U.S. patent application number 14/633463 was filed with the patent office on 2016-02-25 for anti-serum albumin binding variants.
This patent application is currently assigned to GlaxoSmithKline Intellectual Property Development Ltd.. The applicant listed for this patent is GlaxoSmithKline Intellectual Property Development Limited. Invention is credited to Haren ARULANANTHAM, Thil Batuwangala, Elena De Angelis, Carolyn Enever, Haiqun Liu, Oliver Schon.
Application Number | 20160052998 14/633463 |
Document ID | / |
Family ID | 44582954 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160052998 |
Kind Code |
A1 |
ARULANANTHAM; Haren ; et
al. |
February 25, 2016 |
ANTI-SERUM ALBUMIN BINDING VARIANTS
Abstract
The invention relates to improved variants of the anti-serum
albumin immunoglobulin single variable domains, as well as ligands
and drug conjugates comprising such variants, compositions, nucleic
acids, vectors and hosts.
Inventors: |
ARULANANTHAM; Haren;
(Cambridge, GB) ; Batuwangala; Thil; (Cambridge,
GB) ; De Angelis; Elena; (Cambridge, GB) ;
Enever; Carolyn; (Cambridge, GB) ; Liu; Haiqun;
(Cambridge, GB) ; Schon; Oliver; (Cambridge,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GlaxoSmithKline Intellectual Property Development Limited |
Brentford |
|
GB |
|
|
Assignee: |
GlaxoSmithKline Intellectual
Property Development Ltd.
|
Family ID: |
44582954 |
Appl. No.: |
14/633463 |
Filed: |
February 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13816515 |
Feb 12, 2013 |
9012609 |
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PCT/EP2011/063999 |
Aug 12, 2011 |
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14633463 |
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61373397 |
Aug 13, 2010 |
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Current U.S.
Class: |
536/23.53 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 16/2878 20130101; C07K 2317/569 20130101; C07K 2317/56
20130101; C07K 2317/34 20130101; C07K 2317/21 20130101; C07K
2319/21 20130101; C07K 2319/00 20130101; C07K 2317/90 20130101;
C07K 2317/565 20130101; C07K 2317/92 20130101; C07K 2317/94
20130101; C07K 2317/33 20130101; C07K 2319/41 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1. A nucleic acid comprising a nucleotide sequence encoding an
anti-serum albumin (SA) immunoglobulin single variable domain
variant of DOM7h-11-15 (SEQ ID NO: 1), wherein the variant
comprises an amino acid substitution in at least one position
selected from the group consisting of: Leu49, Ala50, and Phe51,
wherein position Met32 is changed to Leu, Phe, or Ile, position
Leu49 is changed to Ile, Val, or Met, position Ala50 is changed to
Val, Leu, or Ile, and/or position Phe51 is changed to Leu, Val,
Ile, Ala, or Tyr.
2. The nucleic acid comprising a nucleotide sequence encoding an
anti-serum albumin (SA) immunoglobulin single variable domain
variant as claimed in claim 1, wherein position Met32 is changed to
Leu, position Leu49 is changed to Ile, position Ala50 is changed to
Val, and/or position Phe51 is changed to Leu.
Description
[0001] The invention relates to improved variants of anti-serum
albumin immunoglobulin single variable domains, as well as ligands
and drug conjugates comprising such variants, compositions, nucleic
acids, vectors and hosts. The invention also relates to the
identification of an epitope in serum albumin that is bound by
these anti-serum albumin immunoglobulin single variable domains and
the specific amino acid residues within those anti-serum albumin
immunoglobulin single variable domains that make contact with serum
albumin.
BACKGROUND OF THE INVENTION
[0002] WO04003019 and WO2008/096158 disclose anti-serum albumin
(SA) binding moieties, such as anti-SA immunoglobulin single
variable domains (dAbs), which have therapeutically-useful
half-lives. These documents disclose monomer anti-SA dAbs as well
as multi-specific ligands comprising such dAbs, e.g., ligands
comprising an anti-SA dAb and a dAb that specifically binds a
target antigen, such as TNFR1. Binding moieties are disclosed that
specifically bind serum albumins from more than one species, e.g.
human/mouse cross-reactive anti-SA dAbs.
[0003] WO05118642 and WO2006/059106 disclose the concept of
conjugating or associating an anti-SA binding moiety, such as an
anti-SA immunoglobulin single variable domain, to a drug, in order
to increase the half-life of the drug. Protein, peptide and NCE
(new chemical entity) drugs are disclosed and exemplified.
WO2006/059106 discloses the use of this concept to increase the
half-life of insulinotropic agents, e.g., incretin hormones such as
glucagon-like peptide (GLP)-1.
[0004] Reference is also made to Holt et al, "Anti-Serum albumin
domain antibodies for extending the half-lives of short lived
drugs", Protein Engineering, Design & Selection, vol 21, no 5,
pp 283-288, 2008.
[0005] WO2008/096158 discloses the molecules given the name
DOM7h-11 and DOM7h-14, which are good anti-SA dAbs.
PCT/EP2010/060112 describes V.sub.H AlbudAbs and affinity matured
derivatives thereof. It would be desirable to provide improved dAbs
that are variants of DOM7h-11 or DOM7h-14, or improved V.sub.H
AlbudAbs.TM., and that specifically bind serum albumin, preferably
albumins from human and non-human species, which would provide
utility in animal models of disease as well as for human therapy
and/or diagnosis. It would also be desirable to provide for the
choice between relatively modest- and high-affinity anti-SA binding
moieties (dAbs). Such moieties could be linked to drugs, the
anti-SA binding moiety being chosen according to the contemplated
end-application. This would allow the drug to be better tailored to
treating and/or preventing chronic or acute indications, depending
upon the choice of anti-SA binding moiety. It would also be
desirable to provide anti-SA dAbs that are monomeric or
substantially so in solution. This would especially be advantageous
when the anti-SA dAb is linked to a binding moiety, e.g., a dAb,
that specifically binds a cell-surface receptor, such as TNFR1,
with the aim of antagonizing the receptor. The monomeric state of
the anti-SA dAb is useful in reducing the chance of receptor
cross-linking, since multimers are less likely to form which could
bind and cross-link receptors (e.g., TNFR1) on the cell surface,
thus increasing the likelihood of receptor agonism and detrimental
receptor signaling.
[0006] SA is an abundant plasma protein and human serum albumin
(HSA) is known to bind to a number of commonly-used drugs (e.g.
warfarin, diazepam, ibuprofen) (as described, for example, by
Ghuman et al. J. Mol. Biol. 2005, 353, 38052). It would be
advantageous to provide an anti-SA binding moiety which does not
interfere with the known HSA-drug interactions.
SUMMARY OF THE INVENTION
[0007] Improved anti-SA dAbs are described in PCT/EP2010/052008 and
PCT/EP2010/052007. PCT/EP2010/060112 describes V.sub.H AlbudAbs and
affinity matured derivatives thereof.
[0008] As described herein, binding interactions between anti-SA
dAbs and SA have been identified using three different techniques.
The present inventors have therefore identified specific
interactions between improved anti-SA dAbs and domain II of HSA
thus identifying the residues within HSA that are involved in
binding by an anti-SA dAb and those residues of an anti-SA dAb that
are involved in the binding interaction. The residues from the
anti-SA dAbs which interact with SA are set out in Tables 22A and
B. Significant interactions are identified in Table 22A while
additional residues at the interface are identified in Table 22B.
Any one of the residues identified in these tables may provide an
interaction with SA. These residues may be modified in order to
modify SA binding of the variants.
[0009] Accordingly, in a first aspect of the invention, there is
provided an anti-serum albumin (SA) immunoglobulin single variable
domain variant of DOM 7h-11 (SEQ ID NO: 125) or DOM 7h-14 (SEQ ID
NO: 123), or a derivative having an amino acid sequence that is at
least 96, 97, 98 or 99% identical to the amino acid sequence of DOM
7h-11 (SEQ ID NO: 125) or DOM7h-14 (SEQ ID NO:123), wherein the
variant comprises an amino acid substitution in at least one of
positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90,
91, 92, 93 or 94 of the amino acid sequence. Positions 28, 29, 30,
31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the
amino acid sequence are those positions in the sequences set out in
the cited SEQ ID NOs: and relative to those residues set out in the
sequences given in these SEQ ID NOs. Suitably, the variant
comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16 or 17 amino acid mutations. Significant interactions are set
out in Table 22A. Accordingly, in one embodiment, the variant
comprises an amino acid substitution in at least one of positions
30, 31, 32, 49, 50, 51, 53, 67, 91 or 94 of the amino acid
sequence. Suitably, the variant comprises at least 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 amino acid mutations. Such a substitution may be
to provide the amino acid that is found at these positions in the
anti-SA immunoglobulin single variable domain DOM7h-11-3 (SEQ ID
NO:2), DOM7h-11-15 (SEQ ID NO:1) or DOM7h14-10 (SEQ ID NO:83) or an
equivalent conservative substitution. Suitably, such a substitution
may serve to improve binding affinity to SA. In one embodiment, the
variant is not a single variable domain selected from DOM7h-11-3,
DOM7h-11-15 or DOM7h-14-10. In another embodiment, the variant is
not a single variable domain as described in PCT/EP2010/052008 and
PCT/EP2010/052007.
[0010] In another aspect, there is provided an anti-serum albumin
(SA) immunoglobulin single variable domain variant of DOM 7r-31
(SEQ ID NO: 71) or DOM 7r-92 (SEQ ID NO: 75), or a derivative
having an amino acid sequence that is at least 96, 97, 98 or 99%
identical to the amino acid sequence of DOM 7r-31 (SEQ ID NO: 71)
or DOM 7r-92 (SEQ ID NO: 75), wherein the variant comprises an
amino acid substitution in at least one of positions 28, 29, 30,
31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the
amino acid sequence. In one embodiment, the variant is not a single
variable domain DOM7r-92-4. In another embodiment, the variant is
not a single variant domain as described in PCT/EP2010/060112.
[0011] In another aspect, the invention provides an anti-serum
albumin (SA) immunoglobulin single variable domain variant of
DOM7h-11-3 (SEQ ID NO: 2) or DOM7h-11-15 (SEQ ID NO: 1), or a
derivative having an amino acid sequence that is at least 96, 97,
98 or 99% identical to the amino acid sequence of DOM7h-11-3 (SEQ
ID NO: 2) or DOM7h-11-15 (SEQ ID NO: 1), wherein the variant
comprises an amino acid substitution in at least one of positions
28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93
or 94 of the amino acid sequence. Suitably, the variant comprises
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or
17 amino acid mutations. In one embodiment of this aspect, the
variant comprises an amino acid substitution in at least one of
positions 30, 31, 32, 49, 50, 51, 53, 67, 91 or 94 of the amino
acid sequence. Suitably, the variant comprises at least 1, 2, 3, 4,
5, 6, 7, 8, 9 or 10 amino acid mutations.
[0012] Amino acid substitution at any of the residues identified in
Tables 22A or 22B may enable modification of the binding properties
of an anti-SA dAb. Importantly, substitutions can be made to modify
the affinity of binding to SA to achieve the desired affinity for a
particular application.
[0013] Thus embodiments of any aspect of the invention provide
anti-SA dAb variants having good anti-serum albumin affinities. The
choice of variant can allow for tailoring of half-life according to
the desired therapeutic and/or prophylactic setting. For example,
in one embodiment, the affinity of the variant for serum albumin is
relatively high, such that the variant would be useful for
inclusion in products that find utility in treating and/or
preventing chronic or persistent diseases, conditions, toxicity or
other chronic indications, for example. In one embodiment, the
affinity of the variant for serum albumin is relatively modest,
such that the variant would be useful for inclusion in products
that find utility in treating and/or preventing acute diseases,
conditions, toxicity or other acute indications, for example. In
one embodiment, the affinity of the variant for serum albumin is
intermediate, such that the variant would be useful for inclusion
in products that find utility in treating and/or preventing acute
or chronic diseases, conditions, toxicity or other acute or chronic
indications, for example.
[0014] It is conceivable that a molecule with an appropriately high
affinity and specificity for serum albumin would stay in
circulation long enough to have the desired therapeutic effect
(Tomlinson, Nature Biotechnology 22, 521-522 (2004)). Here, a high
affinity anti-SA variant would stay in serum circulation matching
that of the species' serum albumin (WO2008096158). Once in
circulation, any fused therapeutic agent to the AlbudAb.TM. variant
(an AlbudAb is an anti-serum albumin dAb or immunoglobulin single
variable domain), be it NCE, peptide or protein, consequently would
be able to act longer on its target and exhibit a longer lasting
therapeutic effect. This would allow for targeting chronic or
persistent diseases without the need of frequent dosing.
[0015] A variant with moderate affinity (but specificity to SA)
would only stay in serum circulation for a short time (e.g., for a
few hours or a few days) allowing for the specific targeting of
therapeutic targets involved in acute diseases by the fused
therapeutic agent.
[0016] This way it is possible to tailor the anti-SA-containing
product to the therapeutic disease area by choosing an anti-SA
variant with the appropriate albumin binding affinity and/or serum
half-life.
[0017] In one embodiment, the invention provides an anti-serum
albumin (SA) immunoglobulin single variable domain variant of
DOM7h-11-15 (SEQ ID NO: 1), or a derivative having an amino acid
sequence that is at least 96, 97, 98 or 99% identical to the amino
acid sequence of DOM7h-11-15 (SEQ ID NO: 1), comprising an amino
acid substitution in at least one of positions Gly30, Thr31, Met32,
Leu49, Ala50, Phe51, Arg53, Ser67, Ala91 or His94 of the amino acid
sequence.
[0018] In one embodiment, there is provided a variant in accordance
with any aspect or embodiment of the invention wherein the
substitution is a conservative amino acid substitution. Suitable
conservative amino acid substitutions are known by those skilled in
the art and are exemplified herein in the following text. Suitably,
a conservative amino acid substitution will maintain similar
contact interactions with SA. Such conservative amino acid
substitution may allow similar binding affinities to the parental
molecule to be maintained.
[0019] Accordingly, in one embodiment, the variant comprises at
least one mutation compared to DOM 7h-11-15 selected from the
following: Position Gly 30=Pro, Ala
[0020] Position Thr 31=Ser
[0021] Position Thr 32=Ser
[0022] Position Leu 49=Norleucine, Ile, Val, Met, Ala, Phe
[0023] Position Trp 50=Tyr, Phe
[0024] Position Asn 51=Gln
[0025] Position Arg 53=Lys, Gln, Asn
[0026] Position Ser 67=Thr, Ala, Cys
[0027] Position Ala 91=Val, Leu, Ile
[0028] Position His 94=Asn, Gln, Lys, Arg
[0029] In another embodiment, there is provided an anti-serum
albumin (SA) immunoglobulin single variable domain variant of DOM
7h-11-3 (SEQ ID NO: 2), or a derivative having an amino acid
sequence that is at least 96, 97, 98 or 99% identical to the amino
acid sequence of DOM 7h-11-3 (SEQ ID NO: 2), wherein the variant
comprises an amino acid substitution in at least one of positions
Gly30, Thr31, Thr32, Leu49, Trp50, Asn51, Arg53, Ser67, Ala91 or
His94 of the amino acid sequence.
[0030] Suitably, the variant comprises a conservative substitution
such that the variant comprises at least one mutation compared to
DOM7h-11-3 selected from the following:
[0031] Position Gly30=Pro, Ala
[0032] Position Thr31=Ser
[0033] Position Thr32=Ser
[0034] Position Leu49=Norleucine, Ile, Val, Met, Ala, Phe
[0035] Position Trp50=Tyr, Phe
[0036] Position Asn51=Gln
[0037] Position Arg53=Lys, Gln, Asn
[0038] Position Ser67=Thr, Ala, Cys
[0039] Position Ala91=Val, Leu, Ile
[0040] Position His94=Asn, Gln, Lys, Arg
[0041] In another embodiment of any aspect or embodiment of the
invention, the substitution is not a conservative substitution.
Introducing a non-conservative substitution/mutation at one of the
residues known to be involved in binding to SA is one way in which
affinity to SA may be decreased or otherwise altered.
[0042] In one embodiment of any aspect or embodiment of the
invention SA is SA from an animal, e.g., a mammal, e.g., a
non-human primate (such as a baboon, rhesus monkey or Cynomolgus
monkey), mouse, human, rabbit, rat, dog, cat or pig. In one
embodiment SA is human SA (HSA).
[0043] In another aspect, the invention provides an HSA binding
moiety which binds to an epitope comprising at least part of the
interface defined by amino acids 227, 228, 229, 230, 232, 233, 263,
307, 308, 309, 314, 317, 318, 321, 322, 325, 326, 329 and 333 of
HSA (wherein sequence is given in SEQ ID NO:81). In one embodiment,
the binding moiety binds to an epitope comprising at least part of
the interface defined by amino acids 228, 230, 308, 309, 317, 318,
321, 322, 325, 326 and 329 of HSA. The residues of HSA which
interact with the anti-HSA binding moieties exemplified by anti-SA
dAbs herein are set out in Tables 22A and B. Significant
interactions are identified in Table 22A while additional residues
at the interface are identified in Table 22B. Any one of the
residues identified in these tables may provide an interaction
between HSA and the HSA binding moiety.
[0044] Suitably, the binding moiety may comprise amino acids
identified in SEQ ID NOs: 1 or 2 at positions 28, 29, 30, 31, 32,
36, 46, 49, 50, 51, 53, 67, 68, 90, 91, 92, 93 or 94 of the amino
acid sequence wherein these amino acids enable binding to SA.
However, these amino acids may be part of a domain which is a
derivative of a non-immunoglobulin protein scaffold. In one
embodiment, the binding moiety is an antibody. Suitably, the
binding moiety is an anti-SA immunoglobulin single variable domain
antibody.
[0045] In one embodiment the variant or binding moiety in
accordance with any aspect or embodiment of the invention comprises
a binding site that specifically binds human SA with a dissociation
constant (KD) of from about 0.1 to about 10000 nM, optionally from
about 1 to about 6000 nM, as determined by surface plasmon
resonance. In another embodiment, the variant or binding moiety in
accordance with any aspect or embodiment of the invention comprises
a binding site that specifically binds human SA with an off-rate
constant (K.sub.d) of from about 1.5.times.10.sup.-4 to about 0.1
sec.sup.-1, optionally from about 3.times.10.sup.-4 to about 0.1
sec.sup.-1 as determined by surface plasmon resonance. In another
embodiment the variant or binding moiety in accordance with any
aspect or embodiment of the invention comprises a binding site that
specifically binds human SA with an on-rate constant (K.sub.a) of
from about 2.times.10.sup.6 to about 1.times.10.sup.4 M.sup.-1
sec.sup.-1, optionally from about 1.times.10.sup.6 to about
2.times.10.sup.4 M.sup.-1 sec.sup.-1 as determined by surface
plasmon resonance. In a further embodiment, the variant or binding
moiety in accordance with any aspect or embodiment of the invention
comprises a binding site that specifically binds Cynomolgus monkey
SA with a dissociation constant (KD) of from about 0.1 to about
10000 nM, optionally from about 1 to about 6000 nM, as determined
by surface plasmon resonance. In yet a further embodiment, the
variant or binding moiety in accordance with any aspect or
embodiment of the invention comprises a binding site that
specifically binds Cynomolgus monkey SA with an off-rate constant
(K.sub.d) of from about 1.5.times.10.sup.-4 to about 0.1
sec.sup.-1, optionally from about 3.times.10.sup.-4 to about 0.1
sec.sup.-1 as determined by surface plasmon resonance. Another
embodiment provides a variant or binding moiety in accordance with
any aspect or embodiment of the invention, wherein the variant
comprises a binding site that specifically binds Cynomolgus monkey
SA with an on-rate constant (K.sub.a) of from about
2.times.10.sup.6 to about 1.times.10.sup.4 M.sup.-1 sec.sup.-1,
optionally from about 1.times.10.sup.6 to about
5.times.10.sup.3M.sup.-1 sec.sup.-1 as determined by surface
plasmon resonance.
[0046] An aspect of the invention provides a multispecific ligand
comprising any anti-SA variant or SA-binding moiety as described
above and a binding moiety that specifically binds a target antigen
other than SA.
[0047] An aspect of the invention provides fusion proteins,
conjugates or compositions comprising any variant or binding moiety
in accordance with the invention. For example, the invention
provides e.g., a fusion protein or fusion with a peptide or NCE
(new chemical entity) drug, comprising a polypeptide, protein,
peptide or NCE drug fused or conjugated (for an NCE) to any variant
or binding moiety as described above. In one embodiment, the
variant or binding moiety gives only a modest drop in affinity when
fused or conjugated to a partner making them useful in fusion
products. An aspect of the invention provides a composition
comprising a variant, fusion protein or ligand of any preceding
aspect and a pharmaceutically acceptable diluent, carrier,
excipient or vehicle.
[0048] Another aspect of the invention provides a nucleic acid
comprising a nucleotide sequence encoding a variant, binding
moiety, multispecific ligand or fusion protein in accordance with
any aspect or embodiment of the invention.
[0049] Another aspect provides a nucleic acid comprising the
nucleotide sequence of a DOM7h-11, DOM7h-14, DOM7h-11-3 or
DOM7h-11-15 variant in accordance with the invention or a
nucleotide sequence that is at least 80% identical to said selected
sequence. Further aspects provide a vector comprising a nucleic
acid of the invention and an isolated host cell comprising such a
vector.
[0050] An aspect of the invention provides a method of treating or
preventing a disease or disorder in a patient, comprising
administering at least one dose of a variant or binding moiety
according to any aspect or embodiment of the invention to said
patient. The invention further provides a variant or binding moiety
in accordance with the present invention for use as a
medicament.
[0051] In another aspect of the invention there is provided a
method for affinity maturation of an anti-SA immunoglobulin single
variable domain comprising taking an anti-SA immunoglobulin single
variable domain and introducing a mutation at an amino acid at any
one of positions 28, 29, 30, 31, 32, 36, 46, 49, 50, 51, 53, 67,
68, 90, 91, 92, 93 or 94 of the amino acid sequence of an anti-SA
immunoglobulin single variable domain. In one embodiment the
anti-SA immunoglobulin single variable domain is, or is derived
from, the amino acid sequence of DOM7h-11 or DOM7h-14. Methods for
obtaining anti-SA immunoglobulin single variable domain molecules
are described, for example, in PCT/EP2010/052008 and
PCT/EP2010/052007. In one embodiment, the maturation may be in
silico. Suitable in silico methods are described, for example, in
Barderas et al. (2008), PNAS, 105, 26, p. 9029-9034. Preferably,
the method comprises introducing a mutation at any one of the amino
acids at positions 30, 31, 32, 49, 50, 51, 53, 67, 91 or 94 or the
amino acid sequence.
[0052] Another aspect provides a method of modifying the binding
affinity of an anti-SA immunoglobulin single variable domain
comprising mutating an amino acid at any one of positions 30, 31,
32, 49, 50, 51, 53, 67, 91 or 94 of the amino acid sequence of an
anti-SA immunoglobulin single variable domain. Suitably the anti-SA
immunoglobulin single variable domain is a DOM7h-11 or DOM7h-14
derivative. Mutations may be to introduce conservative or
non-conservative amino acid substitutions as described above.
[0053] In these aspects, suitably the mutation at any one of these
positions is chosen to modify the affinity of binding to SA of the
matured sequences when compared to the parental anti-SA
immunoglobulin single variable domain.
[0054] The present inventors have identified a specific region of
Serum Albumin that can be bound by an anti-SA binding moiety.
Advantageously, this region is one which can be bound by a binding
moiety, serving to enhance the half life of the bound moiety whilst
not blocking any of the known drug binding sites such that the
binding interaction does not have an effect on the other
drug-binding properties of Serum Albumin. The binding portion
identified can be used to identify binding moieties that
preferentially bind to this region.
[0055] Accordingly, in another aspect of the invention, there is
provided a method of identifying SA binding moieties comprising
taking a portion of HSA defined by amino acids 213-229, 231-238,
321-331, 334-342 or 348-357; or 213-219, 222-228, 231-238, 311-218,
321-324, 329-333 or 347-357; or 321-326 or 329-331 wherein
reference to amino acid residues are references to those amino
acids set out in SEQ ID NO: 81, and using said portions in a
binding assay or screen. In another aspect there is provided a
method for generating an anti-HSA binding agent comprising taking a
portion of HSA defined by amino acids 213-229, 231-238, 321-331,
334-342 or 348-357; or 213-219, 222-228, 231-238, 311-218, 321-324,
329-333 or 347-357; or 321-326 or 329-331 wherein reference to
amino acid residues are references to those amino acids set out in
SEQ ID NO: 81, and using these portions in a screen or assay.
[0056] Suitably an anti-HSA binding moiety may be derived using a
part of HSA comprising the binding epitope described herein. In one
embodiment, the method comprises providing an HSA polypeptide
comprising at least part of the binding interface defined by amino
acids 228, 230, 308, 309, 317, 318, 321, 322, 325, 326 and 329 of
HSA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1: Amino-acid sequence alignment for DOM7h-11 variant
dAbs. A "." at a particular position indicates the same amino as
found in DOM7h-11 at that position. The CDRs are indicated by
underlining and bold text (the first underlined sequence is CDR1,
the second underlined sequence is CDR2 and the third underlined
sequence is CDR3). FIG. 2: Kinetic parameters of DOM7h-11 variants.
KD units=nM; Kd units=sec.sup.-1; Ka units=M.sup.-1 sec.sup.-1. The
notation A e-B means A.times.10.sup.-B and C e D means
C.times.10.sup.D. The overall kinetic ranges in various species, as
supported by the examples below, are indicated. Optional ranges are
also provided for use in particular therapeutic settings (acute or
chronic indications, conditions or diseases and "intermediate" for
use in both chronic and acute settings). High affinity dAbs and
products comprising these are useful for chronic settings. Medium
affinity dAbs and products comprising these are useful for
intermediate settings. Low affinity dAbs and products comprising
these are useful for acute settings. The affinity in this respect
is the affinity for serum albumin. Various example anti-serum dAbs
and fusion proteins are listed, and these support the ranges
disclosed. Many of the examples have favourable kinetics in human
and one or more non-human animals (e.g., in human and Cynomolgus
monkey and/or mouse). Choice of dAb or product comprising this can
be tailored, according to the invention, depending on the setting
(e.g., chronic or acute) to be treated therapeutically.
[0058] FIG. 3: Sequence segments of HSA identified as possible
DOM7h-11-3 epitopes from H/D exchange data
[0059] FIG. 4: Overall structure of HSA in complex with DOM7h11-15.
HSA and DOM7h11-15 are depicted schematically in ribbon
representation. Chains A and C are HSA; Chain B and D are
DOM7h-11-15. FIG. 4A shows the asymmetric unit; FIG. 4B shows
biologically relevant complex in 3 different orientations; FIG. 4C
shows electron density map for DOM7h-11-15 calculated from phases
from the final model contoured at 2.06.
[0060] FIG. 5: Grid showing contacting residues between HSA and
DOM7h-11-15.
[0061] FIG. 6: Alignment of DOM7h-11 lineage AlbudAbs.
[0062] FIG. 7: Detail of interactions between HSA and
DOM7h-11-15.
[0063] FIG. 8: Comparison of epitope data from orthogonal
techniques. HSA is rendered in surface representation and dark
patches depict epitope regions identified using each technique.
[0064] FIG. 9: Positions of lipid carrier pockets relative to
DOM7h-11-15 binding site. The HSA backbone is drawn in tube
representation; lipid molecules are dark spheres; electron density
for DOM7h-11-15 is drawn in dark mesh. 1E7G.pdb was structurally
aligned to the peptide backbone of the HSA/DOM7h-11-15 crystal
structure to superimpose lipid moieties to carrier pockets
(FA1-FA7: fatty acid binding sites).
[0065] FIG. 10: Nucleic and amino acid sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0066] Within this specification the invention has been described,
with reference to embodiments, in a way which enables a clear and
concise specification to be written. It is intended and should be
appreciated that embodiments may be variously combined or separated
without parting from the invention.
[0067] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art (e.g., in cell culture, molecular
genetics, nucleic acid chemistry, hybridization techniques and
biochemistry). Standard techniques are used for molecular, genetic
and biochemical methods (see generally, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short Protocols in Molecular Biology (1999) 4.sup.th Ed, John Wiley
& Sons, Inc. which are incorporated herein by reference) and
chemical methods.
[0068] A "patient" is any animal, e.g., a mammal, e.g., a non-human
primate (such as a baboon, rhesus monkey or Cynomolgus monkey),
mouse, human, rabbit, rat, dog, cat or pig. In one embodiment, the
patient is a human.
[0069] As used herein an antibody refers to IgG, IgM, IgA, IgD or
IgE or a fragment (such as a Fab, Fab', F(ab').sub.2, Fv,
disulphide linked Fv, scFv, closed conformation multispecific
antibody, disulphide-linked scFv, diabody) whether derived from any
species naturally producing an antibody, or created by recombinant
DNA technology; whether isolated from serum, B-cells, hybridomas,
transfectomas, yeast or bacteria.
[0070] As used herein, "antibody format" refers to any suitable
polypeptide structure in which one or more antibody variable
domains can be incorporated so as to confer binding specificity for
antigen on the structure. A variety of suitable antibody formats
are known in the art, such as, chimeric antibodies, humanized
antibodies, human antibodies, single chain antibodies, bispecific
antibodies, antibody heavy chains, antibody light chains,
homodimers and heterodimers of antibody heavy chains and/or light
chains, antigen-binding fragments of any of the foregoing (e.g., a
Fv fragment (e.g., single chain Fv (scFv), a disulfide bonded Fv),
a Fab fragment, a Fab' fragment, a F(ab').sub.2 fragment), a single
antibody variable domain (e.g., a dAb, V.sub.H, V.sub.HH, V.sub.L),
and modified versions of any of the foregoing (e.g., modified by
the covalent attachment of polyethylene glycol or other suitable
polymer or a humanized V.sub.HH).
