope aspartic acid 403 to lysine 425 inclusive,
defined by the following sequence:
[0285] SEQ ID NO.:15:
[0286] Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro Gln
Arg Ile Gly Arg Lys Tyr Lys Lys
[0287] possibly deleted from one or more amino acids of the
tetrapeptide Asp-Asp-Arg-Ser (P3),
[0288] the epitope valine 517 to arginine 527 inclusive, defined by
the following sequence:
[0289] SEQ ID NO.:16:
[0290] Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg
[0291] possibly deleted from one or the two amino acids of the
dipeptide Pro-Arg,
[0292] the epitope tyrosine 555 to glutamine 565 inclusive, defined
by the following sequence:
[0293] SEQ ID NO.:17:
[0294] Tyr Lys Glu Ser Val Asp Gly Arg Gly Asn Gln
[0295] the epitope histidine 693 to glycine 701 inclusive, defined
by the following sequence:
[0296] SEQ ID NO.:18:
[0297] His Asn Ser Asp Phe Arg Asn Arg Gly
[0298] the epitope serine 710 to aspartic acid 725 inclusive,
defined by the following sequence (P4):
[0299] SEQ ID NO.:19:
[0300] Ser Cys Asp Lys Asn Thr Gly Asp Tyr Try Gly Asp Ser Tyr Glu
Asp
[0301] the epitope leucine 730 to serine 741 inclusive, defined by
the following sequence (P4):
[0302] SEQ ID NO.:20:
[0303] Leu Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser
[0304] possibly deleted from the terminal amino acid serine (P4)
and/or the first amino acid leucine
[0305] the epitope serine 817 to serine 830 inclusive, defined by
the following sequence (P5):
[0306] SEQ ID NO.:21:
[0307] Ser Asp Asp Pro Ser Gly Ala Ile Asp Ser Asn Asn Ser
[0308] A final preferred embodiment of the invention relates to the
antigenic polypeptide sequence C of factor VIII, and fragments
and/or epitopes of this sequence. Preferably, the fragments of the
said sequence are histidine 2082 to lysine 2251 inclusive or
leucine 2273 to tyrosine 2332 inclusive, preferably lysine 2085 to
glycine 2121 inclusive and serine 2181 to leucine 2251
inclusive.
[0309] The invention also relates to the sequence epitopes of these
fragments, in particular:
[0310] the epitope isoleucine 2081 to serine 2095 inclusive,
defined by the following sequence:
[0311] SEQ ID NO.:22:
[0312] Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser
possibly deleted from one or more amino acids from the tetrapeptide
Ile-His-Gly-Ile
[0313] the epitope tyrosine 2105 to glycine 2121 inclusive, defined
by the following sequence:
[0314] SEQ ID NO.:23:
[0315] Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser
Thr Gly
[0316] possibly deleted from one or more amino acids of the
tripeptide Tyr-Ser-Leu (P10)
[0317] the epitope asparagine 2128 to asparagine 2138 inclusive,
defined by the following sequence:
[0318] SEQ ID NO.:24:
[0319] Asn Val Asp Ser Ser Gly Ile Lys His Asn
[0320] the epitope histidine 2152 to arginine 2163 inclusive,
defined by the following sequence:
[0321] SEQ ID NO.:25:
[0322] His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg
[0323] the epitope serine 2181 to asparagine 2198 inclusive,
defined by the following sequence:
[0324] SEQ ID NO.:26:
[0325] Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe
Thr Asn
[0326] possibly deleted from one or more amino acids from the
terminal tripeptide Phe-Thr-Asn (P11)
[0327] the epitope serine 2204 to glutamine 2222 inclusive, defined
by the following sequence (P12):
[0328] SEQ ID NO.:27:
[0329] Ser Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala
Trp Arg Pro Gln
[0330] the epitope glutamine 2235 to leucine 2251 inclusive,
defined by the following sequence (P13):
[0331] SEQ ID NO.:28:
[0332] Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
Ser Leu
[0333] possibly deleted from one or two amino acids of the terminal
dipeptide Ser-Leu or one or more amino acids of the tetrapeptide
Val-Lys-Ser-Leu
[0334] the epitope glycine 2242 to leucine 2251 inclusive, defined
by the following sequence:
[0335] SEQ ID NO.:29:
[0336] Gly Val Thr Thr Gln Gly Val Lys Ser Leu
[0337] possibly deleted from one or two amino acids of the terminal
dipeptide Ser-Leu,
[0338] said epitope presenting a possible partial overlapping with
a known monoclonal antibody binding site ESH8 2248-2285
[0339] the epitope isoleucine 2262 to glutamine 2270 inclusive,
defined by the following sequence:
[0340] SEQ ID NO.:30:
[0341] Ile Ser Ser Ser Gln Asp Gly His Gln
[0342] the epitope leucine 2273 to serine 2289 inclusive, defined
by the following sequence (P14):
[0343] SEQ ID NO.:31:
[0344] Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln
Asp Ser
[0345] the epitope proline 2292 to tyrosine 2305 inclusive, defined
by the following sequence (P15):
[0346] SEQ ID NO.:32:
[0347] Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr
[0348] possibly deleted from one or more amino acids of the
terminal tripeptide
[0349] Thr-Arg-Tyr involved in the phospholipid von Willebrand
factor binding site
[0350] the epitope glutamic acid 2322 to tyrosine 2332 inclusive,
defined by the following sequence (P16):
[0351] SEQ ID NO.:33:
[0352] Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr
[0353] The invention also relates to the major parts of the said
epitopes or the said fragments. Said epitopes can be deleted from
one or more terminal amino acids, preferably from one, two or three
amino acids, or can be replaced by one or more amino acids that
present the same characteristic of hydrophilicity, flexibility and
accessibility.
