U.S. patent application number 16/403862 was filed with the patent office on 2019-10-17 for albumin variants.
The applicant listed for this patent is Albumedix Ltd. Invention is credited to Karen Ann Delahay.
Application Number | 20190315836 16/403862 |
Document ID | / |
Family ID | 49160283 |
Filed Date | 2019-10-17 |
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United States Patent
Application |
20190315836 |
Kind Code |
A1 |
Delahay; Karen Ann |
October 17, 2019 |
ALBUMIN VARIANTS
Abstract
The present invention relates to variants of a parent albumin,
the variants having altered plasma half-life compared with the
parent albumin. The present invention also relates to
polynucleotides encoding the variants; nucleic acid constructs,
vectors, and host cells comprising the polynucleotides; and methods
of using the variants.
Inventors: |
Delahay; Karen Ann;
(Nottingham, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Albumedix Ltd |
Nottingham |
|
GB |
|
|
Family ID: |
49160283 |
Appl. No.: |
16/403862 |
Filed: |
May 6, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15915977 |
Mar 8, 2018 |
10329340 |
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16403862 |
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14385631 |
Sep 16, 2014 |
9944691 |
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PCT/EP2013/055487 |
Mar 15, 2013 |
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15915977 |
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61710134 |
Oct 5, 2012 |
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61722544 |
Nov 5, 2012 |
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61724674 |
Nov 9, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/765 20130101;
C07K 2319/00 20130101 |
International
Class: |
C07K 14/765 20060101
C07K014/765 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 16, 2012 |
EP |
12160007.6 |
May 4, 2012 |
EP |
PCT/EP2012/058206 |
Oct 5, 2012 |
EP |
12187326.9 |
Nov 2, 2012 |
EP |
12191086.3 |
Nov 8, 2012 |
EP |
12191854.4 |
Claims
1. (canceled)
2. A conjugate comprising: a polypeptide comprising an albumin
having an amino acid sequence that is at least 90% identical to the
amino acid sequence of SEQ ID NO: 2 and having a substitution in
Domain I of said albumin and having a substitution in Domain III of
said albumin relative to the amino acid sequence set forth in SEQ
ID NO: 2, wherein said albumin has an increased binding affinity to
FcRn relative to the binding affinity of an albumin comprising the
amino acid sequence of SEQ ID NO: 2 to FcRn, and wherein said
substitution in Domain I is selected from amino acids corresponding
to positions 82, 83, 111, 112, or any combination thereof of the
amino acid sequence of SEQ ID NO: 2 and said substitution in Domain
III is selected from amino acids corresponding to positions 425,
505, 510, 512, 524, 527, 531, 534, 569, 573, 575, or any
combination thereof of the amino acid sequence of SEQ ID NO: 2; and
a conjugation partner.
3. The conjugate of claim 2, wherein the conjugation partner is a
therapeutic, prophylactic, diagnostic, imaging, or other
moiety.
4. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions (a) 111 and
573; (b) 82 and 83; (c) 82 and 111; (d) 82 and 112; (e) 82 and 573;
(f) 83 and 111; (g) 83 and 112; (h) 83 and 573; (i) 111 and 112;
(j) 83, 111, and 573; (k) 112 and 573; (l) 82, 83, and 111; (m) 82,
83, and 112; (n) 82, 83, and 573; (o) 82, 111, and 112; (p) 82,
111, and 573; (q) 82, 112, and 573; (r) 83, 111, and 112; (s) 83,
112, and 573; (t) 111, 112, and 573; (u) 82, 83, 111, and 112; (v)
82, 83, 111, and 573; (w) 82, 83, 112, and 573; (x) 82, 111, 112,
and 573; (y) 83, 111, 112, and 573; or (z) 82, 83, 111, 112, and
573 of the amino acid sequence of SEQ ID NO: 2.
5. The conjugate of claim 2, wherein said substitution in Domain I
is selected from amino acids corresponding to positions 83 or 111
of the amino acid sequence of SEQ ID NO: 2.
6. The conjugate of claim 2, wherein said substitution in Domain
III is at the amino acid corresponding to position 573 of the amino
acid sequence of SEQ ID NO: 2.
7. The conjugate of claim 2, wherein said substitution in Domain I
is at the amino acid corresponding to position 83 of the amino acid
sequence of SEQ ID NO: 2 and wherein said substitution is an
asparagine, lysine, or serine.
8. The conjugate of claim 2, wherein said substitution in Domain I
is at the amino acid corresponding to position 111 of the amino
acid sequence of SEQ ID NO: 2 and wherein said substitution is an
aspartic acid, glycine, histidine, arginine, glutamine, or glutamic
acid.
9. The conjugate of claim 2, wherein said substitution in Domain
III is at the amino acid corresponding to position 573 of the amino
acid sequence of SEQ ID NO: 2 and wherein said substitution is a
proline, tyrosine, tryptophan, histidine, phenylalanine, threonine,
isoleucine, or valine.
10. The conjugate of claim 2, wherein said substitution in Domain
III is at the amino acid corresponding to position 573 of the amino
acid sequence of SEQ ID NO: 2 and wherein said substitution is a
proline, tyrosine, or tryptophan.
11. The conjugate of claim 2, wherein said substitution in Domain
III is the amino acid corresponding to at position 573 of the amino
acid sequence of SEQ ID NO: 2 and wherein said substitution is a
proline.
12. The conjugate of claim 2, wherein the polypeptide has an
increased plasma half-life compared with the half-life of the
albumin comprising the amino acid sequence of SEQ ID NO: 2.
13. The conjugate of claim 2, wherein said polypeptide has a
stronger binding affinity to FcRn and optionally, a longer plasma
half-life relative to a polypeptide comprising the amino acid
sequence of SEQ ID NO: 2.
14. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 111 and 573
of the amino acid sequence of SEQ ID NO: 2.
15. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 83, 111,
and 573 of the amino acid sequence of SEQ ID NO: 2.
16. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 82, 111,
and 573 of the amino acid sequence of SEQ ID NO: 2.
17. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 111, 112,
and 573 of the amino acid sequence of SEQ ID NO: 2.
18. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 82, 83,
111, and 573 of the amino acid sequence of SEQ ID NO: 2.
19. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 82, 111,
112, and 573 of the amino acid sequence of SEQ ID NO: 2.
20. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 83, 111,
112, and 573 of the amino acid sequence of SEQ ID NO: 2.
21. The conjugate of claim 2, wherein the polypeptide comprises
substitutions at amino acids corresponding to positions 82, 83,
111, 112, and 573 of the amino acid sequence of SEQ ID NO: 2.
22. The conjugate of claim 2, wherein said substitution in Domain I
is at the amino acid corresponding to position 83 of the amino acid
sequence SEQ ID NO: 2 and wherein said substitution is an
asparagine.
23. The conjugate of claim 2, wherein said substitution in Domain I
is at the amino acid corresponding to position 83 of the amino acid
sequence of SEQ ID NO: 2 and wherein said substitution is a
lysine.
24. The conjugate of claim 2, wherein the said substitution in
Domain I is at the amino acid corresponding to position 111 of the
amino acid sequence of SEQ ID NO: 2 and, wherein said substitution
is a glutamic acid.
25. The conjugate of claim 2, wherein said substitution in Domain I
is at the amino acid corresponding to positions 83 of the amino
acid sequence of SEQ ID NO: 2 and wherein said substitution is a
lysine or asparagine, and the said substitution in Domain III is at
the amino acid corresponding to position 573 of the amino acid
sequence of SEQ ID NO: 2 and wherein said substitution is a
proline.
26. The conjugate of claim 2, wherein said substitutions in Domain
I are at the amino acids corresponding to positions 83 and 111 of
the amino acid sequence of SEQ ID NO: 2 and wherein said
substitutions are an asparagine and a glutamic acid, respectively,
and the said substitution in Domain III is at the amino acid
corresponding to position 573 of the amino acid sequence of SEQ ID
NO: 2 and wherein said substitution is a proline.
27. A method of producing a conjugate comprising: providing an
albumin having an amino acid sequence that is at least 90%
identical to the amino acid sequence of SEQ ID NO: 2 and having a
substitution in Domain I of said albumin and having a substitution
in Domain III of said albumin relative to the amino acid sequence
set forth in SEQ ID NO: 2, wherein said albumin has an increased
binding affinity to FcRn relative to the binding affinity of an
albumin comprising the amino acid sequence of SEQ ID NO: 2 to FcRn,
and wherein said substitution in Domain I is selected from amino
acids corresponding to positions 82, 83, 111, 112, or any
combination thereof of the amino acid sequence of SEQ ID NO: 2 and
said substitution in Domain III is selected from amino acids
corresponding to positions 425, 505, 510, 512, 524, 527, 531, 534,
569, 573, 575, or any combination thereof of the amino acid
sequence of SEQ ID NO: 2; and conjugating the albumin to a
conjugation partner selected from a therapeutic, prophylactic,
diagnostic, imaging or other moiety.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 15/915,977, filed Mar. 8, 2018, which is a
divisional application of U.S. patent application Ser. No.
14/385,631, filed Sep. 16, 2014, which issued as U.S. Pat. No.
9,944,691 on Apr. 17, 2018, and which is a 35 U.S.C. .sctn. 371
national phase application of PCT/EP2013/055487 filed Mar. 15,
2013, which claims priority or the benefit under 35 U.S.C. .sctn.
119 of International Application No. PCT/EP2012/058206 filed May 4,
2012, European Application Nos. 12160007.6, 12187326.9, 12191086.3
and 12191854.4 filed Mar. 16, 2012, Oct. 5, 2012, Nov. 2, 2012 and
Nov. 8, 2012, respectively, and U.S. Provisional Application Nos.
61/710,134, 61/722,544 and 61/724,674 filed Oct. 5, 2012, Nov. 5,
2012 and Nov. 9, 2012, respectively. Each application to which
priority is claimed is hereby incorporated by reference in its
entirety.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The invention relates to variants of albumin or fragments
thereof or fusion polypeptides comprising variant albumin or
fragments thereof having a change in binding affinity to FcRn
and/or a change in half-life compared to the albumin, fragment
thereof or fusion polypeptide comprising albumin or a fragment
thereof. The invention allows tailoring of binding affinity and/or
half-life of an albumin to the requirements and desires of a user
or application.
Description of the Related Art
[0004] Albumin is a protein naturally found in the blood plasma of
mammals where it is the most abundant protein. It has important
roles in maintaining the desired osmotic pressure of the blood and
also in transport of various substances in the blood stream.
Albumins have been characterized from many species including human,
pig, mouse, rat, rabbit and goat and they share a high degree of
sequence and structural homology.
[0005] Albumin binds in vivo to its receptor, the neonatal Fc
receptor (FcRn) "Brambell" and this interaction is known to be
important for the plasma half-life of albumin. FcRn is a membrane
bound protein, expressed in many cell and tissue types. FcRn has
been found to salvage albumin from intracellular degradation
(Roopenian D. C. and Akilesh, S. (2007), Nat. Rev. Immunol 7,
715-725.). FcRn is a bifunctional molecule that contributes to
maintaining a high level of IgGs and albumin in serum in mammals
such as human beings.
[0006] Whilst the FcRn-immunoglobulin (IgG) interaction has been
characterized in the prior art, the FcRn-albumin interaction is
less well characterized. The major FcRn binding site is localized
within DIII (381-585), (Andersen et al (2010), Clinical
Biochemistry 43, 367-372). A number of key amino acids have been
shown to be important in binding, notably histidines H464, H510 and
H536 and Lys500 (Andersen et al (2010), Nat. Commun. 3:610.
DOI:10.1038/ncomms1607). Data indicates that IgG and albumin bind
non-cooperatively to distinct sites on FcRn (Andersen et al.
(2006), Eur. J. Immunol 36, 3044-3051; Chaudhury et al. (2006),
Biochemistry 45, 4983-4990.).
[0007] It is known that mouse FcRn binds IgG from mice and humans
whereas human FcRn appears to be more discriminating (Ober et al.
(2001) Int. Immunol 13, 1551-1559). Andersen et al. (2010) Journal
of Biological Chemistry 285(7):4826-36, describes the affinity of
human and mouse FcRn for each mouse and human albumin (all possible
combinations). No binding of albumin from either species was
observed at physiological pH to either receptor. At acidic pH, a
100-fold difference in binding affinity was observed. In all cases,
binding of albumin and IgG from either species to both receptors
were additive.
[0008] Human serum albumin (HSA) has been well characterized as a
polypeptide of 585 amino acids, the sequence of which can be found
in Peters, T., Jr. (1996) All about Albumin: Biochemistry, Genetics
and Medical, Applications pp10, Academic Press, Inc., Orlando (ISBN
0-12-552110-3). It has a characteristic binding to its receptor
FcRn, where it binds at pH 6.0 but not at pH 7.4.
[0009] The plasma half-life of HSA has been found to be
approximately 19 days. A natural variant having lower plasma
half-life has been identified (Peach, R. J. and Brennan, S. O.,
(1991) Biochim Biophys Acta. 1097:49-54) having the substitution
D494N. This substitution generated an N-glycosylation site in this
variant, which is not present in the wild-type albumin. It is not
known whether the glycosylation or the amino acid change is
responsible for the change in plasma half-life.
[0010] Albumin has a long plasma half-life and because of this
property it has been suggested for use in drug delivery. Albumin
has been conjugated to pharmaceutically beneficial compounds
(WO2000/69902), and it was found that the conjugate maintained the
long plasma half-life of albumin. The resulting plasma half-life of
the conjugate was generally considerably longer than the plasma
half-life of the beneficial therapeutic compound alone.
[0011] Further, albumin has been genetically fused to
therapeutically beneficial peptides (WO 2001/79271 A and
WO2003/59934) with the typical result that the fusion has the
activity of the therapeutically beneficial peptide and a
considerably longer plasma half-life than the plasma half-life of
the therapeutically beneficial peptides alone.
[0012] Otagiri et al (2009), Biol. Pharm. Bull. 32(4), 527-534,
discloses more than 70 albumin variants, of these 25 of these are
found to be mutated in domain III. A natural variant lacking the
last 175 amino acids at the carboxy termini has been shown to have
reduced half-life (Andersen et al (2010), Clinical Biochemistry 43,
367-372). Iwao et al (2007) studied the half-life of naturally
occurring human albumin variants using a mouse model, and found
that K541E and K560E had reduced half-life, E501K and E570K had
increased half-life and K573E had almost no effect on half-life
(Iwao, et. al. (2007) B.B.A. Proteins and Proteomics 1774,
1582-1590).
[0013] Galliano et al (1993) Biochim. Biophys. Acta 1225, 27-32
discloses a natural variant E505K. Minchiotti et al (1990)
discloses a natural variant K536E. Minchiotti et al (1987) Biochim.
Biophys. Acta 916, 411-418, discloses a natural variant K574N.
Takahashi et al (1987) Proc. Natl. Acad. Sci. USA 84, 4413-4417,
discloses a natural variant D550G. Carlson et al (1992). Proc. Nat.
Acad. Sci. USA 89, 8225-8229, discloses a natural variant
D550A.
[0014] WO2011/051489 and WO 2012/150319 (PCT/EP2012/058206)
disclose a number of point mutations in albumin which modulate the
binding of albumin to FcRn, WO2010/092135 discloses a number of
point mutations in albumin which increase the number of thiols
available for conjugation in the albumin, the disclosure is silent
about the effect of the mutations on the binding of the albumin to
FcRn. WO2011/103076 discloses albumin variants, each containing a
substitution in Domain III of HSA. WO2012/112188 discloses albumin
variants containing substitutions in Domain III of HSA.
[0015] Albumin has the ability to bind a number of ligands and
these become associated (associates) with albumin. This property
has been utilized to extend the plasma half-life of drugs having
the ability to non-covalently bind to albumin. This can also be
achieved by binding a pharmaceutical beneficial compound, which has
little or no albumin binding properties, to a moiety having albumin
binding properties, see review article and reference therein, Kratz
(2008) Journal of Controlled Release 132, 171-183.
[0016] Albumin is used in preparations of pharmaceutically
beneficial compounds, in which such a preparation maybe for
example, but not limited to, a nanoparticle or microparticle of
albumin. In these examples the delivery of a pharmaceutically
beneficial compound or mixture of compounds may benefit from
alteration in the albumin's affinity to its receptor where the
beneficial compound has been shown to associate with albumin for
the means of delivery. It is not clear what determines the plasma
half-life of the formed associates (for example but not limited to
Levemir.RTM., Kurtzhals P et al. Biochem. J. 1995; 312:725-731),
conjugates or fusion polypeptides but it appears to be a result of
the combination of the albumin and the selected pharmaceutically
beneficial compound/polypeptide. It would be desirable to be able
to control the plasma half-life of given albumin conjugates,
associates or albumin fusion polypeptides so that a longer or
shorter plasma half-life can be achieved than given by the
components of the association, conjugation or fusion, in order to
be able to design a particular drug according to the particulars of
the indication intended to be treated.
[0017] Albumin is known to accumulate and be catabolized in tumors;
it has also been shown to accumulate in inflamed joints of
rheumatoid arthritis sufferers. See review article and reference
therein, Kratz (2008) Journal of Controlled Release 132, 171-183.
It is envisaged that HSA variants with increased affinity for FcRn
would be advantageous for the delivery of pharmaceutically
beneficial compounds.
[0018] It may even be desirable to have variants of albumin that
have little or no binding to FcRn in order to provide shorter
half-lives or controlled serum pharmacokinetics as described by
Kenanova et al (2009) J. Nucl. Med.; 50 (Supplement 2):1582).
[0019] Kenanova et al (2010, Protein Engineering, Design &
Selection 23(10): 789-798; WO2010/118169) discloses a docking model
comprising a structural model of domain III of HSA (solved at pH 7
to 8) and a structural model of FcRn (solved at pH 6.4). Kenanova
et al discloses that positions 464, 505, 510, 531 and 535 in domain
III potentially interact with FcRn. The histidines at positions
464, 510 and 535 were identified as being of particular interest by
Chaudhury et al., (2006, op. cit.) and these were shown to have a
significant reduction in affinity and shorter half-life in mouse by
Kenanova (2010, op. cit.). However, the studies of Kenanova et al
are limited to domain III of HSA and therefore do not consider HSA
in its native intact configuration. Furthermore, the identified
positions result in a decrease in affinity for the FcRn
receptor.
[0020] The present invention provides further variants having
modulated (i.e. altered) binding affinity to the FcRn receptor. The
albumin moiety or moieties may therefore be used to tailor the
binding affinity to FcRn and/or half-life of fusion polypeptides,
conjugates, associates, nanoparticles and compositions comprising
the albumin moiety.
SUMMARY OF THE INVENTION
[0021] The present invention relates to albumin variants comprising
one or more (several) alterations in Domain I and one or more
(several) alterations in Domain III of the mature polypeptide of
SEQ ID NO: 2 or equivalent positions of other albumins or fragments
thereof.
[0022] The present invention also relates to albumin variants
comprising one or more (several) alterations in Domain I of the
mature polypeptide of SEQ ID NO: 2 or equivalent positions of other
albumins or fragments thereof.
[0023] The present invention also relates to albumin variants
comprising one or more (several) alterations in Domain III of the
mature polypeptide of SEQ ID NO: 2 or equivalent positions of other
albumins or fragments thereof.
[0024] The present invention also relates to isolated
polynucleotides encoding the variants; nucleic acid constructs,
vectors, and host cells comprising the polynucleotides; and methods
of producing the variants.
[0025] The invention also relates to conjugates or associates
comprising the variant albumin or fragment thereof according to the
invention and a beneficial therapeutic moiety or to a fusion
polypeptide comprising a variant albumin or fragment thereof of the
invention and a fusion partner polypeptide.
[0026] The invention further relates to compositions comprising the
variant albumin, fragment thereof, fusion polypeptide comprising
variant albumin or fragment thereof or conjugates comprising the
variant albumin or fragment thereof, according to the invention or
associates comprising the variant albumin or fragment thereof,
according to the invention. The compositions are preferably
pharmaceutical compositions.
[0027] The invention further relates to a pharmaceutical
composition comprising a variant albumin, fragment thereof, fusion
polypeptide comprising variant albumin or fragment thereof or
conjugates comprising the variant albumin or fragment thereof, or
associates comprising the variant albumin or fragment thereof.
[0028] The invention also relates to the use of the variants,
fragments, fusion polypeptides, conjugates, associates,
nanoparticles and microparticles.
[0029] The invention also relates to a method for preparing a
variant albumin, fragment thereof, fusion polypeptide comprising
variant albumin or fragment thereof or conjugates comprising the
variant albumin or fragment thereof, or associates comprising the
variant albumin or fragment thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1: Multiple alignment of amino acid sequences of (i)
full length mature HSA (Hu_1_2_3), (ii) an albumin variant
comprising domain I and domain III of HSA (Hu_1_3), (iii) an
albumin variant comprising domain II and domain III of HSA
(Hu_2_3), (iv) full-length Macaca mulatta albumin (Mac_mul), (v)
full-length Rattus norvegicus albumin (Rat) and (vi) full-length
Mus musculus albumin (Mouse). Positions 500, 550 and 573 (relative
to full length HSA) are indicated by arrows. In FIG. 1 Domains I,
II and III are referred to as 1, 2 and 3 (respectively).
[0031] FIG. 2: Multiple alignment of amino acid sequence of mature
albumin from human, sheep, mouse, rabbit and goat and immature
albumins from chimpanzee ("Chimp"), macaque, hamster, guinea pig,
rat, cow, horse, donkey, dog, chicken, and pig. The Start and End
amino acids of domains 1, 2 and 3 (as defined by Dockal et al (The
Journal of Biological Chemistry, 1999, Vol. 274(41): 29303-29310))
are indicated with respect to mature human albumin.
[0032] FIG. 3: Conserved groups of amino acids based on their
properties.
[0033] FIG. 4: Representation of shFcRn-HSA docking model. (A-B)
Two orientations of the complex are shown. Albumin is shown by a
space-filling diagram, FcRn is shown as a ribbon diagram. The core
binding interface of HSA is highlighted in pink (in grey-scale this
is seen as the darkest (almost black) region; DI (CBI)), while the
area distally localized from the interface is shown as DII (orange)
and DIII is split into sub-domains DIIIe (in colour, this is cyan)
and DIIIb (in colour, this is blue).
[0034] FIG. 5: shFcRn binding of WT HSA, HSA K573P and HSA
N111Q/K573P at pH5.5, samples were injected over immobilized
shFcRn-HIS (.about.1500-2500 RU) at pH 5.5.
[0035] FIG. 6: A proposed shFcRn-HSA docking model, showing the
spatial relationship between shFcRn (space filling diagram) and HSA
(ribbon diagram) DI, DII and DIII including loops of HSA comprising
positions 78 to 88 and 108 to 112.
DEFINITIONS
[0036] Variant: The term "variant" means a polypeptide derived from
a parent albumin by one or more (several) alteration(s), i.e., a
substitution, insertion, and/or deletion, at one or more (several)
positions. A substitution means a replacement of an amino acid
occupying a position with a different amino acid; a deletion means
removal of an amino acid occupying a position; and an insertion
means adding 1 or more (several), such as 1, 2, 3, 4, 5, 6, 7, 8, 9
or 10, preferably 1 to 3 amino acids immediately adjacent an amino
acid occupying a position. In relation to substitutions,
`immediately adjacent` may be to the N-side (`upstream`) or C-side
(`downstream`) of the amino acid occupying a position (`the named
amino acid`). Therefore, for an amino acid named/numbered `X`, the
insertion may be at position `X+1` (`downstream`) or at position
`X-1` (`upstream`).
[0037] Mutant: The term "mutant" means a polynucleotide encoding a
variant.
[0038] Wild-Type Albumin: The term "wild-type" (WT) albumin means
albumin having the same amino acid sequence as naturally found in
an animal or in a human being.
[0039] Parent Albumin: The term "parent" or "parent albumin" means
an albumin to which an alteration is made by the hand of man to
produce the albumin variants of the invention. The parent may be a
naturally occurring (wild-type) polypeptide or an allele thereof,
or even a variant thereof.
[0040] Albumin: Albumins are proteins and constitute the most
abundant protein in plasma in mammals and albumins from a long
number of mammals have been characterized by biochemical methods
and/or by sequence information. Several albumins, e.g., human serum
albumin (HSA), have also been characterized crystallographically
and the structure determined (HSA: He XM, Carter D C (July 1992).
"Atomic structure and chemistry of human serum albumin". Nature 358
(6383): 209-15; horse albumin: Ho, J. X. et al. (2001). X-ray and
primary structure of horse serum albumin (Equus caballus) at
0.27-nm resolution. Eur J Biochem. 215(1):205-12).
[0041] The term "albumin" means a protein having the same and/or
very similar three dimensional (tertiary) structure as HSA or HSA
domains and has similar properties to HSA or to the relevant
domains. Similar three dimensional structures are for example the
structures of the albumins from the species mentioned herein. Some
of the major properties of albumin are i) its ability to regulate
plasma volume (oncotic activity), ii) a long plasma half-life of
around 19 days.+-.5 days, iii) binding to FcRn, iv) ligand-binding,
e.g. binding of endogenous molecules such as acidic, lipophilic
compounds including bilirubin, fatty acids, hemin and thyroxine
(see also Table 1 of Kragh-Hansen et al, 2002, Biol. Pharm. Bull.
25, 695, hereby incorporated by reference), v) binding of small
organic compounds with acidic or electronegative features e.g.
drugs such as warfarin, diazepam, ibuprofen and paclitaxel (see
also Table 1 of Kragh-Hansen et al, 2002, Biol. Pharm. Bull. 25,
695, hereby incorporated by reference). Not all of these properties
need to be fulfilled to in order to characterize a protein or
fragment as an albumin. If a fragment, for example, does not
comprise a domain responsible for binding of certain ligands or
organic compounds the variant of such a fragment will not be
expected to have these properties either.
[0042] Albumins have generally a long plasma half-life of
approximately 20 days or longer, e.g., HSA has a plasma half-life
of 19 days. It is known that the long plasma half-life of HSA is
mediated via interaction with its receptor FcRn, however, an
understanding or knowledge of the exact mechanism behind the long
half-life of HSA is not essential for the invention.
[0043] As examples of albumin proteins according to the invention
can be mentioned human serum albumin (e.g. AAA98797 or P02768-1,
SEQ ID NO: 2 (mature), SEQ ID NO: 4 (immature)), primate serum
albumin, (such as chimpanzee serum albumin (e.g. predicted sequence
XP_517233.2 SEQ ID NO: 5), gorilla serum albumin or macaque serum
albumin (e.g. NP_001182578, SEQ ID NO: 6), rodent serum albumin
(such as hamster serum albumin (e.g. A6YF56, SEQ ID NO: 7), guinea
pig serum albumin (e.g. Q6WDN9-1, SEQ ID NO: 8), mouse serum
albumin (e.g. AAH49971 or P07724-1 Version 3, SEQ ID NO: 9) and rat
serum albumin (e.g. AAH85359 or P02770-1 Version 2, SEQ ID NO:
10))), bovine serum albumin (e.g. cow serum albumin P02769-1, SEQ
ID NO: 11), equine serum albumin such as horse serum albumin (e.g.
P35747-1, SEQ ID NO: 12) or donkey serum albumin (e.g. Q5XLE4-1,
SEQ ID NO: 13), rabbit serum albumin (e.g. P49065-1 Version 2, SEQ
ID NO: 14), goat serum albumin (e.g. ACF10391, SEQ ID NO: 15),
sheep serum albumin (e.g. P14639-1, SEQ ID NO: 16), dog serum
albumin (e.g. P49822-1, SEQ ID NO: 17), chicken serum albumin (e.g.
P19121-1 Version 2, SEQ ID NO: 18) and pig serum albumin (e.g.
P08835-1 Version 2, SEQ ID NO: 19) or a polypeptide having at least
70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99%
amino acid identity to such an albumin. The parent or reference
albumin may be an artificial variant such as HSA K573P (SEQ ID NO:
3) or a chimeric albumin such as the N-terminal of HSA and the
C-terminal of macaca albumin (SEQ ID NO: 20), N-terminal of HSA and
the C-terminal of mouse albumin (SEQ ID NO: 21), N-terminal of HSA
and the C-terminal of rabbit albumin (SEQ ID NO: 22), N-terminal of
HSA and the C-terminal of sheep albumin (SEQ ID NO: 23).
[0044] Other examples of albumin, which are also included in the
scope of this application, include ovalbumin (e.g. P01012.pro:
chicken ovalbumin; O73860.pro: turkey ovalbumin).
[0045] HSA as disclosed in SEQ ID NO: 2 or any naturally occurring
allele thereof, is the preferred albumin (parent albumin) according
to the invention. HSA is a protein consisting of 585 amino acid
residues and has a molecular weight of 67 kDa. In its natural form
it is not glycosylated. The skilled person will appreciate that
natural alleles may exist having essentially the same properties as
HSA but having one or more (several) amino acid changes compared to
SEQ ID NO: 2, and the inventors also contemplate the use of such
natural alleles as parent albumin according to the invention.
[0046] The parent albumin, a fragment thereof, or albumin part of a
fusion polypeptide comprising albumin or a fragment thereof
according to the invention preferably has a sequence identity to
the sequence of HSA shown in SEQ ID NO: 2 of at least 60%,
preferably at least 70%, preferably at least 80%, preferably at
least 85%, preferably at least 86%, preferably at least 87%,
preferably at least 88%, preferably at least 89%, preferably at
least 90%, preferably at least 91%, preferably at least 92%,
preferably at least 93%, preferably at least 94%, preferably at
least 95%, more preferred at least 96%, more preferred at least
97%, more preferred at least 98% and most preferred at least 99%.
It is preferred that the parent albumin maintains at least one of
the major properties of albumin or a similar tertiary structure as
an albumin, such as HSA The sequence identity may be over the
full-length of SEQ ID NO: 2 or over a molecule consisting or
comprising of a fragment such as one or more (several) domains of
SEQ ID NO: 2 such as a molecule consisting of or comprising domain
III (e.g. SEQ ID NO: 27), a molecule consisting of or comprising
domain II and domain III (e.g. SEQ ID NO: 25), a molecule
consisting of or comprising domain I and domain III (e.g. SEQ ID
NO: 24), a molecule consisting of or comprising two copies of
domain III (e.g. SEQ ID NO: 26), a molecule consisting of or
comprising three copies of domain III (e.g. SEQ ID NO: 28) or a
molecule consisting of or comprising domain I and two copies of
domain III (e.g. SEQ ID NO: 29).
[0047] The parent preferably comprises or consists of the amino
acid sequence of SEQ ID NO: 4 (immature sequence of HSA) or SEQ ID
NO: 2 (mature sequence of HSA).
[0048] In another embodiment, the parent is an allelic variant of
the mature polypeptide of SEQ ID NO: 2.
[0049] The parent albumin may be encoded by a polynucleotide that
hybridizes under very low stringency conditions, low stringency
conditions, medium stringency conditions, medium-high stringency
conditions, high stringency conditions, or very high stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1, or (ii) the full-length complementary strand of (i) (J.
Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning,
A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).