[0071] The phrase "immunoglobulin single variable domain" refers to
an antibody variable domain (V.sub.H, V.sub.HH, V.sub.L) that
specifically binds an antigen or epitope independently of different
V regions or domains. An immunoglobulin single variable domain can
be present in a format (e.g., homo- or hetero-multimer) with other
variable regions or variable domains where the other regions or
domains are not required for antigen binding by the single
immunoglobulin variable domain (i.e., where the immunoglobulin
single variable domain binds antigen independently of the
additional variable domains). A "domain antibody" or "dAb" is the
same as an "immunoglobulin single variable domain" as the term is
used herein. A "single immunoglobulin variable domain" is the same
as an "immunoglobulin single variable domain" as the term is used
herein. A "single antibody variable domain" or an "antibody single
variable domain" is the same as an "immunoglobulin single variable
domain" as the term is used herein. An immunoglobulin single
variable domain is in one embodiment a human antibody variable
domain, but also includes single antibody variable domains from
other species such as rodent (for example, as disclosed in WO
00/29004, the contents of which are incorporated herein by
reference in their entirety), nurse shark and Camelid V.sub.HH
dAbs. Camelid V.sub.HH are immunoglobulin single variable domain
polypeptides that are derived from species including camel, llama,
alpaca, dromedary, and guanaco, which produce heavy chain
antibodies naturally devoid of light chains. The V.sub.HH may be
humanized.
[0072] A "domain" is a folded protein structure which has tertiary
structure independent of the rest of the protein. Generally,
domains are responsible for discrete functional properties of
proteins and, in many cases, may be added, removed or transferred
to other proteins without loss of function of the remainder of the
protein and/or of the domain. A "single antibody variable domain"
is a folded polypeptide domain comprising sequences characteristic
of antibody variable domains. It therefore includes complete
antibody variable domains and modified variable domains, for
example, in which one or more loops have been replaced by sequences
which are not characteristic of antibody variable domains, or
antibody variable domains which have been truncated or comprise N-
or C-terminal extensions, as well as folded fragments of variable
domains which retain at least the binding activity and specificity
of the full-length domain.
[0073] In the instant application, the term "prevention" and
"preventing" involves administration of the protective composition
prior to the induction of the disease or condition. "Treatment" and
"treating" involves administration of the protective composition
after disease or condition symptoms become manifest. "Suppression"
or "suppressing" refers to administration of the composition after
an inductive event, but prior to the clinical appearance of the
disease or condition.
[0074] As used herein, the term "dose" refers to the quantity of
ligand administered to a subject all at one time (unit dose), or in
two or more administrations over a defined time interval. For
example, dose can refer to the quantity of ligand (e.g., ligand
comprising an immunoglobulin single variable domain that binds
target antigen) administered to a subject over the course of one
day (24 hours) (daily dose), two days, one week, two weeks, three
weeks or one or more months (e.g., by a single administration, or
by two or more administrations). The interval between doses can be
any desired amount of time. The term "pharmaceutically effective"
when referring to a dose means sufficient amount of the ligand,
domain or pharmaceutically active agent to provide the desired
effect. The amount that is "effective" will vary from subject to
subject, depending on the age and general condition of the
individual, the particular drug or pharmaceutically active agent
and the like. Thus, it is not always possible to specify an exact
"effective" amount applicable for all patients. However, an
appropriate "effective" dose in any individual case may be
determined by one of ordinary skill in the art using routine
experimentation.
[0075] Methods for pharmacokinetic analysis and determination of
ligand (e.g., single variable domain, fusion protein or
multi-specific ligand) half-life will be familiar to those skilled
in the art. Details may be found in Kenneth, A et al: Chemical
Stability of Pharmaceuticals: A Handbook for Pharmacists and in
Peters et al, Pharmacokinetic analysis: A Practical Approach
(1996). Reference is also made to "Pharmacokinetics", M Gibaldi
& D Perron, published by Marcel Dekker, 2.sup.nd Rev. ex
edition (1982), which describes pharmacokinetic parameters such as
t alpha and t beta half lives and area under the curve (AUC).
Optionally, all pharmacokinetic parameters and values quoted herein
are to be read as being values in a human. Optionally, all
pharmacokinetic parameters and values quoted herein are to be read
as being values in a mouse or rat or Cynomolgus monkey.
[0076] Half lives (t1/2 alpha and t1/2 beta) and AUC can be
determined from a curve of serum concentration of ligand against
time. The WinNonlin analysis package, e.g. version 5.1 (available
from Pharsight Corp., Mountain View, Calif. 94040, USA) can be
used, for example, to model the curve. When two-compartment
modeling is used, in a first phase (the alpha phase) the ligand is
undergoing mainly distribution in the patient, with some
elimination. A second phase (beta phase) is the phase when the
ligand has been distributed and the serum concentration is
decreasing as the ligand is cleared from the patient. The t alpha
half life is the half life of the first phase and the t beta half
life is the half life of the second phase. Thus, in one embodiment,
in the context of the present invention, the variable domain,
fusion protein or ligand has a t alpha half life in the range of
(or of about) 15 minutes or more. In one embodiment, the lower end
of the range is (or is about) 30 minutes, 45 minutes, 1 hour, 2
hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11
hours or 12 hours. In addition, or alternatively, the variable
domain, fusion protein or ligand according to the invention will
have a t alpha half life in the range of up to and including 12
hours (or about 12 hours). In one embodiment, the upper end of the
range is (or is about) 11, 10, 9, 8, 7, 6 or 5 hours. An example of
a suitable range is (or is about) 1 to 6 hours, 2 to 5 hours or 3
to 4 hours.
[0077] In one embodiment, the present invention provides the
variable domain, fusion protein or ligand according to the
invention has a t beta half life in the range of (or of about) 2.5
hours or more. In one embodiment, the lower end of the range is (or
is about) 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11
hours, or 12 hours. In addition, or alternatively, the t beta half
life is (or is about) up to and including 21 or 25 days. In one
embodiment, the upper end of the range is (or is about) 12 hours,
24 hours, 2 days, 3 days, 5 days, 10 days, 15 days, 19 days, 20
days, 21 days or 22 days. For example, the variable domain, fusion
protein or ligand according to the invention will have a t beta
half life in the range 12 to 60 hours (or about 12 to 60 hours). In
a further embodiment, it will be in the range 12 to 48 hours (or
about 12 to 48 hours). In a further embodiment still, it will be in
the range 12 to 26 hours (or about 12 to 26 hours).
[0078] As an alternative to using two-compartment modeling, the
skilled person will be familiar with the use of non-compartmental
modeling, which can be used to determine terminal half-lives (in
this respect, the term "terminal half-life" as used herein means a
terminal half-life determined using non-compartmental modeling).
The WinNonlin analysis package, e.g. version 5.1 (available from
Pharsight Corp., Mountain View, Calif. 94040, USA) can be used, for
example, to model the curve in this way. In this instance, in one
embodiment the single variable domain, fusion protein or ligand has
a terminal half life of at least (or at least about) 8 hours, 10
hours, 12 hours, 15 hours, 28 hours, 20 hours, 1 day, 2 days, 3
days, 7 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days,
20 days, 21 days, 22 days, 23 days, 24 days or 25 days. In one
embodiment, the upper end of this range is (or is about) 24 hours,
48 hours, 60 hours or 72 hours or 120 hours. For example, the
terminal half-life is (or is about) from 8 hours to 60 hours, or 8
hours to 48 hours or 12 to 120 hours, e.g., in man.
[0079] In addition, or alternatively to the above criteria, the
variable domain, fusion protein or ligand according to the
invention has an AUC value (area under the curve) in the range of
(or of about) 1 mgmin/ml or more. In one embodiment, the lower end
of the range is (or is about) 5, 10, 15, 20, 30, 100, 200 or 300
mgmin/ml. In addition, or alternatively, the variable domain,
fusion protein or ligand according to the invention has an AUC in
the range of (or of about) up to 600 mgmin/ml. In one embodiment,
the upper end of the range is (or is about) 500, 400, 300, 200,
150, 100, 75 or 50 mgmin/ml. Advantageously the variable domain,
fusion protein or ligand will have an AUC in (or about in) the
range selected from the group consisting of the following: 15 to
150 mgmin/ml, 15 to 100 mgmin/ml, 15 to 75 mgmin/ml, and 15 to 50
mgmin/ml.
[0080] "Surface Plasmon Resonance": Competition assays can be used
to determine if a specific antigen or epitope, such as human serum
albumin, competes with another antigen or epitope, such as
cynomolgus serum albumin, for binding to a serum albumin binding
ligand described herein, such as a specific dAb. Similarly
competition assays can be used to determine if a first ligand, such
as dAb, competes with a second ligand such as a dAb for binding to
a target antigen or epitope. The term "competes" as used herein
refers to substance, such as a molecule, compound, preferably a
protein, which is able to interfere to any extent with the specific
binding interaction between two or more molecules. The phrase "does
not competitively inhibit" means that substance, such as a
molecule, compound, preferably a protein, does not interfere to any
measurable or significant extent with the specific binding
interaction between two or more molecules. The specific binding
interaction between two or more molecules preferably includes the
specific binding interaction between a single variable domain and
its cognate partner or target. The interfering or competing
molecule can be another single variable domain or it can be a
molecule that is structurally and/or functionally similar to a
cognate partner or target.
[0081] The term "binding moiety" refers to a domain that
specifically binds an antigen or epitope independently of a
different epitope or antigen binding domain. A binding moiety may
be a domain antibody (dAb) or may be a domain which is a derivative
of a non-immunoglobulin protein scaffold, e.g., a scaffold selected
from the group consisting of CTLA-4, lipocalin, SpA, an adnectin,
affibody, an avimer, GroEI, transferrin, GroES and fibronectin,
which binds to a ligand other than the natural ligand (in the case
of the present invention, the moiety binds serum albumin). See
WO2008/096158, which discloses examples of protein scaffolds and
methods for selecting antigen or epitope-specific binding domains
from repertoires (see Examples 17 to 25). These specific
disclosures of WO2008/096158 are expressly incorporated herein by
reference as though explicitly written herein and for use with the
present invention, and it is contemplated that any part of such
disclosure can be incorporated into one or more claims herein). In
one aspect, the invention provides a binding moiety comprising the
amino acids which interact with and/or enable binding to SA as
described herein wherein the interacting amino acids are presented
in the context of an alternative or non-immunoglobulin
scaffold.
[0082] In one embodiment, the mutations at any of the positions
identified in accordance with any aspect or embodiment of the
invention are mutations to residues as exemplified in the Examples
section herein. In another embodiment, mutations are to
conservative amino acids substitutions of the exemplified
residues.
[0083] Conservative amino acid substitutions are well know to the
person skilled in the art and are exemplified by the following
table:
TABLE-US-00001 Amino Acid Substitution Original Residues Exemplary
Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg
Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn
Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile
Leu, Val, Met, Ala, Phe, Leu Norleucine Leu Norleucine, Ile, Val,
Met, Ile Ala, Phe Lys Arg, 1,4 Diamino- Arg butyricAcid, Gln, Asn
Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly
Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,
Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Leu Norleucine
[0084] Conservative amino acid substitutions may also relate to
non-naturally occurring amino acid residues, such as
peptidomimetics and other reversed or inverted forms of amino acid
moieties which may be incorporated by chemical peptide
synthesis.
[0085] In one embodiment, the variant comprises one or more of the
following kinetic characteristics:-- [0086] (a) The variant
comprises a binding site that specifically binds human SA with a
dissociation constant (KD) from (or from about) 0.1 to (or to
about) 10000 nM, optionally from (or from about) 1 to (or to about)
6000 nM, as determined by surface plasmon resonance; [0087] (b) The
variant comprises a binding site that specifically binds human SA
with an off-rate constant (K.sub.d) from (or from about)
1.5.times.10.sup.-4 to (or to about) 0.1 sec.sup.-1, optionally
from (or from about) 3.times.10.sup.-4 to (or to about) 0.1
sec.sup.-1 as determined by surface plasmon resonance; [0088] (c)
The variant comprises a binding site that specifically binds human
SA with an on-rate constant (K.sub.a) from (or from about)
2.times.10.sup.6 to (or to about) 1.times.10.sup.4 M.sup.-1
sec.sup.-1, optionally from (or from about) 1.times.10.sup.6 to (or
to about) 2.times.10.sup.4 M.sup.-1 sec.sup.-1 as determined by
surface plasmon resonance; [0089] (d) The variant comprises a
binding site that specifically binds Cynomolgus monkey SA with a
dissociation constant (KD) from (or from about) 0.1 to (or to
about) 10000 nM, optionally from (or from about) 1 to (or to about)
6000 nM, as determined by surface plasmon resonance; [0090] (e) The
variant of any preceding claim, wherein the variant comprises a
binding site that specifically binds Cynomolgus monkey SA with an
off-rate constant (K.sub.d) from (or from about)
1.5.times.10.sup.-4 to (or to about) 0.1 sec.sup.-1, optionally
from (or from about) 3.times.10.sup.-4 to (or to about) 0.1
sec.sup.-1 as determined by surface plasmon resonance; [0091] (f)
The variant of any preceding claim, wherein the variant comprises a
binding site that specifically binds Cynomolgus monkey SA with an
on-rate constant (K.sub.a) from (or from about) 2.times.10.sup.6 to
(or to about) 1.times.10.sup.4 M.sup.-1 sec.sup.-1, optionally from
(or from about) 1.times.10.sup.6 to (or to about) 5.times.10.sup.3
M.sup.-1 sec.sup.-1 as determined by surface plasmon resonance;
[0092] (g) The variant comprises a binding site that specifically
binds rat SA with a dissociation constant (KD) from (or from about)
1 to (or to about) 10000 nM, optionally from (or from about) 20 to
(or to about) 6000 nM, as determined by surface plasmon resonance;
[0093] (h) The variant comprises a binding site that specifically
binds rat SA with an off-rate constant (K.sub.d) from (or from
about) 2.times.10.sup.-3 to (or to about) 0.15 sec.sup.-1,
optionally from (or from about) 9.times.10.sup.-3 to (or to about)
0.14 sec.sup.-1 as determined by surface plasmon resonance; [0094]
(i) The variant comprises a binding site that specifically binds
rat SA with an on-rate constant (K.sub.a) from (or from about)
2.times.10.sup.6 to (or to about) 1.times.10.sup.4 M.sup.-1
sec.sup.-1, optionally from (or from about) 1.times.10.sup.6 to (or
to about) 3.times.10.sup.4 M.sup.-1 sec.sup.-1 as determined by
surface plasmon resonance; [0095] (j) The variant comprises a
binding site that specifically binds mouse SA with a dissociation
constant (KD) from (or from about) 1 to (or to about) 10000 nM as
determined by surface plasmon resonance; [0096] (k) The variant
comprises a binding site that specifically binds mouse SA with an
off-rate constant (K.sub.d) from (or from about) 2.times.10.sup.-3
to (or to about) 0.15 sec.sup.-1 as determined by surface plasmon
resonance; and/or [0097] (l) The variant comprises a binding site
that specifically binds mouse SA with an on-rate constant (K.sub.a)
from (or from about) 2.times.10.sup.6 to (or to about)
1.times.10.sup.4 M.sup.-1 sec.sup.-1, optionally from (or from
about) 2.times.10.sup.6 to (or to about) 1.5.times.10.sup.4
M.sup.-1 sec.sup.-1 as determined by surface plasmon resonance.
[0098] Optionally, the variant has [0099] I: a KD according to (a)
and (d), a K.sub.d according to (b) and (e), and a K.sub.a
according to (c) and (f); or [0100] II: a KD according to (a) and
(g), a K.sub.d according to (b) and (h), and a K.sub.a according to
(c) and (i); or [0101] III: a KD according to (a) and (j), a
K.sub.d according to (b) and (k), and a K.sub.a according to (c)
and (I); or [0102] IV: kinetics according to I and II; or [0103] V:
kinetics according to I and III; or [0104] VI: kinetics according
to I, II and III.
[0105] The invention also provides a ligand comprising a variant of
any preceding aspect or embodiment of the invention. For example,
the ligand can be a dual-specific ligand (see WO04003019 for
examples of dual-specific ligands). In one aspect, the invention
provides a multispecific ligand comprising an anti-SA variant of
any preceding aspect or embodiment of the invention and a binding
moiety that specifically binds a target antigen other than SA. The
binding moiety can be any binding moiety that specifically binds a
target, e.g., the moiety is an antibody, antibody fragment, scFv,
Fab, dAb or a binding moiety comprising a non-immunoglobulin
protein scaffold. Such moieties are disclosed in detail in
WO2008/096158 (see examples 17 to 25, which disclosure is
incorporated herein by reference). Examples of non-immunoglobulin
scaffolds are CTLA-4, lipocallin, staphylococcal protein A (spA),
Affibody.TM., Avimers.TM., adnectins, GroEL and fibronectin.
[0106] In one embodiment, a linker is provided between the
anti-target binding moiety and the anti-SA single variant, the
linker comprising the amino acid sequence AST, optionally ASTSGPS.
Alternative linkers are described in WO2007085814 (incorporated
herein by reference), WO2008/096158 (see the passage at page 135,
line 12 to page 140, line 14, which disclosure and all sequences of
linkers are expressly incorporated herein by reference as though
explicitly written herein and for use with the present invention,
and it is contemplated that any part of such disclosure can be
incorporated into one or more claims herein) and WO2009/068649.
[0107] In one embodiment of the multispecific ligand, the target
antigen may be, or be part of, polypeptides, proteins or nucleic
acids, which may be naturally occurring or synthetic. In this
respect, the ligand of the invention may bind the target antigen
and act as an antagonist or agonist (e.g., EPO receptor agonist).
One skilled in the art will appreciate that the choice is large and
varied. They may be for instance, human or animal proteins,
cytokines and growth factors, cytokine receptors, where cytokine
receptors include receptors for cytokines, enzymes, co-factors for
enzymes or DNA binding proteins.
[0108] As used herein, the term "antagonist of Tumor Necrosis
Factor Receptor 1 (TNFR1)" or "anti-TNFR1 antagonist" or the like
refers to an agent (e.g., a molecule, a compound) which binds TNFR1
and can inhibit a (i.e., one or more) function of TNFR1. For
example, an antagonist of TNFR1 can inhibit the binding of TNF
alpha to TNFR1 and/or inhibit signal transduction mediated through
TNFR1. Accordingly, TNFR1-mediated processes and cellular responses
(e.g., TNF alpha-induced cell death in a standard L929 cytotoxicity
assay) can be inhibited with an antagonist of TNFR1.
[0109] In one embodiment, the multispecific ligand comprises an
anti-SA dAb variant of the invention and an anti-TNFR1 binding
moiety, e.g., an anti-TNFR1 dAb. Optionally, the ligand has only
one anti-TNFR1 binding moiety (e.g., dAb) to reduce the chance of
receptor cross-linking. Anti-TNFR1 dAbs are described, for example,
in WO2006/038027, WO2007/049017, WO2008149148 and WO2010/081787
(the amino acid sequences of which and the nucleotide sequence of
which, as disclosed in those PCT applications, are expressly
incorporated herein by reference as though explicitly written
herein and for use with the present invention, and it is
contemplated that any part of such disclosures can be incorporated
into one or more claims herein).
[0110] In one embodiment, the ligand of the invention is a fusion
protein comprising a variant of the invention fused directly or
indirectly to one or more polypeptides. For example, the fusion
protein can be a "drug fusion" as disclosed in WO2005/118642 (the
disclosure of which is incorporated herein by reference),
comprising a variant of the invention and a polypeptide drug as
defined in that PCT application.
[0111] As used herein, "drug" refers to any compound (e.g., small
organic molecule, nucleic acid, polypeptide) that can be
administered to an individual to produce a beneficial, therapeutic
or diagnostic effect through binding to and/or altering the
function of a biological target molecule in the individual. The
target molecule can be an endogenous target molecule encoded by the
individual's genome (e.g. an enzyme, receptor, growth factor,
cytokine encoded by the individual's genome) or an exogenous target
molecule encoded by the genome of a pathogen (e. g. an enzyme
encoded by the genome of a virus, bacterium, fungus, nematode or
other pathogen). Suitable drugs for use in fusion proteins and
conjugates comprising an anti-SA dAb variant of the invention are
disclosed in WO2005/118642 and WO2006/059106 (the entire
disclosures of which are incorporated herein by reference, and
including the entire list of specific drugs as though this list
were expressly written herein, and it is contemplated that such
incorporation provides disclosure of specific drugs for inclusion
in claims herein). For example, the drug can be glucagon-like
peptide 1 (GLP-1) or a variant, interferon alpha 2b or a variant or
exendin-4 or a variant.
[0112] In one embodiment, the invention provides a drug conjugate
as defined and disclosed in WO2005/118642 and WO2006/059106,
wherein the conjugate comprises a variant of the invention. In one
example, the drug is covalently linked to the variant (e.g., the
variant and the drug are expressed as part of a single
polypeptide). Alternatively, in an example, the drug is
non-covalently bonded or associated with the variant. The drug can
be covalently or noncovalently bonded to the variant directly or
indirectly (e.g., through a suitable linker and/or noncovalent
binding of complementary binding partners (e.g., biotin and
avidin)). When complementary binding partners are employed, one of
the binding partners can be covalently bonded to the drug directly
or through a suitable linker moiety, and the complementary binding
partner can be covalently bonded to the variant directly or through
a suitable linker moiety. When the drug is a polypeptide or
peptide, the drug composition can be a fusion protein, wherein the
polypeptide or peptide, drug and the polypeptide binding moiety are
discrete parts (moieties) of a continuous polypeptide chain. As
described herein, the polypeptide binding moieties and polypeptide
drug moieties can be directly bonded to each other through a
peptide bond, or linked through a suitable amino acid, or peptide
or polypeptide linker.
[0113] A ligand which contains one single variable domain (monomer)
variant of the invention or more than one single variable domain
(multimer, fusion protein, conjugate, and dual specific ligand as
defined herein) which specifically binds to serum albumin, can
further comprise one or more entities selected from, but preferably
not limited to a label, a tag, an additional single variable
domain, a dAb, an antibody, an antibody fragment, a marker and a
drug. One or more of these entities can be located at either the
COOH terminus or at the N terminus or at both the N terminus and
the COOH terminus of the ligand comprising the single variable
domain, (either immunoglobulin or non-immunoglobulin single
variable domain). One or more of these entities can be located at
either the COOH terminus, or the N terminus, or both the N terminus
and the COOH terminus of the single variable domain which
specifically binds serum albumin of the ligand which contains one
single variable domain (monomer) or more than one single variable
domains (multimer, fusion protein, conjugate, and dual specific
ligand as defined herein). Non-limiting examples of tags which can
be positioned at one or both of these termini include a HA, his or
a myc tag. The entities, including one or more tags, labels and
drugs, can be bound to the ligand which contains one single
variable domain (monomer) or more than one single variable domain
(multimer, fusion protein, conjugate, and dual specific ligand as
defined herein), which binds serum albumin, either directly or
through linkers as described above.
[0114] Also encompassed herein is an isolated nucleic acid encoding
any of the variants, fusion proteins, conjugates or ligands
described herein, e.g., a ligand which contains one single variable
domain (monomer) variant of the invention or more than one single
variable domain (e.g., multimer, fusion protein, conjugate, and
dual specific ligand as defined herein) variant which specifically
binds to serum albumin, or which specifically binds both human
serum albumin and at least one non-human serum albumin, or
functionally active fragments thereof. Also encompassed herein is a
vector and/or an expression vector, a host cell comprising the
vector, e.g., a plant or animal cell and/or cell line transformed
with a vector, a method of expressing and/or producing one or more
variants, fusion proteins or ligands which contains one single
variable domain (monomer) variant or more than one single variable
domain variants (e.g., multimer, fusion protein, conjugate, and
dual specific ligand as defined herein) which specifically binds to
serum albumin, or fragment(s) thereof encoded by said vectors,
including in some instances culturing the host cell so that the one
or more variants, fusion proteins or ligands or fragments thereof
are expressed and optionally recovering the ligand which contains
one single variable domain (monomer) or more than one single
variable domain (e.g., multimer, fusion protein, conjugate, and
dual specific ligand as defined herein) which specifically binds to
serum albumin, from the host cell culture medium. Also encompassed
are methods of contacting a ligand described herein with serum
albumin, including serum albumin and/or non-human serum albumin(s),
and/or one or more targets other than serum albumin, where the
targets include biologically active molecules, and include animal
proteins, cytokines as listed above, and include methods where the
contacting is in vitro as well as administering any of the
variants, fusion proteins or ligands described herein to an
individual host animal or cell in vivo and/or ex vivo. Preferably,
administering ligands described herein which comprises a single
variable domain (immunoglobulin or non-immunoglobulin) directed to
serum albumin and/or non-human serum albumin(s), and one or more
domains directed to one or more targets other than serum albumin,
will increase the half life, including the T beta and/or terminal
half life, of the anti-target ligand. Nucleic acid molecules
encoding the variants, fusion proteins or single domain containing
ligands or fragments thereof, including functional fragments
thereof, are contemplated herein. Vectors encoding the nucleic acid
molecules, including but preferably not limited to expression
vectors, are contemplated herein, as are host cells from a cell
line or organism containing one or more of these expression
vectors. Also contemplated are methods of producing any variant,
fusion protein or ligand, including, but preferably not limited to
any of the aforementioned nucleic acids, vectors and host
cells.
[0115] An aspect of the invention provides a nucleic acid
comprising a nucleotide sequence encoding a variant according to
the invention or a multispecific ligand of the invention or fusion
protein of the invention or a nucleotide sequence that is at least
70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to said
selected sequence.
[0116] An aspect of the invention provides a vector comprising the
nucleic acid of the invention. An aspect of the invention provides
an isolated host cell comprising the vector.
[0117] Reference is made to WO2008/096158 for details of library
vector systems, combining single variable domains, characterization
of dual specific ligands, structure of dual specific ligands,
scaffolds for use in constructing dual specific ligands, uses of
anti-serum albumin dAbs and multispecific ligands and
half-life-enhanced ligands, and compositions and formulations of
comprising anti-serum albumin dAbs. These disclosures are
incorporated herein by reference to provide guidance for use with
the present invention, including for variants, ligands, fusion
proteins, conjugates, nucleic acids, vectors, hosts and
compositions of the present invention.