[0354] It is also known that some of the epitopes according to the
invention are comprised in major determinants of human inhibitors
epitopes or several factors binding sites or binding sites of known
monoclonal antibodies, especially the portion C2 that is known to
be the binding site of the monoclonal antibody Mas531P or the
binding site ESH8 as well as phospholipids, Factor Xa or the von
Willebrand factor binding site. However, the specific epitopes
according to the invention or their major parts are preferred
selected portions of said binding sites or may include a possible
overlapping with said binding sites.
[0355] In other cases, the epitopes according to the invention are
more specific portions of known epitopes. Therefore, an artificial
epitope could be easily obtained by synthesis and the specific
above-described fragments can be deleted from non-epitopic portions
such as the fragment described in a C2 fragment (amino acids
phenylalanine 2196 to tryptophan 2203 inclusive and amino acids
valine 2222 to phenylalanine 2234 inclusive, or the sequence
leucine 2252 to threonine 2272 inclusive or the amino acids
phenylalanine 2290 to threonine 2291 inclusive as well as the amino
acids leucine 2306 to methionine 2321 inclusive).
[0356] These sequences, these fragments and these epitopes are
particularly advantageously characterized by high hydrophilicity,
which has been defined by Parker and Hodges (1986), considerable
flexibility, which has been defined by Karplus and Schultz (1985)
and considerable accessibility, which has been defined by Janin
(1979).
[0357] These fragments and these epitopes are, in particular,
exposed on the surface of the factor VIII protein and exhibit
pronounced antigenic and immunogenic characteristics.
[0358] Another aspect of the present invention is related to a
modified (recombinant or transgenic) FVIII, possibly obtained by
genetic engineering, and deleted from one or more of the
above-identified fragments, epitopes or major parts of said
epitopes and/or said fragments.
[0359] Advantageously, said FVIII still allows the binding of
coagulation factor(s), but will be less immunogenic and will not
induce or induce less the formation of inhibitors directed against
said modified FVIII or natural FVIII.
[0360] Advantageously, said polypeptide sequences, fragments or
epitopes are also independently immunogenic (that is to say they
are immunogenic even without being complexed with a protein of
large size such as BSA, KLH haemocyanin, etc.), and preferably
exhibit an immunoaffinity within inhibitors of factor VIII, such as
anti-factor VIII antibodies, and/or exhibit an immunoaffinity for
the receptors of the T lymphocytes and possibly B lymphocytes.
[0361] These sequences, fragments, epitopes and/or major parts of
said fragments or said epitopes induce an immune reaction (antibody
synthesis) when they are injected into a rabbit.
[0362] These characteristics are particularly pronounced in the
case of the epitopes SEQ ID NO.:2 and SEQ ID NO.:5, which comprise
sequences which are relatively "long" in amino acids, i.e. comprise
16 and 22 amino acids, respectively.
[0363] Said sequences are unexpectedly characterized by substantial
immunogenicity towards monoclonal and polyclonal antibodies, but
are sufficiently short to be readily and advantageously obtained by
synthesis.
[0364] The present invention also relates to the conformational
epitopes which comprise at least two different fragments of said
sequence, at least two sequence epitopes and/or at least two major
parts of said epitopes or said different fragments according to the
invention and above identified.
[0365] The conformational epitopes are made up of two or more
different portions of a polypeptide sequence, which portions are
located in proximity to each other when the protein is folded in
its tertiary or quaternary structure.
[0366] These epitopes are capable of being "recognized" (that is to
say of exhibiting an immunoaffinity), preferably simultaneously,
with inhibitors of factor VIII, in particular B and T lymphocytes
(by way of the major histocompatibility locus (MHC I and/or II))
and/or anti-factor VIII antibodies (Scandella et al. (2000); Reding
et al. (2000)).
[0367] Preferably, the said sequence, said fragments, said epitopes
and/or the major parts of said epitopes or said fragments are
complexed with a carrier protein or a carrier peptide, such as BSA,
or KLH haemocyanin, as to form a complex exhibiting a more powerful
immunogenicity.
[0368] The present invention is also related to a pool of more than
three of said fragments, epitopes or major parts of said epitopes
having advantageously important antigenic and/or immunogenic
properties and which may be used advantageously in a diagnostic or
therapeutic method or device such as a kit or a dialysis column,
etc. allowing an efficient, preferably complete, screening and
characterization of the major (if not all) known inhibitors
directed against factor VIII by (human) patients.