[0050] The polynucleotide of SEQ ID NO: 1 or a subsequence thereof,
as well as the amino acid sequence of SEQ ID NO: 2 or a fragment
thereof, may be used to design nucleic acid probes to identify and
clone DNA encoding a parent from strains of different genera or
species according to methods well known in the art. In particular,
such probes can be used for hybridization with the genomic or cDNA
of the genus or species of interest, following standard Southern
blotting procedures, in order to identify and isolate the
corresponding gene therein. Such probes can be considerably shorter
than the entire sequence, but should be at least 14, e.g., at least
25, at least 35, or at least 70 nucleotides in length. Preferably,
the nucleic acid probe is at least 100 nucleotides in length, e.g.,
at least 200 nucleotides, at least 300 nucleotides, at least 400
nucleotides, at least 500 nucleotides, at least 600 nucleotides, at
least 700 nucleotides, at least 800 nucleotides, or at least 900
nucleotides in length. Both DNA and RNA probes can be used. The
probes are typically labelled for detecting the corresponding gene
(for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin).
Such probes are encompassed by the invention.
[0051] A genomic DNA or cDNA library prepared from such other
organisms may be screened for DNA that hybridizes with the probes
described above and encodes a parent. Genomic or other DNA from
such other organisms may be separated by agarose or polyacrylamide
gel electrophoresis, or other separation techniques. DNA from the
libraries or the separated DNA may be transferred to and
immobilized on nitrocellulose or other suitable carrier material.
In order to identify a clone or DNA that is homologous with SEQ ID
NO: 1 or a subsequence thereof, the carrier material is used in a
Southern blot.
[0052] For purposes of the invention, hybridization indicates that
the polynucleotide hybridizes to a labelled nucleotide probe
corresponding to the polynucleotide shown in SEQ ID NO: 1, its
complementary strand, or a subsequence thereof, under low to very
high stringency conditions. Molecules to which the probe hybridizes
can be detected using, for example, X-ray film or any other
detection means known in the art.
[0053] The nucleic acid probe may comprise or consist of the mature
polypeptide coding sequence of SEQ ID NO: 1, i.e. nucleotides 1 to
1785 of SEQ ID NO: 1. The nucleic acid probe may comprise or
consist of a polynucleotide that encodes the polypeptide of SEQ ID
NO: 2 or a fragment thereof.
[0054] For long probes of at least 100 nucleotides in length, very
low to very high stringency conditions are defined as
pre-hybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and either 25% formamide for very low and low
stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally. The carrier material is finally washed three times each
for 15 minutes using 2.times.SSC, 0.2% SDS at 45.degree. C. (very
low stringency), 50.degree. C. (low stringency), 55.degree. C.
(medium stringency), 60.degree. C. (medium-high stringency),
65.degree. C. (high stringency), or 70.degree. C. (very high
stringency).
[0055] For short probes that are about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
pre-hybridization and hybridization at about 5.degree. C. to about
10.degree. C. below the calculated T.sub.m using the calculation
according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA
48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5%
NP-40, 1.times. Denhardt's solution, 1 mM sodium pyrophosphate, 1
mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA
per ml following standard Southern blotting procedures for 12 to 24
hours optimally. The carrier material is finally washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated T.sub.m.
[0056] The parent may be encoded by a polynucleotide with a
sequence identity to the mature polypeptide coding sequence of SEQ
ID NO: 1 of at least 60%, e.g., at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100%,
which encodes a polypeptide which is able to function as an
albumin. In an embodiment, the parent is encoded by a
polynucleotide comprising or consisting of SEQ ID NO: 1.
[0057] Albumin moiety: The albumin part of a fusion polypeptide,
conjugate, associate, nanoparticle or composition comprising the
albumin variant or fragment thereof according to the invention, may
be referred to as an `albumin moiety` or `albumin component`. A
polypeptide according to the invention may comprise or consist of
an albumin moiety.
[0058] FcRn and shFcRn: The term "FcRn" means the human neonatal Fc
receptor (FcRn). shFcRn is a soluble recombinant form of FcRn.
hFcRn is a heterodimer of SEQ ID NO: 30 (truncated heavy chain of
the major histocompatibility complex class I-like Fc receptor
(FCGRT)) and SEQ ID NO: 31 (beta-2-microglobulin). Together, SEQ ID
NO: 30 and 31 form hFcRn.
[0059] Isolated variant: The term "isolated variant" means a
variant that is modified by the hand of man and separated
completely or partially from at least one component with which it
naturally occurs. The term "isolated variant" means a variant in a
form or environment which does not occur in nature. Non-limiting
examples of isolated substances include (1) any non-naturally
occurring variant, (2) any variant that is at least partially
removed from one or more (several) or all of the naturally
occurring constituents with which it is associated in nature; (3)
any variant modified by the hand of man relative to the polypeptide
from which it is derived (e.g. the polypeptide from which it is
derived as found in nature); or (4) any variant modified by
increasing the amount of the variant e relative to other components
with which it is naturally associated (e.g., multiple copies of a
gene encoding the substance; use of a stronger promoter than the
promoter naturally associated with the gene encoding the
substance). An isolated variant may be present in a fermentation
broth sample. The variant may be at least 1% pure, e.g., at least
5% pure, at least 10% pure, at least 20% pure, at least 40% pure,
at least 60% pure, at least 80% pure, and at least 90% pure, as
determined by SDS-PAGE or GP-HPLC.
[0060] Substantially pure variant: The term "substantially pure
variant" means a preparation that contains at most 10%, at most 8%,
at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most
1%, and at most 0.5% by weight of other polypeptide material with
which it is natively or recombinantly associated. Preferably, the
variant is at least 92% pure, e.g., at least 94% pure, at least 95%
pure, at least 96% pure, at least 97% pure, at least 98% pure, at
least 99%, at least 99.5% pure, and 100% pure by weight of the
total polypeptide material present in the preparation. Purity may
be determined by SDS-PAGE or GP-HPLC. The variants of the invention
are preferably in a substantially pure form. This can be
accomplished, for example, by preparing the variant by well-known
recombinant methods and by purification methods.
[0061] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. The
mature polypeptide may be amino acids 1 to 585 of SEQ ID NO: 2,
e.g. with alterations according to the invention and/or with the
inclusion of any post-translational modifications.
[0062] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" means a polynucleotide that encodes a
mature albumin polypeptide. The mature polypeptide coding sequence
may be nucleotides 1 to 1758 of SEQ ID NO: 1 e.g. with inclusions
required to encode a variant according to the invention.
[0063] Sequence Identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0064] For purposes of the invention, the degree of sequence
identity between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 3.0.0 or later, more preferably version 5.0.0 or
later. The optional parameters used are gap open penalty of 10, gap
extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of
BLOSUM62) substitution matrix. The output of Needle labelled
"longest identity" (obtained using the--nobrief option) is used as
the percent identity and is calculated as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0065] For purposes of the invention, the degree of sequence
identity between two deoxyribonucleotide sequences is determined
using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,
supra) as implemented in the Needle program of the EMBOSS package
(EMBOSS: The European Molecular Biology Open Software Suite, Rice
et al., 2000, supra), preferably version 3.0.0 or later, more
preferably version 5.0.0 or later. The optional parameters used are
gap open penalty of 10, gap extension penalty of 0.5, and the
EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The
output of Needle labelled "longest identity" (obtained using
the--nobrief option) is used as the percent identity and is
calculated as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
[0066] Fragment: The term "fragment" means a polypeptide having one
or more (several) amino acids deleted from the amino and/or
carboxyl terminus of an albumin and/or an internal region of
albumin that has retained the ability to bind to FcRn. Fragments
may consist of one uninterrupted sequence derived from HSA or it
may comprise two or more (several) sequences derived from HSA. The
fragments according to the invention have a size of more than
approximately 20 amino acid residues, preferably more than 30 amino
acid residues, more preferred more than 40 amino acid residues,
more preferred more than 50 amino acid residues, more preferred
more than 75 amino acid residues, more preferred more than 100
amino acid residues, more preferred more than 200 amino acid
residues, more preferred more than 300 amino acid residues, even
more preferred more than 400 amino acid residues and most preferred
more than 500 amino acid residues. A fragment may comprise or
consist of one more domains of albumin such as DI+DII, DI+DIII,
DII+DIII, DIII+DIII, DI+DIII+DIII, DIII+DIII+DIII, or fragments of
such domains or combinations of domains.
[0067] Domains I, II and III may be defined with reference to HSA
(SEQ ID NO: 2). For example, HSA domain I may consist of or
comprise amino acids 1 to 194 (.+-.1 to 15 amino acids) of SEQ ID
NO: 2, HSA domain II may consist of or comprise amino acids 192
(.+-.1 to 15 amino acids) to 387 (.+-.1 to 15 amino acids) of SEQ
ID NO: 2 and domain III may consist of or comprise amino acid
residues 381 (.+-.1 to 15 amino acids) to 585 (.+-.1 to 15 amino
acids) of SEQ ID NO: 2. ".+-.1 to 15 amino acids" means that the
residue number may deviate by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, or 15 amino acids to the C-terminus and/or to the
N-terminus of the stated amino acid position. Examples of domains
I, II and III are described by Dockal et al (The Journal of
Biological Chemistry, 1999, Vol. 274(41): 29303-29310) and Kjeldsen
et al (Protein Expression and Purification, 1998, Vol 13: 163-169)
and are tabulated below.
TABLE-US-00001 Amino acid residues of HSA domains I, II and III
with reference to SEQ ID NO: 2 Dockal et al Kjeldsen et al Domain I
1 to 197 1 to 192 Domain II 189 to 385 193 to 382 Domain III 381 to
585 383 to 585
[0068] The skilled person can identify domains I, II and III in
non-human albumins by amino acid sequence alignment with HSA, for
example using the Needleman-Wunsch algorithm (Needleman and Wunsch,
1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle
program of the EMBOSS package (EMBOSS: The European Molecular
Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:
276-277), preferably version 3.0.0 or later, more preferably
version 5.0.0 or later. The optional parameters used are gap open
penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version of BLOSUM62) substitution matrix. Other suitable
software includes MUSCLE ((Multiple sequence comparison by
log-expectation, Robert C. Edgar, Version 3.6,
http://www.drive5.com/muscle; Edgar (2004) Nucleic Acids Research
32(5), 1792-97 and Edgar (2004) BMC Bioinformatics, 5(1):113) which
may be used with the default settings as described in the User
Guide (Version 3.6, September 2005). Versions of MUSCLE later than
3.6 may also be used for any aspect of the invention). Examples of
suitable alignments are provided in FIGS. 1 and 2.
[0069] It is preferred that domains have at least 70, 75, 80, 85,
90, 95, 96, 97, 98, 99, 99.5 identity or 100% identity to Domain I,
II or III of HSA (SEQ ID NO: 2).
[0070] Allelic variant: The term "allelic variant" means any of two
or more (several) alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in polymorphism within populations. Gene
mutations can be silent (no change in the encoded polypeptide) or
may encode polypeptides having altered amino acid sequences. An
allelic variant of a polypeptide is a polypeptide encoded by an
allelic variant of a gene.
[0071] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
its translated polypeptide product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG and ends with a stop codon such as TAA, TAG,
and TGA. The coding sequence may be a DNA, cDNA, synthetic, or
recombinant polynucleotide.
[0072] cDNA: The term "cDNA" means a DNA molecule that can be
prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic cell. cDNA lacks intron
sequences that may be present in the corresponding genomic DNA. The
initial, primary RNA transcript is a precursor to mRNA that is
processed through a series of steps, including splicing, before
appearing as mature spliced mRNA.
[0073] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule, either single- or double-stranded,
which is isolated from a naturally occurring gene or is modified to
contain segments of nucleic acids in a manner that would not
otherwise exist in nature or which is synthetic. The term nucleic
acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences
required for expression of a coding sequence of the invention.
[0074] Control sequences: The term "control sequences" means all
components (e.g. nucleic acid sequences) necessary for the
expression of a polynucleotide encoding a variant of the invention.
Each control sequence may be native (i.e. from the same gene) or
foreign (i.e. from a different gene) to the polynucleotide encoding
the variant or native or foreign to each other. Such control
sequences include, but are not limited to, a leader,
polyadenylation sequence, propeptide sequence, promoter, signal
peptide sequence, and transcription terminator. At a minimum, the
control sequences include a promoter, and transcriptional and
translational stop signals. The control sequences may be provided
with linkers for the purpose of introducing specific restriction
sites facilitating ligation of the control sequences within the
coding region of the polynucleotide encoding a variant.
[0075] Operably linked: The term "operably linked" means a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of a
polynucleotide such that the control sequence directs the
expression of the coding sequence.
[0076] Expression: The term "expression" includes any step involved
in the production of the variant including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0077] Expression vector: The term "expression vector" means a
linear or circular DNA molecule that comprises a polynucleotide
encoding a variant and is operably linked to additional nucleotides
(e.g. control sequences) that provide for its expression.
[0078] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, and/or
the like with a nucleic acid construct or expression vector
comprising a polynucleotide of the present invention. The term
"host cell" encompasses any progeny of a parent cell that is not
identical to the parent cell due to mutations that occur during
replication.
[0079] Plasma half-life: Plasma half-life is ideally determined in
vivo in suitable individuals. However, since it is time consuming
and expensive and there inevitable are ethical concerns connected
with doing experiments in animals or man it is desirable to use an
in vitro assay for determining whether plasma half-life is extended
or reduced. It is known that the binding of albumin to its receptor
FcRn is important for plasma half-life and the correlation between
receptor binding and plasma half-life is that a higher affinity of
albumin to its receptor leads to longer plasma half-life. Thus for
the invention a higher affinity of albumin to FcRn is considered
indicative of an increased plasma half-life and a lower affinity of
albumin to its receptor is considered indicative of a reduced
plasma half-life.
[0080] In this application and claims the binding of albumin to its
receptor FcRn is described using the term affinity and the
expressions "stronger" or "weaker". Thus, it should be understood
that a molecule having a higher affinity to FcRn than HSA is
considered to bind stronger to FcRn than HSA and a molecule having
a lower affinity to FcRn than HSA is considered to bind weaker to
FcRn than HSA.
[0081] The terms "longer plasma half-life" or "shorter plasma
half-life" and similar expressions are understood to be in
relationship to the corresponding parent or reference or
corresponding albumin molecule. Thus, a longer plasma half-life
with respect to a variant albumin of the invention means that the
variant has longer plasma half-life than the corresponding albumin
having the same sequences except for the alteration(s) described
herein, e.g. at one or more (several) positions in Domain I and one
or more (several) positions in Domain III (e.g. in SEQ ID NO:
2).
[0082] Reference: a reference is an albumin, fusion, conjugate,
composition, associate or nanoparticle to which an albumin variant,
fusion, conjugate, composition, associate or nanoparticle is
compared. The reference may comprise or consist of full length
albumin (such as HSA or a natural allele thereof) of a fragment
thereof. A reference may also be referred to as a `corresponding`
albumin, fusion, conjugate, composition, associate or nanoparticle
to which an albumin variant, fusion, conjugate, composition,
associate or nanoparticle is compared. A reference may comprise or
consist of HSA (SEQ ID NO: 2) or a fragment, fusion, conjugate,
associate, nanoparticle or microparticle thereof. Preferably, the
reference is identical to the polypeptide, fusion polypeptide,
conjugate, composition, associate, nanoparticle or microparticle
according to the invention ("being studied") with the exception of
the albumin moiety. Preferably the albumin moiety of the reference
comprises or consists of an albumin (e.g. HSA, SEQ ID NO: 2) or a
fragment thereof. The amino acid sequence of the albumin moiety of
the reference may be longer than, shorter than or, preferably, the
same (.+-.1 to 15 amino acids) length as the amino sequence of the
albumin moiety of the polypeptide, fusion polypeptide, conjugate,
composition, associate, nanoparticle or microparticle according to
the invention ("being studied").
[0083] Equivalent amino acid positions: Throughout this
specification amino acid positions are defined in relation to
full-length mature human serum albumin (i.e. without leader
sequence, SEQ ID NO: 2). However, the skilled person understands
that the invention also relates to variants of non-human albumins
e.g. those disclosed herein) and/or fragments of a human or
non-human albumin. Equivalent positions can be identified in
fragments of human serum albumin, in animal albumins and in
fragments, fusions and other derivative or variants thereof by
comparing amino acid sequences using pairwise (e.g. ClustalW) or
multiple (e.g. MUSCLE) alignments. For example, FIG. 1 shows that
positions equivalent to 500, 550 and 573 in full length human serum
albumin are easily identified in fragments of human serum albumin
and in albumins of other species. Positions 500, 550 and 573 are
indicated by arrows. Further details are provided in the table
below.
Example of Identification of Equivalent Positions in HSA, Animal
Albumins and Albumin Fragments
TABLE-US-00002 [0084] Organism Position equivalent (accession
Albumin to human serum albumin number of Full length Fragment Total
length of (native amino acid): protein) or fragment details mature
protein 500 (K) 550 (D) 573 (K) Homo sapiens Full length -- 585 500
(K) 550 (D) 573 (K) (AAA98797) Homo sapiens Fragment DI, DIII 399
314 (K) 364 (D) 387 (K) Homo sapiens Fragment DI, DIII 403 318 (K)
368 (D) 391 (K) Macaca mulatta Full length -- 584 500 (K) 550 (N)
573 (P) (NP_001182578) Rattus norvegicus Full length -- 584 500 (K)
550 (D) 573 (P) (AAH85359) Mus musculus Full length -- 584 500 (K)
550 (D) 573 (P) (AAH49971)
[0085] FIG. 1 was generated by MUSCLE using the default parameters
including output in ClustalW 1.81 format. The raw output data was
shaded using BoxShade 3.21
(http://www.ch.embnet.orq/software/BOX_form.html) using Output
Format: RTF_new; Font Size: 10; Consensus Line: no consensus line;
Fraction of sequences (that must agree for shading): 0.5; Input
sequence format: ALN. Therefore, throughout this specification
amino acid positions defined in human serum albumin also apply to
equivalent positions in fragments, derivatives or variants and
fusions of human serum albumin, animals from other species and
fragments and fusions thereof. Such equivalent positions may have
(i) a different residue number in its native protein and/or (ii) a
different native amino acid in its native protein.
[0086] Likewise, FIG. 2 shows that equivalent positions can be
identified in fragments (e.g. domains) of an albumin with reference
to SEQ ID NO: 2 (HSA).
Conventions for Designation of Variants
[0087] For purposes of the present invention, the mature
polypeptide disclosed in SEQ ID NO: 2 is used to determine the
corresponding amino acid residue in another albumin. The amino acid
sequence of another albumin is aligned with the mature polypeptide
disclosed in SEQ ID NO: 2, and based on the alignment, the amino
acid position number corresponding to any amino acid residue in the
mature polypeptide disclosed in SEQ ID NO: 2 is determined using
the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 3.0.0 or later, more preferably version 5.0.0 or
later.
[0088] Identification of the corresponding amino acid residue in
another albumin can be determined or confirmed by an alignment of
multiple polypeptide sequences using a suitable computer program
including, but not limited to, "ClustalW" (Larkin et al., 2007,
Bioinformatics 23: 2947-2948), MUSCLE (multiple sequence comparison
by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic
Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh
and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al.,
2005, Nucleic Acids Research 33: 511-518; Katoh and Toh, 2007,
Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in
Molecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics
26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later;
Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using
their respective default parameters.
[0089] When the other polypeptide (or protein) has diverged from
the mature polypeptide of SEQ ID NO: 2 such that traditional
sequence-based comparison fails to detect their relationship
(Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other
pairwise sequence comparison algorithms can be used. Greater
sensitivity in sequence-based searching can be attained using
search programs that utilize probabilistic representations of
polypeptide families (profiles) to search databases. For example,
the PSI-BLAST program generates profiles through an iterative
database search process and is capable of detecting remote homologs
(Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even
greater sensitivity can be achieved if the family or superfamily
for the polypeptide has one or more (several) representatives in
the protein structure databases. Programs such as GenTHREADER
(Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003,
Bioinformatics 19: 874-881) utilize information from a variety of
sources (PSI-BLAST, secondary structure prediction, structural
alignment profiles, and solvation potentials) as input to a neural
network that predicts the structural fold for a query sequence.
Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:
903-919, can be used to align a sequence of unknown structure with
the superfamily models present in the SCOP database. These
alignments can in turn be used to generate homology models for the
polypeptide, and such models can be assessed for accuracy using a
variety of tools developed for that purpose.
[0090] For proteins of known structure, several tools and resources
are available for retrieving and generating structural alignments.
For example the SCOP superfamilies of proteins have been
structurally aligned, and those alignments are accessible and
downloadable. Two or more protein structures can be aligned using a
variety of algorithms such as the distance alignment matrix (Holm
and Sander, 1998, Proteins 33: 88-96) or combinatorial extension
(Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747), and
implementation of these algorithms can additionally be utilized to
query structure databases with a structure of interest in order to
discover possible structural homologs (e.g., Holm and Park, 2000,
Bioinformatics 16: 566-567).
[0091] In describing the albumin variants of the present invention,
the nomenclature described below is adapted for ease of reference.
The accepted IUPAC single letter or three letter amino acid
abbreviation is employed. The term `point mutation` and/or
`alteration` includes deletions, insertions and substitutions.
[0092] Substitutions. For an amino acid substitution, the following
nomenclature is used: Original amino acid, position, substituted
amino acid. Accordingly, for example the substitution of threonine
with alanine at position 226 is designated as "Thr226Ala" or
"T226A". Multiple mutations (or alterations) are separated by
addition marks ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F",
representing substitutions at positions 205 and 411 of glycine (G)
with arginine (R) and serine (S) with phenylalanine (F),
respectively. The Figures also use ("/"), e.g., "E492T/N503D" this
should be viewed as interchangeable with ("+").
[0093] Deletions. For an amino acid deletion, the following
nomenclature is used: Original amino acid, position*. Accordingly,
the deletion of glycine at position 195 is designated as "Gly195*"
or "G195*". Multiple deletions are separated by addition marks
("+"), e.g., "Gly195*+Ser411*" or "G195*+S411*".
[0094] Insertions. As disclosed above, an insertion may be to the
N-side (`upstream`, `X-1`) or C-side (`downstream`, `X+1`) of the
amino acid occupying a position (`the named (or original) amino
acid`, `X`).
[0095] For an amino acid insertion to the C-side (`downstream`,
`X+1`) of the original amino acid (`X`), the following nomenclature
is used: Original amino acid, position, original amino acid,
inserted amino acid. Accordingly the insertion of lysine after
glycine at position 195 is designated "Gly195GlyLys" or "G195GK".
An insertion of multiple amino acids is designated [Original amino
acid, position, original amino acid, inserted amino acid #1,
inserted amino acid #2; etc.]. For example, the insertion of lysine
and alanine after glycine at position 195 is indicated as
"Gly195GlyLysAla" or "G195GKA".
[0096] In such cases the inserted amino acid residue(s) are
numbered by the addition of lower case letters to the position
number of the amino acid residue preceding the inserted amino acid
residue(s). In the above example, the sequence would thus be:
TABLE-US-00003 Parent: Variant: 195 195 195a 195b G G-K-A
[0097] For an amino acid insertion to the N-side (`upstream`,
`X-1`) of the original amino acid (X), the following nomenclature
is used: Original amino acid, position, inserted amino acid,
original amino acid. Accordingly the insertion of lysine (K) before
glycine (G) at position 195 is designated "Gly195LysGly" or
"G195KG". An insertion of multiple amino acids is designated
[Original amino acid, position, inserted amino acid #1, inserted
amino acid #2; etc., original amino acid]. For example, the
insertion of lysine (K) and alanine (A) before glycine at position
195 is indicated as "Gly195LysAlaGly" or "G195KAG". In such cases
the inserted amino acid residue(s) are numbered by the addition of
lower case letters with prime to the position number of the amino
acid residue following the inserted amino acid residue(s). In the
above example, the sequence would thus be:
TABLE-US-00004 Parent: Variant: 195 195a' 195b' 195 G K-A-G
[0098] Multiple alterations. Variants comprising multiple
alterations are separated by addition marks ("+"), e.g.,
"Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution
of tyrosine and glutamic acid for arginine and glycine at positions
170 and 195, respectively.
[0099] Different alterations (e.g. substitutions). Where different
alterations (e.g. substitutions) can be introduced at a position,
the different alterations (e.g. substitutions) are separated by a
comma, e.g., "Arg170Tyr,Glu" represents a substitution of arginine
with tyrosine or glutamic acid at position 170. Thus,
"Tyr167Gly,Ala+Arg170Gly,Ala" designates the following variants:
"Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala",
"Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".
DETAILED DESCRIPTION OF THE INVENTION
[0100] The present invention relates to albumin variants,
comprising an alteration at position in Domain I and an alteration
at a position in Domain III of the mature polypeptide of SEQ ID NO:
2, or at equivalent positions in other albumins or fragments
thereof.
Variants
[0101] A first aspect of the invention provides polypeptides which
are variant albumins or fragments thereof, or fusion polypeptides
comprising the variant albumin or fragment thereof, of a parent
albumin, comprising one or more (several) alterations in Domain I
of albumin, such as HSA (SEQ ID NO: 2) and one or more (several)
alterations in Domain III of albumin, such as HSA (SEQ ID NO:
2).
[0102] It is preferred that the parent albumin and/or the variant
albumin comprises or consists of:
[0103] (a) a polypeptide having at least 60% sequence identity to
the mature polypeptide of SEQ ID NO: 2;
[0104] (b) a polypeptide encoded by a polynucleotide that
hybridizes under low stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 1, or (ii) the
full-length complement of (i);
[0105] (c) a polypeptide encoded by a polynucleotide having at
least 60% identity to the mature polypeptide coding sequence of SEQ
ID NO: 1; and/or
[0106] (d) a fragment of the mature polypeptide of SEQ ID NO:
2.
[0107] The variants of albumin or fragments thereof or fusion
polypeptides comprising albumin or fragments thereof comprise one
or more (several) alterations, such as substitutions, deletions or
insertions at positions in Domain I and one or more (several)
alterations, such as substitutions, deletions or insertions at
positions in Domain III of the mature polypeptide of SEQ ID NO: 2
or in equivalent positions of other albumins or variants or
fragments thereof. A stop codon may be introduced in addition to
the alterations described herein and if introduced is at position
574 or further downstream (e.g. in SEQ ID NO: 2 it is introduced at
from position 574 to 585).
[0108] The variant albumin, a fragment thereof, or albumin part of
a fusion polypeptide comprising variant albumin or a fragment
thereof according to the invention has generally a sequence
identity to the sequence of HSA shown in SEQ ID NO: 2 of at least
60%, preferably at least 70%, preferably at least 80%, preferably
at least 85%, preferably at least 90%, more preferred at least 95%,
more preferred at least 96%, more preferred at least 97%, more
preferred at least 98% and most preferred at least 99%. The variant
has less than 100% identity to SEQ ID NO: 2.
[0109] The variant albumin, a fragment thereof, or albumin part of
a fusion polypeptide comprising variant albumin or a fragment
thereof according to the invention has generally a sequence
identity to the sequence of the parent albumin of at least 60%,
preferably at least 70%, preferably at least 80%, preferably at
least 85%, preferably at least 90%, more preferred at least 95%,
more preferred at least 96%, more preferred at least 97%, more
preferred at least 98% and most preferred at least 99%. The variant
has less than 100% identity to the sequence of the parent
albumin.
[0110] In one aspect, the number of alterations in the variants of
the invention is 1 to 20, e.g., 1 to 10 and 1 to 5, such as 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10 alterations relative to SEQ ID NO: 2 or
relative to the sequence of the parent albumin.
[0111] The one or more (several) alterations in Domain I may be
selected from positions corresponding to positions from 78 to 88
(i.e. 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88) and/or from 105
to 120 (i.e. 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 120) of HSA (SEQ ID NO: 2). In HSA positions 78
to 88 form a loop and positions 105 to 120 form a loop. Therefore,
positions in equivalent loops of other albumins are also included
in the invention. Preferred residues are residues 81 to 85,
particularly 82 and 83, and residues 110 to 114, particularly 111
and 112.
[0112] At position 82 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to Q, D or A, even more preferred to D or A
and most preferred to A. In SEQ ID NO: 2 the native amino acid at
position 82 is glutamic acid, therefore a substitution to glutamic
acid is not preferred.
[0113] At position 83 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to N, K or S, even more preferred to N or K
and most preferred to N. In SEQ ID NO: 2 the native amino acid at
position 82 is threonine, therefore a substitution to threonine is
not preferred.
[0114] At position 111 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to N, E, Q, D, G or H, even more preferred to
E or Q and most preferred to E. In SEQ ID NO: 2 the native amino
acid at position 111 is asparagine, therefore a substitution to
asparagine is not preferred.
[0115] At position 112 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to F, Y or W, even more preferred to F or Y
and most preferred to F. In SEQ ID NO: 2 the native amino acid at
position 112 is leucine, therefore a substitution to leucine is not
preferred.
[0116] At position 573 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to P, Y, W, H, F, T, I or V, even more
preferred to P, Y or W and most preferred to P. In SEQ ID NO: 2 the
native amino acid at position 573 is lysine, therefore a
substitution to lysine is not preferred.
[0117] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82 and 83; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0118] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82 and 111; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0119] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82 and 112; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0120] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0121] An albumin variant may comprise alterations, e.g.
substitutions, at positions 83 and 111; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0122] An albumin variant may comprise alterations, e.g.
substitutions, at positions 83 and 112; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0123] An albumin variant may comprise alterations, e.g.
substitutions, at positions 83 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0124] An albumin variant may comprise alterations, e.g.
substitutions, at positions 111 and 112; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0125] An albumin variant may comprise alterations, e.g.
substitutions, at positions 111 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0126] An albumin variant may comprise alterations, e.g.
substitutions, at positions 112 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0127] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 83, and 111; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0128] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 83, 112; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0129] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 83, and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0130] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 111, and 112; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0131] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 111, and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0132] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 112, and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0133] An albumin variant may comprise alterations, e.g.
substitutions, at positions 83, 111, and 112; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0134] An albumin variant may comprise alterations, e.g.
substitutions, at positions 83, 111, and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0135] An albumin variant may comprise alterations, e.g.
substitutions, at positions 83, 112, and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0136] An albumin variant may comprise alterations, e.g.
substitutions, at positions 111, 112, and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0137] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 83, 111, and 112; of SEQ ID NO: 2
(or equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0138] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 83, 111, and 573; of SEQ ID NO: 2
(or equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0139] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 83, 112, and 573; of SEQ ID NO: 2
(or equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0140] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 111, 112, and 573; of SEQ ID NO: 2
(or equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0141] An albumin variant may comprise alterations, e.g.
substitutions, at positions 83, 111, 112, and 573; of SEQ ID NO: 2
(or equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0142] An albumin variant may comprise alterations, e.g.
substitutions, at positions 82, 83, 111, 112, and 573; of SEQ ID
NO: 2 (or equivalent position of other albumins or variants of
fragments thereof) of an albumin, variant or fragment thereof,
especially SEQ ID NO: 2.
[0143] An albumin variant may comprise alterations, e.g.
substitutions, at positions 425 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0144] An albumin variant may comprise alterations, e.g.
substitutions, at positions 505 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0145] An albumin variant may comprise alterations, e.g.
substitutions, at positions 527 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0146] An albumin variant may comprise alterations, e.g.
substitutions, at positions 534 and 573; of SEQ ID NO: 2 (or
equivalent position of other albumins or variants of fragments
thereof) of an albumin, variant or fragment thereof, especially SEQ
ID NO: 2.