Sequences
TABLE-US-00002 [0118] TABLE 1 Amino Acid Sequences of DOM7h-11
Variant dAbs DOM7h-11-15 (SEQ ID NO: 1)
DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLIL
AFSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF GQGTKVEIKR
DOM7h-11-3 (SEQ ID NO: 2)
DIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIL
WNSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF GQGTKVEIKR
DOM7h-11-12 (SEQ ID NO: 157)
DIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLIL
FGSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTF GQGTKVEIKR
TABLE-US-00003 TABLE 2 Nucleotide Sequences of DOM7h-11 Variant
dAbs DOM7h-11-15 (SEQ ID NO: 3) GACATCCAGA TGACCCAGTC TCCATCCTCC
CTGTCTGCAT CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG TCCGATTGGG
ACGATGTTAA GTTGGTACCA GCAGAAACCA GGGAAAGCCC CTAAGCTCCT GATCCTTGCT
TTTTCCCGTT TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC TGGGACAGAT
TTCACTCTCA CCATCAGCAG TCTGCAACCT GAAGATTTTG CTACGTACTA CTGCGCGCAG
GCTGGGACGC ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA ACGG
DOM7h-11-3 (SEQ ID NO: 4) GACATCCAGA TGACCCAGTC TCCATCCTCC
CTGTCTGCAT CTGTAGGAGA CCGTGTCACC ATCACTTGCC GGGCAAGTCG TCCGATTGGG
ACGACGTTAA GTTGGTACCA GCAGAAACCA GGGAAAGCCC CTAAGCTCCT GATCCTTTGG
AATTCCCGTT TGCAAAGTGG GGTCCCATCA CGTTTCAGTG GCAGTGGATC TGGGACAGAT
TTCACTCTCA CCATCAGCAG TCTGCAACCT GAAGATTTTG CTACGTACTA CTGTGCGCAG
GCTGGGACGC ATCCTACGAC GTTCGGCCAA GGGACCAAGG TGGAAATCAA ACGG
DOM7h-11-12 (SEQ ID NO: 158)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG
ACCGTGTCACCATCACTTGCCGGGCAAGTCGTCCGATTGGGACGATGTT
AAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTTG
TTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTG
GATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGA
TTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTC GGCCAA
GGGACCAAGGTGGAAATCAAACGG
TABLE-US-00004 TABLE 5 Anti-serum albumin dAb (DOM7h) fusions (used
in Rat studies): - DOM7h-14/Exendin-4 fusion DMS number 7138 Amino
acid sequence (SEQ ID NO: 5)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCAQGAALPRTFGQGTKVEIKR Nucleotide sequence
(SEQ ID NO: 6)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAAACCA
GGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCA
TCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGC
AACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTGCGGCGTTGCCTAGGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAACGG DOM7h-14-10/Exendin-4 fusion DMS
number 7139 Amino acid sequence (SEQ ID NO: 7)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPSRF
SGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGTKVEIKR Nucleotide sequence
(SEQ ID NO: 8)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAAACCA
GGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGTCCCA
TCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGC
AACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAACGG DOM7h-11/Exendin-4 fusion DMS
number 7142 Amino acid sequence (SEQ ID NO: 9)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIWFGSRLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR Nucleotide sequence
(SEQ ID NO: 10)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACC
AGGGAAAGCCCCTAAGCTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCC
ATCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG
CAACCTGAAGATTTTGCTACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGT
TCGGCCAAGGGACCAAGGTGGAAATCAAACGG DOM7h-11-15/Exendin-4 fusion DMS
number 7143 Amino acid sequence (SEQ ID NO: 11)
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSGGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILAFSRLQSGVPSRFS
GSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR Nucleotide sequence
(SEQ ID NO: 12)
CATGGTGAAGGAACATTTACCAGTGACTTGTCAAAACAGATGGAAGAGGAGGCAGTG
CGGTTATTTATTGAGTGGCTTAAGAACGGAGGACCAAGTAGCGGGGCACCTCCGCCA
TCGGGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGGACA
TCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCAT
CACTTGCCGGGCAAGTCGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACC
AGGGAAAGCCCCTAAGCTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCA
TCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGC
AACCTGAAGATTTTGCTACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTT
CGGCCAAGGGACCAAGGTGGAAATCAAACGG DOM7h14-10/ G4SC-NCE fusion Amino
acid sequence (SEQ ID NO: 13) encoding DOM7h14-10/G4SC
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGTKVEIKRGGGGSC The
C-terminal cysteine can be linked to a new chemical entity
(pharmaceutical chemical compound, NCE), eg using maleimide
linkage. Nucleotide sequence (SEQ ID NO: 14) encoding
DOM7h14-10/G4SC
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCA
CCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAA
ACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGT
CCCATCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGA
CGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGGTGGCGGAGGGGGTTCCTGT
DOM7h14-10/TVAAPSC fusion Amino acid sequence (SEQ ID NO: 15)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMWRSSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQGTKVEIKRTVAAPSC The
C-terminal cysteine can be linked to a new chemical entity
(pharmaceutical chemical compound, NCE), eg using maleimide
linkage. Nucleotide sequence (SEQ ID NO: 16)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCA
CCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTATCTTGGTACCAGCAGAA
ACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGGCGTTCCTCGTTGCAAAGTGGGGT
CCCATCACGTTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT
CTGCAACCTGAAGATTTTGCTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGA
CGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGACCGTCGCTGCTCCATCTTGT (used in
mouse studies): - DOM7h-11/DOM1m-21-23 fusion DMS number 5515 Amino
acid sequence (SEQ ID NO:17)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIWFG
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR Amino
acid plus myc tag sequence (SEQ ID NO: 18)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTTLSWYQQKPGKAPKLLIWFG
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKRAAA
EQKLISEEDLN Nucleotide sequence (SEQ ID NO: 19)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGG Nucleotide plus myc tag sequence (SEQ ID NO: 20)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGACGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCTGGTTTGGTTCCCGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGTGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGGGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAA TTAA
DOM7h-11-15/DOM1m-21-23 fusion DMS number 5517 Amino acid sequence
(SEQ ID NO: 21)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILAF
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKR Amino
acid plus nucleotide plus myc tag sequence(SEQ ID NO: 22)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNRYSMGWLRQAPGKGLEWVSRIDSYGRGT
YYEDPVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCAKISQFGSNAFDYWGQGTQVT
VSSASTSGPSDIQMTQSPSSLSASVGDRVTITCRASRPIGTMLSWYQQKPGKAPKLLILAF
SRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQAGTHPTTFGQGTKVEIKRAAA
EQKLISEEDLN Nucleotide sequence (SEQ ID NO: 23)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGG Nucleotide plus myc tag sequence (SEQ ID NO: 24)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCG
TCTCTCCTGTGCAGCCTCCGGATTCACCTTTAATAGGTATAGTATGGGGTGGCTCCGC
CAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCACGGATTGATTCTTATGGTCGTGGT
ACATACTACGAAGACCCCGTGAAGGGCCGGTTCAGCATCTCCCGCGACAATTCCAAG
AACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCCGTATATTACT
GTGCGAAAATTTCTCAGTTTGGGTCAAATGCGTTTGACTACTGGGGTCAGGGAACCCA
GGTCACCGTCTCGAGCGCTAGCACCAGTGGTCCATCGGACATCCAGATGACCCAGTC
TCCATCCTCCCTGTCTGCATCTGTAGGAGACCGTGTCACCATCACTTGCCGGGCAAGT
CGTCCGATTGGGACGATGTTAAGTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAG
CTCCTGATCCTTGCTTTTTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCA
GTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGC
TACGTACTACTGCGCGCAGGCTGGGACGCATCCTACGACGTTCGGCCAAGGGACCAA
GGTGGAAATCAAACGGGCGGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAA TTAA
Where a myc-tagged molecule is indicated in this table, this was
the version used in PK studies in the examples. Where no myc-tagged
sequences are given, the PK studies in the examples were not done
with myc-tagged material, ie, the studies were done with the
non-tagged constructs shown.
EXEMPLIFICATION
[0119] All numbering in the experimental section is according to
Kabat (Kabat, E.A. National Institutes of Health (US) &
Columbia University. Sequences of proteins of immunological
interest, edn 5 (US Dept. Of Health and Human Services Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991)).
[0120] Derivation of DOM7h-11, DOM7h-14 and DOM7r variants is
described.
Example 1
Vk Affinity Maturation
Selections:
[0121] HSA (Human Serum Albumin) and RSA (Rat Serum Albumin)
antigens were obtained from Sigma (essentially fatty acid free,
.about.99% (agarose gel electrophoresis), lyophilized powder Cat.
No. A3782 and A6414 respectively)
[0122] Biotinylated products of above two antigens were made by
using EZ Link Sulfo-NHS-SS-Biotin (Pierce, Cat. No. 21331). Free
biotin reagent was removed by passing the samples twice through
PD10 desalting column followed by overnight dialysis against
1000.times. excess volume of PBS at 4.degree. C. Resulting product
was tested by mass spec and 1-2 biotins per molecule were
observed.
Affinity Maturation Libraries:
[0123] Both error-prone and CDR libraries were created using
DOM7h-11 and DOM7h-14 parental dAbs (see WO2008/096158 for the
sequences of DOM7h-11 and DOM7h-14). The CDR libraries were
generated in the pDOM4 vector and the error prone libraries were
generated in the pDOM33 vector (to allow for selection with or
without protease treatment). Vector pDOM4, is a derivative of the
Fd phage vector in which the gene III signal peptide sequence is
replaced with the yeast glycolipid anchored surface protein (GAS)
signal peptide. It also contains a c-myc tag between the leader
sequence and gene III, which puts the gene III back in frame. This
leader sequence functions well both in phage display vectors but
also in other prokaryotic expression vectors and can be universally
used. pDOM33 is a modified version of the pDOM4 vector where the
c-myc tag has been removed which renders the dAb-phage fusion
resistant to the protease trypsin. This allows the use of trypsin
within the phage selection to select for dAbs that are more
protease stable (see WO2008149143).
[0124] For error-prone maturation libraries, plasmid DNA encoding
the dAb to be matured was amplified by PCR, using the
GENEMORPH.RTM. II RANDOM MUTAGENESIS KIT (random, unique
mutagenesis kit, Stratagene). The product was digested with Sal I
and Not I and used in a ligation reaction with cut phage vector
pDOM33. For the CDR libraries, PCR reactions were performed using
degenerate oligonucleotides containing NNK or NNS codons to
diversify the required positions in the dAb to be affinity matured.
Assembly PCR was then used to generate a full length diversified
insert. The insert was digested with Sal I and Not I and used in a
ligation reaction with pDOM4 for mutagenesis of multiple residues
and pDOM5 for mutagenesis of single residues. The pDOM5 vector is a
pUC119-based expression vector where protein expression is driven
by the LacZ promoter. A GAS1 leader sequence (see WO 2005/093074)
ensures secretion of isolated, soluble dAbs into the periplasm and
culture supernatant of E. coli. dAbs are cloned SalI/NotI in this
vector, which appends a myc tag at the C-terminus of the dAb. This
protocol using SalI and Not I results in inclusion of an ST amino
acid sequence at the N-terminus.
[0125] The ligation produced by either method was then used to
transform E. coli strain TB1 by electroporation and the transformed
cells plated on 2.times.TY agar containing 15 .mu.g/ml
tetracycline, yielding library sizes of >5.times.10.sup.7
clones.
[0126] The error-prone libraries had the following average mutation
rate and size: DOM7h-11 (2.5 mutations per dAb), size:
6.1.times.10.sup.8, DOM7h-14 (2.9 mutations per dAb), size:
5.4.times.10.sup.8.
[0127] Each CDR library has four amino acid diversity. Two
libraries were generated for each of CDRs 1 and 3, and one library
for CDR2. The positions diversified within each library are as
follows (amino acids based on VK dummy DPK9 sequence):
TABLE-US-00005 Library size DOM7h-11 DOM7h-14 1--Q27, S28, S30, S31
(CDR1) 8.8 .times. 10.sup.7 5.8 .times. 10.sup.7 2--S30, S31, Y32,
N34 (CDR1) 4.6 .times. 10.sup.8 4.2 .times. 10.sup.8 3--Y49, A50,
A51, S53 (CDR2) 3.9 .times. 10.sup.8 2.4 .times. 10.sup.8 4--Q89,
S91, Y92, S93 (CDR3) 1.8 .times. 10.sup.8 2.5 .times. 10.sup.8
5--Y92, Y93, T94, N96 (CDR3) 4.0 .times. 10.sup.8 3.3 .times.
10.sup.8
Example 2
Selection Strategies
[0128] Three phage selection strategies were adopted for V.kappa.
AlbudAb.TM. (anti-serum albumin dAb) affinity maturation:
[0129] 1) Selections Against HSA Only: [0130] Three rounds of
selection against HSA were carried out. The error prone libraries
and each CDR library were selected as an individual pool in all
rounds. The first round of selection was performed against HSA
passively coated onto an immunotube at 1 mg/ml. Round 2 was
performed against 100 nM HSA and round 3 against 10 nM (CDR
selections) or 20 or 100 nM (Error prone selections) HSA, both as
soluble selections followed by a fourth round of selection with the
error prone libraries against 1.5 nM HSA as a soluble selection.
The error prone libraries were eluted with 0.1M glycine pH 2.0
before neutralisation with 1M Tris pH 8.0 and the CDR libraries
were eluted with 1 mg/ml trypsin before infection into log phase
TG1 cells. The third round of each selection was subcloned into
pDOM5 for screening. Soluble selections used biotinylated HSA.
[0131] 2) Trypsin Selections Against HSA: [0132] In order to select
dAbs with increased protease resistance compared to the parental
clone and with potentially improved biophysical properties, trypsin
was used in phage selections (see WO2008149143). Four rounds of
selection were preformed against HSA. The first round of selection
of error prone libraries was performed against passively coated HSA
at 1 mg/ml without trypsin; the second round against passively
coated HSA at 1 mg/ml with 20 .mu.g/ml trypsin for 1 hour at
37.degree. C.; the third round selection was performed by soluble
selection using biotinylated HSA against 100 nM HSA with 20
.mu.g/ml or 100 .mu.g/ml trypsin for 1 hour at 37.degree. C. The
final round of selection was performed by soluble selection using
biotinylated HSA against 100 nM HSA with 100 .mu.g/ml trypsin
overnight at 37.degree. C.
[0133] 3) Cross-Over Selections Against HSA (Round 1) and RSA
(Rounds 2-4): [0134] The first round selection was carried out
against 1 mg/ml passively coated HSA or 1 .mu.M HSA (soluble
selection), followed by a further three rounds of soluble
selections against biotinylated RSA at concentrations of 1 .mu.M
for round 1, 100 nm for round 2 and 20 nM, 10 nM or 1 nM for round
3.
Screening Strategy and Affinity Determination:
[0135] In each case after selection a pool of phage DNA from the
appropriate round of selection is prepared using a QIAfilter
midiprep kit (Qiagen), the DNA is digested using the restriction
enzymes Sal1 and Not1 and the enriched V genes are ligated into the
corresponding sites in pDOM5 the soluble expression vector which
expresses the dAb with a myc tag (see PCT/EP2008/067789). The
ligated DNA is used to electro-transform E. coli HB 2151 cells
which are then grown overnight on agar plates containing the
antibiotic carbenicillin. The resulting colonies are individually
assessed for antigen binding. In each case at least 96 clones were
tested for binding to HSA, CSA (Cynomolgus monkey Serum Albumin),
MSA (mouse serum albumin) and RSA by BIAcore.TM. (surface plasmon
resonance). MSA antigen was obtained from Sigma (essentially fatty
acid free, .about.99% (agarose gel electrophoresis), lyophilized
powder Cat. No. A3559) and CSA was purified from Cynomolgus serum
albumin using prometic blue resin (Amersham). Soluble dAb fragments
were produced in bacterial culture in ONEX culture media (Novagen)
overnight at 37.degree. C. in 96 well plates. The culture
supernatant containing soluble dAb was centrifuged and analysed by
BIAcore for binding to high density HSA, CSA, MSA and RSA CM5
chips. Clones were found to bind to all these species of serum
albumin by off-rate screening. The clones were sequenced revealing
unique dAb sequences.
[0136] DOM7h11-15 had 96.3% identity to parent (at the amino acid
level). DOM7h-11-3 had 97.2% identity to parent (at the amino acid
level).
[0137] DOM7h-14-10 had 96.3% identity to parent (at the amino acid
level).
[0138] dAbs were expressed as bacterial supernatants in 2.5 L shake
flasks in Onex media at 30.degree. C. for 48 hrs at 250 rpm. dAbs
were purified from the culture media by absorption to protein L
agarose followed by elution with 10 mM glycine pH2.0. Binding to
HSA, CSA, MSA and RSA by BIAcore was confirmed using purified
protein at 3 concentrations 1 .mu.M, 500 nM and 50 nM. To determine
the binding affinity (K.sub.D) of the AlbudAbs to each serum
albumin; purified dAbs were analysed by BIAcore over albumin
concentration range from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250
nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM).
TABLE-US-00006 TABLE 6 Affinity (K.sub.D) AlbudAb to SA (nM) Kd Ka
Rat DOM7h-14 60 2.095E-01 4.00E+06 DOM7h-14-10 4 9.640E-03 4.57E+06
DOM 7h-11 2100 1.00E-01 4.80E+04 DOM 7h-11-3 10000 (7.18e-1)
(8.11e3) (88000) DOM 7h-11-15 20 2.10E-02 1.10E+06 Cyno DOM 7h-14
66 9.65E-02 1.50E+06 DOM 7h-14-10 9 1.15E-02 1.60E+06 DOM 7h-11
1000 6.82E-01 8.00E+05 DOM 7h-11-3 670 9.6E-02 2.90E+05 (200)
(1.5e-1) (7.26e5) DOM 7h-11-15 3 5.57E-03 5.80E+06 Mouse DOM 7h-14
12 4.82E-02 4.10E+06 DOM 7h-14-10 30 3.41E-02 1.29E+06 DOM 7h-11
5000 9.00E-01 DOM 7h-11-3 .gtoreq.10000 (6.12e-1) (1.67e4) (36000)
DOM 7h-11-15 10 9.40E-03 1.10E+06 Human DOM 7h-14 33 4.17E-02
1.43E+06 DOM 7h-14-10 12 1.39E-02 1.50E+06 DOM 7h-11 2800 6.41E-01
7.00E+05 DOM 7h-11-3 32 1.6E-02 6.50E+05 (130) (2.35e-2) (1.86e5)
DOM 7h-11-15 1 1.84E-03 2.00E+06 *: values in brackets were derived
from a second, independent SPR experiment.
[0139] All DOM7h-14 derived variants are cross-reactive to mouse,
rat, human and cyno serum albumin. DOM7h-14-10 has improved
affinity to rat, cyno and human serum albumin compared to
parent.
[0140] DOM7h-11-3 has improved affinity to CSA and HSA. DOM7h-11-15
has improved affinity to RSA, MSA, CSA and HSA.
Example 3
Origins of Key DOM7h-11 Lineage Clones
[0141] DOM7h-11-3: From affinity maturation performed against HSA
using the CDR2 library (Y49, A50, A51, S53), round 3 output 10 nM
HSA
[0142] DOM7h-11-15: From cross-over selections performed against
HSA as round 1 followed by additional 3 rounds of selections
against RSA using the CDR2 library (Y49, A50, A51, S53) at round 3
selection with 1 nM of RSA.
TABLE-US-00007 TABLE 7 CDR sequences (according to Kabat; ref. as
above) CDR AIbudAb CDR1 CDR2 CDR3 DPK9 Vk dummy SQSISSYLN YAASSLQS
QQSYSTPNT (SEQ ID NO: 25) (SEQ ID NO: 26) (SEQ ID NO: 27) DOM7h-11
SRPIGTTLS WFGSRLQS AQAGTHPTT (SEQ ID NO: 28) (SEQ ID NO: 29) (SEQ
ID NO: 30) DOM 7h-11-15 SRPIGTMLS LAFSRLQS AQAGTHPTT (SEQ ID NO:
31) (SEQ ID NO: 32) (SEQ ID NO: 33) DOM 7h-11-3 SRPIGTTLS LWFSRLQS
AQAGTHPTT (SEQ ID NO: 34) (SEQ ID NO: 35) (SEQ ID NO: 36)
Example 4
Origins of Key DOM7h-14 Lineage Clones
[0143] DOM7h-14-10: From affinity maturation performed against HSA
using CDR3 library (Y92, Y93, T94, N96), round 3 output.
TABLE-US-00008 TABLE 8 CDR sequences (according to Kabat; ref. as
above) CDR AIbudAb CDR1 CDR2 CDR3 DOM 7h-14 SQWIGSQLS MWRSSLQS
AQGAALPRT (SEQ ID NO: 37) (SEQ ID NO: 38) (SEQ ID NO: 39) DOM
7h-14-10 SQWIGSQLS MWRSSLQS AQGLRHPKT (SEQ ID NO: 40) (SEQ ID NO:
41) (SEQ ID NO: 42)
Example 5
Expression and Biophysical Characterisation
[0144] The routine bacterial expression level in 2.5 L shake flasks
was determined following culture in Onex media at 30.degree. C. for
48 hrs at 250 rpm. The biophysical characteristics were determined
by SEC MALLS and DSC.
[0145] SEC MALLS (size exclusion chromatography with
multi-angle-LASER-light-scattering) is a non-invasive technique for
the characterizing of macromolecules in solution. Briefly, proteins
(at concentration of 1 mg/mL in buffer Dulbecco's PBS at 0.5 ml/min
are separated according to their hydrodynamic properties by size
exclusion chromatography (column: TSK3000 from TOSOH Biosciences;
S200 from Pharmacia). Following separation, the propensity of the
protein to scatter light is measured using a
multi-angle-LASER-light-scattering (MALLS) detector. The intensity
of the scattered light while protein passes through the detector is
measured as a function of angle. This measurement taken together
with the protein concentration determined using the refractive
index (RI) detector allows calculation of the molar mass using
appropriate equations (integral part of the analysis software Astra
v.5.3.4.12).
[0146] DSC (Differential Scanning calorimetry): briefly, the
protein is heated at a constant rate of 180.degree. C./hrs (at 1
mg/mL in PBS) and a detectable heat change associated with thermal
denaturation measured. The transition midpoint (.sub.appT.sub.m) is
determined, which is described as the temperature where 50% of the
protein is in its native conformation and the other 50% is
denatured. Here, DSC determined the apparent transition midpoint
(appTm) as most of the proteins examined do not fully refold. The
higher the Tm, the more stable the molecule. Unfolding curves were
analysed by non-2-state equations. The software package used was
Origin.RTM. v7.0383.
TABLE-US-00009 TABLE 9 Biophysical parameters AlbudAb SEC MALLS DSC
Tm(.degree. C.) DOM7h-14 M 60 DOM 7h-14-10 M 59 DOM 7h-11 M
66.9-72.2 DOM 7h-11-3 M (95%)* 66.6/70.5 DOM 7h-11-15 M (<5% D)
58.5-60.5 *in one other trial, monomer was primarily seen by SEC
MALLS, although lower than 95%
[0147] We observed expression levels for all clones in Table 9 in
the range from 15 to 119 mg/L in E. coli.
[0148] For DOM7h-14 and DOM7h-11 variants, favorable biophysical
parameters (monomeric in solution as determined by SEC MALLs and
appTm of >55.degree. C. as determined by DSC) and expression
levels were maintained during affinity maturation. Monomeric state
is advantageous because it avoids dimerisation and the risk of
products that may cross-link targets such as cell-surface
receptors.
Example 6
Determination of Serum Half Life in Rat, Mouse and Cynomolgus
Monkey
[0149] AlbudAbs DOM7h-14-10, DOM7h-11 and DOM7h-11-15 were cloned
into the pDOM5 vector. For each AlbudAb.TM., 20-50 mg quantities
were expressed in E. coli and purified from bacterial culture
supernatant using protein L affinity resin and eluted with 100 mM
glycine pH2. The proteins were concentrated to greater than 1
mg/ml, buffer exchanged into PBS and endotoxin depleted using Q
spin columns (Vivascience). For Rat pharmacokinetic (PK) analysis,
AlbudAbs were dosed as single i.v injections at 2.5 mg/kg using 3
rats per compound. Serum samples were taken at 0.16, 1, 4, 12, 24,
48, 72, 120, 168 hrs. Analysis of serum levels was by anti-myc
ELISA as per the method described below.
[0150] For Mouse PK, DOM7h-11 and DOM7h-11-15 were dosed as single
i.v injections at 2.5 mg/kg per dose group of 3 subjects and serum
samples taken at 10 mins; 1 h; 8 h; 24 h; 48 h; 72 h; 96 h.
Analysis of serum levels was by anti-myc ELISA as per the method
described below.
[0151] For Cynomolgus monkey PK DOM7h-14-10 and DOM7h-11-15 were
dosed as single i.v injections at 2.5 mg/kg into 3 female
Cynomolgus monkeys per dose group and serum samples taken at 0.083,
0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, 144, 192, 288, 336, 504 hrs.
Analysis of serum levels was by anti-myc ELISA as per the method
described below.
Anti-Myc ELISA Method
[0152] The AlbudAb concentration in serum was measured by anti-myc
ELISA. Briefly, goat anti-myc polyclonal antibody (1:500; Abcam,
catalogue number ab9132) was coated overnight onto Nunc 96-well
Maxisorp plates and blocked with 5% BSA/PBS+1% Tween. Serum samples
were added at a range of dilutions alongside a standard at known
concentrations. Bound myc-tagged AlbudAb was then detected using a
rabbit polyclonal anti-Vk (1:1000; in-house reagent, bleeds were
pooled and protein A purified before use) followed by an
anti-rabbit IgG HRP antibody (1:10,000; Sigma, catalogue number
A2074). Plates were washed between each stage of the assay with
3.times.PBS+0.1% Tween20 followed by 3.times.PBS. TMB (SureBlue TMB
1-Component Microwell Peroxidase Substrate, KPL, catalogue number
52-00-00) was added after the last wash and was allowed to develop.
This was stopped with 1M HCl and the signal was then measured using
absorbance at 450 nm.
[0153] From the raw ELISA data, the concentration of unknown
samples was established by interpolation against the standard curve
taking into account dilution factors. The mean concentration result
from each time point was determined from replicate values and
entered into WinNonLin analysis package (e.g. version 5.1
(available from Pharsight Corp., Mountain View, Calif. 94040, USA).
The data was fitted using a non-compartmental model, where PK
parameters were estimated by the software to give terminal
half-lives. Dosing information and time points were selected to
reflect the terminal phase of each PK profile.
TABLE-US-00010 TABLE 10 Single AlbudAb .TM. PK PK parameters
Albumin AUC CL t1/2 Vz Species AlbudAb K.sub.D (nM) h .times.
.mu.g/ml ml/h/kg h ml/kg Rat DOM7h-14* 60 DOM7h-14-10 4 2134.6 1.2
42.1 71.2 DOM 7h-11 2100 320.1 7.8 23.3 263.9 DOM 7h-11-15 20 843.4
3.0 30.3 130.7 mouse DOM 7h-11 5000 304.7 8.2 18.3 216.8 DOM
7h-11-15 10 499.2 5.0 33.7 243.4 Cyno DOM 7h-14* 66 217.5 DOM
7h-14-10 9 6174.6 0.4 200.8 117.8 DOM 7h-11* 3300 135.1 DOM
7h-11-15 3 4195 0.6 198.1 170.3 *Historical data
[0154] Pharmacokinetic parameters derived from rat, mouse and
cynomolgus monkey studies were fitted using a non-compartmental
model. Key: AUC: Area under the curve from dosing time extrapolated
to infinity; CL: clearance; t1/2: is the time during which the
blood concentration is halved; Vz: volume of distribution based on
the terminal phase.
[0155] DOM7h-11-15 has an improved AUC and t1/2 in rat and mouse
compared to parent. DOM7h-11-15 also has an improved AUC and t1/2
in cyno compared to parent. This improvement in AUC/t1/2 correlates
with an improved in vitro KD to serum albumin.
Example 7
AlbudAb.TM. IFN Fusions
Cloning and Expression
[0156] As well as single AlbudAbs, the affinity matured Vk Albudabs
were linked to Interferon alpha 2b (IFN.alpha.2b) to determine
whether a useful PK of the AlbudAb was maintained as a fusion
protein.
TABLE-US-00011 Interferon alpha 2b amino acid sequence: (SEQ ID NO:
43) CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKA
ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVI
QGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS FSLSTNLQESLRSKE
Interferon alpha 2b nucleotide sequence: (SEQ ID NO: 44)
TGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAGGAGGACCTTGATGCT
CCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTGAAGGACAGAC
ATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAAAGGCT
GAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTT
CAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAAT
TCTACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATA
CAGGGGGTGGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCT
GGCTGTGAGGAAATACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGA
AATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGATCT
TTTTCTTTGTCAACAAACTTGCAAGAAAGTTTAAGAAGTAAGGAA
[0157] IFNa2b was linked to the AlbudAb via a TVAAPS linker region
(see WO2007085814). The constructs were cloned by SOE-PCR (single
overlap extension according to the method of Horton et al. Gene,
77, p 61 (1989)). PCR amplification of the AlbudAb and IFN
sequences were carried out separately using primers with a
.about.15 base pair overlap at the TVAAPS linker region. The
primers used are as follows:--
TABLE-US-00012 IFN.alpha.2b SOE fragment 5' (SEQ ID NO: 45)
GCCCGGATCCACCGGCTGTGATCTG IFN.alpha.2b SOE fragment 3' (SEQ ID NO:
46) GGAGGATGGAGACTGGGTCATCTGGATGTC Vk SOE fragment 5' (SEQ ID NO:
47) GACATCCAGATGACCCAGTCTCCATCCTCC Vk SOE fragment 3' to also
introduce a myc tag (SEQ ID NO: 48) GCGCAAGCTTTTATTAATTCAGATCCTCTTC
TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT TTGATTTCCACCTTGGTCCC
[0158] The fragments were purified separately and subsequently
assembled in a SOE (single overlap extension PCR extension)
reaction using only the flanking primers.
TABLE-US-00013 IFN.alpha.2b SOE fragment 5' (SEQ ID NO: 49)
GCCCGGATCCACCGGCTGTGATCTG Vk SOE fragment 3' to also introduce a
myc tag (SEQ ID NO: 50) GCGCAAGCTTTTATTAATTCAGATCCTCTTC
TGAGATGAGTTTTTGTTCTGCGGCCGCCCGT TTGATTTCCACCTTGGTCCC
[0159] The assembled PCR product was digested using the restriction
enzymes BamHI and HindIII and the gene ligated into the
corresponding sites in the pDOM50, a mammalian expression vector
which is a pTT5 derivative with an N-terminal V-J2-C mouse IgG
secretory leader sequence to facilitate expression into the cell
media.
TABLE-US-00014 Leader sequence (amino acid): (SEQ ID NO: 51)
METDTLLLWVLLLWVPGSTG Leader sequence (nucleotide): (SEQ ID NO: 52)
ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGG ATCCACCGGGC
[0160] Plasmid DNA was prepared using QIAfilter megaprep (Qiagen).
1 .mu.g DNA/ml was transfected with 293-Fectin into HEK293E cells
and grown in serum free media. The protein is expressed in culture
for 5 days and purified from culture supernatant using protein L
affinity resin and eluted with 100 mM glycine pH2. The proteins
were concentrated to greater than 1 mg/ml, buffer exchanged into
PBS and endotoxin depleted using Q spin columns (Vivascience).