[0369] Another aspect of the present invention relates to an
inhibitor of factor VIII which exhibits an immunoaffinity with
antigenic polypeptide sequences according to the present invention,
with fragments and epitopes of said sequences, with the major parts
of said epitopes or said fragments and/or with the complex
according to the invention.
[0370] An inhibitor is understood to mean any biological molecule
or cell (such as a T-lymphocyte) binding to said FVIII and capable
of giving rise to immune disorders (characterized by humoral immune
response and/or cellular immune response against said FVIII).
[0371] In particular, such an inhibitor can be an anti-factor VIII
monoclonal or polyclonal antibody or antibody fragment (such as the
hypervariable Fab portion of the said antibody) which inactivates
the said factor VIII and/or which inhibits the binding of factor
VIII to the von Willebrand factor and/or to membrane
phospholipids.
[0372] Advantageously, the said inhibitors are synthesized by a
"chimeric" animal which comprises a human immune system, such as an
hu-SCID mouse or transgenic mouse producing human antibodies or
other antibodies production technologies as phage display
technology or immortalized B-cells, by EPV in particular.
[0373] Another aspect of the invention relates to an anti-inhibitor
which is directed against the said previously described factor VIII
inhibitor.
[0374] An anti-inhibitor which is directed against the factor VIII
inhibitor is understood to mean any chemical or biological
molecule, a cell and/or a cell fragment (receptor) which is capable
of interfering with the said inhibitor in such a way as to ensure
its inactivation or avoid or reduce its binding to the factor
VIII.
[0375] Preferably, such an anti-inhibitor is an anti-anti-factor
VIII idiotype (monoclonal or polyclonal) antibody or antibody
fragment, natural or obtained by genetic engineering.
[0376] Another aspect of the invention relates to a pharmaceutical
composition which comprises an adequate pharmaceutical carrier or a
diluent and an element selected from the group consisting of said
antigenic polypeptide sequence of factor VIII, fragments and
epitopes of this sequence or a pool thereof, an inhibitor of factor
VIII which is directed against them, an anti-inhibitor which is
directed against the said inhibitor, and/or a mixture of these.
[0377] The type and amount of adequate pharmaceutical carrier or
diluent (and possibly adjuvant or excipient) present in said
pharmaceutical composition, may vary according to the method of
administration and is possibly combined an adjuvant in order to
improve therapeutical properties of the pharmaceutical composition
according to the invention or to reduce its possible side effects.
Suitable pharmaceutical acceptable carriers used in the
pharmaceutical composition according to the invention are well
known by the person skilled in the art and are selected according
to the methods generally applied by pharmacists and may include
solid, liquid or gaseous non-toxic pharmaceutically acceptable
carriers. The percentage of active product/pharmaceutical
acceptable carrier may vary within very large ranges only limited
by the tolerance and the possible side effects on patients
(including humans), and by frequency and/or mode of
administration.
[0378] Another aspect of the invention relates to a diagnostic
and/or purification device, such as a diagnostic kit, an affinity
filter, or a chromatography column which comprises an element which
is selected from the group consisting of these antigenic
polypeptide sequences, fragments and epitopes and/or major parts of
said epitopes or said fragments, the complex according to the
invention or a pool thereof, an inhibitor which is directed against
them, an anti-inhibitor which is directed against said inhibitor,
and/or a mixture of these. Advantageously, said device comprises a
pool of said epitopes which allow a screening of patients and may
detect the most important inhibitors present in said patients and
which allow a positive test with enough specificity and
sensibility.
[0379] The purification device can therefore consist of a
chromatography column which comprises these sequences of factor
VIII, fragments and epitopes and/or major parts of said fragments
or epitopes, attached to the solid phase of the chromatography
column.
[0380] A physiological liquid (such as serum), which is derived
from a patient and which comprises inhibitors of factor VIII pass
through this chromatography column, with said inhibitors (for
example antibodies) becoming attached specifically to said factor
VIII sequences, fragments, epitopes or said major parts or a pool
thereof. Following elution, it is possible to collect said
inhibitors by causing them to react with anti-inhibitors
(anti-anti-factor VIII idiotype antibodies).
[0381] It is also possible to characterize the anti-anti-factor
VIII idiotype antibodies which are present in a serum by these
anti-inhibitors passed through a chromatography column on which
inhibitors of factor VIII have been attached to the solid
phase.
[0382] It is also possible to reinject (ex vivo treatment) the
physiological liquid (blood or serum or a derived fraction) to said
patient after its inhibitors of factor VIII have been removed by
binding with said factor VIII fragments, epitopes or a pool
thereof; said inhibitors being removed from the physiological fluid
(blood or serum) similarly as proposed for dialysis method applied
to human patients.
[0383] The present invention is also related to a method of
treatment (ex vivo treatment) of a patient suffering from a
pathology induced by inhibitors to the factor VIII which comprises
the steps of extracting said physiological liquid (blood or serum)
from the patient, obtaining its reaction upon a solid support
binding the factor VIII fragments, epitopes or a pool thereof
according to the invention and reinjecting said physiological
liquid to the patient after the removing of the inhibitors having
fixed said factor VIII fragments, epitopes, majors parts or a pool
thereof.