[0147] Particularly preferred albumin variants comprise
substitutions T83N/N111E (e.g. SEQ ID NO: 32); T83N/N111E/K573P
(e.g. SEQ ID NO: 33); T83N/K573P (e.g. SEQ ID NO: 34); T83K/K573P
(e.g. SEQ ID NO: 38); E82A/K573P (e.g. SEQ ID NO: 39); L112F/K573P
(e.g. SEQ ID NO: 40); E82D/K573P (e.g. SEQ ID NO: 43); P110G/K573P
(e.g. SEQ ID NO: 44); N111D/K573P (e.g. SEQ ID NO: 60); N111G/K573P
(e.g. SEQ ID NO: 61); N111H/K573P (e.g. SEQ ID NO: 62); E425A/K573P
(e.g. SEQ ID NO: 64); E505Q/K573P (e.g. SEQ ID NO: 65); T527M/K573P
(e.g. SEQ ID NO: 66); N111E/K573P (e.g. SEQ ID NO: 68); K534V/K573P
(e.g. SEQ ID NO: 73); N111Q/K573P (e.g. SEQ ID NO: 74) which are
described with reference to HSA (SEQ ID NO: 2). Other preferred
albumin variants comprise equivalent substitutions in albumins
other than HSA (SEQ ID NO: 2).
[0148] Also, an albumin variant according to the invention may
comprise one or more (several) alterations at positions selected
from 78 to 88 (78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91) and/or 105 to 120 (105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120) and/or 425, 505, 510, 512, 524,
527, 531, 534, 569, 575 of HSA (SEQ ID NO: 2) or equivalent
positions of other albumins. Preferred alterations are
substitutions such as those described for these positions in the
first aspect of the invention. Particularly preferred substitutions
include D108A (SEQ ID NO: 59); D108E (e.g. SEQ ID NO: 70); N109K
(e.g. SEQ ID NO: 69); P110G (e.g. SEQ ID NO: 42); N111D (e.g. SEQ
ID NO: 46); N111E (e.g. SEQ ID NO: 67); N111G (e.g. SEQ ID NO: 48);
N111H (e.g. SEQ ID NO: 49); N111K (e.g. SEQ ID NO: 54); L112F (e.g.
SEQ ID NO: 37); E425A (e.g. SEQ ID NO: 63); E425K (e.g. SEQ ID NO:
55); E505Q (e.g. SEQ ID NO: 45); H510D (e.g. SEQ ID NO: 57); D512E
(e.g. SEQ ID NO: 50); K524A (e.g. SEQ ID NO: 51); T527A (e.g. SEQ
ID NO: 52); T527M (e.g. SEQ ID NO: 47); E531H (e.g. SEQ ID NO: 53);
K534V (e.g. SEQ ID NO: 56); A569S (e.g. SEQ ID NO: 58); L575F (e.g.
SEQ ID NO: 72); E82A (e.g. SEQ ID NO: 36); E82D (e.g. SEQ ID NO:
41); T83K (e.g. SEQ ID NO: 35); T83N (e.g. SEQ ID NO: 71) which are
described with reference to HSA (SEQ ID NO: 2). Other preferred
albumin variants comprising one or more (several) alterations may
comprise equivalent substitutions in albumins other than HSA (SEQ
ID NO: 2).
[0149] It is preferred that the variant albumin, a fragment thereof
or fusion polypeptide comprising the variant albumin or fragment
thereof has altered binding affinity to FcRn and/or an altered
plasma half-life compared with the corresponding parent or
reference albumin, fragment thereof, or fusion polypeptide
comprising the variant albumin or fragment thereof and/or an
altered binding affinity to FcRn.
[0150] In a particularly preferred embodiment the parent or
reference albumin is HSA (SEQ ID NO: 2) and the variant albumin, a
fragment thereof or fusion polypeptide comprising the variant
albumin or fragment thereof has altered binding affinity to FcRn
and/or an altered plasma half-life compared with the HSA, the
corresponding fragment or fusion polypeptide comprising HSA or
fragment thereof and/or an altered binding affinity to FcRn.
[0151] The correlation between binding of albumin to its receptor
and plasma half-life has been realized by the present inventors
based on the natural occurring allele of HSA D494N. The inventors
have previously analyzed this allele and found that it has a lower
affinity to its receptor FcRn than the affinity of WT HSA to
FcRn.
[0152] Further, it has been disclosed that a transgenic mouse
having the natural mouse FcRn replaced with human FcRn has a higher
serum albumin level than normal mouse (J Exp Med. (2003)
197(3):315-22). The inventors have previously discovered that human
FcRn has a higher affinity to mouse serum albumin than mouse FcRn
has to mouse serum albumin and, therefore, the observed increase in
serum albumin in the transgenic mice corresponds with a higher
affinity between serum albumin and its receptor, confirming the
correlation between albumin binding to FcRn and plasma half-life.
In addition, variants of albumin that have little or no binding to
FcRn have been shown to have reduced half-life in a mouse model,
Kenanova et al (2009) J. Nucl. Med.; 50 (Supplement 2):1582).
[0153] One way to determine whether the affinity of a variant
albumin to FcRn is higher or lower than the parent or reference
albumin is to use the Surface Plasmon Resonance assay (SPR) as
described below. The skilled person will understand that other
methods might be useful to determine whether the affinity of a
variant albumin to FcRn is higher or lower than the affinity of the
parent or reference albumin to FcRn, e.g., determination and
comparison of the binding constants KD. The binding affinity (KD)
between a first molecule (e.g. ligand) and a second molecule (e.g.
receptor) is a function of the kinetic constants for association
(on rate, k.sub.a) and dissociation (off-rate, k.sub.d) according
to KD=k.sub.d/k.sub.a. Thus, according to the invention variant
albumins having a KD that is lower than the KD for natural HSA is
considered to have a higher plasma half-life than HSA and variant
albumins having a KD that is higher than the KD for natural HSA is
considered to have a lower plasma half-life than HSA.
[0154] In an embodiment of the invention, the variants of albumin
or fragments thereof, or fusion polypeptides comprising variant
albumin or a fragment thereof according to the invention have a
plasma half-life that is longer than the plasma half-life of the
parent or reference albumin fragment thereof or fusion polypeptide
comprising the parent or reference albumin or a fragment thereof
and/or an stronger binding affinity to FcRn.
[0155] In a further embodiment the variants of albumin or fragments
thereof, or fusion polypeptides comprising variant albumin or
fragments thereof according to the invention have a plasma
half-life that is shorter than the plasma half-life of the parent
or reference albumin fragment thereof or fusion polypeptide
comprising the parent or reference albumin or a fragment thereof
and/or an weaker binding affinity to FcRn.
[0156] In addition to alterations at positions in Domains I (such
as within loop 78 to 88 and/or within loop 105 to 120 as described
herein) and III (or equivalent position of other albumins or
variants of fragments thereof) the variant albumin or fragments
thereof, or fusion polypeptides comprising variant albumin or
fragments thereof according to the invention may contain additional
substitutions, deletions or insertions in other positions of the
molecules. Such additional substitutions, deletions or insertions
may be useful in order to alter other properties of the molecules
such as but not limited to altered glycosylation; introduction of
reactive groups of the surface such a thiol groups,
removing/generating a carbamoylation site; etc.
[0157] Residues that might be altered in order to provide reactive
residues on the surface and which advantageously could be applied
to the invention has been disclosed in WO2010/092135 (incorporated
herein by reference). Particular preferred residues include the
positions corresponding to positions in SEQ ID NO: 2.
[0158] As examples of alterations that can be made in SEQ ID NO: 2
or in corresponding positions in other albumins in order to provide
a reactive thiol group on the surface includes alterations
corresponding to following alterations in SEQ ID NO: 2: L585C, D1C,
A2C, D562C, A364C, A504C, E505C, T79C, E86C, D129C, D549C, A581C,
D121C, E82C, S270C, A578C, L595LC, D1DC, A2AC, D562DC, A364AC,
A504AC, E505EC, T79TC, E86EC, D129DC, D549DC, A581AC, A581AC,
D121DC, E82EC, S270SC, S579AC, C360*, C316*, C75*, C168*, C558*,
C361*, C91*, C124*, C169* and C567*. Alternatively a cysteine
residue may be added to the N or C terminal of albumin. The term
`reactive thiol` means and/or includes a thiol group provided by a
Cys which is not disulphide bonded to a Cysteine and/or which is
sterically available for binding to a partner such as a conjugation
partner.
Fusion Polypeptides
[0159] A second aspect of the invention relates to fusion
polypeptides. Therefore, the variants of albumin or fragments
thereof according to the invention may be fused with a non-albumin
polypeptide fusion partner. The fusion partner may in principle be
any polypeptide but generally it is preferred that the fusion
partner is a polypeptide having therapeutic, prophylactic
(including vaccine), diagnostic, imaging or other beneficial
properties. Such properties may be referred to as `pharmaceutically
beneficial properties`. Fusion polypeptides comprising albumin or
fragments thereof are known in the art. It has been found that such
fusion polypeptides comprising albumin or a fragment thereof and a
fusion partner polypeptide have a longer plasma half-life compared
to the unfused fusion partner polypeptide alone. According to the
invention it is possible to alter the plasma half-life of the
fusion polypeptides according to the invention compared to the
corresponding fusion polypeptides of the prior art. `Alter`
includes both increasing the plasma half-life and decreasing the
plasma half-life. Increasing the plasma half-life is preferred. The
invention allows tailoring of half-life to a term desired.
[0160] One or more (several) therapeutic, prophylactic (including
vaccine), diagnostic, imaging or other beneficial may be fused to
the N-terminus, the C-terminus of albumin, inserted into a loop in
the albumin structure or any combination thereof. It may or it may
not comprise linker sequences separating the various components of
the fusion polypeptide.
[0161] Teachings relating to fusions of albumin or a fragment
thereof are known in the art and the skilled person will appreciate
that such teachings can also be applied to the invention. WO
2001/79271A (particularly page 9 and/or Table 1), WO 2003/59934
(particularly Table 1), WO03/060071 (particularly Table 1) and
WO01/079480 (particularly Table 1) (each incorporated herein by
reference in their entirety) also contain examples of therapeutic,
prophylactic (including vaccine), diagnostic, imaging or other
beneficial polypeptides that may be fused to albumin or fragments
thereof, and these examples apply also to the invention.
[0162] Further preferences for the second aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Polynucleotides
[0163] A third aspect of the invention relates to isolated
polynucleotides that encode any of the variants or fusion
polypeptides of the invention. The polynucleotide may be an
isolated polynucleotide. The polynucleotide may be comprised a in a
vector (such as a plasmid) and/or in a host cell.
[0164] Further preferences for the third aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Nucleic Acid Constructs
[0165] A fourth aspect of the invention relates to nucleic acid
constructs comprising a polynucleotide encoding a variant or fusion
polypeptide of the invention operably linked to one or more
(several) control sequences that direct the expression of the
coding sequence in a suitable host cell under conditions compatible
with the control sequences.
[0166] A polynucleotide may be manipulated in a variety of ways to
provide for expression of a variant. Manipulation of the
polynucleotide prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotides utilizing recombinant DNA
methods are well known in the art.
[0167] The control sequence may be a promoter sequence, which is
recognized by a host cell for expression of the polynucleotide. The
promoter sequence contains transcriptional control sequences that
mediate the expression of the variant. The promoter may be any
nucleic acid sequence that shows transcriptional activity in the
host cell including mutant, truncated, and hybrid promoters, and
may be obtained from genes encoding extracellular or intracellular
polypeptides either homologous or heterologous to the host
cell.
[0168] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae protease A (PRA1), Saccharomyces cerevisiae protease B
(PRB1), Saccharomyces cerevisiae translation elongation factor
(TEF1), Saccharomyces cerevisiae translation elongation factor
(TEF2), Saccharomyces cerevisiae galactokinase (GAL1),
Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionein (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0169] The skilled person knows useful promoters for use in rice
and mammalian cells, such as CHO or HEK. In a rice host, useful
promoters are obtained from cauliflower mosaic virus 35S RNA gene
(CaMV35S), maize alcohol dehydrogenase (Adh1) and alpha Amy3.
[0170] In a mammalian host cell, such as CHO or HEK, useful
promoters are obtained from Cytomegalovirus (CMV) and CAG hybrid
promoter (hybrid of CMV early enhancer element and chicken
beta-actin promoter), Simian vacuolating virus 40 (SV40).
[0171] The control sequence may also be a suitable transcription
terminator sequence, which is recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3'-terminus of the polynucleotide encoding the variant. Any
terminator that is functional in the host cell may be used.
[0172] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), Saccharomyces cerevisiae alcohol
dehydrogenase (ADH1) and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992, supra.
The skilled person knows useful terminators for use in rice and
mammalian cells, such as CHO or HEK. For example, in a rice host,
preferred terminators are obtained from Agrobacterium tumefaciens
nopaline synthase (Nos) and cauliflower mosaic virus 35S RNA gene
(CaMV35S).
[0173] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA that is important for translation
by the host cell. The leader sequence is operably linked to the
5'-terminus of the polynucleotide encoding the variant. Any leader
sequence that is functional in the host cell may be used.
[0174] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0175] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3'-terminus of the
variant-encoding sequence and, when transcribed, is recognized by
the host cell as a signal to add polyadenosine residues to
transcribed mRNA. Any polyadenylation sequence that is functional
in the host cell may be used.
[0176] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:
5983-5990.
[0177] The control sequence may also be a signal peptide coding
region that encodes a signal peptide linked to the N-terminus of a
variant and directs the variant into the cell's secretory pathway.
The 5'-end of the coding sequence of the polynucleotide may
inherently contain a signal peptide coding region naturally linked
in translation reading frame with the segment of the coding region
that encodes the variant. Alternatively, the 5'-end of the coding
sequence may contain a signal peptide coding region that is foreign
to the coding sequence. The foreign signal peptide coding region
may be required where the coding sequence does not naturally
contain a signal peptide coding region. Alternatively, the foreign
signal peptide coding region may simply replace the natural signal
peptide coding region in order to enhance secretion of the variant.
However, any signal peptide coding region that directs the
expressed variant into the secretory pathway of a host cell may be
used.
[0178] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding sequences are described by Romanos et al., 1992, supra. The
skilled person knows useful signal peptides for use in rice and
mammalian cells, such as CHO or HEK.
[0179] Where both signal peptide and propeptide regions are present
at the N-terminus of a variant, the propeptide region is positioned
next to the N-terminus of the variant and the signal peptide region
is positioned next to the N-terminus of the propeptide region.
[0180] Further preferences for the fourth aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Preparation of Variants
[0181] A fifth aspect of the invention relates to a method for
preparing or obtaining a variant albumin or fragment thereof, or
fusion polypeptides comprising the variant albumin or fragments
thereof, or associates of variant albumin or fragment thereof
comprising:
[0182] (a) introducing into a parent albumin or fragments thereof,
or fusion polypeptides comprising the parent albumin or fragments
thereof one or more (several) alterations in Domain I and one or
more (several) alterations in Domain III; and
[0183] (b) recovering the variant albumin or fragment thereof, or
fusion polypeptides comprising the variant albumin or fragment
thereof.
[0184] Preferred alterations are as described in relation to the
first aspect of the invention. The resultant variant albumin or
fragment thereof may have altered FcRn-binding affinity compared to
the FcRn-binding affinity of a reference such as a parent albumin
or fragment which does not comprise the alterations. More
preferably, the resultant variant albumin or fragment thereof has a
stronger FcRn-binding affinity.
[0185] The invention includes a method for preparing a polypeptide
which is a variant of albumin, fragment thereof or fusion
polypeptide comprising said variant albumin or fragment thereof
having a binding affinity to FcRn which is altered compared to the
binding affinity of a reference albumin, fragment or fusion thereof
to FcRn, comprising:
[0186] (a) providing a nucleic acid encoding a parent albumin such
as an albumin having at least 60% sequence identity to SEQ ID NO:
2;
[0187] (b) modifying the sequence of step (a), to encode a
polypeptide which is a variant albumin, fragment thereof or fusion
polypeptide comprising said variant albumin or fragment thereof
comprising:
[0188] (i) alterations at positions corresponding to one or more
(several) positions in Domain I of the parent albumin and one or
more (several) positions in Domain III (Domain 3), or
[0189] (ii) alterations at positions corresponding to one of more
(several) of any of positions 78 to 120 of Domain I of SEQ ID NO: 2
or at positions corresponding to one or more (several) of any of
positions 425, 505, 510, 512, 524, 527, 531, 534, 569, 573, 575 of
Domain III of SEQ ID NO: 2;
[0190] (c) optionally, introducing the modified sequence of step
(b) in a suitable host cell;
[0191] (d) optionally, growing the cells in a suitable growth
medium under condition leading to expression of the polypeptide;
and
[0192] (e) optionally, recovering the polypeptide from the growth
medium;
[0193] wherein the polypeptide has an altered binding affinity to
FcRn and/or an altered plasma half-life compared with the half-life
of a parent albumin, reference albumin, fragment thereof or fusion
polypeptide comprising said parent albumin, reference albumin or
fragment or fusion thereof.
[0194] It is preferred that the parent albumin and/or the variant
albumin comprises or consists of:
[0195] (a) a polypeptide having at least 60% sequence identity to
the mature polypeptide of SEQ ID NO: 2;
[0196] (b) a polypeptide encoded by a polynucleotide that
hybridizes under low stringency conditions with (i) the mature
polypeptide coding sequence of SEQ ID NO: 1, or (ii) the
full-length complement of (i);
[0197] (c) a polypeptide encoded by a polynucleotide having at
least 60% identity to the mature polypeptide coding sequence of SEQ
ID NO: 1; and/or
[0198] (d) a fragment of the mature polypeptide of SEQ ID NO:
2.
[0199] The variants can be prepared by those skilled persons using
any mutagenesis procedure known in the art, such as site-directed
mutagenesis, synthetic gene construction, semi-synthetic gene
construction, random mutagenesis, shuffling, etc.
[0200] Site-directed mutagenesis is a technique in which one or
more (several) mutations (alterations) are created at one or more
(several) defined sites in a polynucleotide encoding the
parent.
[0201] Site-directed mutagenesis can be accomplished in vitro by
PCR involving the use of oligonucleotide primers containing the
desired mutation. Site-directed mutagenesis can also be performed
in vitro by cassette mutagenesis involving the cleavage by a
restriction enzyme at a site in the plasmid comprising a
polynucleotide encoding the parent and subsequent ligation of an
oligonucleotide containing the mutation in the polynucleotide.
Usually the restriction enzyme that digests at the plasmid and the
oligonucleotide is the same, permitting ligation of the plasmid and
insert to one another. See, e.g., Scherer and Davis, 1979, Proc.
Natl. Acad. Sci. USA 76: 4949-4955; and Barton et al., 1990,
Nucleic Acids Res. 18: 7349-4966.
[0202] Site-directed mutagenesis can also be accomplished in vivo
by methods known in the art. See, e.g., U.S. Patent Application
Publication NO: 2004/0171154; Storici et al., 2001, Nature
Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290;
and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:
15-16.
[0203] Any site-directed mutagenesis procedure can be used in the
invention. There are many commercial kits available that can be
used to prepare variants.
[0204] Synthetic gene construction entails in vitro synthesis of a
designed polynucleotide molecule to encode a polypeptide of
interest. Gene synthesis can be performed utilizing a number of
techniques, such as the multiplex microchip-based technology
described by Tian et al. (2004, Nature 432: 1050-1054) and similar
technologies wherein oligonucleotides are synthesized and assembled
upon photo-programmable microfluidic chips.
[0205] Single or multiple amino acid substitutions, deletions,
and/or insertions can be made and tested using known methods of
mutagenesis, recombination, and/or shuffling, followed by a
relevant screening procedure, such as those disclosed by
Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and
Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413;
or WO 95/22625. Other methods that can be used include error-prone
PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:
10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) and
region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;
Ner et al., 1988, DNA 7: 127).
[0206] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells (Ness et
al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA
molecules that encode active polypeptides can be recovered from the
host cells and rapidly sequenced using standard methods in the art.
These methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide.
[0207] Semi-synthetic gene construction is accomplished by
combining aspects of synthetic gene construction, and/or
site-directed mutagenesis, and/or random mutagenesis, and/or
shuffling. Semi-synthetic construction is typified by a process
utilizing polynucleotide fragments that are synthesized, in
combination with PCR techniques. Defined regions of genes may thus
be synthesized de novo, while other regions may be amplified using
site-specific mutagenic primers, while yet other regions may be
subjected to error-prone PCR or non-error prone PCR amplification.
Polynucleotide sub sequences may then be shuffled.
[0208] Further preferences for the fifth aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Methods of Production
[0209] A sixth aspect of the invention relates to methods of
preparation of a variant according to the invention. The variants
of the invention can be prepared using techniques well known to the
skilled person. One convenient way is by cloning nucleic acid
encoding the parent albumin or a fragment thereof or fusion
polypeptide comprising albumin or a fragment thereof, modifying
said nucleic acid to introduce the desired substitution(s) at
positions in Domain I and Domain III of the mature polypeptide of
SEQ ID NO: 2 (or equivalent positions in other albumins or
fragments thereof), preparing a suitable genetic construct where
the modified nucleic acid is placed in operative connection with
suitable regulatory genetic elements, such as promoter, terminator,
activation sites, ribosome binding sites etc., introducing the
genetic construct into a suitable host organism, culturing the
transformed host organism under conditions leading to expression of
the variant and recovering the variant. All these techniques are
known in the art and it is within the skills of the average
practitioner to design a suitable method for preparing a particular
variant according to the invention.
[0210] The variant polypeptide of the invention may also be
connected to a signal sequence in order to have the variant
polypeptide secreted into the growth medium during culturing of the
transformed host organism. It is generally advantageous to have the
variant polypeptide secreted into the growth medium in order to
ease recovery and purification.
[0211] Techniques for preparing variant polypeptides have also been
disclosed in WO 2009019314 (included by reference) and these
techniques may also be applied to the invention.
[0212] Albumins have been successfully expressed as recombinant
proteins in a range of hosts including fungi (including but not
limited to Aspergillus (WO06066595), Kluyveromyces (Fleer 1991,
Bio/technology 9, 968-975), Pichia (Kobayashi 1998 Therapeutic
Apheresis 2, 257-262) and Saccharomyces (Sleep 1990, Bio/technology
8, 42-46)), bacteria (Pandjaitab 2000, J. Allergy Clin. Immunol.
105, 279-285)), animals (Barash 1993, Transgenic Research 2,
266-276) and plants (including but not limited to potato and
tobacco (Sijmons 1990, Bio/technology 8, 217 and Farran 2002,
Transgenic Research 11, 337-346) and rice e.g. Oryza sativa) and
mammalian cells such as CHO and HEK. The variant polypeptide of the
invention is preferably produced recombinantly in a suitable host
cell. In principle any host cell capable of producing a polypeptide
in suitable amounts may be used and it is within the skills of the
average practitioner to select a suitable host cell according to
the invention. A preferred host organism is yeast, preferably
selected among Saccharomycacae, more preferred Saccharomyces
cerevisiae.
[0213] The variant polypeptides of the invention may be recovered
and purified from the growth medium using a combination of known
separation techniques such as filtration, centrifugation,
chromatography, and affinity separation techniques etc. It is
within the skills of the average practitioner to purify the
variants of the invention using a particular combination of such
known separation steps. As an example of purification techniques
that may be applied to the variants of the invention can be
mentioned the teaching of WO00/44772.
[0214] The variant polypeptides of the invention may be used for
delivering a therapeutically beneficial compound (including
prophylactically beneficial compound such as a vaccine) to an
animal or a human individual in need thereof. Such therapeutically
beneficial compounds include, but are not limited, to labels and
readily detectable compounds for use in diagnostics, such as
various imaging techniques; pharmaceutical active compounds such as
drugs, or specifically binding moieties such as antibodies. The
variants of the invention may even be connected to two or more
(several) different therapeutically beneficial compounds, e.g., an
antibody and a drug, which gives the combined molecule the ability
to bind specifically to a desired target and thereby provide a high
concentration of the connected drug at that particular target.
[0215] Further preferences for the sixth aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Conjugates
[0216] A seventh aspect of the invention relates to conjugates
(conjugations). Therefore, the variants of albumin or fragments
thereof or fusion polypeptides according to the invention may be
conjugated to a second molecule (`conjugation partner`) using
techniques known within the art. The conjugation partner may be a
therapeutic, prophylactic (including vaccine), diagnostic, imaging
or other beneficial moiety. Said conjugation partner may be a
polypeptide or a non-polypeptide chemical. The conjugation partner
may be a polypeptide, a chemical (e.g. chemically synthesized drug)
or a nucleic acid (e.g. DNA, RNA, siRNA).
[0217] Said second molecule may comprise a diagnostic or imaging
moiety, and in this embodiment the conjugate may be useful as a
diagnostic tool such as in imaging; or the second molecule may be a
therapeutic or prophylactic (e.g. vaccine) compound and in this
embodiment the conjugate may be used for therapeutic or
prophylactic (e.g. vaccination) purposes where the conjugate will
have the therapeutic or prophylactic properties of the therapeutic
or prophylactic compound as well as the desirable plasma half-life
provided by the albumin part of the conjugate. Conjugates of
albumin and a therapeutic molecule are known in the art and it has
been verified that such conjugates have long plasma half-life
compared with the non-conjugated, free therapeutic molecule as
such. According to the invention it is possible to alter the
binding affinity to FcRn and/or plasma half-life of the conjugate
according to the invention compared to the corresponding conjugates
of the prior art. `Alter` includes both increasing the plasma
half-life and decreasing the plasma half-life binding affinity to
FcRn and/or increasing the binding affinity and decreasing the
binding affinity to FcRn. Increasing the plasma half-life and/or
binding affinity to FcRn is preferred. The conjugates may
conveniently be linked via a free thiol group present on the
surface of HSA (amino acid residue 34 of mature HSA) using well
known chemistry.
[0218] In one particular preferred aspect the variant albumin or
fragment thereof is conjugated to a beneficial therapeutic or
prophylactic (including vaccine) compound and the conjugate is used
for treatment of a condition in a patient in need thereof, which
condition is responsive to the particular selected therapeutic
compound. Techniques for conjugating such a therapeutically useful
compound to the variant albumin or fragment thereof are known in
the art. WO 2009/019314 (incorporated herein by reference in its
entirety) discloses examples of techniques suitable for conjugating
a therapeutically compound to a polypeptide which techniques can
also be applied to the invention. Further WO 2009/019314 discloses
examples of compounds and moieties that may be conjugated to
substituted transferrin and these examples may also be applied to
the invention. The teaching of WO 2009/019314 is included herein by
reference.
[0219] HSA contains in its natural form one free thiol group (at
Cys34) that conveniently may be used for conjugation. As a
particular embodiment within this aspect the variant albumin or
fragment thereof may comprise further modifications provided to
generate additional free thiol groups on the surface. This has the
benefit that the payload of the variant albumin or fragment thereof
is increased so that more than one molecule of the therapeutic
(e.g. prophylactic) compound can be conjugated to each molecule of
variant albumin or fragment thereof, or two or more (several)
different therapeutic compounds may be conjugated to each molecule
of variant albumin or fragment thereof, e.g., a compound having
targeting properties such as an antibody specific for example a
tumor; and a cytotoxic drug conjugated to the variant albumin or
fragment thereof thereby creating a highly specific drug against a
tumor. Teaching of particular residues that may be modified to
provide for further free thiol groups on the surface can be found
in co-pending patent application WO 2010/092135, which is
incorporated by reference.
[0220] The conjugation partner may alternatively be conjugated to a
fusion polypeptide (described herein), resulting in a molecule
comprising a fusion partner fused to the albumin as well as a
conjugation partner conjugated to the same albumin or even to the
fusion partner. Further preferences for the seventh aspect of the
invention include those of the first aspect of the invention and
those provided below the twelfth aspect of the invention. The
skilled person understands that any aspect of the invention may be
combined with another aspect or aspects of the invention and/or
with one or more (several) of the preferences for the aspects of
the invention and/or other disclosures made herein.
Associates
[0221] An eighth aspect of the invention relates to associates.
Therefore, the variants of albumin or fragments thereof or fusion
polypeptides may further be used in form of "associates". In this
connection the term "associate" is intended to mean a compound
comprising a variant of albumin or a fragment thereof and another
compound bound or associated to the variant albumin or fragment
thereof by non-covalent binding. As an example of such an associate
can be mentioned an associate consisting variant albumin and a
lipid associated to albumin by a hydrophobic interaction. Such
associates are known in the art and they may be prepared using well
known techniques. As an example of a preferred associate according
to the invention, can be mentioned an associate comprising variant
albumin and a taxane, a taxol or taxol derivative (e.g.
paclitaxel). Further examples of associates comprise a therapeutic,
prophylactic (including vaccine), diagnostic, imaging or other
beneficial moiety.
[0222] The half-life of an albumin associate according to the
invention may be longer or shorter than the half-life of the `other
compound` alone. The half-life of an albumin associate according to
the invention may be longer or shorter than the half-life of the
analogous/equivalent albumin associate comprising or consisting of
a reference albumin such as native HSA (instead of an albumin
variant or derivative according to the invention) and the `other
compound`. Likewise, the binding affinity to FcRn an albumin
associate according to the invention may be stronger or weaker than
the binding affinity to FcRn of the analogous/equivalent albumin
associate comprising or consisting of a reference albumin such as
native HSA (instead of an albumin variant or derivative according
to the invention) and the `other compound`. Methods for the
preparation of associates are well-known to the skilled person, for
example, formulation (by association) of HSA with Lipo-compounds is
described in Hussain, R. and Siligardi, G. (2006) International
Journal of Peptide Research and Therapeutics, Vol. 12, NO: 3, pp.
311-315.
[0223] Further preferences for the eighth aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Compositions
[0224] A ninth aspect of the invention relates to compositions.
Therefore the invention is also directed to the use of a variant of
albumin or a fragment thereof or fusion polypeptides comprising
variant albumin or fragments thereof, or a conjugate comprising a
variant of albumin or a fragment thereof, or an associate
comprising a variant of albumin or a fragment thereof for the
manufacture of a pharmaceutical composition, wherein the variant of
albumin or a fragment thereof or fusion polypeptides comprising
variant albumin or fragments thereof, or a conjugate comprising a
variant of albumin or a fragment thereof, or an associate
comprising a variant of albumin or a fragment thereof has an
altered binding affinity to FcRn and/or an altered plasma half-life
compared with HSA or the corresponding fragment thereof or fusion
polypeptide comprising HSA or fragment thereof or conjugate
comprising HSA.
[0225] In this connection the corresponding fragment of HSA is
intended to mean a fragment of HSA that aligns with and has same
number of amino acids as the fragment of the variant albumin with
which it is compared. Similarly the corresponding fusion
polypeptide comprising HSA or conjugate comprising HSA is intended
to mean molecules having same size and amino acid sequence as the
fusion polypeptide of conjugate comprising variant albumin, with
which it is compared.
[0226] The composition may comprise a pharmaceutically acceptable
carrier or excipient such as water, polysorbate 80 or those
specified in the US Pharmacopoeia for human albumin.
[0227] Further preferences for the ninth aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Nanoparticles
[0228] A tenth aspect of the invention relates to a nanoparticle
comprising a variant, fusion, conjugate, associate, nanoparticle,
composition or polynucleotide as disclosed herein.