TABLE-US-00015 TABLE 11 Interferon alpha 2b-AIbudAb sequences with
and without myc-taq (as amino acid- and nucleotide sequence) The
Interferon alpha 2b is N-terminal to the AIbudAb in the following
fusions. aa + myc nt + myc aa no tag nt no tag DMS7321
CDLPQTHSLGSRRTL TGCGACTTGCCA CDLPQTHSLGS TGCGACTTGCCA
(IFN.alpha.2b- MLLAQMRRISLFSCL CAGACACATAGT RRTLMLLAQM CAGACACATAGT
DOM7h- KDRHDFGFPQEEFG TTGGGATCAAGA RRISLFSCLKD TTGGGATCAAGA 14)
NQFQKAETIPVLHEMI AGAACATTGATG RHDFGFPQEE AGAACATTGATG
QQIFNLFSTKDSSAA TTATTAGCACAAA FGNQFQKAETI TTATTAGCACAA
WDETLLDKFYTELYQ TGCGTAGAATTT PVLHEMIQQIF ATGCGTAGAATT
QLNDLEACVIQGVGV CTTTGTTCTCTTG NLFSTKDSSAA TCTTTGTTCTCTT
TETPLMKEDSILAVRK TCTAAAGGACCG WDETLLDKFYT GTCTAAAGGACC
YFQRITLYLKEKKYSP TCACGACTTCGG ELYQQLNDLEA GTCACGACTTCG
CAWEVVRAEIMRSFS ATTCCCTCAGGA CVIQGVGVTET GATTCCCTCAGG
LSTNLQESLRSKETV AGAGTTTGGAAA PLMKEDSILAV AAGAGTTTGGAA
AAPSDIQMTQSPSSL CCAATTCCAAAA RKYFQRITLYLK ACCAATTCCAAA
SASVGDRVTITCRAS AGCAGAAACTAT EKKYSPCAWE AAGCAGAAACTA QWIGSQLSWYQQKP
TCCTGTCTTGCA VVRAEIMRSFS TTCCTGTCTTGC GKAPKLLIMWRSSLQ CGAAATGATCCA
LSTNLQESLRS ACGAAATGATCC SGVPSRFSGSGSGT GCAAATATTCAAT KETVAAPSDIQ
AGCAAATATTCA DFTLTISSLQPEDFAT TTGTTTTCTACAA MTQSPSSLSAS
ATTTGTTTTCTAC YYCAQGAALPRTFGQ AGGACTCATCAG VGDRVTITCRA AAAGGACTCATC
GTKVEIKR CCGCTTGGGATG SQWIGSQLSW AGCCGCTTGGGA AAAEQKLISEEDLN*
AAACTCTGTTAG YQQKPGKAPK TGAAACTCTGTT (SEQ ID NO: 53) ATAAATTCTACAC
LLIMWRSSLQS AGATAAATTCTA TGAACTATATCAA GVPSRFSGSG CACTGAACTATA
CAACTGAACGAT SGTDFTLTISSL TCAACAACTGAA CTAGAGGCTTGC QPEDFATYYCA
CGATCTAGAGGC GTTATTCAGGGT QGAALPRTFG TTGCGTTATTCA GTAGGAGTTACT
QGTKVEIKR GGGTGTAGGAGT GAAACTCCCCTA (SEQ ID NO: 55) TACTGAAACTCC
ATGAAAGAAGAT CCTAATGAAAGA TCAATTCTAGCC AGATTCAATTCTA GTTAGAAAATACT
GCCGTTAGAAAA TTCAGCGTATCA TACTTTCAGCGT CATTGTATTTAAA ATCACATTGTATT
GGAAAAGAAATA TAAAGGAAAAGA CTCCCCATGTGC AATACTCCCCAT ATGGGAGGTGGT
GTGCATGGGAG TAGAGCAGAAAT GTGGTTAGAGCA TATGAGGTCCTT GAAATTATGAGG
CTCTCTTTCTACG TCCTTCTCTCTTT AATTTGCAAGAAT CTACGAATTTGC
CTTTGAGATCTAA AAGAATCTTTGA GGAAACCGTCGC GATCTAAGGAAA TGCTCCATCTGA
CCGTCGCTGCTC CATCCAGATGAC CATCTGACATCC CCAGTCTCCATC AGATGACCCAGT
CTCCCTGTCTGC CTCCATCCTCCC ATCTGTAGGAGA TGTCTGCATCTG CCGTGTCACCAT
TAGGAGACCGTG CACTTGCCGGGC TCACCATCACTT AAGTCAGTGGAT GCCGGGCAAGT
TGGGTCTCAGTT CAGTGGATTGGG ATCTTGGTACCA TCTCAGTTATCTT GCAGAAACCAGG
GGTACCAGCAGA GAAAGCCCCTAA AACCAGGGAAAG GCTCCTGATCAT CCCCTAAGCTCC
GTGGCGTTCCTC TGATCATGTGGC GTTGCAAAGTGG GTTCCTCGTTGC GGTCCCATCACG
AAAGTGGGGTCC TTTCAGTGGCAG CATCACGTTTCA TGGATCTGGGAC GTGGCAGTGGAT
AGATTTCACTCTC CTGGGACAGATT ACCATCAGCAGT TCACTCTCACCA CTGCAACCTGAA
TCAGCAGTCTGC GATTTTGCTACG AACCTGAAGATT TACTACTGTGCT TTGCTACGTACT
CAGGGTGCGGC ACTGTGCTCAGG GTTGCCTAGGAC GTGCGGCGTTG GTTCGGCCAAGG
CCTAGGACGTTC GACCAAGGTGGA GGCCAAGGGAC AATCAAACGGGC CAAGGTGGAAAT
GGCCGCAGAAC CAAACGG (SEQ AAAAACTCATCT ID NO: 56) CAGAAGAGGAT
CTGAATTAA (SEQ ID NO: 54) DMS732 CDLPQTHSLGSRRTL TGCGACTTGCCA
CDLPQTHSLGS TGCGACTTGCCA (IFN.alpha.2b- MLLAQMRRISLFSCL
CAGACACATAGT RRTLMLLAQM CAGACACATAGT DOM7h- KDRHDFGFPQEEFG
TTGGGATCAAGA RRISLFSCLKD TTGGGATCAAGA 14-10) NQFQKAETIPVLHEMI
AGAACATTGATG RHDFGFPQEE AGAACATTGATG QQIFNLFSTKDSSAA TTATTAGCACAAA
FGNQFQKAETI TTATTAGCACAA WDETLLDKFYTELYQ TGCGTAGAATTT PVLHEMIQQIF
ATGCGTAGAATT QLNDLEACVIQGVGV CTTTGTTCTCTTG NLFSTKDSSAA
TCTTTGTTCTCTT TETPLMKEDSILAVRK TCTAAAGGACCG WDETLLDKFYT
GTCTAAAGGACC YFQRITLYLKEKKYSP TCACGACTTCGG ELYQQLNDLEA GTCACGACTTCG
CAWEVVRAEIMRSFS ATTCCCTCAGGA CVIQGVGVTET GATTCCCTCAGG
LSTNLQESLRSKETV AGAGTTTGGAAA PLMKEDSILAV AAGAGTTTGGAA
AAPSDIQMTQSPSSL CCAATTCCAAAA RKYFQRITLYLK ACCAATTCCAAA
SASVGDRVTITCRAS AGCAGAAACTAT EKKYSPCAWE AAGCAGAAACTA QWIGSQLSWYQQKP
TCCTGTCTTGCA VVRAEIMRSFS TTCCTGTCTTGC GKAPKLLIMWRSSLQ CGAAATGATCCA
LSTNLQESLRS ACGAAATGATCC SGVPSRFSGSGSGT GCAAATATTCAAT KETVAAPSDIQ
AGCAAATATTCA DFTLTISSLQPEDFAT TTGTTTTCTACAA MTQSPSSLSAS
ATTTGTTTTCTAC YYCAQGLRHPKTFG AGGACTCATCAG VGDRVTITCRA AAAGGACTCATC
QGTKVEIKR CCGCTTGGGATG SQWIGSQLSW AGCCGCTTGGGA AAAEQKLISEEDLN*
AAACTCTGTTAG YQQKPGKAPK TGAAACTCTGTT (SEQ ID NO: 57) ATAAATTCTACAC
LLIMWRSSLQS AGATAAATTCTA TGAACTATATCAA GVPSRFSGSG CACTGAACTATA
CAACTGAACGAT SGTDFTLTISSL TCAACAACTGAA CTAGAGGCTTGC QPEDFATYYCA
CGATCTAGAGGC GTTATTCAGGGT QGLRHPKTFG TTGCGTTATTCA GTAGGAGTTACT
QGTKVEIKR GGGTGTAGGAGT GAAACTCCCCTA (SEQ ID NO: 59) TACTGAAACTCC
ATGAAAGAAGAT CCTAATGAAAGA TCAATTCTAGCC AGATTCAATTCTA GTTAGAAAATACT
GCCGTTAGAAAA TTCAGCGTATCA TACTTTCAGCGT CATTGTATTTAAA ATCACATTGTATT
GGAAAAGAAATA TAAAGGAAAAGA CTCCCCATGTGC AATACTCCCCAT ATGGGAGGTGGT
GTGCATGGGAG TAGAGCAGAAAT GTGGTTAGAGCA TATGAGGTCCTT GAAATTATGAGG
CTCTCTTTCTACG TCCTTCTCTCTTT AATTTGCAAGAAT CTACGAATTTGC
CTTTGAGATCTAA AAGAATCTTTGA GGAAACCGTCGC GATCTAAGGAAA TGCTCCATCTGA
CCGTCGCTGCTC CATCCAGATGAC CATCTGACATCC CCAGTCTCCATC AGATGACCCAGT
CTCCCTGTCTGC CTCCATCCTCCC ATCTGTAGGAGA TGTCTGCATCTG CCGTGTCACCAT
TAGGAGACCGTG CACTTGCCGGGC TCACCATCACTT AAGTCAGTGGAT GCCGGGCAAGT
TGGGTCTCAGTT CAGTGGATTGGG ATCTTGGTACCA TCTCAGTTATCTT GCAGAAACCAGG
GGTACCAGCAGA GAAAGCCCCTAA AACCAGGGAAAG GCTCCTGATCAT CCCCTAAGCTCC
GTGGCGTTCCTC TGATCATGTGGC GTTGCAAAGTGG GTTCCTCGTTGC GGTCCCATCACG
AAAGTGGGGTCC TTTCAGTGGCAG CATCACGTTTCA TGGATCTGGGAC GTGGCAGTGGAT
AGATTTCACTCTC CTGGGACAGATT ACCATCAGCAGT TCACTCTCACCA CTGCAACCTGAA
TCAGCAGTCTGC GATTTTGCTACG AACCTGAAGATT TACTACTGTGCT TTGCTACGTACT
CAGGGTTTGAGG ACTGTGCTCAGG CATCCTAAGACG GTTTGAGGCATC TTCGGCCAAGGG
CTAAGACGTTCG ACCAAGGTGGAA GCCAAGGGACC ATCAAACGGGCG AAGGTGGAAATC
GCCGCAGAACA AAACGG (SEQ ID AAAACTCATCTC NO: 60) AGAAGAGGATCT
GAATTAA (SEQ ID NO: 58) DMS7325 CDLPQTHSLGSRRTL TGCGACTTGCCA
CDLPQTHSLGS TGCGACTTGCCA (IFN.alpha.2b- MLLAQMRRISLFSCL
CAGACACATAGT RRTLMLLAQM CAGACACATAGT DOM7h- KDRHDFGFPQEEFG
TTGGGATCAAGA RRISLFSCLKD TTGGGATCAAGA 11) NQFQKAETIPVLHEMI
AGAACATTGATG RHDFGFPQEE AGAACATTGATG QQIFNLFSTKDSSAA TTATTAGCACAAA
FGNQFQKAETI TTATTAGCACAA WDETLLDKFYTELYQ TGCGTAGAATTT PVLHEMIQQIF
ATGCGTAGAATT QLNDLEACVIQGVGV CTTTGTTCTCTTG NLFSTKDSSAA
TCTTTGTTCTCTT TETPLMKEDSILAVRK TCTAAAGGACCG WDETLLDKFYT
GTCTAAAGGACC YFQRITLYLKEKKYSP TCACGACTTCGG ELYQQLNDLEA GTCACGACTTCG
CAWEVVRAEIMRSFS ATTCCCTCAGGA CVIQGVGVTET GATTCCCTCAGG
LSTNLQESLRSKETV AGAGTTTGGAAA PLMKEDSILAV AAGAGTTTGGAA
AAPSDIQMTQSPSSL CCAATTCCAAAA RKYFQRITLYLK ACCAATTCCAAA
SASVGDRVTITCRAS AGCAGAAACTAT EKKYSPCAWE AAGCAGAAACTA
RPIGTTLSWYQQKPG TCCTGTCTTGCA VVRAEIMRSFS TTCCTGTCTTGC
KAPKLLIWFGSRLQS CGAAATGATCCA LSTNLQESLRS ACGAAATGATCC
GVPSRFSGSGSGTD GCAAATATTCAAT KETVAAPSDIQ AGCAAATATTCA
FTLTISSLQPEDFATY TTGTTTTCTACAA MTQSPSSLSAS ATTTGTTTTCTAC
YCAQAGTHPTTFGQ AGGACTCATCAG VGDRVTITCRA AAAGGACTCATC GTKVEIKR
CCGCTTGGGATG SRPIGTTLSWY AGCCGCTTGGGA AAAEQKLISEEDLN* AAACTCTGTTAG
QQKPGKAPKLL TGAAACTCTGTT (SEQ ID NO: 61) ATAAATTCTACAC IWFGSRLQSGV
AGATAAATTCTA TGAACTATATCAA PSRFSGSGSG CACTGAACTATA CAACTGAACGAT
TDFTLTISSLQP TCAACAACTGAA CTAGAGGCTTGC EDFATYYCAQA CGATCTAGAGGC
GTTATTCAGGGT GTHPTTFGQG TTGCGTTATTCA GTAGGAGTTACT TKVEIKR (SEQ
GGGTGTAGGAGT GAAACTCCCCTA ID NO: 63) TACTGAAACTCC ATGAAAGAAGAT
CCTAATGAAAGA TCAATTCTAGCC AGATTCAATTCTA GTTAGAAAATACT GCCGTTAGAAAA
TTCAGCGTATCA TACTTTCAGCGT CATTGTATTTAAA ATCACATTGTATT GGAAAAGAAATA
TAAAGGAAAAGA CTCCCCATGTGC AATACTCCCCAT ATGGGAGGTGGT GTGCATGGGAG
TAGAGCAGAAAT GTGGTTAGAGCA TATGAGGTCCTT GAAATTATGAGG CTCTCTTTCTACG
TCCTTCTCTCTTT AATTTGCAAGAAT CTACGAATTTGC CTTTGAGATCTAA AAGAATCTTTGA
GGAAACCGTCGC GATCTAAGGAAA TGCTCCATCTGA CCGTCGCTGCTC CATCCAGATGAC
CATCTGACATCC CCAGTCTCCATC AGATGACCCAGT CTCCCTGTCTGC CTCCATCCTCCC
ATCTGTAGGAGA TGTCTGCATCTG CCGTGTCACCAT TAGGAGACCGTG CACTTGCCGGGC
TCACCATCACTT AAGTCGTCCGAT GCCGGGCAAGT TGGGACGACGTT CGTCCGATTGGG
AAGTTGGTACCA ACGACGTTAAGT GCAGAAACCAGG TGGTACCAGCAG GAAAGCCCCTAA
AAACCAGGGAAA GCTCCTGATCTG GCCCCTAAGCTC GTTTGGTTCCCG CTGATCTGGTTT
GTTGCAAAGTGG GGTTCCCGGTTG GGTCCCATCACG CAAAGTGGGGTC TTTCAGTGGCAG
CCATCACGTTTC TGGATCTGGGAC AGTGGCAGTGGA AGATTTCACTCTC TCTGGGACAGAT
ACCATCAGCAGT TTCACTCTCACC CTGCAACCTGAA ATCAGCAGTCTG GATTTTGCTACG
CAACCTGAAGAT TACTACTGTGCG TTTGCTACGTAC CAGGCTGGGAC TACTGTGCGCAG
GCATCCTACGAC GCTGGGACGCAT GTTCGGCCAAGG CCTACGACGTTC GACCAAGGTGGA
GGCCAAGGGAC AATCAAACGGGC CAAGGTGGAAAT GGCCGCAGAAC CAAACGG (SEQ
AAAAACTCATCT ID NO: 64) CAGAAGAGGAT CTGAATTAA (SEQ ID NO: 62)
DMS7327 CDLPQTHSLGSRRTL TGCGACTTGCCA CDLPQTHSLGS TGCGACTTGCCA
(IFN.alpha.2b- MLLAQMRRISLFSCL CAGACACATAGT RRTLMLLAQM CAGACACATAGT
DOM7h- KDRHDFGFPQEEFG TTGGGATCAAGA RRISLFSCLKD TTGGGATCAAGA 11-15)
NQFQKAETIPVLHEMI AGAACATTGATG RHDFGFPQEE AGAACATTGATG
QQIFNLFSTKDSSAA TTATTAGCACAAA FGNQFQKAETI TTATTAGCACAA
WDETLLDKFYTELYQ TGCGTAGAATTT PVLHEMIQQIF ATGCGTAGAATT
QLNDLEACVIQGVGV CTTTGTTCTCTTG NLFSTKDSSAA TCTTTGTTCTCTT
TETPLMKEDSILAVRK TCTAAAGGACCG WDETLLDKFYT GTCTAAAGGACC
YFQRITLYLKEKKYSP TCACGACTTCGG ELYQQLNDLEA GTCACGACTTCG
CAWEVVRAEIMRSFS ATTCCCTCAGGA CVIQGVGVTET GATTCCCTCAGG
LSTNLQESLRSKETV AGAGTTTGGAAA PLMKEDSILAV AAGAGTTTGGAA
AAPSDIQMTQSPSSL CCAATTCCAAAA RKYFQRITLYLK ACCAATTCCAAA
SASVGDRVTITCRAS AGCAGAAACTAT EKKYSPCAWE AAGCAGAAACTA RPIGTMLSWYQQKP
TCCTGTCTTGCA VVRAEIMRSFS TTCCTGTCTTGC GKAPKLLILAFSRLQS CGAAATGATCCA
LSTNLQESLRS ACGAAATGATCC GVPSRFSGSGSGTD GCAAATATTCAAT KETVAAPSDIQ
AGCAAATATTCA FTLTISSLQPEDFATY TTGTTTTCTACAA MTQSPSSLSAS
ATTTGTTTTCTAC
YCAQAGTHPTTFGQ AGGACTCATCAG VGDRVTITCRA AAAGGACTCATC GTKVEIKR
CCGCTTGGGATG SRPIGTMLSWY AGCCGCTTGGGA AAAEQKLISEEDLN* AAACTCTGTTAG
QQKPGKAPKLL TGAAACTCTGTT (SEQ ID NO: 65) ATAAATTCTACAC ILAFSRLQSGV
AGATAAATTCTA TGAACTATATCAA PSRFSGSGSG CACTGAACTATA CAACTGAACGAT
TDFTLTISSLQP TCAACAACTGAA CTAGAGGCTTGC EDFATYYCAQA CGATCTAGAGGC
GTTATTCAGGGT GTHPTTFGQG TTGCGTTATTCA GTAGGAGTTACT TKVEIKR (SEQ
GGGTGTAGGAGT GAAACTCCCCTA ID NO: 67) TACTGAAACTCC ATGAAAGAAGAT
CCTAATGAAAGA TCAATTCTAGCC AGATTCAATTCTA GTTAGAAAATACT GCCGTTAGAAAA
TTCAGCGTATCA TACTTTCAGCGT CATTGTATTTAAA ATCACATTGTATT GGAAAAGAAATA
TAAAGGAAAAGA CTCCCCATGTGC AATACTCCCCAT ATGGGAGGTGGT GTGCATGGGAG
TAGAGCAGAAAT GTGGTTAGAGCA TATGAGGTCCTT GAAATTATGAGG CTCTCTTTCTACG
TCCTTCTCTCTTT AATTTGCAAGAAT CTACGAATTTGC CTTTGAGATCTAA AAGAATCTTTGA
GGAAACCGTCGC GATCTAAGGAAA TGCTCCATCTGA CCGTCGCTGCTC CATCCAGATGAC
CATCTGACATCC CCAGTCTCCATC AGATGACCCAGT CTCCCTGTCTGC CTCCATCCTCCC
ATCTGTAGGAGA TGTCTGCATCTG CCGTGTCACCAT TAGGAGACCGTG CACTTGCCGGGC
TCACCATCACTT AAGTCGTCCGAT GCCGGGCAAGT TGGGACGATGTT CGTCCGATTGGG
AAGTTGGTACCA ACGATGTTAAGT GCAGAAACCAGG TGGTACCAGCAG GAAAGCCCCTAA
AAACCAGGGAAA GCTCCTGATCCT GCCCCTAAGCTC TGCTTTTTCCCGT CTGATCCTTGCT
TTGCAAAGTGGG TTTTCCCGTTTG GTCCCATCACGT CAAAGTGGGGTC TTCAGTGGCAGT
CCATCACGTTTC GGATCTGGGACA AGTGGCAGTGGA GATTTCACTCTCA TCTGGGACAGAT
CCATCAGCAGTC TTCACTCTCACC TGCAACCTGAAG ATCAGCAGTCTG ATTTTGCTACGTA
CAACCTGAAGAT CTACTGCGCGCA TTTGCTACGTAC GGCTGGGACGC TACTGCGCGCAG
ATCCTACGACGT GCTGGGACGCAT TCGGCCAAGGGA CCTACGACGTTC CCAAGGTGGAAA
GGCCAAGGGAC TCAAACGGGCGG CAAGGTGGAAAT CCGCAGAACAA CAAACGG (SEQ
AAACTCATCTCA ID NO: 68) GAAGAGGATCTG AATTAA (SEQ ID NO: 66)
[0161] The amino acid and nucleotide sequences highlighted in bold
represents the cloning site and MYC tag. * represents the stop
codon at the end of the gene.
Affinity Determination and Biophysical Characterisation:
[0162] To determine the binding affinity (K.sub.D) of the
AlbudAb-IFN.alpha.2b fusion proteins to each serum albumin;
purified fusion proteins were analysed by BIAcore over albumin
(immobilised by primary-amine coupling onto CM5 chips; BIAcore)
using fusion protein concentrations from 5000 nM to 39 nM (5000 nM,
2500 nM, 1250 nM, 625 nM, 312 nM, 156 nM, 78 nM, 39 nM) in HBS-EP
BIAcore buffer.
TABLE-US-00016 TABLE 12 Affinity to SA Affinity to AlbudAb Fusion
SA (nM) Kd Ka Rat DOM7h-14 IFN.alpha.2b 350 4.500E-02 1.28E+05
DOM7h-14-10 IFN.alpha.2b 16 4.970E-03 5.90E+05 DOM 7h-11
IFN.alpha.2b 6000 7.500E-01 nd DOM 7h-11-15 IFN.alpha.2b 200
1.660E-02 1.50E+05 Cyno DOM 7h-14 IFN.alpha.2b 60 1.32E-02 5.0E+05
DOM 7h-14-10 IFN.alpha.2b 19 7.05E-03 4.50E+05 DOM 7h-11
IFN.alpha.2b 3300 3.59E-01 1.20E+05 DOM 7h-11-15 IFN.alpha.2b 15
4.86E-03 3.60E+05 Mouse DOM 7h-14 IFN.alpha.2b 240 3.21E-02
1.50E+06 DOM 7h-14-10 IFN.alpha.2b 60 3.45E-02 6.86E+05 DOM 7h-11
IFN.alpha.2b 6000 1.55E-01 nd DOM 7h-11-15 IFN.alpha.2b 28 6.69E-03
2.80E+05 Human DOM 7h-14 IFN.alpha.2b 244 2.21E-02 9.89E+04 DOM
7h-14-10 IFN.alpha.2b 32 6.58E-03 3.48E+05 DOM 7h-11 IFN.alpha.2b
670 2.02E-01 7.00E+05 DOM 7h-11-15 IFN.alpha.2b 10 1.87E-03
3.50E+05
[0163] When IFNa2b is linked to the AlbudAb variants, in all cases
the affinity of AlbudAb binding to serum albumin is reduced.
DOM7h-14-10 and DOM7-11-15 retain improved binding affinity to
serum albumin across species compared to parent.
TABLE-US-00017 TABLE 13 Biophysical Characterisation Biophysical
Characterisation was carried out by SEC MALLS and DSC as described
above for the single AlbudAbs. Biophysical parameters DMS SEC DSC
AlbudAb Fusion number MALLS Tm(.degree. C.) DOM 7h-14 IFN.alpha.2b
DMS7321 M/D 58-65 DOM 7h-14-10 IFN.alpha.2b DMS7322 M/D 55-65 DOM
7h-11 IFN.alpha.2b DMS7325 M/D 65.8-66.2 DOM 7h-11-15 IFN.alpha.2b
DMS7327 M/D 56.3-66.2
M/D Indicates a Monomer/Dimer Equilibrium as Detected by SEC
MALLS
[0164] We observed expression for all clones in Table 13 in the
range of 17.5 to 54 mg/L in HEK293.
[0165] For IFN.alpha.2b-DOM7h-14 and IFN.alpha.2b-DOM7h-11
variants, favorable biophysical parameters and expression levels
were maintained during affinity maturation.
PK Determination for AlbudAb-IFN.alpha.2bfusions
[0166] AlbudAbs IFN.alpha.2b fusions DMS7321
(IFN.alpha.2b-DOM7h-14) DMS7322 (IFN.alpha.2b-DOM7h-14-10), DMS7325
(IFN.alpha.2b-DOM7h-11), DMS7327 (IFN.alpha.2b-DOM7h-11-15) were
expressed with the myc tag at 20-50 mg quantities in HEK293 cells
and purified from culture supernatant using protein L affinity
resin and eluted with 100 mM glycine pH2. The proteins were
concentrated to greater than 1 mg/ml, buffer exchanged into
Dulbecco's PBS and endotoxin depleted using Q spin columns
(Vivascience).
[0167] For Rat PK, IFN-AlbudAbs were dosed as single i.v injections
at 2.0 mg/kg using 3 rats per compound. Serum samples were taken at
0.16, 1, 4, 8, 24, 48, 72, 120, 168 hrs. Analysis of serum levels
was by EASY ELISA according to manufacturer's instructions (GE
Healthcare, catalogue number RPN5960).
[0168] For Mouse PK, DMS7322 (IFN2b-DOM7h-14-10) DMS7325
(IFN2b-DOM7h-11), DMS7327 (IFN2b-DOM7h-11-15) all with myc tags
were dosed as single i.v injections at 2.0 mg/kg per dose group of
3 subjects and serum samples taken at 10 mins; 1 h; 8 h; 24 h; 48
h; 72 h; 96 h. Analysis of serum levels was by EASY ELISA according
to manufacturer's instructions (GE Healthcare, catalogue number
RPN5960).
TABLE-US-00018 TABLE 14 PK parameters (mean results) AUC CL Albumin
h .times. ml/ t1/2 Vz Species AlbudAb Fusion K.sub.D (nM) ug/ml
h/kg h ml/kg Rat 7h-14 IFN.alpha.2b 350 832.1 2.4 27 94.5 7h-14-10
IFN.alpha.2b 16 1380.7 1.5 35.8 75.2 7h-11 IFN.alpha.2b 6000 327.9
6.5 11 101.9 7h-11-15 IFN.alpha.2b 200 1118.7 1.8 39.5 103.6
7h-11-12 IFN.alpha.2b 1700 747.1 2.8 25.8 104.7 mouse 7h-14
IFN.alpha.2b 240 761.2 2.6 30.4 115.3 7h-14-10 IFN.alpha.2b 60
750.5 2.7 30.9 118.6 7h-11 IFN.alpha.2b 6000 493.9 4.0 8.8 51.2
7h-11-15 IFN.alpha.2b 28 971.8 2.1 33.6 99.6
[0169] Pharmacokinetic parameters derived from rat and mouse
studies were fitted using a non-compartmental model. Key: AUC: Area
under the curve from dosing time extrapolated to infinity; CL:
clearance; t1/2: is the time during which the blood concentration
is halved; Vz: volume of distribution based on the terminal
phase.
[0170] IFN.alpha.2b-AlbudAbs were tested in rat and mouse. For all
IFN.alpha.2b-DOM7h-11 variant fusion proteins in both rat and
mouse, t1/2 is improved compared to parent. The improvement in t1/2
correlates with the improved in vitro K.sub.D to serum albumin. For
IFN.alpha.2b-DOM7h-14-10 variants, the improvement in in vitro
K.sub.D to serum albumin also correlated to an improvement in t1/2
in rat.
[0171] All IFN.alpha.2b-AlbudAb fusion proteins exhibit a 5 to
10-fold decrease in the binding to RSA compared to the single
AlbudAb. This effect is more pronounced (i.e. 10-fold) for the
DOM7h-14 series than the DOM7h-11 series (only 5-fold
decrease).
Example 8
Further AlbudAb Fusions with Proteins, Peptides and NCEs
[0172] Various AlbudAbs fused to other chemical entities namely
domain antibodies (dAbs), peptides and NCEs were tested. The
results are shown in table 15.
TABLE-US-00019 TABLE 15 PK parameters Albumin AUC CL t1/2 Vz
Species AlbudAb Fusion K.sub.D (nM) h .times. ug/ml ml/h/kg h ml/kg
Rat DOM7h-14 Exendin-4 2400 18 57.1 11 901.9 DOM7h-14- Exendin-4 19
43.6 23.1 22.1 740.3 10 DOM7h-11 Exendin-4 2400 6.1 168 7.1 1684.1
DOM7h-11- Exendin-4 273 36.3 27.6 19.3 765.7 15 In a DOM7h-11-
Exendin-4 130 not tested not not not different 15 tested tested
tested experiement DOM7h14- NCE- 62 10 GGGGSC DOM7h14- NCE- 35 10
TVAAPSC Human DOM7h-14 NCE 204 mouse DOM7h-11 DOM1m-21- 234 10.7
4.7 72.5 23 DOM7h-11- DOM1m-21- 1008 2.5 17.4 62.4 15 23
Key: DOM1m-21-23 is an anti-TNFR1 dAb, Exendin-4 is a peptide (a
GLP-1 agonist) of 39 amino acids length. NCE, NCE-GGGGSC and
NCE-TVAAPSC are described below.