[0384] A final aspect of the invention relates to the use of the
pharmaceutical composition according to the invention for preparing
a medicament used for preventing and/or treating immune disorders,
in particular those induced by inhibitors of factor VIII,
inhibitors of the binding of factor VIII to the factor IX and/or
the factor X and/or the von Willebrand factor (vWF) and/or
inhibitors of the binding of factor VIII to membrane
phospholipids.
[0385] The present invention will be described in details in the
following non-limiting examples in reference to the enclosed
figures.
EXAMPLES
[0386] Materials and Methods
[0387] Reagents: MAS530p (Harlan-Seralab, Indianapolis, Ind.) is a
mouse monoclonal antibody specific for the 44-kDa A2 domain of the
factor VIII heavy chain. Biotin-labeled rabbit IgG anti-mouse IgG
was purchased from Dakopatts (Copenhagen, Denmark). Biotin-labeled
goat IgG anti-human IgG and biotin-labeled mouse IgG anti-rabbit
IgG were obtained from Sigma Chemicals (St Louis, Mich.), purified
a-thrombin (3000 IU/mg), streptavidin-peroxidase conjugate,
ovalbumin (OVA), bovine serum albumin (BSA), keyhole limpet
haemocyanin (KLH), and o-phenylenediamine (OPD) were purchased from
Sigma Chemicals (St. Louis, Mich.). Casein was obtained from Merck
(Darmstadt, Germany). 4-chloro-1-naphtol and biotinylated molecular
weight markers were obtained from Bio-Rad Laboratories (Hercules,
Calif.). Freund's adjuvant was from Difco (Detroit, Mich.).
[0388] FVIII Concentrates:
[0389] Plasma FVIII (p-FVIII) was a solvent/detergent-treated FVIII
concentrate (100 IU/mg protein) purified by ion exchange
chromatography (FVIII Conc. SD, CAF-DCF-Red Cross, Brussels,
Belgium). Albumin-free recombinant FVIII (rFVIII) was obtained from
Hyland (Glendale, Calif.).
[0390] Plasma fraction immunoglobulins: Cohn Fraction II+III was
obtained from large plasma pool from 4,800 unpaid donors, after
precipitation in the presence of increasing ethanol concentration.
This fraction contains all Ig classes and subclasses. IgG
composition was determined by nephelometry. The relative percentage
of each subclass was 63,7; 30,1; 3,4 and 2,8 for IgG1, IgG2, IgG3
and IgG4 respectively (average values for 3 different batches of
FII+III).
[0391] Factor VIII Concentrates, Factor VIII Activity and Activity
Inhibition
[0392] Factor VIII activity was determined in a one-stage clotting
assay adapted for use on the Coagulometer KC4A (Sigma Diagnostics).
The assay uses severe hemophilia A plasma (Organon Teknika,
Cambridge, UK) and APPT reagent from Instrumentation Laboratory
(Warrington, UK). Potencies were calculated relative to the
5.sup.th International Standard FVIII concentrate 88/640 (5.4
IU/ml) (NIBSC, Potters Bar, UK). FVIII-inhibitory activity was
measured in purified rabbit and human IgG preparations according to
the modified Bethesda assay. Briefly, affinity-purified IgGs were
serially diluted and incubated for 1 h in the presence of FVIII
concentrate 88/640 (1 IU/ml) at 37.degree. C. The residual FVIII
activity was measured as described above.
[0393] Activation of factor VIII by .alpha.-thrombin and
immunoblotting has been described elsewhere (Peerlinck et al,
1997).
[0394] Synthesis of peptides, conjugation of peptides to carrier
proteins and production of rabbit anti-peptide antisera were
performed by Neosystem (Strasbourg, France).
[0395] Purification of Rabbit and Human Antibodies by Affinity
Chromatography
[0396] For purification of rabbit and human antibodies, 5 mg of
each different peptide was coupled to 1 ml pre-packed NHS-activated
Sepharose (Pharmacia Biotech, Uppsala, Sweden) according to the
manufacturer's instructions. Specific anti-peptide antibodies were
purified with an automated liquid chromatography system
(KTAexplorer 100 A, Pharmacia Uppsala, Sweden) either from 50 ml
rabbit antiserum or from 100 ml of a human plasma fraction,
obtained after Cohn fractionation (fraction II+III; 13 mg
protein/ml). Briefly, samples were dialyzed 3 times against 5
volumes of TE buffer (20 mM Tris-HCl pH 7.2, 150 mM NaCl and 0.02%
NaN.sub.3) and loaded onto the column at a flow rate of 1 ml/min.
The column was sequentially washed at 2 ml/min with 50 ml TE buffer
and 30 ml TE containing 1 M NaCl. After absorption, the material
was eluted (1 ml/min) with 5 ml of 0.1 M citric acid pH 2.5 and
directly recovered in 5 ml of IM Tris-HCl, pH 9.0. Samples were
finally dialyzed versus 10 volumes of equilibration buffer and
concentrated on Centriprep-30 (Amicon, Beverly, Mass.). Ig recovery
was determined by the Bio-Rad protein assay.