[0229] Techniques for incorporation of a molecule into nano- or
microparticles are known in the art. Preferred methods for
preparing nano- or microparticles that may be applied to the
albumin, variant, fragment, fusion, conjugate or associate thereof
according to the invention is disclosed in WO 2004/071536 or
WO2008/007146 or Oner & Groves (Pharmaceutical Research, Vol
10(9), 1993, pages 1387 to 1388) which are incorporated herein by
reference. Preferably the average diameter of a nano-particle is
from 5 to 1000 nm, more preferably 5, 10, 20, 30, 40, 50, 80, 100,
130, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 999 to 5, 10,
20, 30, 40, 50, 80, 100, 130, 150, 200, 300, 400, 500, 600, 700,
800, 900, or 1000 nm. An advantage of a microparticle less than 200
nm diameter, and more particularly less than 130 nm, is that is
amenable to sterilization by filtration through a 0.2 .mu.m
(micron) filter. Preferably, the average diameter of a
micro-particle is from 1000 nm (1 .mu.m (micron)) to 100 .mu.m
(micron), more preferably from 1, 2, 5, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100 to 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100
.mu.m (micron).
[0230] Further preferences for the tenth aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Uses
[0231] An eleventh aspect of the invention relates to use of a
variant albumin, fragment, fusion or conjugate thereof or
nanoparticle or associate thereof. Use may be, for example, in a
method of treatment, prophylaxis, diagnosis or imaging. The variant
albumin or fragments thereof or fusion polypeptides comprising
variant albumin or fragments thereof according to the invention
have the benefit that their binding affinity to FcRn and/or plasma
half-life is altered compared to the parent or reference albumin or
fragments thereof or fusion polypeptides comprising parent or
reference albumin or fragments thereof. This has the advantage that
the binding affinity to FcRn and/or plasma half-life of conjugates
comprising variant albumin or a fragment thereof or fusion
polypeptide comprising variant albumin or a fragment thereof, or an
associate comprising variant albumin or a fragment thereof
according to the invention can be selected in accordance with the
particular therapeutic purpose.
[0232] In some situations, it would be advantageous to use an
albumin, variant, fragment, fusion, conjugate or associate or
composition thereof having a longer plasma half-life than the
reference molecule or composition since this would have the benefit
that the administration of the albumin, variant, fragment, fusion,
conjugate or associate or composition thereof would be needed less
frequently or at a reduced dose (and consequently with fewer side
effects) compared to the situation where the reference molecule or
composition was used. With respect to the use of a variant, fusion,
conjugate, associate, nanoparticle, composition or polynucleotide
the albumin moiety may comprise one more alterations as disclosed
herein.
[0233] In other situations, it would be advantageous to use an
albumin, variant, fragment, fusion, conjugate or associate or
composition thereof having a shorter plasma half-life than the
reference molecule or composition since this would have the benefit
that the administration of the albumin, variant, fragment, fusion,
conjugate or associate or composition thereof can be carried out at
a higher dose compared to the situation where the reference
molecule or composition was used with the benefit that the
administered compound clears from the recipient more quickly than
if the reference molecule or composition was used. With respect to
the use of a variant, fusion, conjugate, associate, nanoparticle,
composition or polynucleotide the albumin moiety may comprise one
more alterations as disclosed herein.
[0234] For example for a conjugate, associate or fusion polypeptide
used for imaging purposes in animals or human beings, where the
imaging moiety has an very short half-life and a conjugate or a
fusion polypeptide comprising HSA has a plasma half-life that is
far longer than needed for the imaging purposes it would be
advantageous to use a variant albumin or fragment thereof of the
invention having a shorter plasma half-life than the parent or
reference albumin or fragment thereof, to provide conjugates of
fusion polypeptides having a plasma half-life that is sufficiently
long for the imaging purpose but sufficiently short to be cleared
form the body of the particular patient on which it is applied.
[0235] In another example for a conjugate, an associate or fusion
polypeptide comprising a therapeutic compound effective to treat or
alleviate a particular condition in a patient in need for such a
treatment it would be advantageous to use the variant albumin or
fragment thereof having a longer plasma half-life than the parent
or reference albumin or fragment thereof, to provide associates or
conjugates or fusion polypeptides having longer plasma half-lives
which would have the benefit that the administration of the
associate or conjugate or fusion polypeptide of the invention would
be needed less frequently or reduced dose with less side effects
compared to the situation where the parent or reference albumin or
associates thereof or fragment thereof was used. For example, the
invention provides a method of treating a proliferative disease in
an individual, comprising administering the individual an effective
amount of an associate according to the invention in which the
associate comprises a taxane, a taxol or taxol derivative (e.g.
paclitaxel).
[0236] In a further aspect the invention relates to compositions
comprising the variant albumin, associates thereof or fragment
thereof, variant albumin fragment or associates thereof or fusion
polypeptide comprising variant albumin or fragment thereof
according to the invention. The compositions are preferably
pharmaceutical compositions. The composition may be prepared using
techniques known in the area such as disclosed in recognized
handbooks within the pharmaceutical field. Since the albumin,
variant, fragment, fusion, conjugate or associate thereof has a
binding affinity to FcRn and/or plasma half-life which is modulated
(i.e. stronger or weaker and/or longer or shorter) than that of a
reference molecule, the composition also has a binding affinity to
FcRn and/or modulated (i.e. altered) plasma half-life relative to
an equivalent composition comprising the reference molecule in
place of the albumin, variant, fragment, fusion, conjugate or
associate thereof as described herein. The composition may be a
vaccine. The polypeptide according to the invention may be an
active pharmaceutical or an excipient. Optionally, the composition
is provided in unit dosage form.
[0237] Preferably the albumin, variant, fragment, fusion, conjugate
or associate thereof has a plasma half-life that is longer than the
plasma half-life of the reference molecule e.g. the same
composition except that the albumin component (e.g. albumin,
variant, fragment, fusion, conjugate or associate) is wild-type
albumin (e.g. HSA) or a variant, fragment, fusion, conjugate or
associate.
[0238] In a particular embodiment the compositions comprise a
variant albumin or a fragment thereof according to the invention
and a compound comprising a pharmaceutically beneficial moiety and
an albumin binding domain (ABD). According to the invention ABD
means a site, moiety or domain capable of binding to circulating
albumin in vivo and thereby conferring transport in the circulation
of the ABD and any compound or moiety bound to said ABD. ABD's are
known in the art and have been shown to bind very tight to albumin
so a compound comprising an ABD bound to albumin will to a certain
extent behave as a single molecule. The inventors have realized by
using the variant albumin or fragment thereof according to the
invention together with a compound comprising a pharmaceutically
beneficial moiety and an ABD makes it possible to alter the binding
affinity to FcRn and/or plasma half-life of the compound comprising
a pharmaceutically beneficial moiety and an ABD compared to the
situation where said compound were injected as such in a patient
having need thereof or administered in a formulation comprising
natural albumin or a fragment thereof.
[0239] The variant albumin or fragments thereof, conjugates
comprising variant albumin or a fragment thereof or fusion
polypeptide comprising variant albumin or a fragment thereof, or an
associate comprising variant albumin or a fragment thereof
according to the invention may also be incorporated into nano- or
microparticles using techniques well known within the art. A
preferred method for preparing nano- or microparticles that may be
applied to the variant albumins or fragments thereof according to
the invention is disclosed in WO 2004/071536 or WO2008/007146 or
Oner & Groves (Pharmaceutical Research, Vol 10(9), 1993, pages
1387 to 1388) which are incorporated herein by reference.
[0240] Further preferences for the eleventh aspect of the invention
include those of the first aspect of the invention and those
provided below the twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
Method for Altering the FcRn-Binding Affinity or Half-Life of a
Molecule
[0241] A twelfth aspect of the invention provides a method for
altering the FcRn-binding affinity or half-life of a molecule
comprising:
[0242] (a) where the molecule is a polypeptide, fusing or
conjugating the molecule to a polypeptide disclosed herein or to a
conjugate disclosed herein; associating the molecule to a
polypeptide disclosed herein or to a conjugate disclosed herein;
incorporating the molecule in a nanoparticle disclosed herein or a
composition disclosed herein;
[0243] (b) where the molecule is not a polypeptide, conjugating the
molecule to a polypeptide disclosed herein or to a conjugate
disclosed herein; associating the molecule to a polypeptide
disclosed herein or to a conjugate a disclosed herein;
incorporating the molecule in a nanoparticle disclosed herein or a
composition disclosed herein.
[0244] Examples of `molecule` include those useful in therapy,
prophylaxis (including those used in vaccines either as an active
pharmaceutical ingredient or as an excipient), imaging and
diagnosis, such as those described herein.
[0245] Further preferences for the twelfth aspect of the invention
include those of the first aspect of the invention and those
provided below this twelfth aspect of the invention. The skilled
person understands that any aspect of the invention may be combined
with another aspect or aspects of the invention and/or with one or
more (several) of the preferences for the aspects of the invention
and/or other disclosures made herein.
[0246] Preferences for all aspects of the invention are provided
below. The skilled person understands that any aspect of the
invention may be combined with another aspect or aspects of the
invention and/or with one or more (several) of the preferences for
the aspects of the invention and/or other disclosures made
herein.
[0247] The variant of albumin or a fragment thereof or fusion
polypeptides comprising variant albumin or fragments thereof,
fragment thereof, conjugate, nanoparticle, associate or composition
may have a plasma half-life that is either longer or shorter,
preferably longer, than the plasma half-life than a corresponding
albumin or a fragment thereof or fusion polypeptides comprising
albumin or fragments thereof, fragment thereof, conjugate,
nanoparticle, associate or composition or a binding to FcRn that is
stronger or weaker, preferably weaker. Preferably the variant of
albumin or a fragment thereof or fusion polypeptides comprising
variant albumin or fragments thereof, fragment thereof, conjugate,
nanoparticle, associate or composition has a plasma half-life that
is longer than the plasma half-life of HSA or the corresponding
albumin or a fragment thereof or fusion polypeptides comprising
albumin or fragments thereof, fragment thereof, conjugate,
nanoparticle, associate or composition.
[0248] Alternatively, this may be expressed as the variant of
albumin or a fragment thereof or fusion polypeptides comprising
variant albumin or fragments thereof, fragment thereof, conjugate,
nanoparticle, associate or composition having a KD to FcRn (e.g.
shFcRn) that is lower than the corresponding KD for HSA to FcRn or
the corresponding fragment thereof or fusion polypeptide comprising
HSA or fragment thereof. Preferably, the KD for the variant of
albumin or a fragment thereof or fusion polypeptides comprising
variant albumin or fragments thereof, fragment thereof, conjugate,
nanoparticle, associate or composition is less than 0.9.times. KD
for HSA to FcRn, more preferred less than 0.5.times. KD for HSA to
FcRn, more preferred less than 0.1.times. KD for HSA to FcRn, even
more preferred less than 0.05.times. KD for HSA to FcRn, even more
preferred less than 0.02.times. KD for HSA to FcRn and most
preferred less than 0.01.times. KD for HSA to FcRn (where X means
`multiplied by`). The KD of the variant of albumin or a fragment
thereof or fusion polypeptides comprising variant albumin or
fragments thereof, fragment thereof, conjugate, nanoparticle,
associate or composition may be between the KD of WT albumin (e.g.
SEQ ID No. 2) for FcRn and the KD of HSA K573P (SEQ ID No. 3) for
FcRn. Such KDs represent binding affinities that are higher than
the binding affinity between HSA and FcRn. A higher binding
affinity indicates a longer half-life, for example plasma
half-life.
[0249] Alternatively, the variant of albumin or a fragment thereof
or fusion polypeptides comprising variant albumin or fragments
thereof, fragment thereof, conjugate, nanoparticle, associate or
composition has a plasma half-life that is shorter than the plasma
half-life of HSA or the corresponding fragment thereof or fusion
polypeptide comprising HSA or fragment thereof.
[0250] This may be expressed as the variant of albumin or a
fragment thereof or fusion polypeptides comprising variant albumin
or fragments thereof, fragment thereof, conjugate, nanoparticle,
associate or composition having a KD to FcRn that is higher than
the corresponding KD for HSA to FcRn or the corresponding of
albumin or a fragment thereof or fusion polypeptides comprising
albumin or fragments thereof, fragment thereof, conjugate,
nanoparticle, associate or composition. Preferably, the KD for the
variant of albumin or a fragment thereof or fusion polypeptides
comprising variant albumin or fragments thereof, fragment thereof,
or a conjugate comprising a variant of albumin or a fragment
thereof is more than 2.times. KD for HSA to FcRn, more preferred
more than 5.times. KD for HSA to FcRn, more preferred more than
10.times. KD for HSA to FcRn, even more preferred more than
25.times. KD for HSA to FcRn, even most preferred more than
50.times. KD for HSA to FcRn. The variant of albumin or a fragment
thereof or fusion polypeptides comprising variant albumin or
fragments thereof, fragment thereof, conjugate, nanoparticle,
associate or composition may be a null binder to FcRn.
[0251] The variant of albumin or a fragment thereof or fusion
polypeptides comprising variant albumin or fragments thereof,
fragment thereof, or a conjugate or nanoparticle or associate or
composition comprising a variant of albumin or a fragment thereof
is preferably the variant of albumin or a fragment thereof or
fusion polypeptides comprising variant albumin or fragments
thereof, fragment thereof, or a conjugate or nanoparticle or
associate or composition comprising a variant of albumin or a
fragment thereof according to the invention. A lower binding
affinity indicates a shorter half-life, for example plasma
half-life.
[0252] One advantage of the invention is that it allows the
half-life of albumin, a variant of albumin or a fragment thereof or
fusion polypeptides comprising variant albumin or fragments
thereof, fragment thereof, conjugate, nanoparticle, associate or
composition to be tailored in order to achieve a binding affinity
or half-life which meets the needs of the user.
[0253] When determining and/or comparing KD, one or more (and
preferably all) of the following parameters may be used:
[0254] Instrument: Biacore 3000 instrument (GE Healthcare)
[0255] Flow cell: CM5 sensor chip
[0256] FcRn: human FcRn, preferably soluble human FcRn, optionally
coupled to a tag such as GST or His, most preferably His such as 6
histidines at the C-terminus of the beta-2-microglobulin (SEQ ID
NO: 31).
[0257] Quantity of FcRn: 1200-2500 RU
[0258] Coupling chemistry: amine coupling chemistry (e.g. as
described in the protocol provided by the manufacturer of the
instrument).
[0259] Coupling method: The coupling may be performed by injecting
20 .mu.g/ml of the protein in 10 mM sodium acetate pH 5.0 (GE
Healthcare). Phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl,
0.005% Tween 20) at pH 5.5) may be used as running buffer and
dilution buffer. Regeneration of the surfaces may be done using
injections of HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA,
0.005% surfactant P20) at pH 7.4 (Biacore AB).
[0260] Quantity of injection of test molecule (e.g. HSA or variant)
20-0.032 .mu.M
[0261] Flow rate of injection: constant, e.g. 30 .mu.l/ml
[0262] Temperature of injection: 25.degree. C.
[0263] Data evaluation software: BIAevaluation 4.1 software
(BIAcore AB).
The preferred method for determining KD is provided in Example
2.
[0264] The invention discloses that one or more (several) positions
in Domain I in combination with one or more (several) positions in
Domain III in SEQ ID NO: 2 (and therefore equivalent positions in
albumins and fragments from human serum and albumin and non-human
serum albumins) may be altered in order to modulate (increase of
decrease) the binding affinity and/or half-life e.g. plasma
half-life of an albumin, fragment, fusion, conjugate, associate,
nanoparticle or composition. An alteration may be a substitution,
insertion or deletion. Substitution is preferred.
[0265] A substitution or insertion may or may not comprise
introduction of a conserved amino acid, i.e. conserved in relation
to the amino acid at the position of interest. Examples of
conserved amino acids are shown by the groups of FIG. 3: aliphatic,
aromatic, hydrophobic, charged, polar, positive, tiny and
small.
[0266] At position 82 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to Q, D, A, even more preferred to D, A and
most preferred to A. In SEQ ID NO: 2 the native amino acid at
position 82 is glutamic acid, therefore a substitution to glutamic
acid is not preferred.
[0267] At position 83 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to N, K, S, even more preferred to N, K and
most preferred to N. In SEQ ID NO: 2 the native amino acid at
position 83 is threonine, therefore a substitution to threonine is
not preferred.
[0268] At position 111 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to N, E, Q, D, G, H, even more preferred to E,
Q and most preferred to E. In SEQ ID NO: 2 the native amino acid at
position 111 is asparagine, therefore a substitution to asparagine
is not preferred.
[0269] At position 112 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to F, Y, W, even more preferred to F, Y and
most preferred to F. In SEQ ID NO: 2 the native amino acid at
position 112 is leucine, therefore a substitution to leucine is not
preferred.
[0270] At position 573 of SEQ ID NO: 2 (or equivalent position of
other albumins or variants of fragments thereof), it is preferred
that the alteration is a substitution, such as from the native
amino acid to A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W, Y, more preferred to P, Y, W, H, F, T, I or V, even more
preferred to P, Y or W and most preferred to P. In SEQ ID NO: 2 the
native amino acid at position 573 is lysine, therefore a
substitution to lysine is not preferred.
[0271] It is preferred that the alteration at position 82 is
conserved relative to A. It is preferred that the alteration at
position 83 is conserved relative to N. It is preferred that the
alteration at position 111 is conserved relative to E. It is
preferred that the alteration at position 112 is conserved relative
to F. It is preferred that the alteration at position 573 is
conserved relative to P.
[0272] Particularly preferred albumin variants comprise
substitutions T83N/N111E (e.g. SEQ ID NO: 32); T83N/N111E/K573P
(e.g. SEQ ID NO: 33); T83N/K573P (e.g. SEQ ID NO: 34); T83K/K573P
(e.g. SEQ ID NO: 38); E82A/K573P (e.g. SEQ ID NO: 39); L112F/K573P
(e.g. SEQ ID NO: 40); E82D/K573P (e.g. SEQ ID NO: 43); P110G/K573P
(e.g. SEQ ID NO: 44); N111D/K573P (e.g. SEQ ID NO: 60); N111G/K573P
(e.g. SEQ ID NO: 61); N111H/K573P (e.g. SEQ ID NO: 62); E425A/K573P
(e.g. SEQ ID NO: 64); E505Q/K573P (e.g. SEQ ID NO: 65); T527M/K573P
(e.g. SEQ ID NO: 66); N111E/K573P (e.g. SEQ ID NO: 68); K534V/K573P
(e.g. SEQ ID NO: 73); N111Q/K573P (e.g. SEQ ID NO: 74) which are
described with reference to HSA (SEQ ID NO: 2). Other preferred
albumin variants comprise equivalent substitutions in albumins
other than HSA (SEQ ID NO: 2).
[0273] Also, an albumin variant according to the invention may
comprise one or more (several) alterations at positions selected
from 78 to 88 and/or 105 to 120 and/or 425, 505, 510, 512, 524,
527, 531, 534, 569, 575 of HSA (SEQ ID NO: 2) or equivalent
positions of other albumins. Preferred alterations are
substitutions such as those described for these positions in the
first aspect of the invention. Particularly preferred substitutions
include D108A (SEQ ID NO: 59); D108E (e.g. SEQ ID NO: 70); N109K
(e.g. SEQ ID NO: 69); P110G (e.g. SEQ ID NO: 42); N111D (e.g. SEQ
ID NO: 46); N111E (e.g. SEQ ID NO: 67); N111G (e.g. SEQ ID NO: 48);
N111H (e.g. SEQ ID NO: 49); N111K (e.g. SEQ ID NO: 54); L112F (e.g.
SEQ ID NO: 37); E425A (e.g. SEQ ID NO: 63); E425K (e.g. SEQ ID NO:
55); E505Q (e.g. SEQ ID NO: 45); H510D (e.g. SEQ ID NO: 57); D512E
(e.g. SEQ ID NO: 50); K524A (e.g. SEQ ID NO: 51); T527A (e.g. SEQ
ID NO: 52); T527M (e.g. SEQ ID NO: 47); E531H (e.g. SEQ ID NO: 53);
K534V (e.g. SEQ ID NO: 56); A569S (e.g. SEQ ID NO: 58); L575F (e.g.
SEQ ID NO: 72); E82A (e.g. SEQ ID NO: 36); E82D (e.g. SEQ ID NO:
41); T83K (e.g. SEQ ID NO: 35); T83N (e.g. SEQ ID NO: 71) which are
described with reference to HSA (SEQ ID NO: 2). Other preferred
albumin variants comprising one or more (several) alterations may
comprise equivalent substitutions in albumins other than HSA (SEQ
ID NO: 2).
[0274] Advantageously, the polypeptide retains substantially the
same tertiary structure (or, for a fragment, the relevant part of
the structure) as a reference or parent albumin such as HSA. The
skilled person understand the term `substantially the same tertiary
structure` bearing in mind that some degree of variation in
tertiary structure is expected as all proteins have some degree of
structural flexibility. This applies particularly to polypeptides
having a higher binding affinity to FcRn than the parent or
reference albumin (e.g. HSA) has to FcRn.
[0275] One or more (several) of the His residues may or may not be
maintained relative to the parent albumin. For example, with
reference to SEQ ID NO: 2, one or more (several) of the following
His residues may be maintained: 3, 9, 39, 67, 105, 128, 146, 242,
247, 288, 338, 367, 440, 464, 510, and/or 535. One or more
(several), preferably all, of the His residues in domain I are
maintained (i.e. 3, 9, 39, 67, 105, 128, 146.). One or more
(several), preferably all, of the His residues in domain II are
maintained (i.e. 242, 247, 288, 338, 367). One or more (several),
preferably all, of the His residues in domain III are maintained
(i.e. 440, 464, 510, 535). One or more (several) or all three of
His 464, 510, 535 may be maintained.
[0276] It is preferred that at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16 or 17 of the disulphide bonds of the albumin
are maintained in the polypeptide. For a polypeptide derived from a
full length albumin, it is preferred that all disulphide bonds
usually present in that albumin are maintained. For a polypeptide
derived from a fragment of albumin, it is preferred that all
disulphide bonds usually present in that fragment are maintained.
It is preferred that Cys34 (or equivalent in non-human albumins) is
maintained.