[0173] Previously we have described the use of genetic fusions with
an albumin-binding dAb (AlbudAb) to extend the PK half-life of
anti-TNFR1 dAbs in vivo (see, e.g., WO04003019, WO2006038027,
WO2008149148). Reference is made to the protocols in these PCT
applications. In the table above, DOM1m-21-23 is an anti-mouse
TNFR1 dAb.
[0174] To produce genetic fusions of exendin-4 or with DOM7h-14 (or
other AlbudAb) which binds serum albumin, the
exendin-4-linker-AlbudAb sequence was cloned into the pTT-5 vector
(obtainable from CNRC, Canada). In each case the exendin-4 was at
the 5' end of the construct and the dAb at the 3' end. The linker
was a (G.sub.4S).sub.3 linker. Endotoxin-free DNA was prepared in
E. coli using alkaline lysis (using the endotoxin-free plasmid Giga
kit, obtainable from Qiagen CA) and used to transfect HEK293E cells
(obtainable from CNRC, Canada). Transfection was into 250 ml/flask
of HEK293E cells at 1.75.times.10.sup.6 cells/ml using 333 ul of
293fectin (Invitrogen) and 250 ug of DNA per flask and expression
was at 30.degree. C. for 5 days. The supernatant was harvested by
centrifugation and purification was by affinity purification on
protein L. Protein was batch bound to the resin, packed on a column
and washed with 10 column volumes of PBS. Protein was eluted with
50 ml of 0.1M glycine pH2 and neutralized with Tris pH8. Protein of
the expected size was identified on an SDS-PAGE gel.
NCE Albudab.TM. Fusions:
[0175] A new chemical entity (NCE) AlbudAb fusion was tested. The
NCE, a small molecule ADAMTS-4 inhibitor was synthesised with a PEG
linker (PEG 4 linker (i.e. 4 PEG molecules before the maleimide)
and a maleimide group for conjugation to the AlbudAb. Conjugation
of the NCE to the AlbudAb is via an engineered cysteine residue at
amino acid position R108C, or following a 5 amino acid (GGGGSC) or
6 amino acid (TVAAPSC) spacer engineered at the end of the AlbudAb.
Briefly, the AlbudAb was reduced with TCEP (Pierce, Catalogue
Number 77720), desalted using a PD10 column (GE healthcare) into 25
mM Bis-Tris, 5 mM EDTA, 10% (v/v) glycerol pH6.5. A 5 fold molar
excess of maleimide activated NCE was added in DMSO not to exceed
10% (V/V) final concentration. The reaction was incubated over
night at room temperature and dialysed extensively into 20 mM Tris
pH7.4
PEG Linker:
##STR00001##
TABLE-US-00020 [0176] Sequences: DOM7h-14 R108C: (SEQ ID NO: 69)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ GTKVEIKC
Nucleotide: (SEQ ID NO: 70)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAATGC
NCE-AlbudAbs DOM7h-14-10 GGGGSC and DOM7h14-10 TVAAPSC, exhibit a 5
to 10 fold decrease in in vitro affinity (K.sub.D) to RSA as
determined by BIAcore when fused to the chemical entity.
[0177] dAb-Albudab fusion: the 2 DOM7h-11 AlbudAbs with the highest
affinity to RSA experience a 2-fold decrease in affinity to RSA as
on BIAcore when fused to a therapeutic domain antibody (DOM1
m-21-23) compared to the unfused AlbudAb. The DOM7h-11 clone shows
a micromolar K.sub.D when fused (2.8 uM) as well as when unfused
(.about.5 uM).
[0178] Exendin 4-AlbudAb fusion: the effect of fusing the AlbudAbs
to a peptide on the binding ability to RSA is about 10-fold, apart
from DOM7h-14-10, which only shows a 4-fold decrease in binding.
The effect, however, is more pronounced for the DOM7h-14 series
(except DOM7h-14-10) than it appears to be for the DOM7h-11
series.
[0179] For all the above data, the T1/2 of the fusion increased
with improved affinity to the species' SA.
[0180] We generally classify Albudab-therapeutics as being
therapeutically amenable (for treatment and/or prophylaxis of
diseases, conditions or indications) when the AlbudAb-drug fusions
show an affinity range (K.sub.D) of from 0.1 nM to 10 mM for serum
albumin binding.
[0181] We define the therapeutic ranges of AlbudAbs and AlbudAb
fusions (Protein-AlbudAbs for example IFNa2b-DOM7h-14-10;
Peptide-AlbudAbs for example Exendin-4-DOM7h-14-10; dAb-AlbudAbs
for example DOM1m21-23-DOM7h11-15; NCE-AlbudAb for example
ADAMTS-4-DOM7h-14-10) as follows: Affinity (K.sub.D) ranges that
are useful for therapy of chronic or acute conditions, diseases or
indications are shown. Also shown are affinity ranges marked as
"intermediate". AlbudAbs and fusions in this range have utility for
chronic or acute diseases, conditions or indications. In this way,
the affinity of the AlbudAb or fusion for serum albumin can be
tailored or chosen according to the disease, condition or
indication to be addressed. As described above, the invention
provides AlbudAbs with affinities that allow for each AlbudAb to be
categorised as "high affinity", "medium affinity" or "low
affinity", thus enabling the skilled person to select the
appropriate AlbudAb of the invention according to the therapy at
hand. See FIG. 2.
Example 9
[0182] PCT/EP2010/060112 describes V.sub.H AlbudAbs and affinity
matured derivatives thereof. V.sub.H AlbudAb sequences are as
follows:
TABLE-US-00021 DOM7r31 amino acid SEQ ID NO: 71
EVQLLESGGGLVQPGGSLRLSCTASGFTFRHYRMGWVRQAPGKGLEWVSW
IRPDGTFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSY
MGDRFDYWGQGTLVTVSS DOM7r31 nucleic acid SEQ ID NO: 72
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGCGTCTCTCCTGTACAGCCTCCGGATTCACCTTTAGGCATTATCGTA
TGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATGG
ATTCGTCCGGATGGTACGTTTACATACTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATCTTAT
ATGGGTGATAGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTC GAGCG
DOM7r-31-14 amino acid SEQ ID NO: 73
EVQLLESGGGLVQPGGSLRLSCTASGFTFRHYRMGWVRQAPGKGLEWVSW
IRPDGTFTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSY
MADRFDYWGQGTLVTVSS DOM7r-31-14 nucleic acid SEQ ID NO: 74
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGCGTCTCTCCTGTACAGCCTCCGGATTCACCTTTAGGCATTATCGTA
TGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCATGG
ATTCGTCCGGATGGTACGTTTACATACTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATCTTAT
ATGGCTGATAGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTC GAGC DOM7h-92
amino acid SEQ ID NO: 75
EVQLLESGGGLVQPGGSLRLSCAASGFTFANATMSWVRQAPGKGLEWVSD
IDQVGHATYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKYS
WHPDLFDYWGQGTLVTVSS DOM7r-92 nucleic acid SEQ ID NO: 76
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGGGAATTATAGGA
TGACTTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAACT
ATTTCTCCTTTGGGTACGTATACATACTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAAGGGCGT
TGGTCGATTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCGAG C DOM7r-92-4
amino acid SEQ ID NO: 77
EVQLLESGGGLVQPGGSLRLSCAASGFTFDTSSMLWVRQAPGKGLEWVSV
IHQSGTPTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFP
STHGKFDYWGQGTLVTVSS DOM7r-92-4 nucleic acid SEQ ID NO: 78
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGATACGAGTAGTA
TGTTGTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTCTCAGTT
ATTCATCAGAGTGGTACGCCTACATACTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGCGTGCCGAGGACACCGCGGTATATTACTGTGCGAAATTTCCG
TCTACTCATGGTAAGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGT CTCGAGC
Affinity Determination:
[0183] To determine the binding affinity (K.sub.D) of the VH
AlbudAbs to each serum albumin; purified fusion proteins were
analysed by BIAcore over albumin (immobilised by primary-amine
coupling onto CM5 chips; BIAcore) using fusion protein
concentrations from 5000 nM to 39 nM (5000 nM, 2500 nM, 1250 nM,
625 nM, 312 nM, 156 nM, 78 nM, 39 nM) in HBS-EP BIAcore buffer, as
described above. MALLS data were obtained as described above.
[0184] Results are shown in the following tables:
TABLE-US-00022 TABLE 16A RSA HSA CSA MSA* KD (M) KD (M) KD (M) KD
(M) DOM7r-92-4 2.6E-07 1.3E-07 9.8E-08 1.1E-07
TABLE-US-00023 TABLE 16B HSA CSA RSA MSA MALLS DOM7r-92 200 nM 170
nM 500 nM 2000 nM 13 + 22 kDa dimer FLAG OD
TABLE-US-00024 TABLE 16 C CSA MSA HSA RSA KD KD KD KD (nM) (nM)
(nM) (nM) MALLS KD KD KD KD 7r-31 fast on/off fast fast fast on/off
on/ on/off M off 7r31- 208, 360, 1330, 103, 90.2, 370 no binding 6,
12, 12 M 14 1950 14.2
Values above represent multiple, independent measurements.
Example 10
Summary of HSA Epitope Mapping with AlbudAbs
[0185] The epitopes of AlbudAb Vk molecules on HSA were determined
using three orthogonal techniques: hydrogen deuterium exchange mass
spectrometry, site directed mutagenesis and structure determination
by x-ray crystallography.
1.0 EPITOPE MAPPING BY HYDROGEN DEUTERIUM (H/D) EXCHANGE
1.1 Protein Preparation
[0186] Domain 2 of HSA (defined as amino acid residues 188-384 of
HSA; SEQ ID NO: 79 and 80 for amino acid and DNA sequences for HSA
Domain 2 respectively; SEQ ID NO: 81 and 82 for amino acid and DNA
sequence of full length HSA respectively) were expressed in Pichia
using the pPICZ.alpha. expression system (Invitrogen) and purified
using Prometic Technologies Mimetic Blue.TM. according to the
manufacturer's instructions. DOM7h-11-3, DOM7h-14-10 and DOM7r-92-4
(SEQ ID NOs: 2, 45, 46 for amino acid and 4, 47, 48 for DNA
sequences respectively). was expressed in E. coli strain HB2151
using an auto-induction expression system. In some instances, the
cloning strategy resulted in additional N and C terminal residues
(see SEQ ID NOs: 121 and 122, for example). The expressed AlbudAbs
were purified from clarified supernatants by Protein-L (DOM7h-11-3
and DOM7h-14-10) or Protein-A (DOM7h-92-4) affinity chromatography
using established protocols.
[0187] Expression and purification of final protein preparations of
HSA Domain 2 and AlbudAbs were confirmed by SDS-PAGE analysis.
1.2 Experimental Method and General Principles
[0188] Methods and principles on using H/D exchange perturbation
for epitope mapping are discussed in a review by Hamuro et al
(2003), J. Biomol. Tech. 2003, 14, 171-182; and Coales et al
(2009), Rapid Communications in Mass Spectrometry 2009 March;
23(5):639-47. For the epitope mapping of HSA, H/D exchange analysis
of the antigen in the presence and absence of AlbudAb was carried
out. The regions of HSA which exchange slower in the presence of
each AlbudAb compared to speed of exchange when the AlbudAb is
absent is considered to define the epitope on HSA. To identify the
epitope one requires firstly the identification of proteolytic
fragments of the antigen and secondly the determination of the
perturbation of the H/D exchange reaction. Suitable methods are
described, for example, in U.S. Pat. No. 6,291,189, U.S. Pat. No.
6,331,400 and U.S. Pat. No. 7,280,923.
[0189] After each H/D exchange reaction HSA was digested with
pepsin. The digested mixture was separated by HPLC. Each HSA peptic
fragment was then analyzed by mass spectrometry to determine the
degree of deuterium incorporation upon the H/D exchange reaction.
To obtain optimal coverage of the HSA sequence a maximum possible
number of peptic fragments were followed. For the H/D exchange
experiments in the presence of AlbudAb, the mixture of antigen and
antibody was digested together. The peptic digest fragment mixture
of the complex contained both AlbudAb and HSA fragments.
[0190] As an excess amount of an AlbudAb over HSA was used, a large
amount of AlbudAb originated peptides may interfere with the mass
detection of antigen originated peptides by ion competition. For
this reason the least possible amount of excess antibody was used
Stock solutions of 199 .mu.M DOM7h-11-3, 199 .mu.M DOM7h-14-10, 547
.mu.M DOM7r-92-4 and 45 .mu.M HSA were used in the H/D exchange
experiments. 24 .mu.l of HSA stock+36 .mu.l of DOM7h-11-3; 49.5
.mu.l of HSA stock+10.5 .mu.l of DOM7r-92-4; 74 .mu.l of HSA
stock+20 .mu.l of DOM7h-14-10 were used to make a complexation
mixtures (final concentrations of HSA:DOM7h-11-3=17.9 .mu.M:119.4
.mu.M; HSA:DOM7h-14-10=35.0 .mu.M:42.0 .mu.M; HSA:DOM7r-92-4=37.1
.mu.M:95.7 .mu.M equivalent to 1:6.7, 1:1.2, 1:2.6 ratio
respectively). For the control reactions either HSA or AlbudAb were
replaced with PBS.
[0191] 10 .mu.l of HSA+DOM7h-11-3 complexation mixture was added to
10 .mu.l of PBS made with D.sub.2O; 5 or 8 .mu.l of HSA:DOM7h-14-10
complexation mixture was added to 12 or 15 .mu.l of PBS made with
D.sub.2O; 5 .mu.l of HSA+DOM7r-92-4 complexation mixture was added
to 15 .mu.l of PBS made with D.sub.2O. All deuteration reactions
were incubated for 500 seconds at 0.degree. C. After incubation all
20 .mu.l of the complexation mixture was mixed with 30 .mu.l of
quenching solution (2M Urea, 1M TCEP pH3.0). 45 .mu.l of the
quenched reaction mixture was injected onto a proprietary
proteolysis/HPLC system and fragments analysed by mass
spectrometry
1.3 Perturbation Results
[0192] Fragments were identified, data deconvoluted and visualized
using proprietary software (ExSAR). A summary of epitope hits based
on H/D exchange perturbation data are highlighted in Table 17
below.
TABLE-US-00025 TABLE 17 Difference in deuteration levels in each
segment of HSA compared with and without (A) DOM7h-11-3, (B)
DOM7h-14-10 and (C) DOM7r-92-4 complexation (A) Incubation Time
(Seconds) Peptide 30 100 300 1000 3000 10000 Average 189-198
##STR00002## 9% ##STR00003## -7% ##STR00004## 7% ##STR00005## -1%
##STR00006## -2% ##STR00007## 6% ##STR00008## 2% 189-200
##STR00009## 2% ##STR00010## 5% ##STR00011## 3% ##STR00012## 3%
##STR00013## -2% ##STR00014## 2% ##STR00015## 2% 203-210
##STR00016## -2% ##STR00017## 2% ##STR00018## 0% ##STR00019## 0%
##STR00020## 5% ##STR00021## 0% ##STR00022## 1% 203-213
##STR00023## 8% ##STR00024## 5% ##STR00025## 1% ##STR00026## 1%
##STR00027## -1% ##STR00028## 6% ##STR00029## 3% 213-219
##STR00030## -9% ##STR00031## -25% ##STR00032## -32% ##STR00033##
-35% ##STR00034## -39% ##STR00035## -32% ##STR00036## -29% 216-219
##STR00037## -12% ##STR00038## -20% ##STR00039## -28% ##STR00040##
-36% ##STR00041## -43% ##STR00042## -42% ##STR00043## -30% 220-228
##STR00044## -35% ##STR00045## -31% ##STR00046## -31% ##STR00047##
-18% ##STR00048## -19% ##STR00049## -8% ##STR00050## -24% 222-228
##STR00051## -38% ##STR00052## -36% ##STR00053## -40% ##STR00054##
-26% ##STR00055## -15% ##STR00056## -3% ##STR00057## -26% 231-238
##STR00058## -17% ##STR00059## -41% ##STR00060## -51% ##STR00061##
-54% ##STR00062## -58% ##STR00063## -33% ##STR00064## -42% 231-250
Not detected Not detected Not detected Not detected Not detected
Not detected Not detected 237-250 ##STR00065## -4% ##STR00066## -8%
##STR00067## -5% ##STR00068## -3% ##STR00069## -1% ##STR00070## -1%
##STR00071## -4% 237-251 ##STR00072## 0% ##STR00073## -5%
##STR00074## -8% ##STR00075## -4% ##STR00076## -1% ##STR00077## 3%
##STR00078## -3% 237-252 ##STR00079## -1% ##STR00080## -5%
##STR00081## -4% ##STR00082## -4% ##STR00083## -1% ##STR00084## -2%
##STR00085## -3% 253-260 ##STR00086## 10% ##STR00087## -6%
##STR00088## 5% ##STR00089## -3% ##STR00090## -1% ##STR00091## -1%
##STR00092## 1% 266-284 Not detected Not detected Not detected Not
detected Not detected Not detected Not detected 269-289 Not
detected Not detected Not detected Not detected Not detected Not
detected Not detected 269-292 Not detected Not detected Not
detected Not detected Not detected Not detected Not detected
292-302 ##STR00093## -2% ##STR00094## -5% ##STR00095## -2%
##STR00096## -3% ##STR00097## 2% ##STR00098## -9% ##STR00099## -3%
292-305 ##STR00100## 5% ##STR00101## 5% ##STR00102## 1%
##STR00103## 3% ##STR00104## -1% ##STR00105## -5% ##STR00106## 1%
295-305 ##STR00107## -2% ##STR00108## -2% ##STR00109## 0%
##STR00110## 4% ##STR00111## -2% ##STR00112## 2% ##STR00113## 0%
301-305 ##STR00114## 2% ##STR00115## -1% ##STR00116## -11%
##STR00117## -1% ##STR00118## -1% ##STR00119## 6% ##STR00120## -1%
311-318 ##STR00121## -24% ##STR00122## -18% ##STR00123## -10%
##STR00124## -1% ##STR00125## 0% ##STR00126## 0% ##STR00127## -9%
311-326 ##STR00128## -24% ##STR00129## -24% ##STR00130## -27%
##STR00131## -19% ##STR00132## -13% ##STR00133## -7% ##STR00134##
-19% 312-326 ##STR00135## -28% ##STR00136## -24% ##STR00137## -29%
##STR00138## -18% ##STR00139## -15% ##STR00140## -9% ##STR00141##
-21% 321-326 ##STR00142## -20% ##STR00143## -20% ##STR00144## -29%
##STR00145## -25% ##STR00146## -23% ##STR00147## -17% ##STR00148##
-22% 329-330 ##STR00149## -1% ##STR00150## -7% ##STR00151## -18%
##STR00152## -29% ##STR00153## -46% ##STR00154## -56% ##STR00155##
-26% 329-331 ##STR00156## -4% ##STR00157## -8% ##STR00158## -9%
##STR00159## -26% ##STR00160## -39% ##STR00161## -48% ##STR00162##
-22% 332-333 ##STR00163## 2% ##STR00164## 5% ##STR00165## -4%
##STR00166## -2% ##STR00167## -14% ##STR00168## -27% ##STR00169##
-7% 334-342 ##STR00170## -8% ##STR00171## -20% ##STR00172## -15%
##STR00173## -22% ##STR00174## -31% ##STR00175## -31% ##STR00176##
-21% 336-345 ##STR00177## 0% ##STR00178## -2% ##STR00179## -8%
##STR00180## -12% ##STR00181## -9% ##STR00182## -11% ##STR00183##
-7% 344-346 ##STR00184## -2% ##STR00185## 4% ##STR00186## -1%
##STR00187## -9% ##STR00188## -5% ##STR00189## -8% ##STR00190## -4%
345-346 ##STR00191## 2% ##STR00192## 0% ##STR00193## 1%
##STR00194## -7% ##STR00195## -6% ##STR00196## -15% ##STR00197##
-4% 347-349 Not detected Not detected Not detected Not detected Not
detected Not detected Not detected 348-349 ##STR00198## -10%
##STR00199## -18% ##STR00200## -30% ##STR00201## -31% ##STR00202##
-30% ##STR00203## -20% ##STR00204## -23% 348-357 ##STR00205## -24%
##STR00206## -33% ##STR00207## -41% ##STR00208## -39% ##STR00209##
-37% ##STR00210## -28% ##STR00211## -34% 349-357 ##STR00212## -27%
##STR00213## -33% ##STR00214## -43% ##STR00215## -41% ##STR00216##
-40% ##STR00217## -30% ##STR00218## -36% 373-384 ##STR00219## 2%
##STR00220## 1% ##STR00221## 0% ##STR00222## 3% ##STR00223## -2%
##STR00224## 6% ##STR00225## 2% 376-384 ##STR00226## 4%
##STR00227## 3% ##STR00228## 1% ##STR00229## -1% ##STR00230## 1%
##STR00231## 3% ##STR00232## 2% 380-384 ##STR00233## -5%
##STR00234## -2% ##STR00235## -4% ##STR00236## 8% ##STR00237## -3%
##STR00238## 5% ##STR00239## 0%
TABLE-US-00026 (B) Incubation Time (Seconds) Peptide 30 100 300
1000 3000 10000 Average 189-198 ##STR00240## -6% ##STR00241## 2%
##STR00242## 6% ##STR00243## 9% ##STR00244## -4% ##STR00245## 8%
##STR00246## 3% 189-200 ##STR00247## -4% ##STR00248## 3%
##STR00249## 4% ##STR00250## 6% ##STR00251## -6% ##STR00252## 4%
##STR00253## 1% 203-210 ##STR00254## -4% ##STR00255## 1%
##STR00256## 4% ##STR00257## 6% ##STR00258## -6% ##STR00259## 3%
##STR00260## 1% 203-213 ##STR00261## 1% ##STR00262## 3%
##STR00263## 5% ##STR00264## 5% ##STR00265## -6% ##STR00266## 4%
##STR00267## 2% 213-219 ##STR00268## -14% ##STR00269## -26%
##STR00270## -32% ##STR00271## -50% ##STR00272## -51% ##STR00273##
-49% ##STR00274## -37% 216-219 ##STR00275## -17% ##STR00276## -28%
##STR00277## -36% ##STR00278## -55% ##STR00279## -60% ##STR00280##
-65% ##STR00281## -44% 220-228 ##STR00282## -63% ##STR00283## -47%
##STR00284## -47% ##STR00285## -51% ##STR00286## -42% ##STR00287##
-25% ##STR00288## -46% 222-228 ##STR00289## -62% ##STR00290## -55%
##STR00291## -52% ##STR00292## -55% ##STR00293## -40% ##STR00294##
-26% ##STR00295## -48% 231-238 ##STR00296## -23% ##STR00297## -28%
##STR00298## -43% ##STR00299## -56% ##STR00300## -55% ##STR00301##
-48% ##STR00302## -42% 231-250 ##STR00303## -4% ##STR00304## -15%
##STR00305## -15% ##STR00306## -23% ##STR00307## -26% ##STR00308##
-22% ##STR00309## -18% 237-250 ##STR00310## -9% ##STR00311## -3%
##STR00312## -1% ##STR00313## -3% ##STR00314## -4% ##STR00315## 3%
##STR00316## -3% 237-251 ##STR00317## -3% ##STR00318## -1%
##STR00319## -2% ##STR00320## -1% ##STR00321## -6% ##STR00322## 2%
##STR00323## -2% 237-252 ##STR00324## -5% ##STR00325## -4%
##STR00326## -4% ##STR00327## 5% ##STR00328## -1% ##STR00329## 5%
##STR00330## -1% 253-260 ##STR00331## -5% ##STR00332## 1%
##STR00333## -1% ##STR00334## -7% ##STR00335## -5% ##STR00336## 7%
##STR00337## -2% 266-284 ##STR00338## -13% ##STR00339## -7%
##STR00340## 2% ##STR00341## 5% ##STR00342## -2% ##STR00343## 10%
##STR00344## -1% 269-289 ##STR00345## -9% ##STR00346## -4%
##STR00347## -8% ##STR00348## 0% ##STR00349## -7% ##STR00350## 4%
##STR00351## -4% 269-292 ##STR00352## 0% ##STR00353## 2%
##STR00354## -3% ##STR00355## 6% ##STR00356## -3% ##STR00357## 3%
##STR00358## 1% 292-302 ##STR00359## -9% ##STR00360## -4%
##STR00361## -3% ##STR00362## 12% ##STR00363## -5% ##STR00364## 6%
##STR00365## -1% 292-305 ##STR00366## -5% ##STR00367## 1%
##STR00368## -4% ##STR00369## 5% ##STR00370## -5% ##STR00371## 3%
##STR00372## -1% 295-305 ##STR00373## -7% ##STR00374## -1%
##STR00375## -1% ##STR00376## 13% ##STR00377## -8% ##STR00378## 7%
##STR00379## 1% 301-305 ##STR00380## 5% ##STR00381## -3%
##STR00382## 1% ##STR00383## 10% ##STR00384## -2% ##STR00385## -5%
##STR00386## 1% 311-318 ##STR00387## -27% ##STR00388## -23%
##STR00389## -17% ##STR00390## -5% ##STR00391## -7% ##STR00392## 7%
##STR00393## -12% 311-326 ##STR00394## -30% ##STR00395## -26%
##STR00396## -25% ##STR00397## -19% ##STR00398## -19% ##STR00399##
-7% ##STR00400## -21% 312-326 ##STR00401## -27% ##STR00402## -22%
##STR00403## -22% ##STR00404## -18% ##STR00405## -20% ##STR00406##
-9% ##STR00407## -20% 321-326 ##STR00408## -9% ##STR00409## -13%
##STR00410## -18% ##STR00411## -13% ##STR00412## -18% ##STR00413##
-11% ##STR00414## -14% 329-330 ##STR00415## -5% ##STR00416## -15%
##STR00417## -13% ##STR00418## -35% ##STR00419## -43% ##STR00420##
-67% ##STR00421## -30% 329-331 ##STR00422## -3% ##STR00423## -7%
##STR00424## -10% ##STR00425## -23% ##STR00426## -31% ##STR00427##
-56% ##STR00428## -22% 332-333 ##STR00429## -2% ##STR00430## 0%
##STR00431## -2% ##STR00432## -14% ##STR00433## -18% ##STR00434##
-55% ##STR00435## -15% 334-342 ##STR00436## -7% ##STR00437## -7%
##STR00438## -11% ##STR00439## -18% ##STR00440## -23% ##STR00441##
-35% ##STR00442## -17% 336-345 ##STR00443## -5% ##STR00444## -7%
##STR00445## -5% ##STR00446## -9% ##STR00447## -15% ##STR00448##
-13% ##STR00449## -9% 344-346 ##STR00450## 4% ##STR00451## 5%
##STR00452## -1% ##STR00453## -7% ##STR00454## -9% ##STR00455##
-14% ##STR00456## -4% 345-346 ##STR00457## 3% ##STR00458## -2%
##STR00459## 2% ##STR00460## -2% ##STR00461## -8% ##STR00462## -29%
##STR00463## -6% 347-349 ##STR00464## -11% ##STR00465## -15%
##STR00466## -15% ##STR00467## -41% ##STR00468## -47% ##STR00469##
-50% ##STR00470## -30% 348-349 ##STR00471## -7% ##STR00472## -18%
##STR00473## -20% ##STR00474## -40% ##STR00475## -43% ##STR00476##
-35% ##STR00477## -27% 348-357 ##STR00478## -28% ##STR00479## -36%
##STR00480## -43% ##STR00481## -60% ##STR00482## -50% ##STR00483##
-44% ##STR00484## -44% 349-357 ##STR00485## -31% ##STR00486## -33%
##STR00487## -41% ##STR00488## -62% ##STR00489## -57% ##STR00490##
-54% ##STR00491## -46% 373-384 ##STR00492## -5% ##STR00493## -1%
##STR00494## -1% ##STR00495## 5% ##STR00496## -5% ##STR00497## 7%
##STR00498## 0% 376-384 ##STR00499## -1% ##STR00500## 1%
##STR00501## 4% ##STR00502## 9% ##STR00503## -4% ##STR00504## 4%
##STR00505## 2% 380-384 ##STR00506## -3% ##STR00507## -2%
##STR00508## -2% ##STR00509## 8% ##STR00510## -7% ##STR00511## 0%
##STR00512## -1%
TABLE-US-00027 (C) Incubation Time (Seconds) Peptide 30 100 300
1000 3000 10000 Average 189-198 Not detected Not detected Not
detected Not detected Not detected Not detected Not detected
189-200 ##STR00513## 1% ##STR00514## -6% ##STR00515## -4%
##STR00516## 1% ##STR00517## -2% ##STR00518## -1% ##STR00519## -2%
203-210 ##STR00520## -20% ##STR00521## -21% ##STR00522## -12%
##STR00523## -16% ##STR00524## -16% ##STR00525## -14% ##STR00526##
-17% 203-213 Not detected Not detected Not detected Not detected
Not detected Not detected Not detected 213-219 ##STR00527## -5%
##STR00528## -9% ##STR00529## -15% ##STR00530## -21% ##STR00531##
-28% ##STR00532## -32% ##STR00533## -18% 216-219 ##STR00534## -8%
##STR00535## -11% ##STR00536## -16% ##STR00537## -24% ##STR00538##
-24% ##STR00539## -30% ##STR00540## -19% 220-228 Not detected Not
detected Not detected Not detected Not detected Not detected Not
detected 222-228 ##STR00541## -29% ##STR00542## -28% ##STR00543##
-22% ##STR00544## -16% ##STR00545## -3% ##STR00546## -5%
##STR00547## -17% 231-238 ##STR00548## -1% ##STR00549## 2%
##STR00550## -6% ##STR00551## -8% ##STR00552## -11% ##STR00553##
-13% ##STR00554## -6% 231-250 Not detected Not detected Not
detected Not detected Not detected Not detected Not detected
237-250 ##STR00555## 2% ##STR00556## 4% ##STR00557## 3%
##STR00558## 0% ##STR00559## -1% ##STR00560## 1% ##STR00561## 2%
237-251 Not detected Not detected Not detected Not detected Not
detected Not detected Not detected 237-252 ##STR00562## 0%
##STR00563## -4% ##STR00564## -2% ##STR00565## -4% ##STR00566## 1%
##STR00567## -2% ##STR00568## -2% 253-260 ##STR00569## 1%
##STR00570## -11% ##STR00571## 2% ##STR00572## -6% ##STR00573## -3%
##STR00574## 4% ##STR00575## -2% 266-284 Not detected Not detected
Not detected Not detected Not detected Not detected Not detected
269-289 Not detected Not detected Not detected Not detected Not
detected Not detected Not detected 269-292 Not detected Not
detected Not detected Not detected Not detected Not detected Not
detected 292-302 ##STR00576## 1% ##STR00577## -5% ##STR00578## -10%
##STR00579## -6% ##STR00580## 6% ##STR00581## 5% ##STR00582## -2%
292-305 ##STR00583## -4% ##STR00584## 0% ##STR00585## 2%
##STR00586## 15% ##STR00587## -4% ##STR00588## -4% ##STR00589## 1%
295-305 ##STR00590## 6% ##STR00591## -1% ##STR00592## -4%
##STR00593## -4% ##STR00594## -4% ##STR00595## 3% ##STR00596## -1%
301-305 ##STR00597## -8% ##STR00598## -4% ##STR00599## 8%
##STR00600## 8% ##STR00601## 4% ##STR00602## -12% ##STR00603## -1%
311-318 ##STR00604## -25% ##STR00605## -19% ##STR00606## -9%
##STR00607## -6% ##STR00608## -8% ##STR00609## -1% ##STR00610##
-11% 311-326 ##STR00611## -26% ##STR00612## -28% ##STR00613## -23%
##STR00614## -21% ##STR00615## -9% ##STR00616## -6% ##STR00617##
-19% 312-326 ##STR00618## -24% ##STR00619## -25% ##STR00620## -20%
##STR00621## -15% ##STR00622## -7% ##STR00623## -6% ##STR00624##
-16% 321-326 ##STR00625## -14% ##STR00626## -22% ##STR00627## -27%
##STR00628## -30% ##STR00629## -16% ##STR00630## -15% ##STR00631##
-21% 329-330 ##STR00632## -26% ##STR00633## -23% ##STR00634## -19%
##STR00635## -36% ##STR00636## -33% ##STR00637## -47% ##STR00638##
-31% 329-331 ##STR00639## -7% ##STR00640## -7% ##STR00641## -8%
##STR00642## -10% ##STR00643## -18% ##STR00644## -44% ##STR00645##
-16% 332-333 ##STR00646## -1% ##STR00647## 5% ##STR00648## -4%
##STR00649## 4% ##STR00650## -1% ##STR00651## -15% ##STR00652## -2%
334-342 ##STR00653## 14% ##STR00654## 9% ##STR00655## 2%
##STR00656## -5% ##STR00657## 4% ##STR00658## -6% ##STR00659## 3%
336-345 ##STR00660## 2% ##STR00661## 4% ##STR00662## -3%
##STR00663## 3% ##STR00664## -1% ##STR00665## 0% ##STR00666## 1%
344-346 Not detected Not detected Not detected Not detected Not
detected Not detected Not detected 345-346 ##STR00667## 4%
##STR00668## 4% ##STR00669## 3% ##STR00670## 3% ##STR00671## 4%
##STR00672## -11% ##STR00673## 1% 347-349 Not detected Not detected
Not detected Not detected Not detected Not detected Not detected
348-349 ##STR00674## 2% ##STR00675## -8% ##STR00676## -14%
##STR00677## 2% ##STR00678## -2% ##STR00679## -13% ##STR00680## -6%
348-357 Not detected Not detected Not detected Not detected Not
detected Not detected Not detected 349-357 ##STR00681## -1%
##STR00682## -7% ##STR00683## -2% ##STR00684## -3% ##STR00685## -2%
##STR00686## -6% ##STR00687## -4% 373-384 ##STR00688## 6%
##STR00689## 0% ##STR00690## 4% ##STR00691## 6% ##STR00692## 3%
##STR00693## 5% ##STR00694## 4% 376-384 ##STR00695## 2%
##STR00696## 1% ##STR00697## -1% ##STR00698## 3% ##STR00699## 0%
##STR00700## -3% ##STR00701## 0% 380-384 Not detected Not detected
Not detected Not detected Not detected Not detected Not
detected
[0193] Based on data summarized in Table 17(A), (B) and (C) it was
concluded that segments which showed significant perturbation (on
average >20%) of deuteration could define the epitope. The
sequence segments identified are highlighted in FIG. 3.