[0397] Selection of Potential Factor-VIII Linear Epitopes
[0398] More than 30 surface regions (linear epitopes) spanning 8 to
25 residues, characterized by a high hydrophilicity, flexibility
and accessibility were identified on the FVIII molecule. On the
basis of their high probability of an outer location (see FIG. 1
for A3), 16 linear peptides (PI to P16) were selected, matching
identified stretches of 13 or more amino-acid residues. These
peptides were synthesized and coupled to ovalbumin for production
of specific antiserum (Table 1, hereafter). P8 includes the epitope
described by Shima et al (1988) and was used as an external
control.
[0399] Experimental Results Obtained from Said Synthesized Linear
Epitopes using the Rabbit Model
[0400] Results are summarized in Table 1 which concerns the
characterization of rabbit anti-FVIII-peptide antisera and
recovered affinity-purified of immunoglobulins. Sixteen synthetic
peptides (from 10 to 20 amino acids) were selected in the A, B, Cl
and C2 domains. After conjugation with ovalbumin, the OVA-peptide
conjugates were injected into rabbits and FVIII anti-peptide
antisera RAP1 to RAP16 were studied. More precisely, two rabbits
were immunized with each FVIII-peptide-ovalbumin preparation.
Specific antisera RAP1 to RAP16 (column b, Table 1) were prepared
and assayed in an ELISA (column c, Table 1) using rFVIII or
FVIII-peptide-KLH as the antigen. ELISA titer is expressed as the
negative log of the reciprocal of the serum dilution giving 50%
binding. The immunoglobulins were then purified by chromatography
on peptide-bound Sepharose. The FVIII domain recognized by the
anti-FVIII peptide Ig after immunoblotting is shown in (column d,
Table 1) and Ig protein recoveries (column e, Table 1) were
measured using immunoglobulins as the standard. The inhibitory
activity, expressed in BU/mg protein, was determined in a FVIII
neutralizing activity assay (column f, Table 1).
[0401] Immunogenicity of FVIII Peptides and Characterization of
Rabbit Anti-FVIII Peptide Antisera
[0402] The reactivity of FVIII anti-peptide antisera was measured
by an ELISA using, as antigen, either the different corresponding
FVIII-peptide coupled to KLH protein or purified rFVIII. The
binding reaction of each anti-FVIII-peptide antiserum was specific
both for the FVIII peptide used to elicit the immune response in
rabbit and for rFVIII (see Table 1).
[0403] To demonstrate the FVIII epitope specificity of the rabbit
anti-peptide antibodies, rFVIII and the rFVIII fragments obtained
after treatment with thrombin were resolved by SDS-PAGE and
analyzed by western blotting with the different preparations of
rabbit IgGs. As expected, most antisera ({fraction (14/16)}, 87%),
showed a strong reaction with the corresponding FVIII fragment
containing the selected linear epitope (see Table 1).
[0404] Purification of Rabbit-anti-FVIII Peptide Antibodies
[0405] The specific rabbit IgG were purified by affinity
chromatography on peptide-Sepharose as described under Methods.
When FVIII-neutralizing activity was measured in a one-stage
clotting assay, significant inhibition was found with two rabbit
IgG purified preparations: RAP2, corresponding to IgG specific for
SEQ ID No. 14 and RAP7 specific for SEQ ID NO.:1.
[0406] Epitope Mapping of Rabbit anti-FVIII Peptide Antibodies by
Immunoblotting with Human rFVIII
[0407] To demonstrate the FVIII epitope specificity of the rabbit
anti-peptide antibodies, rFVIII and the rFVIII fragments obtained
after treatment with thrombin were resolved by SDS-PAGE and
analyzed by western blotting with different preparations of rabbit
IgGs (RAP1 to RAP17 Igs).
[0408] In each run, the rFVIII heavy chain (HC) and light chain
(LC) and their thrombin proteolysis products (44 kDa and 72 kDa)
were identified with a mixture of two monoclonal antibodies, MoAb
530 p and MoAb18, respectively specific for the heavy and light
chain. MoAb18 recognizes the NH.sub.2-terminal light-chain FVIII
fragment obtained after thrombin activation, which proved too small
to remain in the gel after electrophoresis. Fourteen of the 17
rabbit immunoglobulin preparations reacted strongly with both
rFVIII and pFVIII. Antisera RAP1, RAP2, RAP3, RAP4 recognized
exclusively the heavy chains (200 kDa to 92 kDa). Antisera RAP1 and
RAP2 reacted with the 50-kDa A1-domain fragment; RAP3 and RAP4
bound to the 44-kDa fragment (domain A2); RAP5 (specific for the B
domain) bound to the high-molecular-weight FVIII heavy chain (about
200-kDa).
[0409] RAP7, RAP8, and RAP9 reacted with the 80-kDa light-chain
doublet. RAP9 and RAP12 to RAP17 antibodies also detected the
72-kDa FVIII light-chain fragment. As expected, each reactive
antiserum showed a strong reaction with the corresponding FVIII
fragment containing the selected linear epitope. No reaction was
detectable in the gels between RAP6 or RAP10 and the HC or LC FVIII
fragments.