[0277] For all aspects of the invention fusion partner polypeptides
and/or conjugates may comprise one or more (several) of: 4-1BB
ligand, 5-helix, A human C-C chemokine, A human L105 chemokine, A
human L105 chemokine designated huL105_3., A monokine induced by
gamma-interferon (MIG), A partial CXCR4B protein, A platelet basic
protein (PBP), .alpha.1-antitrypsin, ACRP-30 Homologue; Complement
Component C1q C, Adenoid-expressed chemokine (ADEC), aFGF; FGF-1,
AGF, AGF Protein, albumin, an etoposide, angiostatin, Anthrax
vaccine, Antibodies specific for collapsin, antistasin, Anti-TGF
beta family antibodies, antithrombin III, APM-1; ACRP-30; Famoxin,
apo-lipoprotein species, Arylsulfatase B, b57 Protein, BCMA,
Beta-thromboglobulin protein (beta-TG), bFGF; FGF2, Blood
coagulation factors, BMP Processing Enzyme Furin, BMP-10, BMP-12,
BMP-15, BMP-17, BMP-18, BMP-2B, BMP-4, BMP-5, BMP-6, BMP-9, Bone
Morphogenic Protein-2, calcitonin, Calpain-10a, Calpain-10b,
Calpain-10c, Cancer Vaccine, Carboxypeptidase, C-C chemokine, MCP2,
CCR5 variant, CCR7, CCR7, CD11a Mab, CD137; 4-1BB Receptor Protein,
CD20 Mab, CD27, CD27L, CD30, CD30 ligand, CD33 immunotoxin, CD40,
CD40L, CD52 Mab, Cerebus Protein, Chemokine Eotaxin., Chemokine
hIL-8, Chemokine hMCP1, Chemokine hMCP1a, Chemokine hMCP1b,
Chemokine hMCP2, Chemokine hMCP3, Chemokine hSDF1b, Chemokine
MCP-4, chemokine TECK and TECK variant, Chemokine-like protein
IL-8M1 Full-Length and Mature, Chemokine-like protein IL-8M10
Full-Length and Mature, Chemokine-like protein IL-8M3,
Chemokine-like protein IL-8M8 Full-Length and Mature,
Chemokine-like protein IL-8M9 Full-Length and Mature,
Chemokine-like protein PF4-414 Full-Length and Mature,
Chemokine-like protein PF4-426 Full-Length and Mature,
Chemokine-like protein PF4-M2 Full-Length and Mature, Cholera
vaccine, Chondromodulin-like protein, c-kit ligand; SCF; Mast cell
growth factor; MGF; Fibrosarcoma-derived stem cell factor, CNTF and
fragment thereof (such as CNTFAx15`(Axokine.TM.)), coagulation
factors in both pre and active forms, collagens, Complement C5 Mab,
Connective tissue activating protein-III, CTAA16.88 Mab, CTAP-III,
CTLA4-Ig, CTLA-8, CXC3, CXC3, CXCR3; CXC chemokine receptor 3,
cyanovirin-N, Darbepoetin, designated exodus, designated huL105_7.,
DIL-40, DNase, EDAR, EGF Receptor Mab, ENA-78, Endostatin, Eotaxin,
Epithelial neutrophil activating protein-78, EPO receptor; EPOR,
erythropoietin (EPO) and EPO mimics, Eutropin, Exodus protein,
Factor IX, Factor VII, Factor VIII, Factor X and Factor XIII, FAS
Ligand Inhibitory Protein (DcR3), FasL, FasL, FasL, FGF, FGF-12;
Fibroblast growth factor homologous factor-1, FGF-15, FGF-16,
FGF-18, FGF-3; INT-2, FGF-4; gelonin, HST-1; HBGF-4, FGF-5, FGF-6;
Heparin binding secreted transforming factor-2, FGF-8, FGF-9; Glia
activating factor, fibrinogen, flt-1, flt-3 ligand, Follicle
stimulating hormone Alpha subunit, Follicle stimulating hormone
Beta subunit, Follitropin, Fractalkine, fragment. myofibrillar
protein Troponin I, FSH, Galactosidase, Galectin-4, G-CSF, GDF-1,
Gene therapy, Glioma-derived growth factor, glucagon, glucagon-like
peptides, Glucocerebrosidase, glucose oxidase, Glucosidase,
Glycodelin-A; Progesterone-associated endometrial protein, GM-CSF,
gonadotropin, Granulocyte chemotactic protein-2 (GCP-2),
Granulocyte-macrophage colony stimulating factor, growth hormone,
Growth related oncogene-alpha (GRO-alpha), Growth related
oncogene-beta (GRO-beta), Growth related oncogene-gamma
(GRO-gamma), hAPO-4; TROY, hCG, Hepatitus B surface Antigen,
Hepatitus B Vaccine, HER2 Receptor Mab, hirudin, HIV gp120, HIV
gp41, HIV Inhibitor Peptide, HIV Inhibitor Peptide, HIV Inhibitor
Peptide, HIV protease inhibiting peptides, HIV-1 protease
inhibitors, HPV vaccine, Human 6CKine protein, Human Act-2 protein,
Human adipogenesis inhibitory factor, human B cell stimulating
factor-2 receptor, Human beta-chemokine H1305 (MCP-2), Human C-C
chemokine DGWCC, Human CC chemokine ELC protein, Human CC type
chemokine interleukin C, Human CCC3 protein, Human CCF18 chemokine,
Human CC-type chemokine protein designated SLC (secondary lymphoid
chemokine), Human chemokine beta-8 short forms, Human chemokine
C10, Human chemokine CC-2, Human chemokine CC-3, Human chemokine
CCR-2, Human chemokine Ckbeta-7, Human chemokine ENA-78, Human
chemokine eotaxin, Human chemokine GRO alpha, Human chemokine
GROalpha, Human chemokine GRObeta, Human chemokine HCC-1, Human
chemokine HCC-1, Human chemokine I-309, Human chemokine IP-10,
Human chemokine L105_3, Human chemokine L105_7, Human chemokine
MIG, Human chemokine MIG-beta protein, Human chemokine MIP-1alpha,
Human chemokine MIP1beta, Human chemokine MIP-3alpha, Human
chemokine MIP-3beta, Human chemokine PF4, Human chemokine protein
331D5, Human chemokine protein 61164, Human chemokine receptor
CXCR3, Human chemokine SDF1alpha, Human chemokine SDF1beta, Human
chemokine ZSIG-35, Human Chr19Kine protein, Human CKbeta-9, Human
CKbeta-9, Human CX3C 111 amino acid chemokine, Human DNAX
interleukin-40, Human DVic-1 C-C chemokine, Human EDIRF I protein
sequence, Human EDIRF II protein sequence, Human eosinocyte CC type
chemokine eotaxin, Human eosinophil-expressed chemokine (EEC),
Human fast twitch skeletal muscle troponin C, Human fast twitch
skeletal muscle troponin I, Human fast twitch skeletal muscle
Troponin subunit C, Human fast twitch skeletal muscle Troponin
subunit I Protein, Human fast twitch skeletal muscle Troponin
subunit T, Human fast twitch skeletal muscle troponin T, Human
foetal spleen expressed chemokine, FSEC, Human GM-CSF receptor,
Human gro-alpha chemokine, Human gro-beta chemokine, Human
gro-gamma chemokine, Human IL-16 protein, Human IL-1RD10 protein
sequence, Human IL-1RD9, Human IL-5 receptor alpha chain, Human
IL-6 receptor, Human IL-8 receptor protein hIL8RA, Human IL-8
receptor protein hIL8RB, Human IL-9 receptor protein, Human IL-9
receptor protein variant #3, Human IL-9 receptor protein variant
fragment, Human IL-9 receptor protein variant fragment #3, Human
interleukin 1 delta, Human Interleukin 10, Human Interleukin 10,
Human interleukin 18, Human interleukin 18 derivatives, Human
interleukin-1 beta precursor, Human interleukin-1 beta precursor.,
Human interleukin-1 receptor accessory protein, Human interleukin-1
receptor antagonist beta, Human interleukin-1 type-3 receptor,
Human Interleukin-10 (precursor), Human Interleukin-10 (precursor),
Human interleukin-11 receptor, Human interleukin-12 40 kD subunit,
Human interleukin-12 beta-1 receptor, Human interleukin-12 beta-2
receptor, Human Interleukin-12 p35 protein, Human Interleukin-12
p40 protein, Human interleukin-12 receptor, Human interleukin-13
alpha receptor, Human interleukin-13 beta receptor, Human
interleukin-15, Human interleukin-15 receptor from clone P1, Human
interleukin-17 receptor, Human interleukin-18 protein (IL-18),
Human interleukin-3, human interleukin-3 receptor, Human
interleukin-3 variant, Human interleukin-4 receptor, Human
interleukin-5, Human interleukin-6, Human interleukin-7, Human
interleukin-7., Human interleukin-8 (IL-8), Human intracellular
IL-1 receptor antagonist, Human IP-10 and HIV-1 gp120 hypervariable
region fusion protein, Human IP-10 and human Muc-1 core epitope
(VNT) fusion protein, human liver and activation regulated
chemokine (LARC), Human Lkn-1 Full-Length and Mature protein, Human
mammary associated chemokine (MACK) protein Full-Length and Mature,
Human mature chemokine Ckbeta-7, Human mature gro-alpha, Human
mature gro-gamma polypeptide used to treat sepsis, Human MCP-3 and
human Muc-1 core epitope (VNT) fusion protein, Human MI10 protein,
Human MI1A protein, Human monocyte chemoattractant factor hMCP-1,
Human monocyte chemoattractant factor hMCP-3, Human monocyte
chemotactic proprotein (MCPP) sequence, Human neurotactin chemokine
like domain, Human non-ELR CXC chemokine H174, Human non-ELR CXC
chemokine IP10, Human non-ELR CXC chemokine Mig, Human PAI-1
mutants, Human protein with IL-16 activity, Human protein with
IL-16 activity, Human secondary lymphoid chemokine (SLC), Human
SISD protein, Human STCP-1, Human stromal cell-derived chemokine,
SDF-1, Human T cell mixed lymphocyte reaction expressed chemokine
(TMEC), Human thymus and activation regulated cytokine (TARC),
Human thymus expressed, Human TNF-alpha, Human TNF-alpha, Human
TNF-beta (LT-alpha), Human type CC chemokine eotaxin 3 protein
sequence, Human type II interleukin-1 receptor, Human wild-type
interleukin-4 (hIL-4) protein, Human ZCHEMO-8 protein, Humanized
Anti-VEGF Antibodies, and fragments thereof, Humanized Anti-VEGF
Antibodies, and fragments thereof, Hyaluronidase, ICE 10 kD
subunit., ICE 20 kD subunit., ICE 22 kD subunit.,
Iduronate-2-sulfatase, Iduronidase, IL-1 alpha, IL-1 beta, IL-1
inhibitor (IL-1i)., IL-1 mature, IL-10 receptor, IL-11, IL-11,
IL-12 p40 subunit., IL-13, IL-14, IL-15, IL-15 receptor, IL-17,
IL-17 receptor, II-17 receptor, II-17 receptor, IL-19, IL-1i
fragments, IL1-receptor antagonist, IL-21 (TIF), IL-3 containing
fusion protein., IL-3 mutant proteins, IL-3 variants, IL-3
variants, IL-4, IL-4 mutein, IL-4 mutein Y124G, IL-4 mutein Y124X,
IL-4 muteins, II-5 receptor, IL-6, II-6 receptor, IL-7 receptor
clone, IL-8 receptor, IL-9 mature protein variant (Met117 version),
immunoglobulins or immunoglobulin-based molecules or fragment of
either (e.g. a Small Modular ImmunoPharmaceutical.TM. ("SMIP") or
dAb, Fab' fragments, F(ab')2, scAb, scFv or scFv fragment),
including but not limited to plasminogen, Influenza Vaccine,
Inhibin alpha, Inhibin beta, insulin, insulin-like growth factor,
Integrin Mab, inter-alpha trypsin inhibitor, inter-alpha trypsin
inhibitor, Interferon gamma-inducible protein (IP-10), interferons
(such as interferon alpha species and sub-species, interferon beta
species and sub-species, interferon gamma species and sub-species),
interferons (such as interferon alpha species and sub-species,
interferon beta species and sub-species, interferon gamma species
and sub-species), Interleukin 6, Interleukin 8 (IL-8) receptor,
Interleukin 8 receptor B, Interleukin-1alpha, Interleukin-2
receptor associated protein p43, interleukin-3, interleukin-4
muteins, Interleukin-8 (IL-8) protein., interleukin-9,
Interleukin-9 (IL-9) mature protein (Thr117 version), interleukins
(such as IL10, IL11 and IL2), interleukins (such as IL10, IL11 and
IL2), Japanese encephalitis vaccine, Kalikrein Inhibitor,
Keratinocyte growth factor, Kunitz domain protein (such as
aprotinin, amyloid precursor protein and those described in WO
03/066824, with or without albumin fusions), Kunitz domain protein
(such as aprotinin, amyloid precursor protein and those described
in WO 03/066824, with or without albumin fusions), LACI,
lactoferrin, Latent TGF-beta binding protein II, leptin, Liver
expressed chemokine-1 (LVEC-1), Liver expressed chemokine-2
(LVEC-2), LT-alpha, LT-beta, Luteinization Hormone, Lyme Vaccine,
Lymphotactin, Macrophage derived chemokine analogue MDC (n+1),
Macrophage derived chemokine analogue MDC-eyfy, Macrophage derived
chemokine analogue MDC-yl, Macrophage derived chemokine, MDC,
Macrophage-derived chemokine (MDC), Maspin; Protease Inhibitor 5,
MCP-1 receptor, MCP-1a, MCP-1b, MCP-3, MCP-4 receptor, M-CSF,
Melanoma inhibiting protein, Membrane-bound proteins, Met117 human
interleukin 9, MIP-3 alpha, MIP-3 beta, MIP-Gamma, MIRAP, Modified
RANTES, monoclonal antibody, MP52, Mutant Interleukin 6 S176R,
myofibrillar contractile protein Troponin I, Natriuretic Peptide,
Nerve Growth Factor-beta, Nerve Growth Factor-beta2, Neuropilin-1,
Neuropilin-2, Neurotactin, Neurotrophin-3, Neurotrophin-4,
Neurotrophin-4a, Neurotrophin-4b, Neurotrophin-4c, Neurotrophin-4d,
Neutrophil activating peptide-2 (NAP-2), NOGO-66 Receptor, NOGO-A,
NOGO-B, NOGO-C, Novel beta-chemokine designated PTEC, N-terminal
modified chemokine GroHEK/hSDF-1alpha, N-terminal modified
chemokine GroHEK/hSDF-1beta., N-terminal modified chemokine
met-hSDF-1 alpha, N-terminal modified chemokine met-hSDF-1 beta,
OPGL, Osteogenic Protein-1; OP-1; BMP-7, Osteogenic Protein-2,
OX40; ACT-4, OX40L, Oxytocin (Neurophysin I), parathyroid hormone,
Patched, Patched-2, PDGF-D, Pertussis toxoid, Pituitary expressed
chemokine (PGEC), Placental Growth Factor, Placental Growth
Factor-2, Plasminogen Activator Inhibitor-1; PAI-1, Plasminogen
Activator Inhibitor-2; PAI-2, Plasminogen Activator Inhibitor-2;
PAI-2, Platelet derived growth factor, Platelet derived growth
factor Bv-sis, Platelet derived growth factor precursor A, Platelet
derived growth factor precursor B, Platelet Mab, platelet-derived
endothelial cell growth factor (PD-ECGF), Platelet-Derived Growth
Factor A chain, Platelet-Derived Growth Factor B chain, polypeptide
used to treat sepsis, Preproapolipoprotein "milano" variant,
Preproapolipoprotein "paris" variant, pre-thrombin, Primate CC
chemokine "ILINCK", Primate CXC chemokine "IBICK", proinsulin,
Prolactin, Prolactin2, prosaptide, Protease inhibitor peptides,
Protein C, Protein S, pro-thrombin, prourokinase, RANTES, RANTES
8-68, RANTES 9-68, RANTES peptide, RANTES receptor, Recombinant
interleukin-16, Resistin, restrictocin, Retroviral protease
inhibitors, ricin, Rotavirus Vaccine, RSV Mab, saporin, sarcin,
Secreted and Transmembrane polypeptides, Secreted and Transmembrane
polypeptides, serum cholinesterase, serum protein (such as a blood
clotting factor), Soluble BMP Receptor Kinase Protein-3, Soluble
VEGF Receptor, Stem Cell Inhibitory Factor, Staphylococcus Vaccine,
Stromal Derived Factor-1 alpha, Stromal Derived Factor-1 beta,
Substance P (tachykinin), T1249 peptide, T20 peptide, T4
Endonuclease, TACI, Tarc, TGF-beta 1, TGF-beta 2, Thr117 human
interleukin 9, thrombin, thrombopoietin, Thrombopoietin
derivative1, Thrombopoietin derivative2, Thrombopoietin
derivative3, Thrombopoietin derivative4, Thrombopoietin
derivative5, Thrombopoietin derivative6, Thrombopoietin
derivative7, Thymus expressed chemokine (TECK), Thyroid stimulating
Hormone, tick anticoagulant peptide, Tim-1 protein, TNF-alpha
precursor, TNF-R, TNF-RII; TNF p75 Receptor; Death Receptor, tPA,
transferrin, transforming growth factor beta, Troponin peptides,
Truncated monocyte chemotactic protein 2 (6-76), Truncated monocyte
chemotactic protein 2 (6-76), Truncated RANTES protein (3-68),
tumour necrosis factor, Urate Oxidase, urokinase, Vasopressin
(Neurophysin II), VEGF R-3; flt-4, VEGF Receptor; KDR; flk-1,
VEGF-110, VEGF-121, VEGF-138, VEGF-145, VEGF-162, VEGF-165,
VEGF-182, VEGF-189, VEGF-206, VEGF-D, VEGF-E; VEGF-X, von
Willebrand's factor, Wild type monocyte chemotactic protein 2, Wild
type monocyte chemotactic protein 2, ZTGF-beta 9, alternative
antibody scaffolds e.g. anticalin(s), adnectin(s), fibrinogen
fragment(s), nanobodies such as camelid nanobodies, infestin,
and/or any of the molecules mentioned in WO01/79271 (particularly
page 9 and/or Table 1), WO 2003/59934 (particularly Table 1),
WO03/060071 (particularly Table 1) or WO01/079480 (particularly
Table 1) (each incorporated herein by reference in their
entirety).
[0278] Furthermore, conjugates may comprise one or more (several)
of chemotherapy drugs such as: 13-cis-Retinoic Acid, 2-CdA,
2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU,
6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, A, Abraxane,
Accutane.RTM., Actinomycin-D, Adriamycin.RTM., Adrucil.RTM.,
Agrylin.RTM., Ala-Cort.RTM., Aldesleukin, Alemtuzumab, ALIMTA,
Alitretinoin, Alkaban-AQ.RTM., Alkeran.RTM., All-transretinoic
Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine,
Aminoglutethimide, Anagrelide, Anandron.RTM., Anastrozole,
Arabinosylcytosine, Ara-C, Aranesp.RTM., Aredia.RTM.,
Arimidex.RTM., Aromasin.RTM., Arranon.RTM., Arsenic Trioxide,
Asparaginase, ATRA, Avastin.RTM., Azacitidine, BCG, BCNU,
Bevacizumab, Bexarotene, BEXXAR.RTM., Bicalutamide, BiCNU,
Blenoxane.RTM., Bleomycin, Bortezomib, Busulfan, Busulfex.RTM.,
C225, Calcium Leucovorin, Campath.RTM., Camptosar.RTM.,
Camptothecin-11, Capecitabine, Carac.TM., Carboplatin, Carmustine,
Carmustine Wafer, Casodex.RTM., CC-5013, CCNU, CDDP, CeeNU,
Cerubidine.RTM., Cetuximab, Chlorambucil, Cisplatin, Citrovorum
Factor, Cladribine, Cortisone, Cosmegen.RTM., CPT-11,
Cyclophosphamide, Cytadren.RTM., Cytarabine, Cytarabine Liposomal,
Cytosar-U.RTM., Cytoxan.RTM., Dacarbazine, Dacogen, Dactinomycin,
Darbepoetin Alfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin
Hydrochloride, Daunorubicin Liposomal, DaunoXome.RTM., Decadron,
Decitabine, Delta-Cortef.RTM., Deltasone.RTM., Denileukin diftitox,
DepoCyt.TM., Dexamethasone, Dexamethasone acetate, Dexamethasone
Sodium Phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex,
Docetaxel, Doxil.RTM., Doxorubicin, Doxorubicin liposomal,
Droxia.TM., DTIC, DTIC-Dome.RTM., Duralone.RTM., Efudex.RTM.,
Eligard.TM., Ellence.TM., Eloxatin.TM., Elspar.RTM., Emcyt.RTM.,
Epirubicin, Epoetin alfa, Erbitux.TM., Erlotinib, Erwinia
L-asparaginase, Estramustine, Ethyol, Etopophos.RTM., Etoposide,
Etoposide Phosphate, Eulexin.RTM., Evista.RTM., Exemestane,
Fareston.RTM., Faslodex.RTM., Femara.RTM., Filgrastim, Floxuridine,
Fludara.RTM., Fludarabine, Fluoroplex.RTM., Fluorouracil,
Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid,
FUDR.RTM., Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab
ozogamicin, Gemzar.RTM., Gleevec.TM., Gliadel.RTM. Wafer, GM-CSF,
Goserelin, Granulocyte-Colony Stimulating Factor, Granulocyte
Macrophage Colony Stimulating Factor, Halotestin.RTM.,
Herceptin.RTM., Hexadrol, Hexalen.RTM., Hexamethylmelamine, HMM,
Hycamtin.RTM., Hydrea.RTM., Hydrocort Acetate.RTM., Hydrocortisone,
Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate,
Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab
Tiuxetan, Idamycin.RTM., Idarubicin, Ifex.RTM., IFN-alpha,
Ifosfamide, IL-11, IL-2, Imatinib mesylate, Imidazole Carboxamide,
Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin-2,
Interleukin-11, Intron A.RTM. (interferon alfa-2b), Iressa.RTM.,
Irinotecan, Isotretinoin, Kidrolase.RTM., Lanacort.RTM., Lapatinib,
L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran,
Leukine.TM., Leuprolide, Leurocristine, Leustatin.TM., Liposomal
Ara-C, Liquid Pred.RTM., Lomustine, L-PAM, L-Sarcolysin,
Lupron.RTM., Lupron Depot.RTM., M, Matulane.RTM., Maxidex,
Mechlorethamine, Mechlorethamine Hydrochloride, Medralone.RTM.,
Medrol.RTM., Megace.RTM., Megestrol, Megestrol Acetate, Melphalan,
Mercaptopurine, Mesna, Mesnex.TM., Methotrexate, Methotrexate
Sodium, Methylprednisolone, Meticorten.RTM., Mitomycin,
Mitomycin-C, Mitoxantrone, M-Prednisol.RTM., MTC, MTX,
Mustargen.RTM., Mustine, Mutamycin.RTM., Myleran.RTM., Mylocel.TM.,
Mylotarg.RTM., Navelbine.RTM., Nelarabine, Neosar.RTM.,
Neulasta.TM., Neumega.RTM., Neupogen.RTM., Nexavar.RTM.,
Nilandron.RTM., Nilutamide, Nipent.RTM., Nitrogen Mustard,
Nolvadex.RTM., Novantrone.RTM., Octreotide, Octreotide acetate,
Oncospar.RTM., Oncovin.RTM., Ontak.RTM., Onxal.TM., Oprelvekin,
Orapred.RTM., Orasone.RTM., Oxaliplatin, a taxol or taxol
derivative e.g. Paclitaxel or Paclitaxel Protein-bound,
Pamidronate, Panitumumab, Panretin.RTM., Paraplatin.RTM.,
Pediapred.RTM., PEG Interferon, Pegaspargase, Pegfilgrastim,
PEG-INTRON.TM., PEG-L-asparaginase, PEMETREXED, Pentostatin,
Phenylalanine Mustard, Platinol.RTM., Platinol-AQ.RTM.,
Prednisolone, Prednisone, Prelone.RTM., Procarbazine, PROCRIT.RTM.,
Proleukin.RTM., Prolifeprospan 20 with Carmustine Implant,
Purinethol.RTM., R, Raloxifene, Revlimid.RTM., Rheumatrex.RTM.,
Rituxan.RTM., Rituximab, Roferon-A.RTM. (Interferon Alfa-2a),
Rubex.RTM., Rubidomycin hydrochloride, Sandostatin.RTM.,
Sandostatin LAR.RTM., Sargramostim, Solu-Cortef.RTM.,
Solu-Medrol.RTM., Sorafenib, SPRYCEL.TM., STI-571, Streptozocin,
SU11248, Sunitinib, Sutent.RTM., Tamoxifen, Tarceva.RTM.,
Targretin.RTM., Taxol.RTM., Taxotere.RTM., Temodar.RTM.,
Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid.RTM.,
TheraCys.RTM., Thioguanine, Thioguanine Tabloid.RTM.,
Thiophosphoamide, Thioplex.RTM., Thiotepa, TICE.RTM., Toposar.RTM.,
Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin,
Trexall.TM., Trisenox.RTM., TSPA, TYKERB.RTM., VCR, Vectibix.TM.,
Velban.RTM., Velcade.RTM., VePesid.RTM., Vesanoid.RTM., Viadur.TM.,
Vidaza.RTM., Vinblastine, Vinblastine Sulfate, Vincasar Pfs.RTM.,
Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26,
Vorinostat, VP-16, Vumon.RTM., Xeloda.RTM., Zanosar.RTM.,
Zevalin.TM., Zinecard.RTM., Zoladex.RTM., Zoledronic acid, Zolinza,
Zometa.RTM.; radiopharmaceuticals such as: Carbon-11, Carbon-14,
Chromium-51, Cobalt-57, Cobalt-58, Erbium-169, Fluorine-18,
Gallium-67, Gold-198, Indium-111, Indium-113m, Iodine-123,
Iodine-125, Iodine-131, Iron-59, Krypton-81m, Nitrogen-13,
Oxygen-15, Phosphorous-32, Rhenium-186, Rubidium-82, Samarium-153,
Selenium-75, Strontium-89, Technetium-99m, Thallium-201, Tritium,
Xenon-127, Xenon-133, Yttrium-90; imaging agents such as
Gadolinium, magnetite, manganese, technetium, I125, I131, P32,
TI201, Iopamidol, PET-FDG.
[0279] Further fusion partners, conjugation partners and/or
molecules for inclusion in a nanoparticle, associate or composition
according to the invention include: acromegaly drugs e.g.
somatuline, lanreotide, octreotide, Sandostatin; antithrombotics
e.g. bivalirudin, Angiomax, dalteparin, Fragmin, enoxaparin,
Lovenox, Drotrecogin alfa (e.g. Activated), Xigris, heparin;
assisted reproductive therapy compounds e.g. choriogonadotropin,
Ovidrel, follitropin, alpha/beta; enzymes e.g. hyaluronidase,
Hylenex; diabetes drugs e.g. exenatide, Byetta, glucagon, insulin,
liraglutide, albiglutide, GLP-1 agonists, exendin or an exendin
analog; compounds useful in diagnosis e.g. protirelin, Thyrel TRH
Thypinone, secretin (e.g. synthetic human), Chirhostim, thyrotropin
(e.g. alpha), Thyrogen' erythropoiesis drugs e.g. Darbepoetin alfa,
Aranesp, Epoetin alfa, Epogen, Eprex, drugs for the treatment of
genetic defects e.g. pegademase, drugs for the treatment of growth
failure e.g. Adagen, mecasermin, rinfabate, drugs for the treatment
of cystic fibrosis e.g. Dornase alfa, Pulmozyme, drugs for the
treatment of metabolic disorders e.g. Agalsidase beta, Fabrazyme,
alglucosidase alpha, Myozyme, Laronidase, Aldurazyme, drugs for the
treatment of genital wart intralesional e.g. Interferon alfa-n3,
Alferon N, drugs for the treatment of granulomatous disease e.g.
Interferon gamma-1b, Actimmune; drugs for the treatment of growth
failure e.g. pegvisomant, Somavert, somatropin, Genotropin,
Nutropin, Humatrope, Serostim, Protropin; drugs for the treatment
of heart failure e.g. nesiritide, Natrecor; drugs for the treatment
of hemophilia e.g. a coagulation factor e.g. Factor VIII, Helixate
FS, Kogenate FS, Factor IX, BeneFIX, Factor VIIa, Novoseven,
desmopressin, Stimate, DDAVP; hematopoietic drugs e.g. Filgrastim
(G-CSF), Neupogen, Oprelvekin, Neumega, Pegfilgrastim, Neulasta,
Sargramostim, Leukine; drugs for the treatment of hepatitis C e.g.
Interferon alfa-2a, Roferon A, Interferon alfa-2b, Intron A,
Interferon alfacon-1, Infergen, Peginterferon alfa-2a, Pegasys,
Peginterferon alfa-2b, PEG-Intron; drugs for the treatment of HIV
e.g. enfuvirtide, Fuzeon; Fabs e.g. Fab (antithrombin), Abciximab,
ReoPro; monoclonal antibodies e.g. Daclizumab, Zenapax; antiviral
monoclonal antibodies e.g. Palivizumab, Synagis; monoclonal
antibodies for the treatment of asthma e.g. Omalizumab, Xolair;
monoclonal antibodies for use in diagnostic imaging e.g.
Arcitumomab, CEA-Scan, Capromab Pendetide, ProstaScint, Satumomab
Pendetide, OncoScint CR/OV, Fabs for use in diagnostic imaging e.g.
Nofetumomab, Verluma; immuno-supressant monoclonal antibodies e.g.
Basiliximab, Simulect, Muromonab-CD3, Orthoclone OKT3; monoclonal
antibodies for the treatment of malignancy e.g. Alemtuzumab,
Campath, Ibritumomab tiuxetan, Zevalin, Rituximab, Rituxan,
Trastuzumab, Herceptin; monoclonal antibodies for the treatment of
rheumatoid arthritis (RA) e.g. Adalimumab, Humira, Infliximab,
Remicade; monoclonal antibodies for use as a
radio-immuno-therapeutic e.g. Tositumomab and Iodine I.sup.131,
Tositumomab, Bexxar; drugs for the treatment of macular
degeneration e.g. pegaptanib, Macugen; drugs for the treatment of
malignancy e.g. Aldesleukin, Proleukin, Interleukin-2,
Asparaginase, Elspar, Rasburicase, Elitek, Denileukin diftitox,
Ontak, Pegaspargase, Oncaspar, goserelin, leuprolide; drugs for the
treatment of multiple sclerosis (MS) e.g. Glatiramer acetate (e.g.
copolymer-1), Copaxone, Interferon beta-1a, Avonex, Interferon
beta-1a, Rebif, Interferon beta-1b, Betaseron; drugs for the
treatment of mucositis e.g. palifermin, Kepivance; drug for the
treatment of dystonia e.g., neurotoxin, Botulinum Toxin Type A,
BOTOX, BOTOX Cosmetic, Botulinum Toxin Type B, MYOBLOC; drugs for
the treatment of osteoporosis e.g. teriparatide, Forteo; drugs for
the treatment of psoriasis e.g. Alefacept, Amevive; drugs for the
treatment of RA e.g. abatacept, Orencia, Anakinra, Kineret,
Etanercept, Enbrel; thrombolytics e.g. Alteplase, Activase,
recombinant tissue plasminogen activator (rtPA), Anistreplase,
Eminase, Reteplase, Retavase, Streptokinase, Streptase,
Tenecteplase, TNKase (tenecteplase), Urokinase, Abbokinase,
Kinlytic; drugs for the treatment of osteoporosis e.g. calcitonin
(e.g. salmon), Miacalcin, Fortical, drugs for the treatment of skin
ulcers e.g. Becaplermin, Regranex, Collagenase, Santyl.
[0280] Such polypeptides and chemical compounds may be referred to
as diagnostic moieties, therapeutic moieties, prophylactic moieties
or beneficial moieties.
[0281] Preferably the fusion partner and/or conjugation partner is
not an albumin, variant or fragment thereof.
[0282] One or more (several) therapeutic or prophylactic
polypeptides may be fused to the N-terminus, the C-terminus of
albumin, inserted into a loop in the albumin structure or any
combination thereof. It may or it may not comprise linker sequences
separating the various components of the fusion polypeptide.
[0283] Teachings relating to fusions of albumin or a fragment
thereof are known in the art and the skilled person will appreciate
that such teachings can also be applied to the invention. WO
2001/79271A and WO 2003/59934 (incorporated herein by reference)
also contain examples of therapeutic and prophylactic polypeptides
that may be fused to albumin or fragments thereof, and these
examples apply also to the invention.
[0284] The invention is further described by the following examples
that should not be construed as limiting the scope of the
invention.
EXAMPLES
Example 1: Preparation of HSA Mutein Expression Plasmids
[0285] HSA variants were expressed using standard molecular biology
techniques, such as described in Sambrook, J. and D. W. Russell,
2001 (Molecular Cloning: a laboratory manual, 3.sup.rd ed. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y).
[0286] Construction of the K573P expression plasmid is described in
WO2011/051489 (incorporated herein by reference). Construction of
the remaining expression plasmids was performed as described in WO
2012/150319 (PCT/EP12/058206, incorporated herein by reference).
Variants HSA T83K, HSA E82A, HSA E82D, HSA P110G, HSA L112F and HSA
T83N/N111E were produced as described in Example 6, Method 2 of WO
2012/150319 (PCT/EP12/058206, incorporated herein by reference).
Combination mutants containing the K573P substitution were produced
as described in "Production of combination mutants with K573P" (WO
2012/150319 (PCT/EP12/058206)), where the required fragments were
inserted into appropriately digested pDB4852 (described in WO
2012/150319 (PCT/EP12/058206, incorporated herein by reference)).
Fragments containing T83N/N111E, T83K, E82A, E82D, P110G and L112F
were removed from synthetic constructs via the indicated
restriction sites (Table 1). The fragment containing the T83N
substitution was removed from pDB4874 (described in WO 2012/150319
(PCT/EP12/058206, incorporated herein by reference)). Ligation of
the polynucleotides encoding HSA variants and plasmids
pDB3964/pDB4852 produced plasmids, which were used to express the
desired mutants (Table 1). All plasmids were sequenced to confirm
that the HSA sequence was only mutated at the desired
position(s).
[0287] Construction of HSA T83N, HSA N111E and HSA N111E/K573P was
as described in WO 2012/150319 (PCT/EP12/058206, incorporated
herein by reference).
[0288] Transformation of S. cerevisiae was performed as described
in WO 2012/150319 (PCT/EP12/058206, incorporated herein by
reference), employing the 24 hour stocking method described in WO
2011/051489, with the exception that the host strain was S.
cerevisiae DYB7 (Payne et al (2008) Applied and Environmental
Microbiology Vol. 74(24): 7759-7766) with four copies of PDI
integrated into the genome.
TABLE-US-00005 TABLE 1 Construction of HSA mutein expression
plasmids. Digested Restriction fragment Variant enzymes size (kb)
Plasmid SEQ ID NO HSA T83N/N111E SacII/NheI 0.395 pDB4966 32 HSA
SacII/NheI 0.395 pDB4967 33 T83N/N111E/K573P HSA T83N/K573P
SacII/NheI 0.395 pDB4968 34 HSA T83K SacII/NheI 0.395 pDB4903 35
HSA E82A SacII/NheI 0.395 pDB4904 36 HSA L112F SacII/NheI 0.395
pDB4907 37 HSA T83K/K573P SacII/NheI 0.395 pDB4908 38 HSA
E82A/K573P SacII/NheI 0.395 pDB4909 39 HSA L112F/K573P SacII/NheI
0.395 pDB4912 40 HSA E82D SacII/NheI 0.395 pDB4905 41 HSA P110G
SacII/NheI 0.395 pDB4906 42 HSA E82D/K573P SacII/NheI 0.395 pDB4910
43 HSA P110G/K573P SacII/NheI 0.395 pDB4911 44
Example 2: SPR Analysis of Binding Affinity of Albumin Variants to
FcRn
[0289] SPR analyses were performed as described in WO 2012/150319
(PCT/EP12/058206, incorporated herein by reference).
[0290] The variants were albumin (SEQ ID NO: 2), each with one
point mutation selected from: D108A, N111D, N111G, N111H, N111K,
K190A, R197A, K276N, R410A, Y411A, P416A, E425A, E425K, K466A,
D471A, R472A, N503D, N503K, E505K, E505Q, H510D, H510E, D512A,
D512E, K524A, K525A, T527A, T527D, T527M, E531A, E531H, K534V,
H535F, E565V, A569L, A569S, A569V, and V576F.
[0291] Firstly, the variants were analyzed by SPR to determine
their binding response (RU) to shFcRn. Only variants showing a
binding response more than 20% higher or lower than the binding
response of wild-type albumin were analyzed to identify the KD
(Table 2, below). Wild-type HSA and HSA with mutation K573P were
used as controls.
TABLE-US-00006 TABLE 2 Binding affinity of albumin variants to
shFcRn Molecule SEQ ID NO: Ka (10.sup.3/Ms) Kd (10.sup.-3/s) KD
(.mu.M) WT rHSA 2 -- -- 3.1 .+-. 0.4* HSA K573P 3 -- -- 0.4 .+-.
0.1* HSA E505Q 45 2.1 2.9 1.4 HSA N111D 46 0.8 4.4 5.2 HSA T527M 47
2.7 3.3 1.2 HSA N111G 48 1.6 5.2 3.3 HSA N111H 49 0.5 2.4 5.0 HSA
D512E 50 2.7 10.9 4.1 HSA K524A 51 3.3 11.6 3.5 HSA T527A 52 2.6
13.7 5.2 HSA E531H 53 3.5 20.8 6.2 HSA N111K 54 0.5 8.3 17.3 HSA
E425K 55 3.6 12.4 3.5 HSA K534V 56 4.8 5.5 1.1 HSA H510D 57 0.2 0.4
0.2 HSA A569S 58 0.7 4.8 6.8 HSA D108A 59 0.9 12.7 13.7 *Mean of
five repeats, therefore Ka and Kd data are not provided
[0292] Variants with a lower KD than wild-type HSA have a higher
binding affinity to shFcRn. Conversely, variants with a higher KD
than wild-type HSA have a lower binding affinity to shFcRn.
[0293] The data for positions 108 and 111 support the involvement
of a loop including positions 105 to 120 in interaction with FcRn
and therefore that alteration at any position within this loop will
modulate the binding affinity of albumin to FcRn.
Example 3. SPR Analysis of Binding Affinity of Albumin Variants to
FcRn
[0294] The variants were albumin (SEQ ID NO: 2), each with one
point mutation selected from: N111D, N111G, N111H, N111D/K573P,
N111G/K573P, N111H/K573P, E505Q, E425A, T527M, E505Q/K573P,
E425A/K573P and T527M/K573P were prepared as described in
above.
TABLE-US-00007 TABLE 3 Binding affinity of albumin variants to
shFcRn-HIS SEQ ID Molecule NO: Ka (10.sup.3/Ms) Kd (10.sup.-3/s) KD
(.mu.M) WT rHSA 2 -- -- 3.6 .+-. 0.54* HSA K573P 3 -- -- 0.6 .+-.
0.12** HSA N111D 46 9.8 9.1 17.9 17.9 1.8 2.0 HSA N111G 48 7.4 7.4
20.5 19.2 2.7 2.6 HSA N111H 49 4.4 4.0 15.6 14.2 3.5 3.6 HSA
N111D/K573P 60 4.0 4.2 1.9 2.2 0.5 0.5 HSA N111G/K573P 61 4.1 4.7
1.7 2.3 0.4 0.5 HSA N111H/K573P 62 2.9 3.0 1.7 2.2 0.6 0.7 HSA
E505Q 45 5.1 5.0 4.9 6.0 1.0 1.2 HSA E425A 63 6.6 7.9 34.1 28.1 5.1
3.6 HSA T527M 47 4.9 4.8 4.4 5.1 0.9 1.1 HSA E425A/K573P 64 3.4 3.6
2.5 3.2 0.7 0.9 HSA E505Q/K573P 65 0.4 0.4 0.5 1.1 1.6 2.5 HSA
T527M/K573P 66 2.6 2.8 1.2 2.2 0.5 0.8 *Mean of 8 and standard
deviation **Mean of 5 and standard deviation.