2.0 ALANINE SCANNING SITE DIRECTED MUTAGENESIS (SDM)
[0194] Target residues for SDM for Alanine scanning were selected
based on 3 criteria: (1) H/D exchange perturbation data above, (2)
surface accessibility of side chains based on a previously
published crystal structure of HSA (1BKE.pdb (RCSB Protein
DataBank)) and (3) charge or size of side chains.
2.1 Cloning and Expression of Alanine Mutants
[0195] The wild type template of HSA was PCR-cloned into a
mammalian expression vector using standard molecular biology
protocols. A 6-Histidine tag was fused to the C-terminus of the
sequence (SEQ ID NOS: 25 and 34 for amino acid and DNA sequences
respectively for the WT HSA-His.sub.6 construct) for nickel
affinity purification. Primer pairs used for PCR amplification to
make the WT expression construct were TB147 and TB148 (SEQ ID NOS:
85 and 86 respectively).
[0196] Mutants were made following standard molecular biology
protocols using the WT HSA-His.sub.6 construct as a template for
mutagenesis. The list of Alanine mutants and mutagenesis oligo
pairs used to construct them are listed in Table 18 below.
TABLE-US-00028 TABLE 18 Primers pairs for making HSA mutants K225A
TB153 GAGCCAGAGATTTCCCGCCGCTGAGTTT sense SEQ ID NO: GCAGAAG 87
TB154 CTTCTGCAAACTCAGCGGCGGGAAATCT anti- SEQ ID NO: CTGGCTC sense
88 E227A TB155 GAGATTTCCCAAAGCTGCCTTTGCAGAA sense SEQ ID NO:
GTTTCCAAG 89 TB156 CTTGGAAACTTCTGCAAAGGCAGCTTTG anti- SEQ ID NO:
GGAAATCTC sense 90 E230A TB157 CCAAAGCTGAGTTTGCAGCCGTTTCCAA sense
SEQ ID NO: GTTAGTGAC 91 TB158 GTCACTAACTTGGAAACGGCTGCAAACT anti-
SEQ ID NO: CAGCTTTGG sense 92 D314A TB159
GATTTTGTTGAAAGTAAGGCCGTTTGCA sense SEQ ID NO: AAAACTATG 93 TB160
CATAGTTTTTGCAAACGGCCTTACTTTCA anti- SEQ ID NO: ACAAAATC sense 94
K317A TB161 GAAAGTAAGGATGTTTGCGCCAACTATG sense SEQ ID NO:
CTGAGGCAAAGG 95 TB162 CCTTTGCCTCAGCATAGTTGGCGCAAAC anti- SEQ ID NO:
ATCCTT AC TTTC sense 96 V325A TB163 GCTGAGGCAAAGGATGCCTTCCTGGGC
sense SEQ ID NO: ATGTTTTTG 97 TB164 CAAAAACATGCCCAGGAAGGCATCCTTT
anti- SEQ ID NO: GCCTCAGC sense 98 M329A TB165
GGATGTCTTCCTGGGCGCCTTTTTGTAT sense SEQ ID NO: GAATATG 99 TB166
CATATTCATACAAAAAGGCGCCCAGGAA anti- SEQ ID NO: GACATCC sense 100
K351A TB167 GCTGCTGCTGAGACTTGCCGCCACATAT sense SEQ ID NO:
GAAACCACTCTAG 101 TB168 CTAGAGTGGTTTCATATGTGGCGGCAAG anti- SEQ ID
NO: TCTCAGCAGCAGC sense 102
[0197] Sequence verified clones were selected from plasmid DNA
minipreps made using Millipore Montage kits following the
manufacturers protocols. Amino acid and DNA sequences of constructs
are summarized in Table 19 below.
TABLE-US-00029 TABLE 19 List of HSA mutants made HSA-His6 Amino
Acid DNA WT SEQ ID 103 SEQ ID 112 K225A SEQ ID 104 SEQ ID 113 E227A
SEQ ID 105 SEQ ID 114 E230A SEQ ID 106 SEQ ID 115 D314A SEQ ID 107
SEQ ID 116 K317A SEQ ID 108 SEQ ID 117 V325A SEQ ID 109 SEQ ID 118
M329A SEQ ID 110 SEQ ID 119 K351A SEQ ID 111 SEQ ID 120
[0198] His.sub.6-tagged WT HSA and mutants were expressed in
mammalian HEK293-6E cells using transient transfection techniques.
Mutants and WT HSA were purified from clarified expression
supernatants using nickel affinity chromatography according to
established protocols. SDS-PAGE analysis of the purified mutants
showed >95% purity.
2.2 BIAcore Analysis of Alanine Mutants
2.2.1 Experimental Method for BIAcore
[0199] Briefly, WT HSA and mutants were immobilised onto CM5
Biacore chips on a Biacore 3000 (GE Healthcare). This was performed
by first activating all four flow cells with EDC/NHS and then
injecting WT HSA or mutants in acetate buffer pH 4.5. Any free
sites on the chip were then blocked with an injection of
ethanolamine across all four flow cells. Levels of immobilization
are for each sample are summarized in Table 20 below.
TABLE-US-00030 TABLE 20 BIAcore CM5 chip preparation. Chip1 Fc2
E227A HSA 312 RUs Fc4 WT HSA 293 RUs Chip2 Fc2 D314A HSA 369 RU Fc3
K225A HSA 143 RU Fc4 E230A HSA 339 RU Chip3 Fc2 M324A HSA 167 RU
Fc3 V325A HSA 147 RU Fc4 WT HSA 304 RU Chip4 Fc2 K317A HSA 305 RU
Fc3 K351A HSA 165 RU Fc4 WT HSA 223 RU
2.2.2 BIAcore Data
[0200] Flow rate using HBS-EP buffer was 40 uL/min and the purified
dAb proteins were injected for 1 minute at concentrations 5000 nM
followed by 7 further injections at a sequential 1:2 dilution in
mobile phase.
[0201] Analysis and determination of equilibrium binding constants
(KD) was performed using standard procedures.
TABLE-US-00031 TABLE 21 A DOM7h- DOM7h- 14-10myc 11-15myc
DOM7h-11-3myc x-fold x-fold x-fold decrease decrease decrease HSA
in in in mutant nM binding nM binding nM binding K225A 2.34E- 0.31
1.17E- 0.53 2.41E-08 0.90375 09 09 E227A 3.33E- 4.44 7.91E- 3.58
1.46E-07 5.475 08 09 E230A 1.50E- 2.00 2.45E- 11.09 8.00E-07 30 08
08 D314A 5.35E- 0.71 4.45E- 2.01 1.78E-08 0.6675 09 09 M329A 5.65E-
0.75 5.72 E- 2.59 6.52E-07 24.45 09 09 WT 7.5E- 2.21E- 2.66667E- 09
09 08
TABLE-US-00032 TABLE 21 B DOM7r31-14myc DOM7r92-4myc x-fold x-fold
decrease HSA decrease in mutant nM in binding nM binding K225A
3.49E-08 0.71 2.49E-08 0.44 E227A 1.28E-07 2.62 no 88.81 binding
E230A 4.06E-06 83.03 3.97E-07 7.05 D314A 3.45E-08 0.71 5.20E-08
0.92 M329A 2.75E-08 0.56 4.93E-07 8.76 WT 4.89E-08 5.63E-08
2.2.3 BIAcore Analysis and Conclusions
[0202] DOM7h-14-10: No significant decrease in binding was observed
upon mutagenesis of any of the above residues in isolation.
[0203] DOM7h-11-15: Some significant decrease in binding of
DOM7h-11-15 to E230A is observed (11-fold decrease in binding over
WT). This suggests that residue 230 on HSA plays a significant
contribution in the specific binding to human serum albumin.
[0204] DOM7h-11-3: A significant decrease in binding was observed
for two residues (E230 and M324) upon mutagenesis to alanine. This
suggests that these two residues play an important contribution to
the Antibody/Antigen interaction.
[0205] DOM7r92-4: A significant decrease in binding for E230A was
observed.
[0206] DOM7r31-14: A significant decrease in binding for E227A was
observed.
3 CRYSTAL STRUCTURE OF THE DOM7H-11-15/HSA COMPLEX
3.1 Protein Preparation
[0207] Fatty acid free HSA from a commercial source was purified by
size exclusion chromatography to >95% purity as judged by
SDS-PAGE.
[0208] DOM7h-11-15 (SEQ ID NO: 1 and SEQ ID NO: 2 for amino acid
and DNA sequences respectively) was expressed in E. coli strain
BL21 DE3 using an auto-induction expression system. DOM7h-11-15 was
purified from clarified supernatants by Protein-L affinity
chromatography using established protocols. It was further purified
by ion exchange chromatography using a Hi-Trap SP column using
established protocols.
[0209] HSA was mixed with DOM7h-11-15 and the complex purified by
size exclusion chromatography. Protein was concentrated in 20 mM
Tris-Cl pH 8.0 prior to crystallization screening.
3.2 Crystallization
[0210] The HSA/DOM7h-11-15 complex was put into a crystallization
screen with approximately 1200 conditions using the sitting drop
method.
[0211] 3.3 X-ray Diffraction Data Collection and Processing
[0212] HSA/DOM7h-11-15 crystals were flash frozen in liquid
nitrogen after cryoprotection. The crystal was maintained at 100K
during data collection. X-ray diffraction data were collected at
the SWISS LIGHT SOURCE (SLS, Villigen, Switzerland).
[0213] Data were processed using XDS and XSCALE (Kabsch) reviewed,
for example, in Acta Crystallogr D Biol Crystallogr. 2010 Feb. 1;
66(Pt 2): 125-132). The crystals belonged to the space group
P2.sub.12.sub.12.sub.1 with two complexes HSA/DOM7h-11-15 in the
asymmetric unit.
[0214] Data collection statistics are summarised in Table 22
below.
TABLE-US-00033 TABLE 22 Data Collection and Processing Statistics
for HSA/DOM7h-11-15 complex Crystal Complex HSA-DOM7h11-15 X-ray
source PXI/X06SA (SLS .sup.1) Wavlength [.ANG.] 1.0000 Detector
PILATUS 6M Temperature [K] 100 Space group P 2.sub.1 2.sub.2
2.sub.3 Cell: a; b; c [.ANG.] 102,15; 110.00; 141.34
.alpha.:.beta.:.gamma. [.degree.] 90.0; 90,0; 90.0 Resolution
[.ANG.] .sup.2 2.50 (2.91-2.66) Unique reflections .sup.2 55,052
(10,796) Multiplicity .sup.2 5.3 (5.5) Completeness [%] .sup.2 98.7
(100.0) R.sub.sym[%] .sup.2, 3 10.0 (43.9) R.sub.meas [%] .sup.2,4
11.1 (48.3) mean(I)/sigma .sup.2,5 12.17 (4.63) .sup.1 SWISS LIGHT
SOURCE (SLS, Villigen, Switzerland) .sup.2 Numbers in brackets
correspond to the resolution bin with R.sub.sym = 43.9%. 3 R sym =
h i n h I ^ h - I h , i h i n h I h , i with I ^ h = 1 n h i n h I
h , i ##EQU00001## where I.sub.h,i is the intensity value of the
ith measurement of h R meas 4 = h n h n h - 1 i n h I ^ h - I h , i
h i n h I h , i with I ^ h = 1 n h i n h I h , i ##EQU00002## where
I.sub.h,i is the intensity value of the ith measurement of h .sup.5
Calculated from independent reflections
3.4 Structure Determination and Model Refinement
[0215] Structure determination and model refinement was carried out
to generate a representation of HSA in complex with DOM
7h-11-15.
[0216] The structure of the complex was determined by molecular
replacement. DOM7h11-15 bound to HSA showed clear electron density
in the initial maps from phases determined from the HSA molecules
only and allowed unambiguous placement of the antibody domain using
a difference maps. Subsequent model building and refinement was
performed according to standard protocols with the software
packages in CCP4 and COOT (see Collaborative Computational Project,
Number 4. 1994.
"The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst.
D50, 760-763; and "Coot: model-building tools for molecular
graphics" Emsley P, Cowtan K Acta Crystallographica Section
D-Biological Crystallography 60: 2126-2132 Part 12 Sp. Iss. 1 Dec.
2004, for example). Refinement statistics are summarized in Table
23
[0217] The Ramachandran Plot of the final model shows 91.5% of all
residues in the most favoured region, 8.1% in the additionally
allowed region, 0.4% of the residues in the generously allowed, and
no residues in the disallowed regions (Modelling statistics are
summarized in Table 23).
TABLE-US-00034 TABLE 23 Refinement and Modelling Statistics for the
HSA/DOM7h-11-15 Structure Complex HSA-DOM7h11-15 Resolution [.ANG.]
86.71-2.50 Number of reflections (working/test) 54,062/989
R.sub.cryst [%] 23.8 R.sub.free.sup.2 [%] 28.5 Total number of
atoms: Protein 10,881 Water 103 Ligand -- Sulphate 60
1,2-Ethanediol 16 Deviation from ideal geometry: .sup.3 Bond
lengths [.ANG.] 0.008 Bond angles [.degree.] 1.06 Bonded B's .sup.4
[.ANG..sup.2] 1.7 Ramachandran Plot: .sup.5 Most flavoured regions
92.0 Additional allowed regions 7.7 Generously allowed regions 0.3
Disallowed regions 0.0 .sup.1 Vaules as defined in REFMAC5, without
sigma cut-off .sup.2 Test-set contains 1.8% of measured reflections
.sup.3 Root mean square deviations from geometric target values
.sup.4 Calculated with programme MOLEMAN .sup.5 Calculated with
programme PROCHECK
3.5 Structure Analysis
3.5.1 Overall Structure
[0218] The structure of HSA in complex with DOM7h11-15 is
represented in FIG. 4. FIG. 4A shows the asymmetric unit containing
2 copies each of HSA and DOM7h-11-15. The biologically relevant
unit which consists of one molecule each of HSA and DOM7h-11-15 is
shown in two orientations in FIG. 4B.
3.5.2 The Epitope and Paratope
[0219] Residues on the HSA-Albudab binding interface are displayed
in a format by residue number in FIG. 5 (this figure lists all
residue to residue contacts within 4.5 A between chains A (HSA) and
chain B (DOM7h-11-15)). All significant interaction pairs are
marked as solid diamonds. Types of interactions for those
considered to significant are listed in Table 22.
TABLE-US-00035 TABLE 22A List of interactions between HSA and
DOM7h-11-15 HSA DOM7h-11-15 Residue Residue Residue Residue Chain
Number Type Chain Number Type Interaction A 228 (PHE) B 49 (LEU)
hydrophobic A 228 (PHE) B 91 (ALA) hydrophobic A 230 (GLU) B 94
(HIS) 1 H-bond SC-SC A 308 (ASP) B 53 (ARG) 3 H-bonds all SC- SC A
309 (PHE) B 51 (PHE) hydrophobic A 317 (LYS) B 67 (SER) 1 H-bond
SC-MC A 318 (ASN) B 31 (THR) 2 H-bond MC- SC, SC-SC A 321 (GLU) B
30 (GLY) 1 H-bond MC-SC A 322 (ALA) B 32 (MET) hydrophobic A 325
(VAL) B 32 (MET) hydrophobic A 325 (VAL) B 91 (ALA) hydrophobic A
326 (PHE) B 32 (MET) hydrophobic A 326 (PHE) B 51 (PHE) hydrophobic
A 329 (MET) B 32 (MET) hydrophobic A 329 (MET) B 49 (LEU)
hydrophobic A 329 (MET) B 50 (ALA) hydrophobic A 329 (MET) B 51
(PHE) hydrophobic A 329 (MET) B 91 (ALA) hydrophobic
[0220] All but two DOM7h-11-15 residues binding HSA are from CDR1,
2 and 3. Residues forming the paratope are showing in the alignment
in FIG. 6 below where DOM7h-11-3, DOM7h11-15 are aligned against Vk
dummy (VKDUM-1). Table 22B below lists additional residues on the
AlbudAb-HSA interface within 4.5 A.
TABLE-US-00036 TABLE 22B Table of additional residues on interface
HSA DOM7h-11-15 Residue Residue Residue Residue Chain number number
Chain number number A 227 (GLU) B 36 (TYR) A 227 (GLU) B 46 (LEU) A
228 (PHE) B 92 (GLY) A 229 (ALA) B 36 (TYR) A 229 (ALA) B 92 (GLY)
A 229 (ALA) B 93 (THR) A 229 (ALA) B 94 (HIS) A 232 (SER) B 92
(GLY) A 233 (LYS) B 94 (HIS) A 263 (TYR) B 94 (HIS) A 307 (ALA) B
53 (ARG) A 308 (ASP) B 51 (PHE) A 314 (ASP) B 31 (THR) A 317 (LYS)
B 30 (GLY) A 317 (LYS) B 68 (GLY) A 318 (ASN) B 29 (ILE) A 318
(ASN) B 30 (GLY) A 318 (ASN) B 32 (MET) A 318 (ASN) B 51 (PHE) A
321 (GLU) B 28 (PRO) A 321 (GLU) B 29 (ILE) A 321 (GLU) B 68 (GLY)
A 322 (ALA) B 29 (ILE) A 325 (VAL) B 90 (GLN) A 332 (TYR) B 49
(LEU) A 333 (GLU) B 49 (LEU)
Significant interactions listed in Table 22 are detailed further in
FIG. 7 and panels within. In these figures, interacting residues
are drawn in stick representation with any hydrogen bonds drawn as
dashed lines. Corresponding electron density for those interacting
side chains are also show depicted in mesh (contoured at 1.56).
4.0 CONCLUSIONS
[0221] Three orthogonal techniques have been used to determine the
epitope of the DOM7h-11 lineage on HSA. The results from all
techniques provide information about the region of HSA which forms
the epitope. Whilst H/D exchange perturbation data give a range of
possible residues, Alanine scanning data and the crystal structure
provide more detailed information on a single residue level. FIG. 8
below summarizes level of detail and specificity the data from each
technique has provided.
[0222] Based on the crystal structure, it is also possible to state
that the bind of DOM7h-11-15 to HSA does not block or obstruct any
lipid carrier pockets on HSA. This is particularly relevant for
therapeutic applications since these binding pockets are utilized
by a number of therapeutic compounds for systemic transport.
Therefore any potential biopharmaceutical formatted with
DOM7h-11-15 would not be expected to interfere with HSA-drug
interactions and transport. Drug/lipid carrier pocket positions
relative to the DOM7h-11-15 epitope are detailed in FIG. 9. Based
on the similar epitope observed with DOM7h-14-10 and DOM7r-92-4 H/D
exchange perturbation data, these AlbudAbs.TM. would also not be
expected to interfere with HSA-drug interactions and transport.
TABLE-US-00037 TABLE OF SEQUENCES SEQ ID NO: IDENTIFIER Amino acid
Nucleic acid DOM7h-11-15 1 3 DOM7h-11-3 2 4 DOM7h-14/Exendin-4
fusion 5 6 DOM7h-14-10/Exendin-4 fusion 7 8 DOM7h-11/Exendin-4
fusion 9 10 DOM7h-11-15/Exendin-4 fusion 11 12 DOM7h14-10/G4SC-NCE
fusion 13 14 DOM7h14-10/TVAAPSC fusion 15 16 DOM7h-11/DOM1m-21-23
fusion 17 19 DOM7h-11/DOM1m-21-23 fusion + myc tag 18 20
DOM7h-11-15/DOM1m-21-23 fusion 21 23 DOM7h-11-15/DOM1m-21-23 fusion
+ myc tag 22 24 DPK9 Vk dummy CDRs 1-3 25-27 -- DOM7h-11 CDRs 1-3
28-30 -- DOM 7h-11-15 CDRs 1-3 31-33 -- DOM 7h-11-3 CDRs 1-3 34-36
-- DOM 7h-14 CDRs 1-3 37-39 -- DOM 7h-14-10 CDRs 1-3 40-42 --
Interferon alpha 2b 43 44 IFN.alpha.2b SOE fragment 5' -- 45
IFN.alpha.2b SOE fragment 3' -- 46 Vk SOE fragment 5' -- 47 Vk SOE
fragment 3' to also introduce a myc tag -- 48 IFN.alpha.2b SOE
fragment 5' -- 49 Vk SOE fragment 3' to also introduce a myc tag --
50 Leader sequence 51 52 DMS7321 53 54 (IFN.alpha.2b-DOM7h-14) +
myc DMS7321 (IFN.alpha.2b-DOM7h-14) no tag 55 56 DMS732
(IFN.alpha.2b-DOM7h-14-10) + myc 57 58 DMS732
(IFN.alpha.2b-DOM7h-14-10) no tag 59 60 DMS 7325
(IFN.alpha.2b-DOM7h-11) + myc 61 62 DMS 7325
(IFN.alpha.2b-DOM7h-11) no tag 63 64 DMS 7327
(IFN.alpha.2b-DOM7h-11-15) + myc 65 66 DMS 7327
(IFN.alpha.2b-DOM7h-11-15) no tag 67 68 DOM7h-14 R108C 69 70
DOM7r31 71 72 DOM7r-31-14 73 74 DOM7h-94 75 76 DOM7r-92-4 77 78
DOMAIN 2 OF HSA 79 80 FULL LENGTH HSA 81 82 DOM 7H-14-10 83 84
PRIMER TB147 -- 85 PRIMER TB148 -- 86 TB153 -- 87 TB154 -- 88 TB155
-- 89 TB156 -- 90 TB157 -- 91 TB158 -- 92 TB159 -- 93 TB160 -- 94
TB161 -- 95 TB162 -- 96 TB163 -- 97 TB164 -- 98 TB165 -- 99 TB166
-- 100 TB167 -- 101 TB168 -- 102 HSA-His6 WT 103 112 HSA-His6 K225A
104 113 HSA-His6 E227A 105 114 HSA-His6 E230A 106 115 HSA-His6
D314A 107 116 HSA-His6 K317A 108 117 HSA-His6 V325A 109 118
HSA-His6 M329A 110 119 HSA-His6 K351A 111 120 DOM 7h-11-13 121 122
DOM7H-14 123 124 DOM7H-11 125 126
Example 11
Sequences of DOM7h-14-10 Variants
[0223] In another embodiment of the invention, listed below are the
amino acid and nucleotide sequences for some variants of the
anti-serum albumin immunoglobulin single variable domain
DOM7h-14.