[0410] Experimental Results Obtained from said Synthesized Linear
Epitopes to Purify and Characterize Human Autoantibodies
[0411] Table 2 concerns the characterization of human anti-FVIII
antibodies from Cohn fraction II+III of healthy individuals.
[0412] Human anti-peptide IgG preparations (HAP1 through HAP17)
were so far purified on Sepharose coupled to 13 different FVIII
peptides (column a, Table 2). The Igs (column b, Table 2) were
analyzed by immunoblotting. Binding to the rFVIII HC or LC chains
and to the rFVIII thrombin fragment is shown respectively in
columns c and d, Table 2. FVIII-domain reactivity is shown in
column e, Table 2. Arrows indicate a decrease in 80-kDa band
intensity. Ig recovery (column f, Table 2) after affinity
purification is expressed in .mu.g/10 mg loaded FII+III (see
Materials and Methods). Inhibition of the clotting assay was
determined after incubation in the presence of each of the 13 Ig
preparations in the Bethesda assay (column g, Table 2).
[0413] Use of FVIII Peptides for the Immunopurification of Human
Anti-FVIII Antibodies in Healthy Donors
[0414] To prepare and characterize human anti-FVIII antibodies
present in healthy individuals, we analyzed Cohn fraction II+III,
rich in immunoglobulins, for the presence of selected specific
anti-peptide antibodies. Human anti-FVIII-peptide antibodies (HAP 1
to HAP 1, HAP 16 and HAP 17) were purified by affinity
chromatography on Sepharose coupled to the appropriate peptide (see
Table 2). As a typical example, FIG. 2 shows the chromatographic
profile obtained with SEQ ID 32, a sequence found in C2 domain.
Table 2 summarizes the results obtained with 17 epitopic sequences
selected in each FVIII domain (Al, A2, A3, B, Cl and C2).
Significant amounts of immunoglobulins, specific for each of the 13
FVIII peptides used, were obtained from the starting plasma
fraction II+III. The specificity of the resulting purified human
antibodies was directly tested by immunoblotting with plasma FVIII,
recombinant FVIII, and the fragments obtained after thrombin
proteolysis (see Table 2).
[0415] The IgG isotype distribution in the human purified antibody
preparations was found to be quite heterogeneous. Interestingly, 40
to 79% of the recovered IgGs belonged to the IgG2 subclass. In most
preparations, IgG4 appeared to be over-represented (up to 25%).
[0416] All the human anti-FVIII-peptide antibody preparations were
tested for the capacity to inhibit FVIII activity in a one-stage
clotting assay. Table 2 shows that seven out of 13 preparations
tested (54%) displayed inhibitory activity, SEQ ID NO.: 14, SEQ ID
NO.: 19, SEQ ID NO.: 2, SEQ ID NO.: 5, SEQ ID NO.: 22, SEQ ID NO.:
32 and SEQ ID NO.: 33, respectively. As a typical example, the
inhibition of FVIII activity in function of anti-SEQ ID 32 Ig
concentration is shown in FIG. 2.
[0417] Human Anti-FVIII-peptide Ig Immunospecificity Towards
FVIII
[0418] The specificity of the resulting purified human antibodies
was tested by immunoblotting with plasma FVIII, recombinant FVIII,
and the fragments obtained after thrombin proteolysis. Again, the
FVIII fragments were identified with either FVIII-HC- or
FVIII-LC-specific mouse monoclonal antibodies or
FVIII-peptide-specific rabbit polyclonal antibodies. The human
antibodies were identified after binding of biotinylated goat
anti-human IgG. FIG. 3 shows the immunoreaction of
high-molecular-weight FVIII (.gtoreq.92-kDa) with four human
antibody preparations, purified on Sepharose coupled to FVIII
peptide SEQ ID NO.: 11 (Ser.sup.109-Lys.sup.127), SEQ ID NO.: 14
(Cys.sup.329-Asp.sup.348), SEQ ID NO.: 15 (Tyr.sup.407-Lys.sup.425)
or SEQ ID NO.: 19 (Cys.sup.711-Asp.sup.725). The 50-kDa FVIII
fragment (domain A1) was recognized by human antibodies purified on
Ser.sup.109-Lys.sup.127 or Cys.sup.329-Asp.sup.348-Sepharose and
the 44-kDa FVIII fragment (A2) by immunoglobulins purified on
Tyr.sup.407-Lys.sup.425 and Cys.sup.711-Asp.sup.725-Sepharose. The
lack of reactivity of the anti-(Ser.sup.817-Ser.sup.830)
immunoglobulin preparation (HAP5) with the FVIII fragments confirms
that this epitope is located in the amino-terminal end of domain B
(FIG. 3). Human antibodies purified on Sepharose coupled to peptide
SEQ ID NO.: 1 (Arg.sup.1652-Tyr.sup.1664) or SEQ ID NO.: 2
(Asp.sup.1681-Arg.sup.1696) reacted strongly with the 80-kDa FVIII
light chain (FIG. 3). For both preparations, the reaction with the
80-kDa band disappeared after thrombin proteolysis, indicating that
the epitopes, as expected, are located in the a3 acidic peptide at
the NH.sub.2-terminal part of the FVIII A3 domain. When human
antibodies specific for peptide SEQ ID NO.: 5
(Arg.sup.1797-Tyr.sup.1815 in A3 domain) were analyzed by
immunoblotting, their specificity for rFVIII appeared restricted to
the 80-kDa FVIII light chain and its 72-kDa thrombin fragment.