Variants with a lower KD than wild-type HSA have a higher binding
affinity to shFcRn. Conversely, variants with a higher KD than
wild-type HSA have a lower binding affinity to shFcRn.
[0295] The data for variants including K573P generate increases in
affinity consistent with the K573P substitution only.
Example 4. SPR Analysis of Binding Affinity of Albumin Variants to
FcRn
[0296] The variants were albumin (SEQ ID NO: 2), each with one
point mutation selected from: N111R, N111Q, N111E, N111R/K573P,
N111Q/K573P, N111E/K573P, N109D, N109E, N109Q, N109R, N109K, N109H,
N109G, D108E, T83N, L575F and K534V/K573P were prepared as
described above.
TABLE-US-00008 TABLE 4a Binding affinity of albumin variants to
shFcRn-HIS SEQ ID Molecule NO: Ka (10.sup.3/Ms) Kd (10.sup.-3/s) KD
(.mu.M) WT HSA 2 -- -- 2.0 .+-. 0.3* HSA K573P 3 -- -- 0.3 .+-.
0.0** HSA N111E 67 15.3 14.3 13.1 15.2 0.8 1.1 HSA N111E/K573P 68
4.2 -- 2.4 -- 0.6 -- HSA N109K 69 9.7 6.3 18.3 21.6 1.9 3.4 HSA
D108E 70 13.9 7.5 16.6 19.5 1.2 2.6 HSA T83N 71 17.7 15.2 15.6 16.8
0.9 1.1 HSA L575F 72 11.8 8.3 31.3 32.2 2.7 4.0 HSA K534V/K573P 73
4.7 4.5 6.9 6.9 1.5 1.5 *Mean of 11 and standard deviation **Mean
of 5 and standard deviation.
TABLE-US-00009 TABLE 4b SEQ Molecule ID NO: Ka (10.sup.3/Ms) Kd
(10.sup.-3/s) KD (.mu.M) WT rHSA 2 -- -- 3.6 .+-. 0.54* HSA K573P 3
-- -- 0.6 .+-. 0.12** HSA N111D 46 9.8 9.1 17.9 17.9 1.8 2.0 HSA
N111G 48 7.4 7.4 20.5 19.2 2.7 2.6 HSA N111H 49 4.4 4.0 15.6 14.2
3.5 3.6 *Mean of 8 and standard deviation **Mean of 5 and standard
deviation.
The data demonstrate a role for the 108 to 111 loop in binding of
HSA to FcRn, with reduced binding affinity observed in the D108A
and N111K variants (Table 2). Additional mutations at position 111
demonstrated a range of binding affinities, from the reduced
affinity observed for the N111K variant through to the N111E
variant, which displayed an increased affinity for FcRn as compared
to WT HSA (Table 4). Variant N111Q/K573P (FIG. 5, SEQ ID NO: 74)
shows a binding curve with increased response compared to wt HSA
and slower dissociation compared to wt HSA, this is consistent with
the K573P substitution. The relative position of loop region 108 to
112 of HSA and FcRn (FIG. 6) suggests that this region has
potential to contribute to FcRn binding as predicted in Example 2.
Further details regarding FIGS. 5 and 6 are provided in WO
2012/150319 (PCT/EP12/058206, incorporated herein by
reference).
[0297] The relative position of adjacent loop region of Domain I
(domain 1), comprising residues 78 to 88 (FIG. 6), suggests that
this region has potential to contribute to FcRn binding. This is
supported by the observation that the T83N variant shows increased
affinity for FcRn compared to WT HSA (Table 4).
[0298] Mutation of the adjacent residues, particularly E82, P110
and L112 (FIG. 6), would be predicted to alter the binding affinity
of HSA for FcRn.
Example 5: SPR Analysis of Binding Affinity of Albumin Variants to
FcRn
[0299] SPR analyses were performed on a Biacore 3000 instrument (GE
Healthcare). Immobilization was carried out on CM5 chips coupled
with shFcRn (GeneArt 1177525) using GE Healthcare amine coupling
chemistry as per manufacturer's instructions. Immobilized levels of
shFcRn-HIS (shFcRn with a 6-His tail on the C-terminus of
beta-2-microglobulin) were .about.1200 RU and achieved by injecting
20 .mu.g/mL shFcRn in sodium acetate pH4.5 (GE Healthcare). Chip
surface was left to stabilize with a constant flow (5 .mu.L/min) of
running buffer--Di-basic/Mono-basic phosphate buffer pH5.5 at
25.degree. C. overnight. After ligand stabilization, the chip
surface was conditioned by injecting 3.times.45 .mu.L
Di-basic/Mono-basic phosphate buffer at 30 .mu.L/min followed by
HBS_EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant
P20) at pH 7.4 (GE Healthcare)) regeneration steps (12 s) in
between each injection. Surfaces were then checked for activity by
injecting 3.times.45 .mu.L positive control at 30 .mu.L/min,
followed by 12 s regeneration pulse.
[0300] pH 5.5 Binding Analysis: Sensorgrams for binding data were
obtained by injecting 45 .mu.L of 20 .mu.M (diluted in pH 5.5
buffer) of analytes in pH 5.5 running buffer at 30 .mu.L/min in
duplicate. 2.times.12 s regeneration pulses post injection were
performed to restore the baseline (HBS-EP pH 7.4; 10 .mu.L at 50
.mu.L/min). The reference was then subtracted and BiaEvaluation
software 4.1 used to obtain binding analysis data.
[0301] pH 5.5 Kinetic Analysis: Sensorgrams for kinetic data were
obtained by injecting 45 .mu.L of five concentrations: 20 .mu.M, 4
.mu.M, 0.8 .mu.M 0.16 .mu.M and 0.032 .mu.M of analytes in pH 5.5
running buffer at 30 .mu.L/min with a 90 s delay post injection (to
allow smooth dissociation for kinetic modelling). 2.times.12 s
regeneration pulses post injection were performed to restore the
baseline (HBS-EP pH 7.4; 10 .mu.L at 50 .mu.L/min). Analysis was
performed on two separate occasions. The reference cell value was
then subtracted and Biaevaluation software 4.1 used to obtain
kinetic data and confirm KD values.
[0302] SPR was used to identify the binding response of variants to
FcRn, the results are shown in Tables 5a and 5b.
TABLE-US-00010 TABLE 5a Molecule SEQ ID NO Binding Response (RU) WT
rHSA 2 229 HSA K573P 3 300 HSA T83K 35 194 HSA T83K/K573P 38 285
HSA E82A 36 221 HSA E82A/K573P 39 275 HSA E82D 41 227 HSA
E82D/K573P 43 269 HSA P110G 42 235 HSA P110G/K573P 44 284 HSA L112F
37 253 HSA L112F/K573P 40 290
Values shown are a mean of two runs.
TABLE-US-00011 TABLE 5b Molecule SEQ ID NO Binding Response (RU) WT
rHSA 2 148 HSA K573P 3 181 HSA T83N/N111E 32 167
Values shown are a mean of two runs.
[0303] KD analysis was performed on variants to assess variant-FcRn
binding affinity relative to HSA-K573-FcRn binding affinity. The
results are shown in Table 6. Further analysis was carried out to
calculate binding affinities (Table 7).
TABLE-US-00012 TABLE 6 Binding affinity (fold SEQ ID difference,
relative to HSA Molecule NO: KD (.mu.M) wild-type) WT rHSA 2 3.82
-- HSA L112F 37 1.44 2.7 HSA T83K 35 1.42 2.7 HSA E82A 36 2.81 1.4
HSA K573P 3 0.18 21.2 HSA L112F/K573P 40 0.108 35.4 HSA T83K/K573P
38 0.147 26.0 HSA E82A/K573P 39 0.174 22.0
TABLE-US-00013 TABLE 7 Fold difference SEQ Mean compared ID KD KD
to HSA Molecule NO: Ka (1/Ms) Kd (1/s) (.mu.M) (.mu.M) wild-type WT
rHSA 2 0.63 .times. 10.sup.4 0.0133 2.11 1.97 -- 0.78 .times.
10.sup.4 0.0141 1.83 HSA K573P 3 0.81 .times. 10.sup.4 1.32 .times.
10.sup.-3 0.162 0.20 9.9 0.74 .times. 10.sup.4 1.77 .times.
10.sup.-3 0.238 HSA T83N/ 33 2.28 .times. 10.sup.4 1.16 .times.
10.sup.-3 0.051 0.061 32.3 N111E/ 2.28 .times. 10.sup.4 1.59
.times. 10.sup.-3 0.070 K573P HSA T83N/ 34 1.55 .times. 10.sup.4
1.3 .times. 10.sup.-3 0.084 0.12 16.4 K573P 1.22 .times. 10.sup.4
1.84 .times. 10.sup.-3 0.15
[0304] The data show that HSA T83N/N111E/K573P and HSA T83N/K573P
have high FcRn binding affinities relative to wild-type HSA. HSA
E82A and HSA L112F both show improved binding to FcRn compared to
wild-type HSA binding to FcRn and this suggests that the loops
comprising amino acids 78 to 88 of HSA (SEQ ID NO: 2) and 105 to
120 of HSA (SEQ ID NO: 2) are involved in the binding of HSA to
FcRn.
[0305] HSA with single mutations at position L112 or T83 show
similar FcRn binding affinities to each other. However, the double
mutation of L112 and K573 has a stronger binding affinity to FcRn
than the double mutation of T83 and K573.
TABLE-US-00014 TABLE 8 SEQ Ka KD Mean KD Molecule ID NO:
(10.sup.3/MS) Kd (10.sup.3/s) (.mu.M) (.mu.M) WT HSA 2 4.3 63.6
13.7 13.8 5.6 77.6 13.9 HSA-K573P 3 4.3 6.2 1.4 1.1 5.2 4.6 0.89
HSA-E82D 41 2.3 84.3 36.9 24.1 6.5 73.0 11.3 HSA-E82D/K573P 43 4.9
6.7 1.4 1.1 5.5 5.0 0.9
[0306] The data of Table 8 show that HSA-E82D has a low FcRn
binding affinity relative to wild-type albumin and HSA-K573P has a
high FcRn binding relative to wild-type albumin. However, the
double mutant HSA-E82D/K573P shows the same FcRn binding affinity
as HSA-K573P, i.e. inclusion of the E82D substitution does not
adversely affect FcRn binding.
[0307] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
Sequence CWU 1
1
7411758DNAHomo sapiensmisc_feature(1)..(1758)cDNA encoding HSA
1gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa
60gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta
120aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga
gtcagctgaa 180aattgtgaca aatcacttca tacccttttt ggagacaaat
tatgcacagt tgcaactctt 240cgtgaaacct atggtgaaat ggctgactgc
tgtgcaaaac aagaacctga gagaaatgaa 300tgcttcttgc aacacaaaga
tgacaaccca aacctccccc gattggtgag accagaggtt 360gatgtgatgt
gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat
420gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt
tgctaaaagg 480tataaagctg cttttacaga atgttgccaa gctgctgata
aagctgcctg cctgttgcca 540aagctcgatg aacttcggga tgaagggaag
gcttcgtctg ccaaacagag actcaagtgt 600gccagtctcc aaaaatttgg
agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660cagagatttc
ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa
720gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag
ggcggacctt 780gccaagtata tctgtgaaaa tcaagattcg atctccagta
aactgaagga atgctgtgaa 840aaacctctgt tggaaaaatc ccactgcatt
gccgaagtgg aaaatgatga gatgcctgct 900gacttgcctt cattagctgc
tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960gaggcaaagg
atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat
1020tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct
agagaagtgc 1080tgtgccgctg cagatcctca tgaatgctat gccaaagtgt
tcgatgaatt taaacctctt 1140gtggaagagc ctcagaattt aatcaaacaa
aattgtgagc tttttgagca gcttggagag 1200tacaaattcc agaatgcgct
attagttcgt tacaccaaga aagtacccca agtgtcaact 1260ccaactcttg
tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat
1320cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct
gaaccagtta 1380tgtgtgttgc atgagaaaac gccagtaagt gacagagtca
ccaaatgctg cacagaatcc 1440ttggtgaaca ggcgaccatg cttttcagct
ctggaagtcg atgaaacata cgttcccaaa 1500gagtttaatg ctgaaacatt
caccttccat gcagatatat gcacactttc tgagaaggag 1560agacaaatca
agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca
1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa
gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa
aacttgttgc tgcaagtcaa 1740gctgccttag gcttataa 17582585PRTHomo
sapiens 2Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu
Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln
Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn
Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala
Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu
Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala
Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu
Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg
Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu
Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg
His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150
155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala
Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly
Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu
Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala
Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val
Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu
Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala
Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265
270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu
Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys
Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met
Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val
Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu
Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala
Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln
Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390
395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val
Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn
Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu
Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp
Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg
Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val
Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505
510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu
Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu
Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala
Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu
Gly Leu 580 5853585PRTArtificial SequenceHSA K573P 3Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 5854609PRTHomo
sapiens 4Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser
Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser
Glu Val Ala 20 25 30His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys
Ala Leu Val Leu 35 40 45Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro
Phe Glu Asp His Val 50 55 60Lys Leu Val Asn Glu Val Thr Glu Phe Ala
Lys Thr Cys Val Ala Asp65 70 75 80Glu Ser Ala Glu Asn Cys Asp Lys
Ser Leu His Thr Leu Phe Gly Asp 85 90 95Lys Leu Cys Thr Val Ala Thr
Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110Asp Cys Cys Ala Lys
Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125His Lys Asp
Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140Asp
Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys145 150
155 160Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala
Pro 165 170 175Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe
Thr Glu Cys 180 185 190Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu
Pro Lys Leu Asp Glu 195 200 205Leu Arg Asp Glu Gly Lys Ala Ser Ser
Ala Lys Gln Arg Leu Lys Cys 210 215 220Ala Ser Leu Gln Lys Phe Gly
Glu Arg Ala Phe Lys Ala Trp Ala Val225 230 235 240Ala Arg Leu Ser
Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255Lys Leu
Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265
270Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile
275 280 285Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys
Cys Glu 290 295 300Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu
Val Glu Asn Asp305 310 315 320Glu Met Pro Ala Asp Leu Pro Ser Leu
Ala Ala Asp Phe Val Glu Ser 325 330 335Lys Asp Val Cys Lys Asn Tyr
Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350Met Phe Leu Tyr Glu
Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365Leu Leu Leu
Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 370 375 380Cys
Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu385 390
395 400Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn
Cys 405 410 415Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn
Ala Leu Leu 420 425 430Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser
Thr Pro Thr Leu Val 435 440 445Glu Val Ser Arg Asn Leu Gly Lys Val
Gly Ser Lys Cys Cys Lys His 450 455 460Pro Glu Ala Lys Arg Met Pro
Cys Ala Glu Asp Tyr Leu Ser Val Val465 470 475 480Leu Asn Gln Leu
Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495Val Thr
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505
510Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala
515 520 525Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu
Lys Glu 530 535 540Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu
Val Lys His Lys545 550 555 560Pro Lys Ala Thr Lys Glu Gln Leu Lys
Ala Val Met Asp Asp Phe Ala 565 570 575Ala Phe Val Glu Lys Cys Cys
Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590Ala Glu Glu Gly Lys
Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 595 600
605Leu5621PRTPan troglodytes 5Met Asn Glu Ser Ser Cys Cys Ser Thr
Ser Leu Pro Ala Phe Gly Val1 5 10 15Ser Val Leu Asp Ser Gly His Ser
Ser Ser Ser Ala Tyr Ser Arg Gly 20 25 30Val Phe Arg Arg Asp Ala His
Lys Ser Glu Val Ala His Arg Phe Lys 35 40 45Asp Leu Gly Glu Glu Asn
Phe Lys Ala Leu Val Leu Val Ala Phe Ala 50 55 60Gln Tyr Leu Gln Gln
Cys Pro Phe Glu Asp His Val Lys Leu Val Asn65 70 75 80Glu Val Thr
Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu 85 90 95Asn Cys
Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr 100 105
110Val Ala Thr Leu Arg Glu Lys Tyr Gly Glu Met Ala Asp Cys Cys Ala
115 120 125Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys
Asp Asp 130 135 140Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val
Asp Val Met Cys145 150 155 160Thr Ala Phe His Asp Asn Glu Gly Thr
Phe Leu Lys Lys Tyr Leu Tyr 165 170 175Glu Val Ala Arg Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe 180 185 190Phe Ala Glu Arg Tyr
Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala 195 200 205Asp Lys Ala
Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu 210 215 220Gly
Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln225 230
235 240Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu
Ser 245 250 255Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys
Leu Val Thr 260 265 270Asp Leu Thr Lys Val His Thr Glu Cys Cys His
Gly Asp Leu Leu Glu 275 280 285Cys Ala Asp Asp Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln 290 295 300Asp Ser Ile Ser Ser Lys Leu
Lys Glu Cys Cys Glu Lys Pro Leu Leu305 310 315 320Glu Lys Ser His
Cys Leu Ala Glu Val Glu Asn Asp Glu Met Pro Ala 325 330 335Asp Leu
Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Glu Val Cys 340 345
350Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
355 360 365Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu
Leu
Leu Arg 370 375 380Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala385 390 395 400Asp Pro His Glu Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu 405 410 415Val Glu Glu Pro Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu 420 425 430Gln Leu Gly Glu Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr 435 440 445Lys Lys Val
Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 450 455 460Asn
Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys465 470
475 480Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
Leu 485 490 495Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val
Thr Lys Cys 500 505 510Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu 515 520 525Val Asp Glu Thr Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr 530 535 540Phe His Ala Asp Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys545 550 555 560Lys Gln Thr Ala
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr 565 570 575Lys Glu
Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu 580 585
590Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
595 600 605Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 610
615 6206608PRTMacaca mulatta 6Met Lys Trp Val Thr Phe Ile Ser Leu
Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe Arg Arg
Asp Thr His Lys Ser Glu Val Ala 20 25 30His Arg Phe Lys Asp Leu Gly
Glu Glu His Phe Lys Gly Leu Val Leu 35 40 45Val Ala Phe Ser Gln Tyr
Leu Gln Gln Cys Pro Phe Glu Glu His Val 50 55 60Lys Leu Val Asn Glu
Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp65 70 75 80Glu Ser Ala
Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95Lys Leu
Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105
110Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln
115 120 125His Lys Asp Asp Asn Pro Asn Leu Pro Pro Leu Val Arg Pro
Glu Val 130 135 140Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Ala
Thr Phe Leu Lys145 150 155 160Lys Tyr Leu Tyr Glu Val Ala Arg Arg
His Pro Tyr Phe Tyr Ala Pro 165 170 175Glu Leu Leu Phe Phe Ala Ala
Arg Tyr Lys Ala Ala Phe Ala Glu Cys 180 185 190Cys Gln Ala Ala Asp
Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205Leu Arg Asp
Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220Ala
Ser Leu Gln Lys Phe Gly Asp Arg Ala Phe Lys Ala Trp Ala Val225 230
235 240Ala Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Ala Glu Val
Ser 245 250 255Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys
Cys His Gly 260 265 270Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp
Leu Ala Lys Tyr Met 275 280 285Cys Glu Asn Gln Asp Ser Ile Ser Ser
Lys Leu Lys Glu Cys Cys Asp 290 295 300Lys Pro Leu Leu Glu Lys Ser
His Cys Leu Ala Glu Val Glu Asn Asp305 310 315 320Glu Met Pro Ala
Asp Leu Pro Ser Leu Ala Ala Asp Tyr Val Glu Ser 325 330 335Lys Asp
Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345
350Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Met
355 360 365Leu Leu Leu Arg Leu Ala Lys Ala Tyr Glu Ala Thr Leu Glu
Lys Cys 370 375 380Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys
Val Phe Asp Glu385 390 395 400Phe Gln Pro Leu Val Glu Glu Pro Gln
Asn Leu Val Lys Gln Asn Cys 405 410 415Glu Leu Phe Glu Gln Leu Gly
Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430Val Arg Tyr Thr Lys
Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445Glu Val Ser
Arg Asn Leu Gly Lys Val Gly Ala Lys Cys Cys Lys Leu 450 455 460Pro
Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val465 470
475 480Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu
Lys 485 490 495Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg
Pro Cys Phe 500 505 510Ser Ala Leu Glu Leu Asp Glu Ala Tyr Val Pro
Lys Ala Phe Asn Ala 515 520 525Glu Thr Phe Thr Phe His Ala Asp Met
Cys Thr Leu Ser Glu Lys Glu 530 535 540Lys Gln Val Lys Lys Gln Thr
Ala Leu Val Glu Leu Val Lys His Lys545 550 555 560Pro Lys Ala Thr
Lys Glu Gln Leu Lys Gly Val Met Asp Asn Phe Ala 565 570 575Ala Phe
Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Ala Cys Phe 580 585
590Ala Glu Glu Gly Pro Lys Phe Val Ala Ala Ser Gln Ala Ala Leu Ala
595 600 6057608PRTMesocricetus auratus 7Met Lys Trp Val Thr Phe Leu
Leu Leu Leu Phe Val Ser Asp Ser Ala1 5 10 15Phe Ser Arg Gly Leu Phe
Arg Arg Asp Ala His Lys Ser Glu Ile Ala 20 25 30His Arg Phe Lys Asp
Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45Ile Ala Phe Ser
Gln Phe Leu Gln Lys Cys Pro Tyr Glu Glu His Val 50 55 60Lys Leu Val
Asn Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp65 70 75 80Glu
Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90
95Lys Leu Cys Ala Ile Pro Thr Leu Arg Asp Ser Tyr Gly Glu Leu Ala
100 105 110Asp Cys Cys Ala Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe
Leu Lys 115 120 125His Lys Asp Asp His Pro Asn Leu Pro Pro Phe Val
Arg Pro Asp Ala 130 135 140Glu Ala Met Cys Thr Ser Phe Gln Glu Asn
Ala Val Thr Phe Met Gly145 150 155 160His Tyr Leu His Glu Val Ala
Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175Glu Leu Leu Tyr Tyr
Ala Glu Lys Tyr Ser Ala Ile Met Thr Glu Cys 180 185 190Cys Gly Glu
Ala Asp Lys Ala Ala Cys Ile Thr Pro Lys Leu Asp Ala 195 200 205Leu
Lys Glu Lys Ala Leu Ala Ser Ser Val Asn Gln Arg Leu Lys Cys 210 215
220Ser Ser Leu Gln Arg Phe Gly Gln Arg Ala Phe Lys Ala Trp Ala
Val225 230 235 240Ala Arg Met Ser Gln Lys Phe Pro Lys Ala Asp Phe
Ala Glu Ile Thr 245 250 255Lys Leu Ala Thr Asp Leu Thr Lys Leu Thr
Glu Glu Cys Cys His Gly 260 265 270Asp Leu Leu Glu Cys Ala Asp Asp
Arg Ala Glu Leu Ala Lys Tyr Met 275 280 285Cys Glu Asn Gln Ala Ser
Ile Ser Ser Lys Leu Gln Ala Cys Cys Asp 290 295 300Lys Pro Val Leu
Lys Lys Ser His Cys Leu Ser Glu Val Glu Asn Asp305 310 315 320Asp
Leu Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Asp 325 330
335Lys Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly
340 345 350Thr Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser
Val Ala 355 360 365Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr
Leu Glu Lys Cys 370 375 380Cys Ala Glu Ala Asp Pro Ser Ala Cys Tyr
Gly Lys Val Leu Asp Glu385 390 395 400Phe Gln Pro Leu Val Glu Glu
Pro Lys Asn Leu Val Lys Ala Asn Cys 405 410 415Glu Leu Phe Glu Lys
Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile 420 425 430Val Arg Tyr
Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445Glu
Ala Ala Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Val Leu 450 455
460Pro Glu Ala Gln Arg Leu Pro Cys Val Glu Asp Tyr Ile Ser Ala
Ile465 470 475 480Leu Asn Arg Val Cys Val Leu His Glu Lys Thr Pro
Val Ser Glu Gln 485 490 495Val Thr Lys Cys Cys Thr Gly Ser Val Val
Glu Arg Arg Pro Cys Phe 500 505 510Ser Ala Leu Pro Val Asp Glu Thr
Tyr Val Pro Lys Glu Phe Lys Ala 515 520 525Glu Thr Phe Thr Phe His
Ala Asp Ile Cys Ser Leu Pro Glu Lys Glu 530 535 540Lys Gln Met Lys
Lys Gln Ala Ala Leu Val Glu Leu Val Lys His Lys545 550 555 560Pro
Lys Ala Thr Gly Pro Gln Leu Arg Thr Val Leu Gly Glu Phe Thr 565 570
575Ala Phe Leu Asp Lys Cys Cys Lys Ala Glu Asp Lys Glu Ala Cys Phe
580 585 590Ser Glu Asp Gly Pro Lys Leu Val Ala Ser Ser Gln Ala Ala
Leu Ala 595 600 6058608PRTCavia porcellus 8Met Lys Trp Val Thr Phe
Ile Ser Leu Leu Phe Leu Phe Ser Ser Val1 5 10 15Tyr Ser Arg Gly Val
Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30His Arg Phe Asn
Asp Leu Gly Glu Gly His Phe Lys Gly Leu Val Leu 35 40 45Ile Thr Leu
Ser Gln His Leu Gln Lys Ser Pro Phe Glu Glu His Val 50 55 60Lys Leu
Val Asn Glu Val Thr Asp Phe Ala Lys Ala Cys Val Ala Asp65 70 75
80Glu Ser Ala Gln Asn Cys Gly Lys Ala Ile Ala Thr Leu Phe Gly Asp
85 90 95Lys Val Cys Ala Ile Pro Ser Leu Arg Glu Thr Tyr Gly Glu Leu
Ala 100 105 110Asp Cys Cys Ala Lys Glu Asp Pro Asp Arg Val Glu Cys
Phe Leu Gln 115 120 125His Lys Asp Asp Asn Pro Asn Leu Pro Pro Phe
Glu Arg Pro Glu Pro 130 135 140Glu Ala Leu Cys Thr Ala Phe Lys Glu
Asn Asn Asp Arg Phe Ile Gly145 150 155 160His Tyr Leu Tyr Glu Val
Ser Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175Glu Leu Leu Tyr
Tyr Ala Glu Lys Tyr Lys Asn Ala Leu Thr Glu Cys 180 185 190Cys Glu
Ala Ala Asp Lys Ala Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200
205Ile Lys Glu Lys Ala Leu Val Ser Ser Ala Gln Gln Arg Leu Lys Cys
210 215 220Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp
Ser Val225 230 235 240Ala Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu
Phe Ala Glu Ile Ser 245 250 255Thr Ile Val Thr Ser Leu Thr Lys Val
Thr Lys Glu Cys Cys His Gly 260 265 270Asp Leu Leu Glu Cys Ala Asp
Asp Arg Gln Glu Leu Ala Lys Tyr Met 275 280 285Cys Glu His Gln Asp
Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Val 290 295 300Lys Pro Thr
Leu Gln Lys Ala His Cys Ile Leu Glu Ile Gln Arg Asp305 310 315
320Glu Leu Pro Thr Glu Leu Pro Asp Leu Ala Val Asp Phe Val Glu Asp
325 330 335Lys Glu Val Cys Lys Asn Phe Ala Glu Ala Lys Asp Val Phe
Leu Gly 340 345 350Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu
Tyr Ser Ile Gly 355 360 365Met Leu Leu Arg Ile Ala Lys Gly Tyr Glu
Ala Lys Leu Glu Lys Cys 370 375 380Cys Ala Glu Ala Asp Pro His Ala
Cys Tyr Ala Lys Val Phe Asp Glu385 390 395 400Leu Gln Pro Leu Ile
Asp Glu Pro Lys Lys Leu Val Gln Gln Asn Cys 405 410 415Glu Leu Phe
Asp Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ala 420 425 430Val
Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440
445Glu Tyr Ala Arg Lys Leu Gly Ser Val Gly Thr Lys Cys Cys Ser Leu
450 455 460Pro Glu Thr Glu Arg Leu Ser Cys Thr Glu Asn Tyr Leu Ala
Leu Ile465 470 475 480Leu Asn Arg Leu Cys Ile Leu His Glu Lys Thr
Pro Val Ser Glu Arg 485 490 495Val Thr Lys Cys Cys Thr Glu Ser Leu
Val Asn Arg Arg Pro Cys Phe 500 505 510Ser Ala Leu His Val Asp Glu
Thr Tyr Val Pro Lys Pro Phe His Ala 515 520 525Asp Ser Phe Thr Phe
His Ala Asp Ile Cys Thr Leu Pro Glu Lys Glu 530 535 540Lys Gln Val
Lys Lys Gln Met Ala Leu Val Glu Leu Val Lys His Lys545 550 555
560Pro Lys Ala Ser Glu Glu Gln Met Lys Thr Val Met Gly Asp Phe Ala
565 570 575Ala Phe Leu Lys Lys Cys Cys Asp Ala Asp Asn Lys Glu Ala
Cys Phe 580 585 590Thr Glu Asp Gly Pro Lys Leu Val Ala Lys Cys Gln
Ala Thr Leu Ala 595 600 6059608PRTMus musculus 9Met Lys Trp Val Thr
Phe Leu Leu Leu Leu Phe Val Ser Gly Ser Ala1 5 10 15Phe Ser Arg Gly
Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30His Arg Tyr
Asn Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45Ile Ala
Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala 50 55 60Lys
Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp65 70 75
80Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp
85 90 95Lys Leu Cys Ala Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu
Ala 100 105 110Asp Cys Cys Thr Lys Gln Glu Pro Glu Arg Asn Glu Cys
Phe Leu Gln 115 120 125His Lys Asp Asp Asn Pro Ser Leu Pro Pro Phe
Glu Arg Pro Glu Ala 130 135 140Glu Ala Met Cys Thr Ser Phe Lys Glu
Asn Pro Thr Thr Phe Met Gly145 150 155 160His Tyr Leu His Glu Val
Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175Glu Leu Leu Tyr
Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys 180 185 190Cys Ala
Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly 195 200
205Val Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys
210 215 220Ser Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp
Ala Val225 230 235 240Ala Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp
Phe Ala Glu Ile Thr 245 250 255Lys Leu Ala Thr Asp Leu Thr Lys Val
Asn Lys Glu Cys Cys His Gly 260 265 270Asp Leu Leu Glu Cys Ala Asp
Asp Arg Ala Glu Leu Ala Lys Tyr Met 275 280 285Cys Glu Asn Gln Ala
Thr Ile Ser Ser Lys Leu Gln Thr Cys Cys Asp 290 295 300Lys Pro Leu
Leu Lys Lys Ala His Cys Leu Ser Glu Val Glu His Asp305 310 315
320Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp Phe Val Glu Asp
325 330 335Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe
Leu Gly 340 345 350Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp
Tyr Ser Val Ser 355 360 365Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu
Ala Thr Leu Glu Lys Cys 370 375 380Cys Ala Glu Ala Asn Pro Pro Ala
Cys Tyr Gly Thr Val Leu Ala
Glu385 390 395 400Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val
Lys Thr Asn Cys 405 410 415Asp Leu Tyr Glu Lys Leu Gly Glu Tyr Gly
Phe Gln Asn Ala Ile Leu 420 425 430Val Arg Tyr Thr Gln Lys Ala Pro
Gln Val Ser Thr Pro Thr Leu Val 435 440 445Glu Ala Ala Arg Asn Leu
Gly Arg Val Gly Thr Lys Cys Cys Thr Leu 450 455 460Pro Glu Asp Gln
Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile465 470 475 480Leu
Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His 485 490
495Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe
500 505 510Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe
Lys Ala 515 520 525Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu
Pro Glu Lys Glu 530 535 540Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala
Glu Leu Val Lys His Lys545 550 555 560Pro Lys Ala Thr Ala Glu Gln
Leu Lys Thr Val Met Asp Asp Phe Ala 565 570 575Gln Phe Leu Asp Thr
Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe 580 585 590Ser Thr Glu
Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala 595 600
60510608PRTRattus norvegicus 10Met Lys Trp Val Thr Phe Leu Leu Leu
Leu Phe Ile Ser Gly Ser Ala1 5 10 15Phe Ser Arg Gly Val Phe Arg Arg
Glu Ala His Lys Ser Glu Ile Ala 20 25 30His Arg Phe Lys Asp Leu Gly
Glu Gln His Phe Lys Gly Leu Val Leu 35 40 45Ile Ala Phe Ser Gln Tyr
Leu Gln Lys Cys Pro Tyr Glu Glu His Ile 50 55 60Lys Leu Val Gln Glu
Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp65 70 75 80Glu Asn Ala
Glu Asn Cys Asp Lys Ser Ile His Thr Leu Phe Gly Asp 85 90 95Lys Leu
Cys Ala Ile Pro Lys Leu Arg Asp Asn Tyr Gly Glu Leu Ala 100 105
110Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln
115 120 125His Lys Asp Asp Asn Pro Asn Leu Pro Pro Phe Gln Arg Pro
Glu Ala 130 135 140Glu Ala Met Cys Thr Ser Phe Gln Glu Asn Pro Thr
Ser Phe Leu Gly145 150 155 160His Tyr Leu His Glu Val Ala Arg Arg
His Pro Tyr Phe Tyr Ala Pro 165 170 175Glu Leu Leu Tyr Tyr Ala Glu
Lys Tyr Asn Glu Val Leu Thr Gln Cys 180 185 190Cys Thr Glu Ser Asp
Lys Ala Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200 205Val Lys Glu
Lys Ala Leu Val Ala Ala Val Arg Gln Arg Met Lys Cys 210 215 220Ser
Ser Met Gln Arg Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val225 230
235 240Ala Arg Met Ser Gln Arg Phe Pro Asn Ala Glu Phe Ala Glu Ile
Thr 245 250 255Lys Leu Ala Thr Asp Val Thr Lys Ile Asn Lys Glu Cys
Cys His Gly 260 265 270Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu
Leu Ala Lys Tyr Met 275 280 285Cys Glu Asn Gln Ala Thr Ile Ser Ser
Lys Leu Gln Ala Cys Cys Asp 290 295 300Lys Pro Val Leu Gln Lys Ser
Gln Cys Leu Ala Glu Ile Glu His Asp305 310 315 320Asn Ile Pro Ala
Asp Leu Pro Ser Ile Ala Ala Asp Phe Val Glu Asp 325 330 335Lys Glu
Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345
350Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser
355 360 365Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu
Lys Cys 370 375 380Cys Ala Glu Gly Asp Pro Pro Ala Cys Tyr Gly Thr
Val Leu Ala Glu385 390 395 400Phe Gln Pro Leu Val Glu Glu Pro Lys
Asn Leu Val Lys Thr Asn Cys 405 410 415Glu Leu Tyr Glu Lys Leu Gly
Glu Tyr Gly Phe Gln Asn Ala Val Leu 420 425 430Val Arg Tyr Thr Gln
Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445Glu Ala Ala
Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu 450 455 460Pro
Glu Ala Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile465 470
475 480Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu
Lys 485 490 495Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg
Pro Cys Phe 500 505 510Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro
Lys Glu Phe Lys Ala 515 520 525Glu Thr Phe Thr Phe His Ser Asp Ile
Cys Thr Leu Pro Asp Lys Glu 530 535 540Lys Gln Ile Lys Lys Gln Thr
Ala Leu Ala Glu Leu Val Lys His Lys545 550 555 560Pro Lys Ala Thr
Glu Asp Gln Leu Lys Thr Val Met Gly Asp Phe Ala 565 570 575Gln Phe
Val Asp Lys Cys Cys Lys Ala Ala Asp Lys Asp Asn Cys Phe 580 585
590Ala Thr Glu Gly Pro Asn Leu Val Ala Arg Ser Lys Glu Ala Leu Ala
595 600 60511607PRTBos taurus 11Met Lys Trp Val Thr Phe Ile Ser Leu
Leu Leu Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe Arg Arg
Asp Thr His Lys Ser Glu Ile Ala 20 25 30His Arg Phe Lys Asp Leu Gly
Glu Glu His Phe Lys Gly Leu Val Leu 35 40 45Ile Ala Phe Ser Gln Tyr
Leu Gln Gln Cys Pro Phe Asp Glu His Val 50 55 60Lys Leu Val Asn Glu
Leu Thr Glu Phe Ala Lys Thr Cys Val Ala Asp65 70 75 80Glu Ser His
Ala Gly Cys Glu Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95Glu Leu
Cys Lys Val Ala Ser Leu Arg Glu Thr Tyr Gly Asp Met Ala 100 105
110Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Ser
115 120 125His Lys Asp Asp Ser Pro Asp Leu Pro Lys Leu Lys Pro Asp
Pro Asn 130 135 140Thr Leu Cys Asp Glu Phe Lys Ala Asp Glu Lys Lys
Phe Trp Gly Lys145 150 155 160Tyr Leu Tyr Glu Ile Ala Arg Arg His
Pro Tyr Phe Tyr Ala Pro Glu 165 170 175Leu Leu Tyr Tyr Ala Asn Lys
Tyr Asn Gly Val Phe Gln Glu Cys Cys 180 185 190Gln Ala Glu Asp Lys
Gly Ala Cys Leu Leu Pro Lys Ile Glu Thr Met 195 200 205Arg Glu Lys
Val Leu Ala Ser Ser Ala Arg Gln Arg Leu Arg Cys Ala 210 215 220Ser
Ile Gln Lys Phe Gly Glu Arg Ala Leu Lys Ala Trp Ser Val Ala225 230
235 240Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Val Glu Val Thr
Lys 245 250 255Leu Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys
His Gly Asp 260 265 270Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu
Ala Lys Tyr Ile Cys 275 280 285Asp Asn Gln Asp Thr Ile Ser Ser Lys
Leu Lys Glu Cys Cys Asp Lys 290 295 300Pro Leu Leu Glu Lys Ser His
Cys Ile Ala Glu Val Glu Lys Asp Ala305 310 315 320Ile Pro Glu Asn
Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Lys 325 330 335Asp Val
Cys Lys Asn Tyr Gln Glu Ala Lys Asp Ala Phe Leu Gly Ser 340 345
350Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Glu Tyr Ala Val Ser Val
355 360 365Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Glu
Cys Cys 370 375 380Ala Lys Asp Asp Pro His Ala Cys Tyr Ser Thr Val
Phe Asp Lys Leu385 390 395 400Lys His Leu Val Asp Glu Pro Gln Asn
Leu Ile Lys Gln Asn Cys Asp 405 410 415Gln Phe Glu Lys Leu Gly Glu
Tyr Gly Phe Gln Asn Ala Leu Ile Val 420 425 430Arg Tyr Thr Arg Lys
Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445Val Ser Arg
Ser Leu Gly Lys Val Gly Thr Arg Cys Cys Thr Lys Pro 450 455 460Glu
Ser Glu Arg Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu465 470
475 480Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys
Val 485 490 495Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro
Cys Phe Ser 500 505 510Ala Leu Thr Pro Asp Glu Thr Tyr Val Pro Lys
Ala Phe Asp Glu Lys 515 520 525Leu Phe Thr Phe His Ala Asp Ile Cys
Thr Leu Pro Asp Thr Glu Lys 530 535 540Gln Ile Lys Lys Gln Thr Ala
Leu Val Glu Leu Leu Lys His Lys Pro545 550 555 560Lys Ala Thr Glu
Glu Gln Leu Lys Thr Val Met Glu Asn Phe Val Ala 565 570 575Phe Val
Asp Lys Cys Cys Ala Ala Asp Asp Lys Glu Ala Cys Phe Ala 580 585
590Val Glu Gly Pro Lys Leu Val Val Ser Thr Gln Thr Ala Leu Ala 595
600 60512607PRTEquus caballus 12Met Lys Trp Val Thr Phe Val Ser Leu
Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Val Leu Arg Arg
Asp Thr His Lys Ser Glu Ile Ala 20 25 30His Arg Phe Asn Asp Leu Gly
Glu Lys His Phe Lys Gly Leu Val Leu 35 40 45Val Ala Phe Ser Gln Tyr
Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60Lys Leu Val Asn Glu
Val Thr Glu Phe Ala Lys Lys Cys Ala Ala Asp65 70 75 80Glu Ser Ala
Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95Lys Leu
Cys Thr Val Ala Thr Leu Arg Ala Thr Tyr Gly Glu Leu Ala 100 105
110Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Thr
115 120 125His Lys Asp Asp His Pro Asn Leu Pro Lys Leu Lys Pro Glu
Pro Asp 130 135 140Ala Gln Cys Ala Ala Phe Gln Glu Asp Pro Asp Lys
Phe Leu Gly Lys145 150 155 160Tyr Leu Tyr Glu Val Ala Arg Arg His
Pro Tyr Phe Tyr Gly Pro Glu 165 170 175Leu Leu Phe His Ala Glu Glu
Tyr Lys Ala Asp Phe Thr Glu Cys Cys 180 185 190Pro Ala Asp Asp Lys
Leu Ala Cys Leu Ile Pro Lys Leu Asp Ala Leu 195 200 205Lys Glu Arg
Ile Leu Leu Ser Ser Ala Lys Glu Arg Leu Lys Cys Ser 210 215 220Ser
Phe Gln Asn Phe Gly Glu Arg Ala Val Lys Ala Trp Ser Val Ala225 230
235 240Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Glu Val Ser
Lys 245 250 255Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys
His Gly Asp 260 265 270Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu
Ala Lys Tyr Ile Cys 275 280 285Glu His Gln Asp Ser Ile Ser Gly Lys
Leu Lys Ala Cys Cys Asp Lys 290 295 300Pro Leu Leu Gln Lys Ser His
Cys Ile Ala Glu Val Lys Glu Asp Asp305 310 315 320Leu Pro Ser Asp
Leu Pro Ala Leu Ala Ala Asp Phe Ala Glu Asp Lys 325 330 335Glu Ile
Cys Lys His Tyr Lys Asp Ala Lys Asp Val Phe Leu Gly Thr 340 345
350Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu
355 360 365Leu Leu Arg Ile Ala Lys Thr Tyr Glu Ala Thr Leu Glu Lys
Cys Cys 370 375 380Ala Glu Ala Asp Pro Pro Ala Cys Tyr Arg Thr Val
Phe Asp Gln Phe385 390 395 400Thr Pro Leu Val Glu Glu Pro Lys Ser
Leu Val Lys Lys Asn Cys Asp 405 410 415Leu Phe Glu Glu Val Gly Glu
Tyr Asp Phe Gln Asn Ala Leu Ile Val 420 425 430Arg Tyr Thr Lys Lys
Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445Ile Gly Arg
Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro 450 455 460Glu
Ser Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu Ala Leu465 470
475 480Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys
Ile 485 490 495Thr Lys Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro
Cys Phe Ser 500 505 510Ala Leu Glu Leu Asp Glu Gly Tyr Val Pro Lys
Glu Phe Lys Ala Glu 515 520 525Thr Phe Thr Phe His Ala Asp Ile Cys
Thr Leu Pro Glu Asp Glu Lys 530 535 540Gln Ile Lys Lys Gln Ser Ala
Leu Ala Glu Leu Val Lys His Lys Pro545 550 555 560Lys Ala Thr Lys
Glu Gln Leu Lys Thr Val Leu Gly Asn Phe Ser Ala 565 570 575Phe Val
Ala Lys Cys Cys Gly Arg Glu Asp Lys Glu Ala Cys Phe Ala 580 585
590Glu Glu Gly Pro Lys Leu Val Ala Ser Ser Gln Leu Ala Leu Ala 595
600 60513607PRTEquus asinus 13Met Lys Trp Val Thr Phe Val Ser Leu
Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Phe Arg Gly Val Leu Arg Arg
Asp Thr His Lys Ser Glu Ile Ala 20 25 30His Arg Phe Asn Asp Leu Gly
Glu Lys His Phe Lys Gly Leu Val Leu 35 40 45Val Ala Phe Ser Gln Tyr
Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60Lys Leu Val Asn Glu
Val Thr Glu Phe Ala Lys Lys Cys Ala Ala Asp65 70 75 80Glu Ser Ala
Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95Lys Leu
Cys Thr Val Ala Thr Leu Arg Ala Thr Tyr Gly Glu Leu Ala 100 105
110Asp Cys Cys Glu Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Thr
115 120 125His Lys Asp Asp His Pro Asn Leu Pro Lys Leu Lys Pro Glu
Pro Asp 130 135 140Ala Gln Cys Ala Ala Phe Gln Glu Asp Pro Asp Lys
Phe Leu Gly Lys145 150 155 160Tyr Leu Tyr Glu Val Ala Arg Arg His
Pro Tyr Phe Tyr Gly Pro Glu 165 170 175Leu Leu Phe His Ala Glu Glu
Tyr Lys Ala Asp Phe Thr Glu Cys Cys 180 185 190Pro Ala Asp Asp Lys
Ala Gly Cys Leu Ile Pro Lys Leu Asp Ala Leu 195 200 205Lys Glu Arg
Ile Leu Leu Ser Ser Ala Lys Glu Arg Leu Lys Cys Ser 210 215 220Ser
Phe Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ser Val Ala225 230
235 240Arg Leu Ser Gln Lys Phe Pro Lys Ala Asp Phe Ala Glu Val Ser
Lys 245 250 255Ile Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys
His Gly Asp 260 265 270Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu
Thr Lys Tyr Ile Cys 275 280 285Glu His Gln Asp Ser Ile Ser Gly Lys
Leu Lys Ala Cys Cys Asp Lys 290 295 300Pro Leu Leu Gln Lys Ser His
Cys Ile Ala Glu Val Lys Glu Asp Asp305 310 315 320Leu Pro Ser Asp
Leu Pro Ala Leu Ala Ala Asp Phe Ala Glu Asp Lys 325 330 335Glu Ile
Cys Lys His Tyr Lys Asp Ala Lys Asp Val Phe Leu Gly Thr 340 345
350Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser Leu
355 360 365Leu Leu Arg Ile Ala Lys Thr Tyr Glu Ala Thr Leu Glu Lys
Cys Cys 370 375 380Ala Glu Ala Asp Pro Pro Ala Cys Tyr Ala Thr Val
Phe Asp Gln Phe385 390 395 400Thr Pro Leu Val Glu Glu Pro Lys Ser
Leu Val Lys Lys Asn Cys Asp 405 410 415Leu Phe Glu Glu Val Gly
Glu
Tyr Asp Phe Gln Asn Ala Leu Ile Val 420 425 430Arg Tyr Thr Lys Lys
Ala Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445Ile Gly Arg
Thr Leu Gly Lys Val Gly Ser Arg Cys Cys Lys Leu Pro 450 455 460Glu
Ser Glu Arg Leu Pro Cys Ser Glu Asn His Leu Ala Leu Ala Leu465 470
475 480Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys
Ile 485 490 495Thr Lys Cys Cys Thr Asp Ser Leu Ala Glu Arg Arg Pro
Cys Phe Ser 500 505 510Ala Leu Glu Leu Asp Glu Gly Tyr Ile Pro Lys
Glu Phe Lys Ala Glu 515 520 525Thr Phe Thr Phe His Ala Asp Ile Cys
Thr Leu Pro Glu Asp Glu Lys 530 535 540Gln Ile Lys Lys Gln Ser Ala
Leu Ala Glu Leu Val Lys His Lys Pro545 550 555 560Lys Ala Thr Lys
Glu Gln Leu Lys Thr Val Leu Gly Asn Phe Ser Ala 565 570 575Phe Val
Ala Lys Cys Cys Gly Ala Glu Asp Lys Glu Ala Cys Phe Ala 580 585
590Glu Glu Gly Pro Lys Leu Val Ala Ser Ser Gln Leu Ala Leu Ala 595
600 60514608PRTOryctolagus cuniculus 14Met Lys Trp Val Thr Phe Ile
Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe
Arg Arg Glu Ala His Lys Ser Glu Ile Ala 20 25 30His Arg Phe Asn Asp
Val Gly Glu Glu His Phe Ile Gly Leu Val Leu 35 40 45Ile Thr Phe Ser
Gln Tyr Leu Gln Lys Cys Pro Tyr Glu Glu His Ala 50 55 60Lys Leu Val
Lys Glu Val Thr Asp Leu Ala Lys Ala Cys Val Ala Asp65 70 75 80Glu
Ser Ala Ala Asn Cys Asp Lys Ser Leu His Asp Ile Phe Gly Asp 85 90
95Lys Ile Cys Ala Leu Pro Ser Leu Arg Asp Thr Tyr Gly Asp Val Ala
100 105 110Asp Cys Cys Glu Lys Lys Glu Pro Glu Arg Asn Glu Cys Phe
Leu His 115 120 125His Lys Asp Asp Lys Pro Asp Leu Pro Pro Phe Ala
Arg Pro Glu Ala 130 135 140Asp Val Leu Cys Lys Ala Phe His Asp Asp
Glu Lys Ala Phe Phe Gly145 150 155 160His Tyr Leu Tyr Glu Val Ala
Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175Glu Leu Leu Tyr Tyr
Ala Gln Lys Tyr Lys Ala Ile Leu Thr Glu Cys 180 185 190Cys Glu Ala
Ala Asp Lys Gly Ala Cys Leu Thr Pro Lys Leu Asp Ala 195 200 205Leu
Glu Gly Lys Ser Leu Ile Ser Ala Ala Gln Glu Arg Leu Arg Cys 210 215
220Ala Ser Ile Gln Lys Phe Gly Asp Arg Ala Tyr Lys Ala Trp Ala
Leu225 230 235 240Val Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe
Thr Asp Ile Ser 245 250 255Lys Ile Val Thr Asp Leu Thr Lys Val His
Lys Glu Cys Cys His Gly 260 265 270Asp Leu Leu Glu Cys Ala Asp Asp
Arg Ala Asp Leu Ala Lys Tyr Met 275 280 285Cys Glu His Gln Glu Thr
Ile Ser Ser His Leu Lys Glu Cys Cys Asp 290 295 300Lys Pro Ile Leu
Glu Lys Ala His Cys Ile Tyr Gly Leu His Asn Asp305 310 315 320Glu
Thr Pro Ala Gly Leu Pro Ala Val Ala Glu Glu Phe Val Glu Asp 325 330
335Lys Asp Val Cys Lys Asn Tyr Glu Glu Ala Lys Asp Leu Phe Leu Gly
340 345 350Lys Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser
Val Val 355 360 365Leu Leu Leu Arg Leu Gly Lys Ala Tyr Glu Ala Thr
Leu Lys Lys Cys 370 375 380Cys Ala Thr Asp Asp Pro His Ala Cys Tyr
Ala Lys Val Leu Asp Glu385 390 395 400Phe Gln Pro Leu Val Asp Glu
Pro Lys Asn Leu Val Lys Gln Asn Cys 405 410 415Glu Leu Tyr Glu Gln
Leu Gly Asp Tyr Asn Phe Gln Asn Ala Leu Leu 420 425 430Val Arg Tyr
Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445Glu
Ile Ser Arg Ser Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455
460Pro Glu Ala Glu Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Val
Val465 470 475 480Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro
Val Ser Glu Lys 485 490 495Val Thr Lys Cys Cys Ser Glu Ser Leu Val
Asp Arg Arg Pro Cys Phe 500 505 510Ser Ala Leu Gly Pro Asp Glu Thr
Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525Glu Thr Phe Thr Phe His
Ala Asp Ile Cys Thr Leu Pro Glu Thr Glu 530 535 540Arg Lys Ile Lys
Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys545 550 555 560Pro
His Ala Thr Asn Asp Gln Leu Lys Thr Val Val Gly Glu Phe Thr 565 570
575Ala Leu Leu Asp Lys Cys Cys Ser Ala Glu Asp Lys Glu Ala Cys Phe
580 585 590Ala Val Glu Gly Pro Lys Leu Val Glu Ser Ser Lys Ala Thr
Leu Gly 595 600 60515583PRTCapra hircus 15Asp Thr His Lys Ser Glu
Ile Ala His Arg Phe Asn Asp Leu Gly Glu1 5 10 15Glu Asn Phe Gln Gly
Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Asp Glu His Val Lys Leu Val Lys Glu Leu Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser His Ala Gly Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Glu Leu Cys Lys Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Asp Met Ala Asp Cys Cys Glu Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Lys His Lys Asp Asp Ser Pro Asp
Leu 100 105 110Pro Lys Leu Lys Pro Glu Pro Asp Thr Leu Cys Ala Glu
Phe Lys Ala 115 120 125Asp Glu Lys Lys Phe Trp Gly Lys Tyr Leu Tyr
Glu Val Ala Arg Arg 130 135 140His Pro Tyr Phe Tyr Ala Pro Glu Leu
Leu Tyr Tyr Ala Asn Lys Tyr145 150 155 160Asn Gly Val Phe Gln Glu
Cys Cys Gln Ala Glu Asp Lys Gly Ala Cys 165 170 175Leu Leu Pro Lys
Ile Glu Thr Met Arg Glu Lys Val Leu Ala Ser Ser 180 185 190Ala Arg
Gln Arg Leu Arg Cys Ala Ser Ile Gln Lys Phe Gly Glu Arg 195 200
205Ala Leu Lys Ala Trp Ser Val Ala Arg Leu Ser Gln Lys Phe Pro Lys
210 215 220Ala Asp Phe Thr Asp Val Thr Lys Ile Val Thr Asp Leu Thr
Lys Val225 230 235 240His Lys Glu Cys Cys His Gly Asp Leu Leu Glu
Cys Ala Asp Asp Arg 245 250 255Ala Asp Leu Ala Lys Tyr Ile Cys Asp
His Gln Asp Thr Leu Ser Ser 260 265 270Lys Leu Lys Glu Cys Cys Asp
Lys Pro Val Leu Glu Lys Ser His Cys 275 280 285Ile Ala Glu Ile Asp
Lys Asp Ala Val Pro Glu Asn Leu Pro Pro Leu 290 295 300Thr Ala Asp
Phe Ala Glu Asp Lys Glu Val Cys Lys Asn Tyr Gln Glu305 310 315
320Ala Lys Asp Val Phe Leu Gly Ser Phe Leu Tyr Glu Tyr Ser Arg Arg
325 330 335His Pro Glu Tyr Ala Val Ser Val Leu Leu Arg Leu Ala Lys
Glu Tyr 340 345 350Glu Ala Thr Leu Glu Asp Cys Cys Ala Lys Glu Asp
Pro His Ala Cys 355 360 365Tyr Ala Thr Val Phe Asp Lys Leu Lys His
Leu Val Asp Glu Pro Gln 370 375 380Asn Leu Ile Lys Lys Asn Cys Glu
Leu Phe Glu Lys His Gly Glu Tyr385 390 395 400Gly Phe Gln Asn Ala
Leu Ile Val Arg Tyr Thr Arg Lys Ala Pro Gln 405 410 415Val Ser Thr
Pro Thr Leu Val Glu Ile Ser Arg Ser Leu Gly Lys Val 420 425 430Gly
Thr Lys Cys Cys Ala Lys Pro Glu Ser Glu Arg Met Pro Cys Thr 435 440
445Glu Asp Tyr Leu Ser Leu Ile Leu Asn Arg Leu Cys Val Leu His Glu
450 455 460Lys Thr Pro Val Ser Glu Lys Val Thr Lys Cys Cys Thr Glu
Ser Leu465 470 475 480Val Asn Arg Arg Pro Cys Phe Ser Asp Leu Thr
Leu Asp Glu Thr Tyr 485 490 495Val Pro Lys Pro Phe Asp Gly Glu Ser
Phe Thr Phe His Ala Asp Ile 500 505 510Cys Thr Leu Pro Asp Thr Glu
Lys Gln Ile Lys Lys Gln Thr Ala Leu 515 520 525Val Glu Leu Leu Lys
His Lys Pro Lys Ala Thr Asp Glu Gln Leu Lys 530 535 540Thr Val Met
Glu Asn Phe Val Ala Phe Val Asp Lys Cys Cys Ala Ala545 550 555
560Asp Asp Lys Glu Gly Cys Phe Leu Leu Glu Gly Pro Lys Leu Val Ala
565 570 575Ser Thr Gln Ala Ala Leu Ala 58016607PRTOvis aries 16Met
Lys Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala1 5 10
15Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Ile Ala
20 25 30His Arg Phe Asn Asp Leu Gly Glu Glu Asn Phe Gln Gly Leu Val
Leu 35 40 45Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu
His Val 50 55 60Lys Leu Val Lys Glu Leu Thr Glu Phe Ala Lys Thr Cys
Val Ala Asp65 70 75 80Glu Ser His Ala Gly Cys Asp Lys Ser Leu His
Thr Leu Phe Gly Asp 85 90 95Glu Leu Cys Lys Val Ala Thr Leu Arg Glu
Thr Tyr Gly Asp Met Ala 100 105 110Asp Cys Cys Glu Lys Gln Glu Pro
Glu Arg Asn Glu Cys Phe Leu Asn 115 120 125His Lys Asp Asp Ser Pro
Asp Leu Pro Lys Leu Lys Pro Glu Pro Asp 130 135 140Thr Leu Cys Ala
Glu Phe Lys Ala Asp Glu Lys Lys Phe Trp Gly Lys145 150 155 160Tyr
Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 165 170
175Leu Leu Tyr Tyr Ala Asn Lys Tyr Asn Gly Val Phe Gln Glu Cys Cys
180 185 190Gln Ala Glu Asp Lys Gly Ala Cys Leu Leu Pro Lys Ile Asp
Ala Met 195 200 205Arg Glu Lys Val Leu Ala Ser Ser Ala Arg Gln Arg
Leu Arg Cys Ala 210 215 220Ser Ile Gln Lys Phe Gly Glu Arg Ala Leu
Lys Ala Trp Ser Val Ala225 230 235 240Arg Leu Ser Gln Lys Phe Pro
Lys Ala Asp Phe Thr Asp Val Thr Lys 245 250 255Ile Val Thr Asp Leu
Thr Lys Val His Lys Glu Cys Cys His Gly Asp 260 265 270Leu Leu Glu
Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280 285Asp
His Gln Asp Ala Leu Ser Ser Lys Leu Lys Glu Cys Cys Asp Lys 290 295
300Pro Val Leu Glu Lys Ser His Cys Ile Ala Glu Val Asp Lys Asp
Ala305 310 315 320Val Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe
Ala Glu Asp Lys 325 330 335Glu Val Cys Lys Asn Tyr Gln Glu Ala Lys
Asp Val Phe Leu Gly Ser 340 345 350Phe Leu Tyr Glu Tyr Ser Arg Arg
His Pro Glu Tyr Ala Val Ser Val 355 360 365Leu Leu Arg Leu Ala Lys
Glu Tyr Glu Ala Thr Leu Glu Asp Cys Cys 370 375 380Ala Lys Glu Asp
Pro His Ala Cys Tyr Ala Thr Val Phe Asp Lys Leu385 390 395 400Lys
His Leu Val Asp Glu Pro Gln Asn Leu Ile Lys Lys Asn Cys Glu 405 410
415Leu Phe Glu Lys His Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val
420 425 430Arg Tyr Thr Arg Lys Ala Pro Gln Val Ser Thr Pro Thr Leu
Val Glu 435 440 445Ile Ser Arg Ser Leu Gly Lys Val Gly Thr Lys Cys
Cys Ala Lys Pro 450 455 460Glu Ser Glu Arg Met Pro Cys Thr Glu Asp
Tyr Leu Ser Leu Ile Leu465 470 475 480Asn Arg Leu Cys Val Leu His
Glu Lys Thr Pro Val Ser Glu Lys Val 485 490 495Thr Lys Cys Cys Thr
Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505 510Asp Leu Thr
Leu Asp Glu Thr Tyr Val Pro Lys Pro Phe Asp Glu Lys 515 520 525Phe
Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Lys 530 535
540Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His Lys
Pro545 550 555 560Lys Ala Thr Asp Glu Gln Leu Lys Thr Val Met Glu
Asn Phe Val Ala 565 570 575Phe Val Asp Lys Cys Cys Ala Ala Asp Asp
Lys Glu Gly Cys Phe Val 580 585 590Leu Glu Gly Pro Lys Leu Val Ala
Ser Thr Gln Ala Ala Leu Ala 595 600 60517608PRTcanis lupus
familiaris 17Met Lys Trp Val Thr Phe Ile Ser Leu Phe Phe Leu Phe
Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Leu Val Arg Arg Glu Ala Tyr Lys
Ser Glu Ile Ala 20 25 30His Arg Tyr Asn Asp Leu Gly Glu Glu His Phe
Arg Gly Leu Val Leu 35 40 45Val Ala Phe Ser Gln Tyr Leu Gln Gln Cys
Pro Phe Glu Asp His Val 50 55 60Lys Leu Ala Lys Glu Val Thr Glu Phe
Ala Lys Ala Cys Ala Ala Glu65 70 75 80Glu Ser Gly Ala Asn Cys Asp
Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95Lys Leu Cys Thr Val Ala
Ser Leu Arg Asp Lys Tyr Gly Asp Met Ala 100 105 110Asp Cys Cys Glu
Lys Gln Glu Pro Asp Arg Asn Glu Cys Phe Leu Ala 115 120 125His Lys
Asp Asp Asn Pro Gly Phe Pro Pro Leu Val Ala Pro Glu Pro 130 135
140Asp Ala Leu Cys Ala Ala Phe Gln Asp Asn Glu Gln Leu Phe Leu
Gly145 150 155 160Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr
Phe Tyr Ala Pro 165 170 175Glu Leu Leu Tyr Tyr Ala Gln Gln Tyr Lys
Gly Val Phe Ala Glu Cys 180 185 190Cys Gln Ala Ala Asp Lys Ala Ala
Cys Leu Gly Pro Lys Ile Glu Ala 195 200 205Leu Arg Glu Lys Val Leu
Leu Ser Ser Ala Lys Glu Arg Phe Lys Cys 210 215 220Ala Ser Leu Gln
Lys Phe Gly Asp Arg Ala Phe Lys Ala Trp Ser Val225 230 235 240Ala
Arg Leu Ser Gln Arg Phe Pro Lys Ala Asp Phe Ala Glu Ile Ser 245 250
255Lys Val Val Thr Asp Leu Thr Lys Val His Lys Glu Cys Cys His Gly
260 265 270Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys
Tyr Met 275 280 285Cys Glu Asn Gln Asp Ser Ile Ser Thr Lys Leu Lys
Glu Cys Cys Asp 290 295 300Lys Pro Val Leu Glu Lys Ser Gln Cys Leu
Ala Glu Val Glu Arg Asp305 310 315 320Glu Leu Pro Gly Asp Leu Pro
Ser Leu Ala Ala Asp Phe Val Glu Asp 325 330 335Lys Glu Val Cys Lys
Asn Tyr Gln Glu Ala Lys Asp Val Phe Leu Gly 340 345 350Thr Phe Leu
Tyr Glu Tyr Ala Arg Arg His Pro Glu Tyr Ser Val Ser 355 360 365Leu
Leu Leu Arg Leu Ala Lys Glu Tyr Glu Ala Thr Leu Glu Lys Cys 370 375
380Cys Ala Thr Asp Asp Pro Pro Thr Cys Tyr Ala Lys Val Leu Asp
Glu385 390 395 400Phe Lys Pro Leu Val Asp Glu Pro Gln Asn Leu Val
Lys Thr Asn Cys 405 410 415Glu Leu Phe Glu Lys Leu Gly Glu Tyr Gly
Phe Gln Asn Ala Leu Leu 420 425 430Val Arg Tyr Thr Lys Lys Ala Pro
Gln Val Ser Thr Pro Thr Leu Val 435 440 445Glu Val Ser Arg Lys Leu
Gly Lys Val Gly Thr Lys Cys Cys Lys Lys 450 455 460Pro Glu Ser Glu
Arg Met Ser Cys Ala Glu Asp Phe Leu Ser Val Val465 470
475 480Leu Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu
Arg 485 490 495Val Thr Lys Cys Cys Ser Glu Ser Leu Val Asn Arg Arg
Pro Cys Phe 500 505 510Ser Gly Leu Glu Val Asp Glu Thr Tyr Val Pro
Lys Glu Phe Asn Ala 515 520 525Glu Thr Phe Thr Phe His Ala Asp Leu
Cys Thr Leu Pro Glu Ala Glu 530 535 540Lys Gln Val Lys Lys Gln Thr
Ala Leu Val Glu Leu Leu Lys His Lys545 550 555 560Pro Lys Ala Thr
Asp Glu Gln Leu Lys Thr Val Met Gly Asp Phe Gly 565 570 575Ala Phe
Val Glu Lys Cys Cys Ala Ala Glu Asn Lys Glu Gly Cys Phe 580 585
590Ser Glu Glu Gly Pro Lys Leu Val Ala Ala Ala Gln Ala Ala Leu Val
595 600 60518615PRTGallus gallus 18Met Lys Trp Val Thr Leu Ile Ser
Phe Ile Phe Leu Phe Ser Ser Ala1 5 10 15Thr Ser Arg Asn Leu Gln Arg
Phe Ala Arg Asp Ala Glu His Lys Ser 20 25 30Glu Ile Ala His Arg Tyr
Asn Asp Leu Lys Glu Glu Thr Phe Lys Ala 35 40 45Val Ala Met Ile Thr
Phe Ala Gln Tyr Leu Gln Arg Cys Ser Tyr Glu 50 55 60Gly Leu Ser Lys
Leu Val Lys Asp Val Val Asp Leu Ala Gln Lys Cys65 70 75 80Val Ala
Asn Glu Asp Ala Pro Glu Cys Ser Lys Pro Leu Pro Ser Ile 85 90 95Ile
Leu Asp Glu Ile Cys Gln Val Glu Lys Leu Arg Asp Ser Tyr Gly 100 105
110Ala Met Ala Asp Cys Cys Ser Lys Ala Asp Pro Glu Arg Asn Glu Cys
115 120 125Phe Leu Ser Phe Lys Val Ser Gln Pro Asp Phe Val Gln Pro
Tyr Gln 130 135 140Arg Pro Ala Ser Asp Val Ile Cys Gln Glu Tyr Gln
Asp Asn Arg Val145 150 155 160Ser Phe Leu Gly His Phe Ile Tyr Ser
Val Ala Arg Arg His Pro Phe 165 170 175Leu Tyr Ala Pro Ala Ile Leu
Ser Phe Ala Val Asp Phe Glu His Ala 180 185 190Leu Gln Ser Cys Cys
Lys Glu Ser Asp Val Gly Ala Cys Leu Asp Thr 195 200 205Lys Glu Ile
Val Met Arg Glu Lys Ala Lys Gly Val Ser Val Lys Gln 210 215 220Gln
Tyr Phe Cys Gly Ile Leu Lys Gln Phe Gly Asp Arg Val Phe Gln225 230
235 240Ala Arg Gln Leu Ile Tyr Leu Ser Gln Lys Tyr Pro Lys Ala Pro
Phe 245 250 255Ser Glu Val Ser Lys Phe Val His Asp Ser Ile Gly Val
His Lys Glu 260 265 270Cys Cys Glu Gly Asp Met Val Glu Cys Met Asp
Asp Met Ala Arg Met 275 280 285Met Ser Asn Leu Cys Ser Gln Gln Asp
Val Phe Ser Gly Lys Ile Lys 290 295 300Asp Cys Cys Glu Lys Pro Ile
Val Glu Arg Ser Gln Cys Ile Met Glu305 310 315 320Ala Glu Phe Asp
Glu Lys Pro Ala Asp Leu Pro Ser Leu Val Glu Lys 325 330 335Tyr Ile
Glu Asp Lys Glu Val Cys Lys Ser Phe Glu Ala Gly His Asp 340 345
350Ala Phe Met Ala Glu Phe Val Tyr Glu Tyr Ser Arg Arg His Pro Glu
355 360 365Phe Ser Ile Gln Leu Ile Met Arg Ile Ala Lys Gly Tyr Glu
Ser Leu 370 375 380Leu Glu Lys Cys Cys Lys Thr Asp Asn Pro Ala Glu
Cys Tyr Ala Asn385 390 395 400Ala Gln Glu Gln Leu Asn Gln His Ile
Lys Glu Thr Gln Asp Val Val 405 410 415Lys Thr Asn Cys Asp Leu Leu
His Asp His Gly Glu Ala Asp Phe Leu 420 425 430Lys Ser Ile Leu Ile
Arg Tyr Thr Lys Lys Met Pro Gln Val Pro Thr 435 440 445Asp Leu Leu
Leu Glu Thr Gly Lys Lys Met Thr Thr Ile Gly Thr Lys 450 455 460Cys
Cys Gln Leu Gly Glu Asp Arg Arg Met Ala Cys Ser Glu Gly Tyr465 470
475 480Leu Ser Ile Val Ile His Asp Thr Cys Arg Lys Gln Glu Thr Thr
Pro 485 490 495Ile Asn Asp Asn Val Ser Gln Cys Cys Ser Gln Leu Tyr
Ala Asn Arg 500 505 510Arg Pro Cys Phe Thr Ala Met Gly Val Asp Thr
Lys Tyr Val Pro Pro 515 520 525Pro Phe Asn Pro Asp Met Phe Ser Phe
Asp Glu Lys Leu Cys Ser Ala 530 535 540Pro Ala Glu Glu Arg Glu Val
Gly Gln Met Lys Leu Leu Ile Asn Leu545 550 555 560Ile Lys Arg Lys
Pro Gln Met Thr Glu Glu Gln Ile Lys Thr Ile Ala 565 570 575Asp Gly
Phe Thr Ala Met Val Asp Lys Cys Cys Lys Gln Ser Asp Ile 580 585
590Asn Thr Cys Phe Gly Glu Glu Gly Ala Asn Leu Ile Val Gln Ser Arg
595 600 605Ala Thr Leu Gly Ile Gly Ala 610 61519607PRTSus scrofa
19Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1
5 10 15Tyr Ser Arg Gly Val Phe Arg Arg Asp Thr Tyr Lys Ser Glu Ile
Ala 20 25 30His Arg Phe Lys Asp Leu Gly Glu Gln Tyr Phe Lys Gly Leu
Val Leu 35 40 45Ile Ala Phe Ser Gln His Leu Gln Gln Cys Pro Tyr Glu
Glu His Val 50 55 60Lys Leu Val Arg Glu Val Thr Glu Phe Ala Lys Thr
Cys Val Ala Asp65 70 75 80Glu Ser Ala Glu Asn Cys Asp Lys Ser Ile
His Thr Leu Phe Gly Asp 85 90 95Lys Leu Cys Ala Ile Pro Ser Leu Arg
Glu His Tyr Gly Asp Leu Ala 100 105 110Asp Cys Cys Glu Lys Glu Glu
Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125His Lys Asn Asp Asn
Pro Asp Ile Pro Lys Leu Lys Pro Asp Pro Val 130 135 140Ala Leu Cys
Ala Asp Phe Gln Glu Asp Glu Gln Lys Phe Trp Gly Lys145 150 155
160Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu
165 170 175Leu Leu Tyr Tyr Ala Ile Ile Tyr Lys Asp Val Phe Ser Glu
Cys Cys 180 185 190Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys
Ile Glu His Leu 195 200 205Arg Glu Lys Val Leu Thr Ser Ala Ala Lys
Gln Arg Leu Lys Cys Ala 210 215 220Ser Ile Gln Lys Phe Gly Glu Arg
Ala Phe Lys Ala Trp Ser Leu Ala225 230 235 240Arg Leu Ser Gln Arg
Phe Pro Lys Ala Asp Phe Thr Glu Ile Ser Lys 245 250 255Ile Val Thr
Asp Leu Ala Lys Val His Lys Glu Cys Cys His Gly Asp 260 265 270Leu
Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280
285Glu Asn Gln Asp Thr Ile Ser Thr Lys Leu Lys Glu Cys Cys Asp Lys
290 295 300Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Ala Lys Arg
Asp Glu305 310 315 320Leu Pro Ala Asp Leu Asn Pro Leu Glu His Asp
Phe Val Glu Asp Lys 325 330 335Glu Val Cys Lys Asn Tyr Lys Glu Ala
Lys His Val Phe Leu Gly Thr 340 345 350Phe Leu Tyr Glu Tyr Ser Arg
Arg His Pro Asp Tyr Ser Val Ser Leu 355 360 365Leu Leu Arg Ile Ala
Lys Ile Tyr Glu Ala Thr Leu Glu Asp Cys Cys 370 375 380Ala Lys Glu
Asp Pro Pro Ala Cys Tyr Ala Thr Val Phe Asp Lys Phe385 390 395
400Gln Pro Leu Val Asp Glu Pro Lys Asn Leu Ile Lys Gln Asn Cys Glu
405 410 415Leu Phe Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu
Ile Val 420 425 430Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro
Thr Leu Val Glu 435 440 445Val Ala Arg Lys Leu Gly Leu Val Gly Ser
Arg Cys Cys Lys Arg Pro 450 455 460Glu Glu Glu Arg Leu Ser Cys Ala
Glu Asp Tyr Leu Ser Leu Val Leu465 470 475 480Asn Arg Leu Cys Val
Leu His Glu Lys Thr Pro Val Ser Glu Lys Val 485 490 495Thr Lys Cys
Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505 510Ala
Leu Thr Pro Asp Glu Thr Tyr Lys Pro Lys Glu Phe Val Glu Gly 515 520
525Thr Phe Thr Phe His Ala Asp Leu Cys Thr Leu Pro Glu Asp Glu Lys
530 535 540Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Leu Lys His
Lys Pro545 550 555 560His Ala Thr Glu Glu Gln Leu Arg Thr Val Leu
Gly Asn Phe Ala Ala 565 570 575Phe Val Gln Lys Cys Cys Ala Ala Pro
Asp His Glu Ala Cys Phe Ala 580 585 590Val Glu Gly Pro Lys Phe Val
Ile Glu Ile Arg Gly Ile Leu Ala 595 600 60520584PRTartificial
sequenceN terminal is residues 1 to 572 of HSA. C terminal is
residues 573 to 584 of Macaque albumin. 20Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Phe Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Ala 58021584PRTArtificial
SequenceN terminal is residues 1 to 572 from HSA. C terminal is
residues 573 to 584 from mouse albumin. 21Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys
Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys
Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Pro Asn Leu Val 565 570 575Thr Arg Cys Lys Asp Ala Leu Ala
58022584PRTArtificial SequenceN terminal is residues 1 to 572 of
HSA. C terminal is residues 573 to 584 of rabbit albumin. 22Asp Ala
His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu
Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25
30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp
Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala
Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp
Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe
Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys
Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser
Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His
Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu
Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys
Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295
300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile
Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410
415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535
540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Pro Lys Leu Val 565 570 575Glu Ser Ser Lys Ala Thr Leu Gly
58023584PRTArtificial SequenceN-terminal is residues 1 to 572 of
HSA. C- terminal is residues 573 to 583 of sheep albumin. 23Asp Ala
His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu
Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25
30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp
Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala
Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp
Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe
Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys
Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser
Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His
Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu
Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys
Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295
300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile
Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410
415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535
540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Pro Lys Leu Val 565 570 575Ala Ser Thr Gln Ala Ala Leu Ala
58024399PRTArtificial sequenceArtificial albumin variant human
serum albumin domain 1 and human serum albumin domain 3 24Asp Ala
His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu
Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25
30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu
35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp
Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala
Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala
Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp
Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe
Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys
Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190Ser Ala Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu 195 200 205Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala
Leu Leu Val Arg 210 215 220Tyr Thr Lys Lys Val Pro Gln Val Ser Thr
Pro Thr Leu Val Glu Val225 230 235 240Ser Arg Asn Leu Gly Lys Val
Gly Ser Lys Cys Cys Lys His Pro Glu 245 250 255Ala Lys Arg Met Pro
Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 260 265 270Gln Leu Cys
Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 275 280 285Lys
Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 290 295
300Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr305 310 315 320Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu
Lys Glu Arg Gln 325 330 335Ile Lys Lys Gln Thr Ala Leu Val Glu Leu
Val Lys His Lys Pro Lys 340 345 350Ala Thr Lys Glu Gln Leu Lys Ala
Val Met Asp Asp Phe Ala Ala Phe 355 360 365Val Glu Lys Cys Cys Lys
Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 370 375 380Glu Gly Lys Lys
Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu385 390
39525403PRTArtificial sequenceArtificial albumin variant human
serum albumin domain 2 and human serum albumin domain 3 25Asp Glu
Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu1 5 10 15Lys
Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp 20 25
30Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu
35 40 45Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys
Cys 50 55 60His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu
Ala Lys65 70 75 80Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys
Leu Lys Glu Cys 85 90 95Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys
Ile Ala Glu Val Glu 100 105 110Asn Asp Glu Met Pro Ala Asp Leu Pro
Ser Leu Ala Ala Asp Phe Val 115 120 125Glu Ser Lys Asp Val Cys Lys
Asn Tyr Ala Glu Ala Lys Asp Val Phe 130 135 140Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser145 150 155 160Val Val
Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu 165 170
175Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe
180 185 190Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile
Lys Gln 195 200 205Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys
Phe Gln Asn Ala 210 215 220Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
Gln Val Ser Thr Pro Thr225 230 235 240Leu Val Glu Val Ser Arg Asn
Leu Gly Lys Val Gly Ser Lys Cys Cys 245 250 255Lys His Pro Glu Ala
Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser 260 265 270Val Val Leu
Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser 275 280 285Asp
Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro 290 295
300Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu
Phe305 310 315 320Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys
Thr Leu Ser Glu 325 330 335Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
Leu Val Glu Leu Val Lys 340 345 350His Lys Pro Lys Ala Thr Lys Glu
Gln Leu Lys Ala Val Met Asp Asp 355 360 365Phe Ala Ala Phe Val Glu
Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr 370 375 380Cys Phe Ala Glu
Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala385 390 395 400Leu
Gly Leu26410PRTArtificial sequenceArtificial albumin variant two
consecutive copies of human serum albumin domain 3 26Val Glu Glu
Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu1 5 10 15Gln Leu
Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr 20 25 30Lys
Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 35 40
45Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
50 55 60Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
Leu65 70 75 80Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val
Thr Lys Cys 85 90 95Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe
Ser Ala Leu Glu 100 105 110Val Asp Glu Thr Tyr Val Pro Lys Glu Phe
Asn Ala Glu Thr Phe Thr 115 120 125Phe His Ala Asp Ile Cys Thr Leu
Ser Glu Lys Glu Arg Gln Ile Lys 130 135 140Lys Gln Thr Ala Leu Val
Glu Leu Val Lys His Lys Pro Lys Ala Thr145 150 155 160Lys Glu Gln
Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu 165 170 175Lys
Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly 180 185
190Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Val Glu Glu
195 200 205Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln
Leu Gly 210 215 220Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
Thr Lys Lys Val225 230 235 240Pro Gln Val Ser Thr Pro Thr Leu Val
Glu Val Ser Arg Asn Leu Gly 245 250 255Lys Val Gly Ser Lys Cys Cys
Lys His Pro Glu Ala Lys Arg Met Pro 260 265 270Cys Ala Glu Asp Tyr
Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu 275 280 285His Glu Lys
Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu 290 295 300Ser
Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu305 310
315 320Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala 325 330 335Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr
340 345 350Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys
Glu Gln 355 360 365Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val
Glu Lys Cys Cys 370 375 380Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala
Glu Glu Gly Lys Lys Leu385 390 395 400Val Ala Ala Ser Gln Ala Ala
Leu Gly Leu 405 41027205PRTArtificial sequenceArtificial albumin
variant human serum albumin domain 3 27Val Glu Glu Pro Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu1 5 10 15Gln Leu Gly Glu Tyr Lys
Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr 20 25 30Lys Lys Val Pro Gln
Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg 35 40 45Asn Leu Gly Lys
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys 50 55 60Arg Met Pro
Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu65 70 75 80Cys
Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys 85 90
95Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu
100 105 110Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr
Phe Thr 115 120 125Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu
Arg Gln Ile Lys 130 135 140Lys Gln Thr Ala Leu Val Glu Leu Val Lys
His Lys Pro Lys Ala Thr145 150 155 160Lys Glu Gln Leu Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu 165 170 175Lys Cys Cys Lys Ala
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly 180 185 190Lys Lys Leu
Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 195 200
20528615PRTArtificial SequenceHSA Domain III + HSA Domain III + HSA
Domain III 28Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu
Leu Phe Glu1 5 10 15Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu
Val Arg Tyr Thr 20 25 30Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu
Val Glu Val Ser Arg 35 40 45Asn Leu Gly Lys Val Gly Ser Lys Cys Cys
Lys His Pro Glu Ala Lys 50 55 60Arg Met Pro Cys Ala Glu Asp Tyr Leu
Ser Val Val Leu Asn Gln Leu65 70 75 80Cys Val Leu His Glu Lys Thr
Pro Val Ser Asp Arg Val Thr Lys Cys 85 90 95Cys Thr Glu Ser Leu Val
Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu 100 105 110Val Asp Glu Thr
Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr 115 120 125Phe His
Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys 130 135
140Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala
Thr145 150 155 160Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu 165 170 175Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr
Cys Phe Ala Glu Glu Gly 180 185 190Lys Lys Leu Val Ala Ala Ser Gln
Ala Ala Leu Gly Leu Val Glu Glu 195 200 205Pro Gln Asn Leu Ile Lys
Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly 210 215 220Glu Tyr Lys Phe
Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val225 230 235 240Pro
Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly 245 250
255Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro
260 265 270Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys
Val Leu 275 280 285His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
Cys Cys Thr Glu 290 295 300Ser Leu Val Asn Arg Arg Pro Cys Phe Ser
Ala Leu Glu Val Asp Glu305 310 315 320Thr Tyr Val Pro Lys Glu Phe
Asn Ala Glu Thr Phe Thr Phe His Ala 325 330 335Asp Ile Cys Thr Leu
Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr 340 345 350Ala Leu Val
Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln 355 360 365Leu
Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys 370 375
380Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys
Leu385 390 395 400Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Val Glu
Glu Pro Gln Asn 405 410 415Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu
Gln Leu Gly Glu Tyr Lys 420 425 430Phe Gln Asn Ala Leu Leu Val Arg
Tyr Thr Lys Lys Val Pro Gln Val 435 440 445Ser Thr Pro Thr Leu Val
Glu Val Ser Arg Asn Leu Gly Lys Val Gly 450 455 460Ser Lys Cys Cys
Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu465 470 475 480Asp
Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys 485 490
495Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val
500 505 510Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
Tyr Val 515 520 525Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp Ile Cys 530 535 540Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala Leu Val545 550 555 560Glu Leu Val Lys His Lys Pro
Lys Ala Thr Lys Glu Gln Leu Lys Ala 565 570 575Val Met Asp Asp Phe
Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp 580 585 590Asp Lys Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala 595 600 605Ser
Gln Ala Ala Leu Gly Leu 610 61529604PRTArtificial SequenceHSA
Domain I + HSA Domain III + HSA Domain III 29Asp Ala His Lys Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys
Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro
Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser
Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 195 200
205Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
210 215 220Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val
Glu Val225 230 235 240Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys
Cys Lys His Pro Glu 245 250 255Ala Lys Arg Met Pro Cys Ala Glu Asp
Tyr Leu Ser Val Val Leu Asn 260 265 270Gln Leu Cys Val Leu His Glu
Lys Thr Pro Val Ser Asp Arg Val Thr 275 280 285Lys Cys Cys Thr Glu
Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 290 295 300Leu Glu Val
Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr305 310 315
320Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln
325 330 335Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys
Pro Lys 340 345 350Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp
Phe Ala Ala Phe 355 360 365Val Glu Lys Cys Cys Lys Ala Asp Asp Lys
Glu Thr Cys Phe Ala Glu 370 375 380Glu Gly Lys Lys Leu Val Ala Ala
Ser Gln Ala Ala Leu Gly Leu Val385 390 395 400Glu Glu Pro Gln Asn
Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 405 410 415Leu Gly Glu
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 420 425 430Lys
Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 435 440
445Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg
450 455 460Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
Leu Cys465 470 475 480Val Leu His Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys 485 490 495Thr Glu Ser Leu Val Asn Arg Arg Pro
Cys Phe Ser Ala Leu Glu Val 500 505 510Asp Glu Thr Tyr Val Pro Lys
Glu Phe Asn Ala Glu Thr Phe Thr Phe 515 520 525His Ala Asp Ile Cys
Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 530 535 540Gln Thr Ala
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys545 550 555
560Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys
565 570 575Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Lys 580 585 590Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
595 60030290PRTHomo sapiensmisc_feature(1)..(290)Truncated heavy
chain of the major histocompatibility complex class I-like Fc
receptor (FCGRT) (together, SEQ ID No. 30 and SEQ ID No. 31 form
FcRN) 30Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu
Phe1 5 10 15Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu
Leu Tyr 20 25 30His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro
Ala Phe Trp 35 40 45Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser
Tyr Asn Ser Leu 50 55 60Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val
Trp Glu Asn Gln Val65 70 75 80Ser Trp Tyr Trp Glu Lys Glu Thr Thr
Asp Leu Arg Ile Lys Glu Lys 85 90 95Leu Phe Leu Glu Ala Phe Lys Ala
Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110Leu Gln Gly Leu Leu Gly
Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125Pro Thr Ala Lys
Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140Leu Lys
Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile145 150 155
160Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr
165 170 175Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu
Glu Arg 180 185 190Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser
Met Arg Leu Lys 195 200 205Ala Arg Pro Ser Ser Pro Gly Phe Ser Val
Leu Thr Cys Ser Ala Phe 210 215 220Ser Phe Tyr Pro Pro Glu Leu Gln
Leu Arg Phe Leu Arg Asn Gly Leu225 230 235 240Ala Ala Gly Thr Gly
Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255Phe His Ala
Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270Tyr
Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280
285Glu Leu 29031119PRTHomo
sapiensmisc_feature(1)..(119)Beta-2-microglobulin (together, SEQ ID
No. 30 and SEQ ID No. 31 form FcRN) 31Met Ser Arg Ser Val Ala Leu
Ala Val Leu Ala Leu Leu Ser Leu Ser1 5 10 15Gly Leu Glu Ala Ile Gln
Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30His Pro Ala Glu Asn
Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45Gly Phe His Pro
Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50 55 60Arg Ile Glu
Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp65 70 75 80Ser
Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp 85 90
95Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile
100 105 110Val Lys Trp Asp Arg Asp Met 11532585PRTArtificial
SequenceHSA T83N, N111E 32Asp Ala His Lys Ser Glu Val Ala His Arg
Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile
Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val
Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala
Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe
Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Asn Tyr
Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn
Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Glu Leu 100 105 110Pro
Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120
125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala
Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu
Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys
Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala
Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu
Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235
240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser
Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu
Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met
Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser
Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val
Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro
Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr
Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360
365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu
Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu
Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys
His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu
Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr
Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475
480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
Thr
485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys
Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser
Gln Ala Ala Leu Gly Leu 580 58533585PRTArtificial SequenceHSA T83N,
N111E, K573P 33Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala
Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val
Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser
Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys
Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Asn Tyr Gly Glu Met
Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Glu Leu 100 105 110Pro Arg Leu Val
Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn
Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135
140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys
Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala
Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys
Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp
Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe
Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val
His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250
255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala
Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp
Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu
Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr
Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr
Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys
Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375
380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr
Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val
Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His
Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser
Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro
Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu
Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490
495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr
Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys
Phe Ala Glu Glu Gly Pro Lys Leu Val 565 570 575Ala Ala Ser Gln Ala
Ala Leu Gly Leu 580 58534585PRTArtificial SequenceHSA T83N, K573P
34Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1
5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu
Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn
Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr
Val Ala Thr Leu65 70 75 80Arg Glu Asn Tyr Gly Glu Met Ala Asp Cys
Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His
Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155
160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280
285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu
Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg
Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro
Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Pro Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 58535585PRTArtificial SequenceHSA T83K 35Asp Ala His Lys Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys
Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro
Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser
Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Lys Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58536585PRTArtificial SequenceHSA E82A 36Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Ala Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58537585PRTArtificial SequenceHSA L112F 37Asp Ala His Lys Ser Glu
Val Ala
His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val
Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp
His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys
Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr
Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu
Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu
Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Phe 100 105
110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe
Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys
Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp
Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg
Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe
Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys
Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230
235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp
Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu
Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu
Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu
Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp
Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His
Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345
350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu
355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu
Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu
Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val
Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu
Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys
Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp
Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu
Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470
475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe
His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro
Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe
Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys
Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala
Ser Gln Ala Ala Leu Gly Leu 580 58538585PRTArtificial SequenceHSA
T83K, K573P 38Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala
Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val
Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser
Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys
Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Lys Tyr Gly Glu Met
Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val
Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn
Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135
140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys
Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala
Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys
Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp
Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe
Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val
His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250
255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala
Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp
Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu
Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr
Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr
Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys
Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375
380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr
Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val
Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His
Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser
Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro
Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu
Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490
495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr
Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys
Phe Ala Glu Glu Gly Pro Lys Leu Val 565 570 575Ala Ala Ser Gln Ala
Ala Leu Gly Leu 580 58539585PRTArtificial SequenceHSA E82A, K573P
39Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1
5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu
Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn
Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr
Val Ala Thr Leu65 70 75 80Arg Ala Thr Tyr Gly Glu Met Ala Asp Cys
Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His
Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155
160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280
285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu
Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg
Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro
Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Pro Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 58540585PRTArtificial SequenceHSA L112F, K573P 40Asp Ala His
Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn
Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln
Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40
45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr
Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys
Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Phe 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310
315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val
Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425
430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val
Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys
Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser
Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn
Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser
Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu
Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys
Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550
555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu
Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58541585PRTArtificial SequenceHSA E82D 41Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Asp Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg
Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120
125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala
Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu
Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys
Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala
Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu
Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235
240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser
Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu
Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met
Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser
Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val
Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro
Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr
Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360
365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro
370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu
Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu
Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys
His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu
Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr
Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475
480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys
Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser
Gln Ala Ala Leu Gly Leu 580 58542585PRTArtificial SequenceHSA P110G
42Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1
5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu
Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn
Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr
Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys
Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His
Lys Asp Asp Asn Gly Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155
160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280
285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu
Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg
Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro
Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 58543585PRTArtificial SequenceHSA E82D, K573P 43Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Asp Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58544585PRTArtificial SequenceHSA P110G, K573P 44Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Gly
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58545585PRTArtificial sequenceHSA E505Q 45Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215
220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr
Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu
Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu
Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu
Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu
Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp
Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu
Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330
335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr
340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro
His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu
Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu
Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro
Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser
Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala
Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455
460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu
Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu
Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Gln Thr
Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu
Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys
His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met
Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570
575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 58546585PRTArtificial
sequenceHSA N111D 46Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys
Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu
Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu
Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp
Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu
Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys
Phe Leu Gln His Lys Asp Asp Asn Pro Asp Leu 100 105 110Pro Arg Leu
Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp
Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135
140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys
Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala
Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys
Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp
Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe
Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val
His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250
255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser
260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys
Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala
Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp
Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu
Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr
Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr
Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys
Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375
380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr
Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val
Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His
Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser
Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro
Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu
Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490
495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr
Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys
Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala
Ala Leu Gly Leu 580 58547585PRTArtificial sequenceHSA T527M 47Asp
Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10
15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr
Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys
Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val
Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys
Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys
Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val
Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe
Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr
Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr
Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser
Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His
Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu
Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys
Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295
300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile
Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410
415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Met Ala 515 520 525Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535
540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58548585PRTArtificial sequenceHSA N111G 48Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Gly
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58549585PRTArtificial sequenceHSA N111H 49Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro His
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58550585PRTArtificial sequenceHSA H512E 50Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Glu 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58551585PRTArtificial sequenceHSA K524A 51Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Ala Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58552585PRTArtificial sequenceHSA T527A 52Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Ala Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58553585PRTArtificial sequenceHSA E531H 53Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg
Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro
Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520
525Leu Val His Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 58554585PRTArtificial sequenceHSA N111K 54Asp Ala His Lys Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys
Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro
Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser
Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Lys
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58555585PRTArtificial sequenceHSA E425K 55Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Lys Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58556585PRTArtificial sequenceHSA K534V 56Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Val His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58557585PRTArtificial sequenceHSA H510D 57Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe Asp Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys
Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala
Ala Leu Gly Leu 580 58558585PRTArtificial sequenceHSA A569S 58Asp
Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10
15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr
Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys
Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val
Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys
Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys
Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val
Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe
Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr
Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr
Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser
180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe
Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser
Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His
Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala
Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu
Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys
Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295
300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile
Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410
415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535
540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ser Glu Glu Gly
Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58559585PRTArtificial sequenceHSA D108A 59Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Ala Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58560585PRTArtificial sequenceHSA N111D, K573P 60Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asp Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58561585PRTArtificial sequenceHSA N111G, K573P 61Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Gly Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58562585PRTArtificial sequenceHSA N111H, K573P 62Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50
55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
His Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58563585PRTArtificial sequenceHSA E425A 63Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Ala Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58564585PRTArtificial sequenceHSA E425A, K573P 64Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Ala Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58565585PRTArtificial sequenceHSA E505Q, K573P 65Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Gln
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58566585PRTArtificial sequenceHSA T527M, K573P 66Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280
285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu
Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395
400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro
405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu
Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys
Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg
Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg
Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro
Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile
Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Met Ala 515 520
525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu
530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys
Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
Gly Pro Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 58567585PRTArtificial sequenceHSA N111E 67Asp Ala His Lys Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys
Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro
Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser
Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Glu
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58568585PRTArtificial sequenceHSA N111E, K573P 68Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Glu Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58569585PRTArtificial sequenceHSA N109K 69Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Lys Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58570585PRTArtificial sequenceHSA N108E 70Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Glu Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu
Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys
Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met
Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val
Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu
Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala
Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln
Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390
395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val
Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn
Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu
Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp
Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg
Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val
Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505
510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala
515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu
Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu
Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala
Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu
Gly Leu 580 58571585PRTArtificial sequenceHSA T83N 71Asp Ala His
Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn
Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln
Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40
45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys
50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr
Leu65 70 75 80Arg Glu Asn Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys
Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310
315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val
Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425
430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys
435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val
Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys
Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser
Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn
Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser
Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu
Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys
Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550
555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu
Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58572585PRTArtificial sequenceHSA L575F 72Asp Ala His Lys Ser Glu
Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu
His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75
80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro
85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn
Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr
Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu
Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr
Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro
Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala
Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Phe Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58573585PRTArtificial sequenceHSA K534V, K573P 73Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn
Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser
Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val
Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440
445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His
450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr
Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu
Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val
Val His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val
Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val
565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580
58574585PRTArtificial SequenceHSA N111Q, K573P 74Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe
Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys
Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro
Gln Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys
Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro
Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe
Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu
Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser
Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200
205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro
210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu
Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys
Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys
Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val
Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala
Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315
320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg
325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala
Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala
Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys
Glu Leu
Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu
Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro
Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser
Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala
Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455
460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu
Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu
Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr
Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu
Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys
His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met
Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala
Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Pro Lys Leu Val 565 570
575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585
* * * * *
References