TABLE-US-00038 DOM7h-14-56. (SEQ ID NO: 127)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPMLLIMW
SSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ GTKVEIKW
DOM7h-14-65. (SEQ ID NO: 128)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ GTKVEIKW
DOM7h-14-74. (SEQ ID NO: 129)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGK GTKVENKW
DOM7h-14-76. (SEQ ID NO: 130)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLKHPKTYGQ GTKVEIKW
DOM7h-14-82. (SEQ ID NO: 131)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGMRHPKTFGQ GTKVEIKW
DOM7h-14-100. (SEQ ID NO: 132)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ GTKVENKW
DOM7h-14-101. (SEQ ID NO: 133)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSALQNGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ GTKVEIKW
DOM7h-14-109. (SEQ ID NO: 134)
DIQMTQSPSSLFASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRKPKTFGQ GTKVKIKW
DOM7h-14-115. (SEQ ID NO: 135)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ GTKVEIKW
DOM7h-14-116. (SEQ ID NO: 136)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRYPKTFGQ GTKVEIKW
DOM7h-14-119. (SEQ ID NO: 137)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ GTKVEIKR
DOM7h-14-120. (SEQ ID NO: 138)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGQ GTKVENKR
DOM7h-14-121. (SEQ ID NO: 139)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSALQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTFGQ GTKVEIKR
DOM7h-14-122. (SEQ ID NO: 140)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGK GTKVEIKR
DOM7h-14-123. (SEQ ID NO: 141)
DIQMTQSPSSLSASVGDRVTITCRASQWIGSQLSWYQQKPGKAPKLLIMW
RSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAQGLRHPKTYGK GTKVENKR
DOM7h-14-56. (SEQ ID NO: 142)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTATGCTCCTGATCATGTGG
AGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAATGG DOM7h-14-65. (SEQ ID NO: 143)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAATGG DOM7h-14-74. (SEQ ID NO: 144)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTACGGCAAA
GGGACCAAGGTGGAAAACAAATGG DOM7h-14-76. (SEQ ID NO: 145)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAAGCATCCTAAGACGTACGGCCAA
GGGACCAAGGTGGAAATCAAATGG DOM7h-14-82. (SEQ ID NO: 146)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTATGAGGCATCCTAAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAATGG DOM7h-14-100. (SEQ ID NO: 147)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGCGGCATCCTAAGACGTACGGCCAA
GGGACCAAGGTGGAAAACAAATGG DOM7h-14-101. (SEQ ID NO: 148)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCGCGTTACAAAATGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAATGG DOM7h-14-109. (SEQ ID NO: 149)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTTTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGAAACCTAAGACTTTCGGCCAA
GGGACCAAGGTGAAAATCAAATGG DOM7h-14-115. (SEQ ID NO: 150)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAAACGTACGGCCAA
GGGACCAAGGTGGAAATCAAATGG DOM7h-14-116. (SEQ ID NO: 151)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGTATCCTAAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAATGG DOM7h-14-119. (SEQ ID NO: 152)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGCGGCATCCTAAGACGTACGGCCAA
GGGACCAAGGTGGAAATCAAACGG DOM7h-14-120. (SEQ ID NO: 153)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGCGGCATCCTAAGACGTACGGCCAA
GGGACCAAGGTGGAAAACAAACGG DOM7h-14-121. (SEQ ID NO: 154)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCGCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG DOM7h-14-122. (SEQ ID NO: 155)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTACGGCAAA
GGGACCAAGGTGGAAATCAAACGG DOM7h-14-123. (SEQ ID NO: 156)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGTGGATTGGGTCTCAGTTAT
CTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCATGTGG
CGTTCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTGCTCAGGGTTTGAGGCATCCTAAGACGTACGGCAAA
GGGACCAAGGTGGAAAACAAACGG
Sequence CWU 1
1
1581108PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 1Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Arg Pro Ile Gly Thr Met 20 25 30 Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Leu Ala Phe
Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
2108PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 2Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Arg Pro Ile Gly Thr Thr 20 25 30 Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Leu Trp Asn
Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
3324DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 3gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtcg tccgattggg
acgatgttaa gttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatccttgct ttttcccgtt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgcgcgcag gctgggacgc atcctacgac
gttcggccaa 300gggaccaagg tggaaatcaa acgg 3244324DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 4gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtcg tccgattggg acgacgttaa gttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcctttgg aattcccgtt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgcgcag gctgggacgc atcctacgac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 3245163PRTmus musculus 5His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Pro Pro Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 50 55
60 Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala65 70 75 80 Ser Gln Trp Ile Gly Ser Gln Leu Ser Trp Tyr Gln Gln
Lys Pro Gly 85 90 95 Lys Ala Pro Lys Leu Leu Ile Met Trp Arg Ser
Ser Leu Gln Ser Gly 100 105 110 Val Pro Ser Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu 115 120 125 Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Ala 130 135 140 Gln Gly Ala Ala Leu
Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu145 150 155 160 Ile Lys
Arg6489DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 6catggtgaag gaacatttac cagtgacttg
tcaaaacaga tggaagagga ggcagtgcgg 60ttatttattg agtggcttaa gaacggagga
ccaagtagcg gggcacctcc gccatcgggt 120ggtggaggcg gttcaggcgg
aggtggcagc ggcggtggcg ggtcggacat ccagatgacc 180cagtctccat
cctccctgtc tgcatctgta ggagaccgtg tcaccatcac ttgccgggca
240agtcagtgga ttgggtctca gttatcttgg taccagcaga aaccagggaa
agcccctaag 300ctcctgatca tgtggcgttc ctcgttgcaa agtggggtcc
catcacgttt cagtggcagt 360ggatctggga cagatttcac tctcaccatc
agcagtctgc aacctgaaga ttttgctacg 420tactactgtg ctcagggtgc
ggcgttgcct aggacgttcg gccaagggac caaggtggaa 480atcaaacgg
4897163PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 7His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro
Pro Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 50 55 60 Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala65 70 75
80 Ser Gln Trp Ile Gly Ser Gln Leu Ser Trp Tyr Gln Gln Lys Pro Gly
85 90 95 Lys Ala Pro Lys Leu Leu Ile Met Trp Arg Ser Ser Leu Gln
Ser Gly 100 105 110 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu 115 120 125 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Ala 130 135 140 Gln Gly Leu Arg His Pro Lys Thr
Phe Gly Gln Gly Thr Lys Val Glu145 150 155 160 Ile Lys
Arg8489DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 8catggtgaag gaacatttac cagtgacttg
tcaaaacaga tggaagagga ggcagtgcgg 60ttatttattg agtggcttaa gaacggagga
ccaagtagcg gggcacctcc gccatcgggt 120ggtggaggcg gttcaggcgg
aggtggcagc ggcggtggcg ggtcggacat ccagatgacc 180cagtctccat
cctccctgtc tgcatctgta ggagaccgtg tcaccatcac ttgccgggca
240agtcagtgga ttgggtctca gttatcttgg taccagcaga aaccagggaa
agcccctaag 300ctcctgatca tgtggcgttc ctcgttgcaa agtggggtcc
catcacgttt cagtggcagt 360ggatctggga cagatttcac tctcaccatc
agcagtctgc aacctgaaga ttttgctacg 420tactactgtg ctcagggttt
gaggcatcct aagacgttcg gccaagggac caaggtggaa 480atcaaacgg
4899163PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 9His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro
Pro Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 50 55 60 Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala65 70 75
80 Ser Arg Pro Ile Gly Thr Thr Leu Ser Trp Tyr Gln Gln Lys Pro Gly
85 90 95 Lys Ala Pro Lys Leu Leu Ile Trp Phe Gly Ser Arg Leu Gln
Ser Gly 100 105 110 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu 115 120 125 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Ala 130 135 140 Gln Ala Gly Thr His Pro Thr Thr
Phe Gly Gln Gly Thr Lys Val Glu145 150 155 160 Ile Lys
Arg10489DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 10catggtgaag gaacatttac
cagtgacttg tcaaaacaga tggaagagga ggcagtgcgg 60ttatttattg agtggcttaa
gaacggagga ccaagtagcg gggcacctcc gccatcgggt 120ggtggaggcg
gttcaggcgg aggtggcagc ggcggtggcg ggtcggacat ccagatgacc
180cagtctccat cctccctgtc tgcatctgta ggagaccgtg tcaccatcac
ttgccgggca 240agtcgtccga ttgggacgac gttaagttgg taccagcaga
aaccagggaa agcccctaag 300ctcctgatct ggtttggttc ccggttgcaa
agtggggtcc catcacgttt cagtggcagt 360ggatctggga cagatttcac
tctcaccatc agcagtctgc aacctgaaga ttttgctacg 420tactactgtg
cgcaggctgg gacgcatcct acgacgttcg gccaagggac caaggtggaa 480atcaaacgg
48911163PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 11His Gly Glu Gly Thr Phe Thr Ser Asp
Leu Ser Lys Gln Met Glu Glu1 5 10 15 Glu Ala Val Arg Leu Phe Ile
Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala Pro Pro
Pro Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser Gly
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser 50 55 60 Ser
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala65 70 75
80 Ser Arg Pro Ile Gly Thr Met Leu Ser Trp Tyr Gln Gln Lys Pro Gly
85 90 95 Lys Ala Pro Lys Leu Leu Ile Leu Ala Phe Ser Arg Leu Gln
Ser Gly 100 105 110 Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu 115 120 125 Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
Ala Thr Tyr Tyr Cys Ala 130 135 140 Gln Ala Gly Thr His Pro Thr Thr
Phe Gly Gln Gly Thr Lys Val Glu145 150 155 160 Ile Lys
Arg12489DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 12catggtgaag gaacatttac
cagtgacttg tcaaaacaga tggaagagga ggcagtgcgg 60ttatttattg agtggcttaa
gaacggagga ccaagtagcg gggcacctcc gccatcgggt 120ggtggaggcg
gttcaggcgg aggtggcagc ggcggtggcg ggtcggacat ccagatgacc
180cagtctccat cctccctgtc tgcatctgta ggagaccgtg tcaccatcac
ttgccgggca 240agtcgtccga ttgggacgat gttaagttgg taccagcaga
aaccagggaa agcccctaag 300ctcctgatcc ttgctttttc ccgtttgcaa
agtggggtcc catcacgttt cagtggcagt 360ggatctggga cagatttcac
tctcaccatc agcagtctgc aacctgaaga ttttgctacg 420tactactgcg
cgcaggctgg gacgcatcct acgacgttcg gccaagggac caaggtggaa 480atcaaacgg
48913114PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 13Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30 Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Met Trp Arg
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly Leu Arg His Pro Lys
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly
Gly Gly 100 105 110 Ser Cys14345DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
14gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc
60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtgctcag
ggtttgaggc atcctaagac gttcggccaa 300gggaccaagg tggaaatcaa
acggggtggc ggagggggtt cctgt 34515115PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 15Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln
20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Ala Gln Gly Leu Arg His Pro Lys 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Cys 115
16345DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 16gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg
tctcagttat cttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatcatgtgg cgttcctcgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtgctcag ggtttgaggc atcctaagac
gttcggccaa 300gggaccaagg tggaaatcaa acggaccgtc gctgctccat cttgt
34517235PRTmus musculus 17Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Asn Arg Tyr 20 25 30 Ser Met Gly Trp Leu Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asp
Ser Tyr Gly Arg Gly Thr Tyr Tyr Glu Asp Pro Val 50 55 60 Lys Gly
Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Ser Gly
Pro Ser Asp 115 120 125 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly Asp 130 135 140 Arg Val Thr Ile Thr Cys Arg Ala Ser
Arg Pro Ile Gly Thr Thr Leu145 150 155 160 Ser Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Trp 165 170 175 Phe Gly Ser Arg
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 180 185 190 Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 195 200 205
Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr Thr 210
215 220 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg225 230 235
18249PRTArtificial SequenceAmino acid plus myc tag 18Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg Tyr 20 25
30 Ser Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Arg Ile Asp Ser Tyr Gly Arg Gly Thr Tyr Tyr Glu Asp
Pro Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln Phe Gly Ser
Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val
Ser Ser Ala Ser Thr Ser Gly Pro Ser Asp 115 120 125 Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 130 135 140 Arg Val
Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Gly Thr Thr Leu145 150 155
160 Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Trp
165 170 175 Phe Gly Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser 180 185 190 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu 195 200
205 Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr Thr
210 215 220 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Ala Ala
Glu Gln225 230 235 240 Lys Leu Ile Ser Glu Glu Asp Leu Asn 245
19705DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 19gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttaat
aggtatagta tggggtggct ccgccaggct 120ccagggaagg gtctagagtg
ggtctcacgg attgattctt atggtcgtgg tacatactac 180gaagaccccg
tgaagggccg gttcagcatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggac accgccgtat attactgtgc
gaaaatttct 300cagtttgggt caaatgcgtt tgactactgg ggtcagggaa
cccaggtcac cgtctcgagc 360gctagcacca gtggtccatc ggacatccag
atgacccagt ctccatcctc cctgtctgca 420tctgtaggag accgtgtcac
catcacttgc cgggcaagtc gtccgattgg gacgacgtta 480agttggtacc
agcagaaacc agggaaagcc cctaagctcc tgatctggtt tggttcccgg
540ttgcaaagtg gggtcccatc acgtttcagt ggcagtggat ctgggacaga
tttcactctc 600accatcagca gtctgcaacc tgaagatttt gctacgtact
actgtgcgca ggctgggacg 660catcctacga cgttcggcca agggaccaag
gtggaaatca aacgg 70520750DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
20gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtgcag cctccggatt cacctttaat aggtatagta tggggtggct ccgccaggct
120ccagggaagg gtctagagtg ggtctcacgg attgattctt atggtcgtgg
tacatactac 180gaagaccccg tgaagggccg gttcagcatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgccgtat attactgtgc gaaaatttct 300cagtttgggt caaatgcgtt
tgactactgg ggtcagggaa cccaggtcac cgtctcgagc 360gctagcacca
gtggtccatc ggacatccag atgacccagt ctccatcctc cctgtctgca
420tctgtaggag accgtgtcac catcacttgc cgggcaagtc gtccgattgg
gacgacgtta 480agttggtacc agcagaaacc agggaaagcc cctaagctcc
tgatctggtt tggttcccgg 540ttgcaaagtg gggtcccatc acgtttcagt
ggcagtggat ctgggacaga tttcactctc 600accatcagca gtctgcaacc
tgaagatttt gctacgtact actgtgcgca ggctgggacg 660catcctacga
cgttcggcca agggaccaag gtggaaatca aacgggcggc cgcagaacaa
720aaactcatct cagaagagga tctgaattaa 75021235PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 21Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asn Arg Tyr 20 25 30 Ser Met Gly Trp Leu Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Asp Ser Tyr Gly
Arg Gly Thr Tyr Tyr Glu Asp Pro Val 50 55 60 Lys Gly Arg Phe Ser
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Ile Ser Gln Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105
110 Gly Thr Gln Val Thr Val Ser Ser Ala Ser Thr Ser Gly Pro Ser Asp
115 120 125 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp 130 135 140 Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile
Gly Thr Met Leu145 150 155 160 Ser Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Leu 165 170 175 Ala Phe Ser Arg Leu Gln Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 180 185 190 Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 195 200 205 Asp Phe Ala
Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr Thr 210 215 220 Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg225 230 235
22249PRTArtificial SequenceAmino acid plus nucleotide plus myc tag
22Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Arg
Tyr 20 25 30 Ser Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Arg Ile Asp Ser Tyr Gly Arg Gly Thr Tyr
Tyr Glu Asp Pro Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ile Ser Gln
Phe Gly Ser Asn Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln
Val Thr Val Ser Ser Ala Ser Thr Ser Gly Pro Ser Asp 115 120 125 Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 130 135
140 Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Gly Thr Met
Leu145 150 155 160 Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Leu 165 170 175 Ala Phe Ser Arg Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly Ser 180 185 190 Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu 195 200 205 Asp Phe Ala Thr Tyr Tyr
Cys Ala Gln Ala Gly Thr His Pro Thr Thr 210 215 220 Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Ala Ala Ala Glu Gln225 230 235 240 Lys
Leu Ile Ser Glu Glu Asp Leu Asn 245 23705DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 23gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttaat aggtatagta
tggggtggct ccgccaggct 120ccagggaagg gtctagagtg ggtctcacgg
attgattctt atggtcgtgg tacatactac 180gaagaccccg tgaagggccg
gttcagcatc tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgcg tgccgaggac accgccgtat attactgtgc gaaaatttct
300cagtttgggt caaatgcgtt tgactactgg ggtcagggaa cccaggtcac
cgtctcgagc 360gctagcacca gtggtccatc ggacatccag atgacccagt
ctccatcctc cctgtctgca 420tctgtaggag accgtgtcac catcacttgc
cgggcaagtc gtccgattgg gacgatgtta 480agttggtacc agcagaaacc
agggaaagcc cctaagctcc tgatccttgc tttttcccgt 540ttgcaaagtg
gggtcccatc acgtttcagt ggcagtggat ctgggacaga tttcactctc
600accatcagca gtctgcaacc tgaagatttt gctacgtact actgcgcgca
ggctgggacg 660catcctacga cgttcggcca agggaccaag gtggaaatca aacgg
70524750DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 24gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt
cacctttaat aggtatagta tggggtggct ccgccaggct 120ccagggaagg
gtctagagtg ggtctcacgg attgattctt atggtcgtgg tacatactac
180gaagaccccg tgaagggccg gttcagcatc tcccgcgaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac accgccgtat
attactgtgc gaaaatttct 300cagtttgggt caaatgcgtt tgactactgg
ggtcagggaa cccaggtcac cgtctcgagc 360gctagcacca gtggtccatc
ggacatccag atgacccagt ctccatcctc cctgtctgca 420tctgtaggag
accgtgtcac catcacttgc cgggcaagtc gtccgattgg gacgatgtta
480agttggtacc agcagaaacc agggaaagcc cctaagctcc tgatccttgc
tttttcccgt 540ttgcaaagtg gggtcccatc acgtttcagt ggcagtggat
ctgggacaga tttcactctc 600accatcagca gtctgcaacc tgaagatttt
gctacgtact actgcgcgca ggctgggacg 660catcctacga cgttcggcca
agggaccaag gtggaaatca aacgggcggc cgcagaacaa 720aaactcatct
cagaagagga tctgaattaa 750259PRTArtificial SequenceCDR 1 amino acid
sequence identified using molecular biology techniques. 25Ser Gln
Ser Ile Ser Ser Tyr Leu Asn1 5 268PRTArtificial SequenceCDR 2 amino
acid sequence identified using molecular biology techniques. 26Tyr
Ala Ala Ser Ser Leu Gln Ser1 5 279PRTArtificial SequenceCDR 3 amino
acid sequence identified using molecular biology techniques. 27Gln
Gln Ser Tyr Ser Thr Pro Asn Thr1 5 289PRTArtificial SequenceCDR 1
amino acid sequence identified using molecular biology techniques.
28Ser Arg Pro Ile Gly Thr Thr Leu Ser1 5 298PRTArtificial
SequenceCDR 2 amino acid sequence identified using molecular
biology techniques. 29Trp Phe Gly Ser Arg Leu Gln Ser1 5
309PRTArtificial SequenceCDR 3 amino acid sequence identified using
molecular biology techniques. 30Ala Gln Ala Gly Thr His Pro Thr
Thr1 5 319PRTArtificial SequenceCDR 1 amino acid sequence
identified using molecular biology techniques. 31Ser Arg Pro Ile
Gly Thr Met Leu Ser1 5 328PRTArtificial SequenceCDR 2 amino acid
sequence identified using molecular biology techniques. 32Leu Ala
Phe Ser Arg Leu Gln Ser1 5 339PRTArtificial SequenceCDR 3 amino
acid sequence identified using molecular biology techniques. 33Ala
Gln Ala Gly Thr His Pro Thr Thr1 5 349PRTArtificial SequenceCDR 1
amino acid sequence identified using molecular biology techniques.
34Ser Arg Pro Ile Gly Thr Thr Leu Ser1 5 358PRTArtificial
SequenceCDR 2 amino acid sequence identified using molecular
biology techniques. 35Leu Trp Phe Ser Arg Leu Gln Ser1 5
369PRTArtificial SequenceCDR 3 amino acid sequence identified using
molecular biology techniques. 36Ala Gln Ala Gly Thr His Pro Thr
Thr1 5 379PRTArtificial SequenceCDR 1 amino acid sequence
identified using molecular biology techniques. 37Ser Gln Trp Ile
Gly Ser Gln Leu Ser1 5 388PRTArtificial SequenceCDR 2 amino acid
sequence identified using molecular biology techniques. 38Met Trp
Arg Ser Ser Leu Gln Ser1 5 399PRTArtificial SequenceCDR 3 amino
acid sequence identified using molecular biology techniques. 39Ala
Gln Gly Ala Ala Leu Pro Arg Thr1 5 409PRTArtificial SequenceCDR 1
amino acid sequence identified using molecular biology techniques.
40Ser Gln Trp Ile Gly Ser Gln Leu Ser1 5 418PRTArtificial
SequenceCDR 2 amino acid sequence identified using molecular
biology techniques. 41Met Trp Arg Ser Ser Leu Gln Ser1 5
429PRTArtificial SequenceCDR 3 amino acid sequence identified using
molecular biology techniques. 42Ala Gln Gly Leu Arg His Pro Lys
Thr1 5 43165PRTArtificial SequenceInterferon alpha 2b amino acid
sequence identified using molecular biology techniques. 43Cys Asp
Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20
25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe
Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln
Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr Leu65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val
Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150
155 160 Leu Arg Ser Lys Glu 165 44495DNAArtificial
SequenceInterferon alpha 2b nucleic acid sequence identified using
molecular biology techniques. 44tgtgatctgc ctcaaaccca cagcctgggt
agcaggagga ccttgatgct cctggcacag 60atgaggagaa tctctctttt ctcctgcttg
aaggacagac atgactttgg atttccccag 120gaggagtttg gcaaccagtt
ccaaaaggct gaaaccatcc ctgtcctcca tgagatgatc 180cagcagatct
tcaatctctt cagcacaaag gactcatctg ctgcttggga tgagaccctc
240ctagacaaat tctacactga actctaccag cagctgaatg acctggaagc
ctgtgtgata 300cagggggtgg gggtgacaga gactcccctg atgaaggagg
actccattct ggctgtgagg 360aaatacttcc aaagaatcac tctctatctg
aaagagaaga aatacagccc ttgtgcctgg 420gaggttgtca gagcagaaat
catgagatct ttttctttgt caacaaactt gcaagaaagt 480ttaagaagta aggaa
4954525DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 45gcccggatcc accggctgtg atctg
254630DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 46ggaggatgga gactgggtca tctggatgtc
304730DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 47gacatccaga tgacccagtc tccatcctcc
304882DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 48gcgcaagctt ttattaattc agatcctctt
ctgagatgag tttttgttct gcggccgccc 60gtttgatttc caccttggtc cc
824925DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 49gcccggatcc accggctgtg atctg
255082DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 50gcgcaagctt ttattaattc agatcctctt
ctgagatgag tttttgttct gcggccgccc 60gtttgatttc caccttggtc cc
825120PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 51Met Glu Thr Asp Thr Leu Leu Leu Trp
Val Leu Leu Leu Trp Val Pro1 5 10 15 Gly Ser Thr Gly 20
5261DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 52atggagaccg acaccctgct gctgtgggtg
ctgctgctgt gggtgcccgg atccaccggg 60c 6153293PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 53Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg
Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val
Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80 Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105
110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu Thr Val Ala Ala
Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr Cys Arg Ala Ser
Gln Trp Ile Gly Ser Gln Leu Ser Trp Tyr Gln 195 200 205 Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Met Trp Arg Ser Ser 210 215 220 Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr225 230
235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr 245 250 255 Tyr Tyr Cys Ala Gln Gly Ala Ala Leu Pro Arg Thr Phe
Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg Ala Ala Ala Glu
Gln Lys Leu Ile Ser 275 280 285 Glu Glu Asp Leu Asn 290
54882DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 54tgcgacttgc cacagacaca tagtttggga
tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt
ctcttgtcta
aaggaccgtc acgacttcgg attccctcag 120gaagagtttg gaaaccaatt
ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc 180cagcaaatat
tcaatttgtt ttctacaaag gactcatcag ccgcttggga tgaaactctg
240ttagataaat tctacactga actatatcaa caactgaacg atctagaggc
ttgcgttatt 300cagggtgtag gagttactga aactccccta atgaaagaag
attcaattct agccgttaga 360aaatactttc agcgtatcac attgtattta
aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta gagcagaaat
tatgaggtcc ttctctcttt ctacgaattt gcaagaatct 480ttgagatcta
aggaaaccgt cgctgctcca tctgacatcc agatgaccca gtctccatcc
540tccctgtctg catctgtagg agaccgtgtc accatcactt gccgggcaag
tcagtggatt 600gggtctcagt tatcttggta ccagcagaaa ccagggaaag
cccctaagct cctgatcatg 660tggcgttcct cgttgcaaag tggggtccca
tcacgtttca gtggcagtgg atctgggaca 720gatttcactc tcaccatcag
cagtctgcaa cctgaagatt ttgctacgta ctactgtgct 780cagggtgcgg
cgttgcctag gacgttcggc caagggacca aggtggaaat caaacgggcg
840gccgcagaac aaaaactcat ctcagaagag gatctgaatt aa
88255279PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 55Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu
Thr Val Ala Ala Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr
Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln Leu Ser Trp Tyr Gln 195 200
205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Met Trp Arg Ser Ser
210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Ala Gln Gly Ala Ala Leu
Pro Arg Thr Phe Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg
275 56837DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 56tgcgacttgc cacagacaca
tagtttggga tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt
ctcttgtcta aaggaccgtc acgacttcgg attccctcag 120gaagagtttg
gaaaccaatt ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc
180cagcaaatat tcaatttgtt ttctacaaag gactcatcag ccgcttggga
tgaaactctg 240ttagataaat tctacactga actatatcaa caactgaacg
atctagaggc ttgcgttatt 300cagggtgtag gagttactga aactccccta
atgaaagaag attcaattct agccgttaga 360aaatactttc agcgtatcac
attgtattta aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta
gagcagaaat tatgaggtcc ttctctcttt ctacgaattt gcaagaatct
480ttgagatcta aggaaaccgt cgctgctcca tctgacatcc agatgaccca
gtctccatcc 540tccctgtctg catctgtagg agaccgtgtc accatcactt
gccgggcaag tcagtggatt 600gggtctcagt tatcttggta ccagcagaaa
ccagggaaag cccctaagct cctgatcatg 660tggcgttcct cgttgcaaag
tggggtccca tcacgtttca gtggcagtgg atctgggaca 720gatttcactc
tcaccatcag cagtctgcaa cctgaagatt ttgctacgta ctactgtgct
780cagggtgcgg cgttgcctag gacgttcggc caagggacca aggtggaaat caaacgg
83757293PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 57Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu
Thr Val Ala Ala Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr
Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln Leu Ser Trp Tyr Gln 195 200
205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Met Trp Arg Ser Ser
210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Ala Gln Gly Leu Arg His
Pro Lys Thr Phe Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg
Ala Ala Ala Glu Gln Lys Leu Ile Ser 275 280 285 Glu Glu Asp Leu Asn
290 58882DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 58tgcgacttgc cacagacaca
tagtttggga tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt
ctcttgtcta aaggaccgtc acgacttcgg attccctcag 120gaagagtttg
gaaaccaatt ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc
180cagcaaatat tcaatttgtt ttctacaaag gactcatcag ccgcttggga
tgaaactctg 240ttagataaat tctacactga actatatcaa caactgaacg
atctagaggc ttgcgttatt 300cagggtgtag gagttactga aactccccta
atgaaagaag attcaattct agccgttaga 360aaatactttc agcgtatcac
attgtattta aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta
gagcagaaat tatgaggtcc ttctctcttt ctacgaattt gcaagaatct
480ttgagatcta aggaaaccgt cgctgctcca tctgacatcc agatgaccca
gtctccatcc 540tccctgtctg catctgtagg agaccgtgtc accatcactt
gccgggcaag tcagtggatt 600gggtctcagt tatcttggta ccagcagaaa
ccagggaaag cccctaagct cctgatcatg 660tggcgttcct cgttgcaaag
tggggtccca tcacgtttca gtggcagtgg atctgggaca 720gatttcactc
tcaccatcag cagtctgcaa cctgaagatt ttgctacgta ctactgtgct
780cagggtttga ggcatcctaa gacgttcggc caagggacca aggtggaaat
caaacgggcg 840gccgcagaac aaaaactcat ctcagaagag gatctgaatt aa
88259279PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 59Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu
Thr Val Ala Ala Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr
Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln Leu Ser Trp Tyr Gln 195 200
205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Met Trp Arg Ser Ser
210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Ala Gln Gly Leu Arg His
Pro Lys Thr Phe Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg
275 60837DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 60tgcgacttgc cacagacaca
tagtttggga tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt
ctcttgtcta aaggaccgtc acgacttcgg attccctcag 120gaagagtttg
gaaaccaatt ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc
180cagcaaatat tcaatttgtt ttctacaaag gactcatcag ccgcttggga
tgaaactctg 240ttagataaat tctacactga actatatcaa caactgaacg
atctagaggc ttgcgttatt 300cagggtgtag gagttactga aactccccta
atgaaagaag attcaattct agccgttaga 360aaatactttc agcgtatcac
attgtattta aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta
gagcagaaat tatgaggtcc ttctctcttt ctacgaattt gcaagaatct
480ttgagatcta aggaaaccgt cgctgctcca tctgacatcc agatgaccca
gtctccatcc 540tccctgtctg catctgtagg agaccgtgtc accatcactt
gccgggcaag tcagtggatt 600gggtctcagt tatcttggta ccagcagaaa
ccagggaaag cccctaagct cctgatcatg 660tggcgttcct cgttgcaaag
tggggtccca tcacgtttca gtggcagtgg atctgggaca 720gatttcactc
tcaccatcag cagtctgcaa cctgaagatt ttgctacgta ctactgtgct
780cagggtttga ggcatcctaa gacgttcggc caagggacca aggtggaaat caaacgg
83761293PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 61Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu
Thr Val Ala Ala Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr
Cys Arg Ala Ser Arg Pro Ile Gly Thr Thr Leu Ser Trp Tyr Gln 195 200
205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Trp Phe Gly Ser Arg
210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Ala Gln Ala Gly Thr His
Pro Thr Thr Phe Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg
Ala Ala Ala Glu Gln Lys Leu Ile Ser 275 280 285 Glu Glu Asp Leu Asn
290 62882DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 62tgcgacttgc cacagacaca
tagtttggga tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt
ctcttgtcta aaggaccgtc acgacttcgg attccctcag 120gaagagtttg
gaaaccaatt ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc
180cagcaaatat tcaatttgtt ttctacaaag gactcatcag ccgcttggga
tgaaactctg 240ttagataaat tctacactga actatatcaa caactgaacg
atctagaggc ttgcgttatt 300cagggtgtag gagttactga aactccccta
atgaaagaag attcaattct agccgttaga 360aaatactttc agcgtatcac
attgtattta aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta
gagcagaaat tatgaggtcc ttctctcttt ctacgaattt gcaagaatct
480ttgagatcta aggaaaccgt cgctgctcca tctgacatcc agatgaccca
gtctccatcc 540tccctgtctg catctgtagg agaccgtgtc accatcactt
gccgggcaag tcgtccgatt 600gggacgacgt taagttggta ccagcagaaa
ccagggaaag cccctaagct cctgatctgg 660tttggttccc ggttgcaaag
tggggtccca tcacgtttca gtggcagtgg atctgggaca 720gatttcactc
tcaccatcag cagtctgcaa cctgaagatt ttgctacgta ctactgtgcg
780caggctggga cgcatcctac gacgttcggc caagggacca aggtggaaat
caaacgggcg 840gccgcagaac aaaaactcat ctcagaagag gatctgaatt aa
88263279PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 63Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu
Thr Val Ala Ala Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr
Cys Arg Ala Ser Arg Pro Ile Gly Thr Thr Leu Ser Trp Tyr Gln 195 200
205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Trp Phe Gly Ser Arg
210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Ala Gln Ala Gly Thr His
Pro Thr Thr Phe Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg
275 64837DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 64tgcgacttgc cacagacaca
tagtttggga tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt
ctcttgtcta aaggaccgtc acgacttcgg attccctcag 120gaagagtttg
gaaaccaatt ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc
180cagcaaatat tcaatttgtt ttctacaaag gactcatcag ccgcttggga
tgaaactctg 240ttagataaat tctacactga actatatcaa caactgaacg
atctagaggc ttgcgttatt 300cagggtgtag gagttactga aactccccta
atgaaagaag attcaattct agccgttaga 360aaatactttc agcgtatcac
attgtattta aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta
gagcagaaat tatgaggtcc ttctctcttt ctacgaattt gcaagaatct
480ttgagatcta aggaaaccgt cgctgctcca tctgacatcc agatgaccca
gtctccatcc 540tccctgtctg
catctgtagg agaccgtgtc accatcactt gccgggcaag tcgtccgatt
600gggacgacgt taagttggta ccagcagaaa ccagggaaag cccctaagct
cctgatctgg 660tttggttccc ggttgcaaag tggggtccca tcacgtttca
gtggcagtgg atctgggaca 720gatttcactc tcaccatcag cagtctgcaa
cctgaagatt ttgctacgta ctactgtgcg 780caggctggga cgcatcctac
gacgttcggc caagggacca aggtggaaat caaacgg 83765293PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 65Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg
Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val
Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80 Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105
110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu Thr Val Ala Ala
Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr Cys Arg Ala Ser
Arg Pro Ile Gly Thr Met Leu Ser Trp Tyr Gln 195 200 205 Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Leu Ala Phe Ser Arg 210 215 220 Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr225 230
235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr 245 250 255 Tyr Tyr Cys Ala Gln Ala Gly Thr His Pro Thr Thr Phe
Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg Ala Ala Ala Glu
Gln Lys Leu Ile Ser 275 280 285 Glu Glu Asp Leu Asn 290
66882DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 66tgcgacttgc cacagacaca tagtttggga
tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt ctcttgtcta
aaggaccgtc acgacttcgg attccctcag 120gaagagtttg gaaaccaatt
ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc 180cagcaaatat
tcaatttgtt ttctacaaag gactcatcag ccgcttggga tgaaactctg
240ttagataaat tctacactga actatatcaa caactgaacg atctagaggc
ttgcgttatt 300cagggtgtag gagttactga aactccccta atgaaagaag
attcaattct agccgttaga 360aaatactttc agcgtatcac attgtattta
aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta gagcagaaat
tatgaggtcc ttctctcttt ctacgaattt gcaagaatct 480ttgagatcta
aggaaaccgt cgctgctcca tctgacatcc agatgaccca gtctccatcc
540tccctgtctg catctgtagg agaccgtgtc accatcactt gccgggcaag
tcgtccgatt 600gggacgatgt taagttggta ccagcagaaa ccagggaaag
cccctaagct cctgatcctt 660gctttttccc gtttgcaaag tggggtccca
tcacgtttca gtggcagtgg atctgggaca 720gatttcactc tcaccatcag
cagtctgcaa cctgaagatt ttgctacgta ctactgcgcg 780caggctggga
cgcatcctac gacgttcggc caagggacca aggtggaaat caaacgggcg
840gccgcagaac aaaaactcat ctcagaagag gatctgaatt aa
88267279PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 67Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15 Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160 Leu Arg Ser Lys Glu
Thr Val Ala Ala Pro Ser Asp Ile Gln Met Thr 165 170 175 Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 180 185 190 Thr
Cys Arg Ala Ser Arg Pro Ile Gly Thr Met Leu Ser Trp Tyr Gln 195 200
205 Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Leu Ala Phe Ser Arg
210 215 220 Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr225 230 235 240 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr 245 250 255 Tyr Tyr Cys Ala Gln Ala Gly Thr His
Pro Thr Thr Phe Gly Gln Gly 260 265 270 Thr Lys Val Glu Ile Lys Arg
275 68837DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 68tgcgacttgc cacagacaca
tagtttggga tcaagaagaa cattgatgtt attagcacaa 60atgcgtagaa tttctttgtt
ctcttgtcta aaggaccgtc acgacttcgg attccctcag 120gaagagtttg
gaaaccaatt ccaaaaagca gaaactattc ctgtcttgca cgaaatgatc
180cagcaaatat tcaatttgtt ttctacaaag gactcatcag ccgcttggga
tgaaactctg 240ttagataaat tctacactga actatatcaa caactgaacg
atctagaggc ttgcgttatt 300cagggtgtag gagttactga aactccccta
atgaaagaag attcaattct agccgttaga 360aaatactttc agcgtatcac
attgtattta aaggaaaaga aatactcccc atgtgcatgg 420gaggtggtta
gagcagaaat tatgaggtcc ttctctcttt ctacgaattt gcaagaatct
480ttgagatcta aggaaaccgt cgctgctcca tctgacatcc agatgaccca
gtctccatcc 540tccctgtctg catctgtagg agaccgtgtc accatcactt
gccgggcaag tcgtccgatt 600gggacgatgt taagttggta ccagcagaaa
ccagggaaag cccctaagct cctgatcctt 660gctttttccc gtttgcaaag
tggggtccca tcacgtttca gtggcagtgg atctgggaca 720gatttcactc
tcaccatcag cagtctgcaa cctgaagatt ttgctacgta ctactgcgcg
780caggctggga cgcatcctac gacgttcggc caagggacca aggtggaaat caaacgg
83769108PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 69Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30 Leu Ser Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Met Trp Arg
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly Leu Arg His Pro Lys
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Cys 100 105
70324DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 70gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg
tctcagttat cttggtacca gcagaaacca 120gggaaagccc ctaagctcct
gatcatgtgg cgttcctcgt tgcaaagtgg ggtcccatca 180cgtttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg ctacgtacta ctgtgctcag ggtttgaggc atcctaagac
gttcggccaa 300gggaccaagg tggaaatcaa atgc 32471118PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 71Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe
Thr Phe Arg His Tyr 20 25 30 Arg Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Arg Pro Asp Gly
Thr Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Ser Tyr Met Gly Asp Arg Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110 Leu Val Thr Val Ser Ser 115 72355DNAArtificial SequenceNucleic
acid sequence identified using molecular biology techniques.
72gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtacag cctccggatt cacctttagg cattatcgta tgggttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcatgg attcgtccgg atggtacgtt
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaatcttat 300atgggtgata ggtttgacta
ctggggtcag ggaaccctgg tcaccgtctc gagcg 35573118PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 73Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe
Thr Phe Arg His Tyr 20 25 30 Arg Met Gly Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Arg Pro Asp Gly
Thr Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Ser Tyr Met Ala Asp Arg Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110 Leu Val Thr Val Ser Ser 115 74354DNAArtificial SequenceNucleic
acid sequence identified using molecular biology techniques.
74gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc
60tcctgtacag cctccggatt cacctttagg cattatcgta tgggttgggt ccgccaggct
120ccagggaagg gtctagagtg ggtctcatgg attcgtccgg atggtacgtt
tacatactac 180gcagactccg tgaagggccg gttcaccatc tcccgcgaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgcg tgccgaggac
accgcggtat attactgtgc gaaatcttat 300atggctgata ggtttgacta
ctggggtcag ggaaccctgg tcaccgtctc gagc 35475119PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 75Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ala Asn Ala 20 25 30 Thr Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Asp Ile Asp Gln Val Gly
His Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Tyr Ser Trp His Pro Asp Leu Phe Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 76351DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 76gaggtgcagc tgttggagtc tgggggaggc ttggtacagc
ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttggg aattatagga
tgacttgggt ccgccaggct 120ccagggaagg gtctagagtg ggtctcaact
atttctcctt tgggtacgta tacatactac 180gcagactccg tgaagggccg
gttcaccatc tcccgcgaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgcg tgccgaggac accgcggtat attactgtgc gaaagggcgt
300tggtcgattt ttgactactg gggtcaggga accctggtca ccgtctcgag c
35177119PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 77Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Asp Thr Ser 20 25 30 Ser Met Leu Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile
His Gln Ser Gly Thr Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Lys Phe Pro Ser Thr His Gly Lys Phe Asp Tyr Trp Gly
Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
78357DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 78gaggtgcagc tgttggagtc tgggggaggc
ttggtacagc ctggggggtc cctgcgtctc 60tcctgtgcag cctccggatt cacctttgat
acgagtagta tgttgtgggt ccgccaggct 120ccagggaagg gtctagagtg
ggtctcagtt attcatcaga gtggtacgcc tacatactac 180gcagactccg
tgaagggccg gttcaccatc tcccgcgaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgcg tgccgaggac accgcggtat attactgtgc
gaaatttccg 300tctactcatg gtaagtttga ctactggggt cagggaaccc
tggtcaccgt ctcgagc 35779197PRTArtificial SequenceAmino acid
sequence identified using molecular biology techniques. 79Glu Gly
Lys Val Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu1 5 10 15
Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu 20
25 30 Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
Val 35 40 45 Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly
Asp Leu Leu 50 55 60 Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys
Tyr Ile Cys Glu Asn65 70 75 80 Gln Asp Ser Ile Ser Ser Lys Leu Lys
Glu Cys Cys Glu Lys Pro Leu 85 90 95 Leu Glu Lys Ser His Cys Ile
Ala Glu Val Glu Asn Asp Glu Met Pro 100 105 110 Ala Asp Leu Pro Ser
Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val 115 120 125 Cys Lys Asn
Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu 130 135 140 Tyr
Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu145 150
155 160 Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala
Ala 165 170 175 Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu
Phe Lys Pro 180 185 190 Leu Val Glu Glu Pro 195 80591DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 80gaagggaagg tttcgtctgc caaacagaga ctcaagtgtg
ccagtctcca aaaatttgga 60gaaagagctt tcaaagcatg ggcagtagct cgcctgagcc
agagatttcc caaagctgag 120tttgcagaag tttccaagtt agtgacagat
cttaccaaag tccacacgga atgctgccat 180ggagatctgc ttgaatgtgc
tgatgacagg gcggaccttg ccaagtatat ctgtgaaaat 240caagattcga
tctccagtaa actgaaggaa tgctgtgaaa aacctctgtt ggaaaaatcc
300cactgcattg ccgaagtgga aaatgatgag atgcctgctg acttgccttc
attagctgct 360gattttgttg aaagtaagga tgtttgcaaa aactatgctg
aggcaaagga tgtcttcctg 420ggcatgtttt tgtatgaata tgcaagaagg
catcctgatt actctgtcgt gctgctgctg 480agacttgcca agacatatga
aaccactcta gagaagtgct gtgccgctgc agatcctcat 540gaatgctatg
ccaaagtgtt cgatgaattt aaacctcttg tggaagagcc t 59181585PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 81Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
Leu Gly Glu1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys
Leu Val
Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu
Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly
Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80 Arg Glu Thr Tyr Gly
Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn
Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro
Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120
125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg
130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala
Lys Arg145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu
Arg Asp Glu Gly Lys Val Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys
Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala
Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu
Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240
Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245
250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile
Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu
Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro
Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys
Asp Val Cys Lys Asn Tyr Ala305 310 315 320 Glu Ala Lys Asp Val Phe
Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp
Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu
Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365
Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370
375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr
Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val
Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His
Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser
Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro
Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480 Leu
Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490
495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp
500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln
Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr
Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe
Val Glu Lys Cys Cys Lys545 550 555 560 Ala Asp Asp Lys Glu Thr Cys
Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala
Ala Leu Gly Leu 580 585 821755DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
82gatgcacaca 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
gtttcgtctg 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 gagcttgtga aacacaagcc caaggcaaca
1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa
gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa
aacttgttgc tgcaagtcaa 1740gctgccttag gctta 175583108PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 83Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Trp Ile Gly Ser Gln 20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Met Trp Arg Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Ala Gln Gly Leu Arg His Pro Lys 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
84330DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 84tcgacggaca tccagatgac ccagtctcca
tcctccctgt ctgcatctgt aggagaccgt 60gtcaccatca cttgccgggc aagtcagtgg
attgggtctc agttatcttg gtaccagcag 120aaaccaggga aagcccctaa
gctcctgatc atgtggcgtt cctcgttgca aagtggggtc 180ccatcacgtt
tcagtggcag tggatctggg acagatttca ctctcaccat cagcagtctg
240caacctgaag attttgctac gtactactgt gctcagggtt tgaggcatcc
taagacgttc 300ggccaaggga ccaaggtgga aatcaaacgg 3308556DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 85taacaagaat aatgggatcc accggcgatg cacacaagag
tgaggttgct catcgg 568688DNAArtificial SequenceNucleic acid sequence
identified using molecular biology techniques. 86gcgcgcgcgc
gcttcaagct ttcattaatg gtgatggtga tgatgtaagc ctaaggcagc 60ttgacttgca
gcaacaagtt ttttaccc 888735DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
87gagccagaga tttcccgccg ctgagtttgc agaag 358835DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 88cttctgcaaa ctcagcggcg ggaaatctct ggctc
358937DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 89gagatttccc aaagctgcct ttgcagaagt
ttccaag 379037DNAArtificial SequenceNucleic acid sequence
identified using molecular biology techniques. 90cttggaaact
tctgcaaagg cagctttggg aaatctc 379137DNAArtificial SequenceNucleic
acid sequence identified using molecular biology techniques.
91ccaaagctga gtttgcagcc gtttccaagt tagtgac 379237DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 92gtcactaact tggaaacggc tgcaaactca gctttgg
379337DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 93gattttgttg aaagtaaggc cgtttgcaaa
aactatg 379437DNAArtificial SequenceNucleic acid sequence
identified using molecular biology techniques. 94catagttttt
gcaaacggcc ttactttcaa caaaatc 379540DNAArtificial SequenceNucleic
acid sequence identified using molecular biology techniques.
95gaaagtaagg atgtttgcgc caactatgct gaggcaaagg 409640DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 96cctttgcctc agcatagttg gcgcaaacat ccttactttc
409736DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 97gctgaggcaa aggatgcctt cctgggcatg
tttttg 369836DNAArtificial SequenceNucleic acid sequence identified
using molecular biology techniques. 98caaaaacatg cccaggaagg
catcctttgc ctcagc 369935DNAArtificial SequenceNucleic acid sequence
identified using molecular biology techniques. 99ggatgtcttc
ctgggcgcct ttttgtatga atatg 3510035DNAArtificial SequenceNucleic
acid sequence identified using molecular biology techniques.
100catattcata caaaaaggcg cccaggaaga catcc 3510141DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 101gctgctgctg agacttgccg ccacatatga aaccactcta g
4110241DNAArtificial SequenceNucleic acid sequence identified using
molecular biology techniques. 102ctagagtggt ttcatatgtg gcggcaagtc
tcagcagcag c 41103591PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 103Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15 Glu Asn
Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35
40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp
Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val
Ala Thr Leu65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys
Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His
Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165
170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Val
Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys
Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu
Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys
Leu Val Thr Asp Leu Thr Lys225 230 235 240 Val His Thr Glu Cys Cys
His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu
Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys
Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290
295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile
Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400 Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410
415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475 480 Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535
540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu His
His His His His His 580 585 590 104591PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 104Asp
Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10
15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu
Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu
Cys Thr Val Ala Thr Leu65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala
Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val
Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn
Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145
150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys
Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu
Gly Lys Val Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser
Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val
Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Ala Ala Glu Phe Ala Glu
Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230
235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp
Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu
Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu
Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu
Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320 Glu Ala Lys Asp
Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His
Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355
360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu
Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln
Leu Gly Glu385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val
Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys
Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr
Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys
Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475
480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys
Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560 Ala Asp Asp Lys Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser
Gln Ala Ala Leu Gly Leu His His His His His His 580 585 590
105591PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 105Asp Ala His Lys Ser Glu Val Ala
His Arg Phe Lys Asp Leu Gly Glu1 5 10 15 Glu Asn Phe Lys Ala Leu
Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160 Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Val Ser 180 185
190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220 Lys Ala Ala Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310
315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400 Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val
Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435
440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu
His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys
Thr Glu Ser465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala
Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu
Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu
Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala
Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu His His His His His
His 580 585 590 106591PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 106Asp Ala His Lys
Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15 Glu Asn
Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30
Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35
40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp
Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val
Ala Thr Leu65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys
Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His
Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu
Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr
Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro
Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160
Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165
170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Val
Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys
Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu
Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Ala Val Ser Lys
Leu Val Thr Asp Leu Thr Lys225 230 235 240 Val His Thr Glu Cys Cys
His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu
Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys
Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285
Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290
295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys
Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe
Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile
Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400 Tyr
Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410
415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met
Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr
Lys Cys Cys Thr Glu Ser465 470 475 480 Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535
540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu His
His His His His His 580 585 590 107591PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 107Asp
Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10
15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu
Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu
Cys Thr Val Ala Thr Leu65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala
Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val
Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn
Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145
150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys
Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu
Gly Lys Val Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser
Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val
Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu
Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240 Val His Thr
Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg
Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265
270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu
Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Ala Val Cys
Lys Asn Tyr Ala305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met
Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val
Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu
Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala
Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln
Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390
395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val
Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn
Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu
Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp
Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480 Leu Val Asn Arg
Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val
Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515
520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln
Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys
Cys Cys Lys545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly
Leu His His His His His His 580 585 590 108591PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 108Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
Leu Gly Glu1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys
Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala
Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu
Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80 Arg Glu Thr
Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu
Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105
110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125 Asp Asn Glu Glu
Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His
Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155
160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala
165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys
Val Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240 Val His Thr Glu Cys
Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp
Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser
Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280
285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Ala Asn
Tyr Ala305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu
Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu
Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405
410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly
Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg
Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val
Thr Lys Cys Cys Thr Glu Ser465 470 475 480 Leu Val Asn Arg Arg Pro
Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys
Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys
Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525
Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530
535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys
Lys545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly
Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu His
His His His His His 580 585 590 109591PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 109Asp
Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10
15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln
20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val
Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu
Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu
Cys Thr Val Ala Thr Leu65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala
Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe
Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val
Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn
Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140
Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145
150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys
Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu
Gly Lys Val Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser
Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val
Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu
Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240 Val His Thr
Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg
Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265
270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His
275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu
Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys
Lys Asn Tyr Ala305 310 315 320 Glu Ala Lys Asp Ala Phe Leu Gly Met
Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val
Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu
Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala
Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln
Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390
395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val
Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn
Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu
Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp
Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480 Leu Val Asn Arg
Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val
Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510
Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515
520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln
Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys
Cys Cys Lys545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly
Leu His His His His His His 580 585 590 110591PRTArtificial
SequenceAmino acid sequence identified using molecular biology
techniques. 110Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp
Leu Gly Glu1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys
Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala
Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu
Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80 Arg Glu Thr
Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu
Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105
110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His
115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe
Phe Ala Lys Arg145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys
Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp
Glu Leu Arg Asp Glu Gly Lys Val Ser 180 185 190 Ser Ala Lys Gln Arg
Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe
Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys
Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230
235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp
Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp
Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu
Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu
Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu
Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320 Glu Ala Lys Asp
Val Phe Leu Gly Ala Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His
Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350
Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355
360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu
Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln
Leu Gly Glu385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val
Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys
Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr
Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys
Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475
480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr
485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His
Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys
Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys
Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala
Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560 Ala Asp Asp Lys Glu
Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser
Gln Ala Ala Leu Gly Leu His His His His His His 580 585 590
111591PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 111Asp Ala His Lys Ser Glu Val Ala
His Arg Phe Lys Asp Leu Gly Glu1 5 10 15 Glu Asn Phe Lys Ala Leu
Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe
Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala
Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60
Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65
70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160 Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Val Ser 180 185
190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310
315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Ala Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400 Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val
Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435
440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu
His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys
Thr Glu Ser465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala
Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu
Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu
Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala
Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555
560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu His His His His His
His 580 585 590 1121774DNAArtificial SequenceNucleic acid sequence
identified using molecular biology techniques. 112gatgcacaca
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 gtttcgtctg 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
gagcttgtga aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt
tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata
aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa
1740gctgccttag gcttacatca tcaccatcac catt 17741131774DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 113gatgcacaca 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 gtttcgtctg ccaaacagag actcaagtgt
600gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc
tcgcctgagc 660cagagatttc ccgccgctga 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 gagcttgtga
aacacaagcc caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat
ttcgcagctt ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg
ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag
gcttacatca tcaccatcac catt 17741141774DNAArtificial SequenceNucleic
acid sequence identified using molecular biology techniques.
114gatgcacaca 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 gtttcgtctg ccaaacagag actcaagtgt 600gccagtctcc
aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc
660cagagatttc ccaaagctgc ctttgcagaa 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 gagcttgtga aacacaagcc
caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt
ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag
gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttacatca
tcaccatcac catt 17741151774DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
115gatgcacaca 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 gtttcgtctg ccaaacagag actcaagtgt 600gccagtctcc
aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc
660cagagatttc ccaaagctga gtttgcagcc 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 gagcttgtga aacacaagcc
caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt
ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag
gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttacatca
tcaccatcac catt 17741161774DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
116gatgcacaca 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 gtttcgtctg 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 ccgtttgcaa 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 gagcttgtga aacacaagcc
caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt
ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag
gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttacatca
tcaccatcac catt 17741171774DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
117gatgcacaca 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 gtttcgtctg 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 atgtttgcgc caactatgct
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 gagcttgtga aacacaagcc
caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt
ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag
gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttacatca
tcaccatcac catt 17741181774DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
118gatgcacaca 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 gtttcgtctg 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 atgccttcct 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 gagcttgtga aacacaagcc
caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt
ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag
gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttacatca
tcaccatcac catt 17741191774DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
119gatgcacaca 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 gtttcgtctg 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 gggcgccttt 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 gagcttgtga aacacaagcc
caaggcaaca 1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt
ttgtagagaa gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag
gagggtaaaa aacttgttgc tgcaagtcaa 1740gctgccttag gcttacatca
tcaccatcac catt 17741201774DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
120gatgcacaca
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
gtttcgtctg 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 gccacatatg 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 gagcttgtga aacacaagcc caaggcaaca
1620aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa
gtgctgcaag 1680gctgacgata aggagacctg ctttgccgag gagggtaaaa
aacttgttgc tgcaagtcaa 1740gctgccttag gcttacatca tcaccatcac catt
1774121113PRTArtificial SequenceAmino acid sequence identified
using molecular biology techniques. 121Ser Thr Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser1 5 10 15 Val Gly Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Gly 20 25 30 Thr Thr Leu
Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu
Ile Leu Trp Asn Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe 50 55
60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala
Gly Thr His 85 90 95 Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Ala Ala 100 105 110 Ala122339DNAArtificial
SequenceNucleic acid sequence identified using molecular biology
techniques. 122tcgacggaca tccagatgac ccagtctcca tcctccctgt
ctgcatctgt aggagaccgt 60gtcaccatca cttgccgggc aagtcgtccg attgggacga
cgttaagttg gtaccagcag 120aaaccaggga aagcccctaa gctcctgatc
ctttggaatt cccgtttgca aagtggggtc 180ccatcacgtt tcagtggcag
tggatctggg acagatttca ctctcaccat cagcagtctg 240caacctgaag
attttgctac gtactactgt gcgcaggctg ggacgcatcc tacgacgttc
300ggccaaggga ccaaggtgga aatcaaacgg gcggccgca
339123108PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 123Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30 Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Met Trp
Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly Ala Ala Leu
Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 124324DNAArtificial SequenceNucleic acid sequence
identified using molecular biology techniques. 124gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtca gtggattggg tctcagttat cttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtgctcag
ggtgcggcgt tgcctaggac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324125108PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 125Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Arg Pro Ile Gly Thr Thr 20 25 30 Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Trp Phe
Gly Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala Gly Thr His
Pro Thr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 126324DNAArtificial SequenceNucleic acid sequence
identified using molecular biology techniques. 126gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc
gggcaagtcg tccgattggg acgacgttaa gttggtacca gcagaaacca
120gggaaagccc ctaagctcct gatctggttt ggttcccggt tgcaaagtgg
ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg ctacgtacta ctgtgcgcag
gctgggacgc atcctacgac gttcggccaa 300gggaccaagg tggaaatcaa acgg
324127108PRTArtificial SequenceAmino acid sequence identified using
molecular biology techniques. 127Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30 Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Met Leu Leu Ile 35 40 45 Met Trp
Ser Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly Leu Arg His
Pro Lys 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Trp
100 105 128108PRTArtificial SequenceAmino acid sequence identified
using molecular biology techniques. 128Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30 Leu Ser Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Met
Trp Arg Ser Ala Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly Leu Arg
His Pro Lys 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Trp 100 105 129108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 129Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Arg His Pro Lys 85 90 95 Thr Tyr Gly Lys Gly Thr Lys Val Glu
Asn Lys Trp 100 105 130108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 130Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Lys His Pro Lys 85 90 95 Thr Tyr Gly Gln Gly Thr Lys Val Glu
Ile Lys Trp 100 105 131108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 131Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Met Arg His Pro Lys 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Trp 100 105 132108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 132Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Arg His Pro Lys 85 90 95 Thr Tyr Gly Gln Gly Thr Lys Val Glu
Asn Lys Trp 100 105 133108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 133Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ala Leu Gln Asn Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Arg His Pro Lys 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Trp 100 105 134107PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 134Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Phe Ala Ser Val Gly Asp1 5 10 15 Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln Leu 20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Met 35
40 45 Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Glu65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly Leu
Arg Lys Pro Lys Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Lys Ile
Lys Trp 100 105 135108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 135Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ala Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Arg His Pro Lys 85 90 95 Thr Tyr Gly Gln Gly Thr Lys Val Glu
Ile Lys Trp 100 105 136108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 136Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ala Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Arg Tyr Pro Lys 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Trp 100 105 137108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 137Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Arg His Pro Lys 85 90 95 Thr Tyr Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 138108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 138Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Gly
Leu Arg His Pro Lys 85 90 95 Thr Tyr Gly Gln Gly Thr
Lys Val Glu Asn Lys Arg 100 105 139108PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 139Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln
20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Met Trp Arg Ser Ala Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Ala Gln Gly Leu Arg His Pro Lys 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 140108PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 140Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln
20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Ala Gln Gly Leu Arg His Pro Lys 85 90 95 Thr Tyr Gly Lys Gly Thr
Lys Val Glu Ile Lys Arg 100 105 141108PRTArtificial SequenceAmino
acid sequence identified using molecular biology techniques. 141Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Trp Ile Gly Ser Gln
20 25 30 Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Met Trp Arg Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Ala Gln Gly Leu Arg His Pro Lys 85 90 95 Thr Tyr Gly Lys Gly Thr
Lys Val Glu Asn Lys Arg 100 105 142324DNAArtificial SequenceNucleic
acid sequence identified using molecular biology techniques.
142gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctatgctcct gatcatgtgg agttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaagac gttcggccaa 300gggaccaagg
tggaaatcaa atgg 324143324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
143gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttccgcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaagac gttcggccaa 300gggaccaagg
tggaaatcaa atgg 324144324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
144gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaagac gtacggcaaa 300gggaccaagg
tggaaaacaa atgg 324145324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
145gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaagc atcctaagac gtacggccaa 300gggaccaagg
tggaaatcaa atgg 324146324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
146gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtatgaggc atcctaagac gttcggccaa 300gggaccaagg
tggaaatcaa atgg 324147324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
147gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgcggc atcctaagac gtacggccaa 300gggaccaagg
tggaaaacaa atgg 324148324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
148gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttccgcgt
tacaaaatgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaagac gttcggccaa 300gggaccaagg
tggaaatcaa atgg 324149324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
149gacatccaga tgacccagtc tccatcctcc ctgtttgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgagga aacctaagac tttcggccaa 300gggaccaagg
tgaaaatcaa atgg 324150324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
150gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttccgcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaaaac gtacggccaa 300gggaccaagg
tggaaatcaa atgg 324151324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
151gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttccgcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggt atcctaagac gttcggccaa 300gggaccaagg
tggaaatcaa atgg 324152324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
152gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgcggc atcctaagac gtacggccaa 300gggaccaagg
tggaaatcaa acgg 324153324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
153gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgcggc atcctaagac gtacggccaa 300gggaccaagg
tggaaaacaa acgg 324154324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
154gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttccgcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaagac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324155324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
155gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaagac gtacggcaaa 300gggaccaagg
tggaaatcaa acgg 324156324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
156gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtca gtggattggg tctcagttat cttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcatgtgg cgttcctcgt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgctcag ggtttgaggc atcctaagac gtacggcaaa 300gggaccaagg
tggaaaacaa acgg 324157108PRTArtificial SequenceAmino acid sequence
identified using molecular biology techniques. 157Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Arg Pro Ile Gly Thr Met 20 25 30
Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Leu Phe Gly Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln Ala
Gly Thr His Pro Thr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 158324DNAArtificial SequenceNucleic acid
sequence identified using molecular biology techniques.
158gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
ccgtgtcacc 60atcacttgcc gggcaagtcg tccgattggg acgatgttaa gttggtacca
gcagaaacca 120gggaaagccc ctaagctcct gatcttgttt ggttcccggt
tgcaaagtgg ggtcccatca 180cgtttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg ctacgtacta
ctgtgcgcag gctgggacgc atcctacgac gttcggccaa 300gggaccaagg
tggaaatcaa acgg 324
* * * * *