[0419] No immunoreaction with the rFVIII chains or fragments was
detected with antibody preparations specific for FVIII peptides SEQ
C and SEQ ID NO.: 23, although a positive reaction was obtained in
the ELISA using rFVIII. This could mean that these immunoglobulin
preparations recognize a conformational epitope.
[0420] Use of FVIII Synthetic Peptides to Characterize Human
Anti-FVIII Antibodies in Hemophilia A Patient Plasmas
[0421] The selected peptides were used in ELISA experiment to
determine the anti-FVIII antibody specificity's present in
hemophilia A plasmas. The peptides were coated on microplate (25
.mu.g/ml in PBS buffer during 16 h at 4.degree. C.). A {fraction
(1/10)} to {fraction (1/1000)} dilution of plasma patient in
Tris-casein buffer was reacted with the coated peptide for 2 h at
37.degree. C. The bound human IgG was measured as described in
Methods. Control samples were plasma pools of healthy donors. FIG.
4 shows the results obtained with the plasma of 4 hemophilia A
patients. The optical densities are corrected average values (OD
patient-OD normal plasma pool) of two independent experiments.
[0422] Molecular Model Epitope Prediction
[0423] Pemberton et al (1997) have built a molecular model of the A
domains of FVIII. This 3-D model makes it possible to explore
predictions for important regions of FVIII activity. The model was
used to locate the FVIII-peptide epitopes identified by the Parker
and Hodge algorithms. As predicted by these algorithms, all
peptides located in the A domains were found on the FVIII surface
and were fully accessible to specific.
[0424] The overlap between the epitope and the FIXa-binding loop (5
common residues spanning Glu.sup.1811-Tyr.sup.1815) may explain the
inhibitory action of the corresponding
anti-(Arg.sup.1797-Tyr.sup.1815) antibodies on formation of the
fibrin clot.
[0425] Analysis
[0426] In the clotting test, significant inhibition of FVIII
activity was recorded in the presence of rabbit
anti-(Cys.sup.329-Asp.sup.348) and anti-(Arg.sup.1653-Tyr.sup.1664)
antibodies, but different inhibition patterns were observed.
Inhibition by anti-(Arg.sup.1653-Tyr.sup.1664) follows second-order
kinetics with a drastic reduction in FVIII activity.
Anti-(Cys.sup.329-Asp.sup.348) Ig is less efficient and shows a
more complex type of reaction, with a non-linear dependence on the
antibody concentration. Epitope Arg.sup.1652-Tyr.sup.1664 and the
adjacent major binding site vWF (residues
Glu.sup.1675-Glu.sup.1684) are located in the acidic light-chain
peptide a3. As shown by western blotting, a3 is released from the
A3 domain after thrombin treatment, preventing further binding of
anti-(Arg.sup.1652-Tyr.sup.1664) Ig to activated FVIII. Similar
results have been reported by Shima et al (1991), who described the
FVIII sequence Asp.sup.1663-Ser.sup.1669 as a binding site of
rabbit polyclonal antibodies neutralizing FVIII activity. Epitope
Cys.sup.329-Asp.sup.348 overlapped the acidic Asp.sup.348-Lys 362
sequence (in al) described as adjacent to the activated protein C
(Arg.sup.336) and thrombin (Arg.sup.372) cleavage sites. It is the
target of human hemophilic inhibitors.
Anti-(Asp.sup.348-Lys.sup.362) antibodies may interfere with
proteolysis or with the FX interaction site
(Met.sup.337-Arg.sup.372) (Saenko et al., 1999 and Scandella et
al., 2000).
[0427] FVIII-neutralizing activity was measured in all 13 Ig
preparations. Seven human Ig preparations displayed inhibition of
procoagulant activity, these being specific for amino-acid residues
Cys.sup.711-Asp.sup.725, Tyr.sup.1681-Arg.sup.1696 and
Arg.sup.1797-Tyr.sup.1815 respectively The Cys.sup.711-Asp.sup.725
sequence contains sulfated tyrosines at Tyr.sup.718, Tyr.sup.719,
and Tyr.sup.723, and overlaps with the FVIII HC region
Lys.sup.713-Arg.sup.740 described as promoting both activation and
HC proteolysis. The additional sulfated groups may be required for
proper interaction with thrombin or another component as in the
FX-activating complex. The sequence also overlaps with region
Gly.sup.701-Ser.sup.750, recognized by a weakly inhibitory mouse
monoclonal antibody. Peptide P8 (Tyr.sup.1681-Arg.sup.1696) (FVIII
LC) includes the sequence Glu.sup.1684-Arg.sup.1689 already
described by Shima et al, 1991. It contains the thrombin activation
site Arg.sup.1689-Ser.sup.1690. P4 (Cys.sup.711-Asp.sup.725) is
also included in the Asp.sup.712-Ala.sup.736 sequence detected by
analysis of the patient antibody repertoire by gene phage display
technology. It is proposed as a possible additional inhibitor in
patients (van den Brink et al, 2000). Peptide P9
(Arg.sup.1797-Tyr.sup.1815) contains the FXa binding site (see
below).
[0428] Of the 16 anti-FVIII-peptide immunoglobulins purified from
humans or produced in rabbits, 7 did neutralize FVIII activity
under the tested conditions. Using small peptide sequences and
immunobinding assays, we have provided evidence for additional new
epitopes. We have located new epitopes in the A1 domain (residues
Ser.sup.109-Cys.sup.127), the A2 domain (Cys.sup.407-Lys.sup.425),
and the B domain (Ser.sup.817-Ser.sup.830 and
Glu.sup.1078-Pro.sup.1092).
[0429] Autoantibodies immunopurified with denatured FVIII have been
reported in healthy subjects and in pools of normal human
immunoglobulins (processed fraction II, see above) (Algiman et al.,
1992 and Moreau et al., 2000). A possible role in clearance of
denatured FVIII or its fragments from the bloodstream and/or in the
immunotolerance was suggested.
[0430] Identification of the FVIII epitopes is a major challenge to
be met in order to improve FVIII treatment and the quality of
therapeutic FVIII concentrates. FVIII epitope sequences help to
determine the contribution of patient polyclonal anti-FVIII Igs to
overall inhibitory and regulatory activity. They could also be used
to monitor the usual switch in anti-FVIII specificity in a patient
during treatment. Said characterization of FVIII epitopes and a
model of their locations on the folded molecule improves the
treatment of inhibitors in both hemophilic and non-hemophilic
patients (detection, follow-up, therapeutic use of FVIII epitope
peptides . . . ).
1TABLE 1 Characterization of rabbit anti-FVIII peptides antibodies
ELISA domain FVIII RAP-IgG Inhibitor Rabbit Titer(c) i. Recovery(e)
Titer(f) SEQ ID(a) Antiserum(b) P-KLH r-FVIII recognize(d) .mu.g/ml
serum BU/mg SEQ ID 11 RAP1 2.5 2.2 A1 27 -- SEQ ID 14 RAP2 3.6 2.5
A1/a1 55 1,5 SEQ ID 15 SEQ RAP3 2.5 3.2 A2 268 -- ID 19 SEQ ID 21
RAP4 2.5 1.3 A2/a2 12 -- SEQ C RAP5 4.6 3.9 B 106 -- SEQ ID 01 SEQ
RAP6 3.8 2.9 -- 14 -- ID 02 SEQ ID 05 RAP7 3.9 3.9 a3 .dwnarw. 35
0,5 SEQ ID 23 SEQ RAP8 1.9 0.9 a3/A3 .dwnarw. 3 -- ID 22 RAP9 3.8
2.6 A3 42 -- SEQ ID 26 SEQ RAP10 3.9 0.8 -- 65 -- ID 27 SEQ ID 28
RAP11 ND ND ND ND ND SEQ ID 31 SEQ RAP12 4.1 1,1 C2 ND ND ID 32 SEQ
ID 33 RAP13 3.7 1,1 C2 ND ND RAP14 3.8 0,9 C2 ND ND RAP15 3.2 0,7
C2 ND ND RAP16 3.5 1,8 C2 ND ND RAP17 4.8 1,2 C2 ND ND
[0431]
2TABLE 2 Characterization of human anti-FVIII peptides
autoantibodies FVIII FVIII reactivity HAP-IgG inhibitory i. on
immunoblot FVHI Recovery(f) Activity(g) SEQ ID(a) Anti-peptide
Ig(b) (-thrombin) (c)(+thrombin)(d) domain(e) .mu.g/10 mgIgG BU/mg
SEQ ID 11 HAP1 >92kDa 50kDa A1 0,27 -- SEQ ID 14 HAP2 >92kDa
50kDa A1/a1 1,07 3,4 SEQ ID 15 HAP3 >92kDa 44kDa A2 0,06 -- SEQ
ID 19 HAP4 92kDa 44kDa A2/a2 0,12 + SEQ ID 21 HAP5 >100kDa -- B
0,26 -- SEQ C HAP6 -- -- -- 0,03 -- SEQ ID 01 HAP7 80kDa 80kDa a3
.dwnarw. 0,20 -- SEQ ID 02 HAP8 80kDa 80kDa a3/A3 .dwnarw. 0,01 +
SEQ ID 05 HAP9 80kDa 72kDa A3 0,08 + SEQ ID 23 HAP10 -- -- -- 0,11
-- SEQ ID 22 HAP11 ND ND ND 0,98 4,3 SEQ ID 26 HAP12 ND ND ND ND ND
SEQ ID 27 HAP13 ND ND ND ND ND SEQ ID 28 HAP14 ND ND ND ND ND SEQ
ID 31 HAP15 ND ND ND ND ND SEQ ID 32 HAP16 80kDa 72kDa A3C1C2 2,40
6,3 SEQ ID 33 HAP17 ND ND ND 1,06 2,4 +: Inhibition >25% at 100
.mu.g/ml
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* * * * *