U.S. patent application number 13/078241 was filed with the patent office on 2011-07-28 for single domain antibodies directed against tumor necrosis factor-alpha and uses therefor.
This patent application is currently assigned to Ablynx N.V.. Invention is credited to Hans De Haard, Marc Lauwereys, Karen Silence.
Application Number | 20110184150 13/078241 |
Document ID | / |
Family ID | 35800205 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110184150 |
Kind Code |
A1 |
Silence; Karen ; et
al. |
July 28, 2011 |
SINGLE DOMAIN ANTIBODIES DIRECTED AGAINST TUMOR NECROSIS
FACTOR-ALPHA AND USES THEREFOR
Abstract
The present invention relates to polypeptides derived from
single domain heavy chain antibodies directed to Tumor Necrosis
Factor-alpha. It further relates to single domain antibodies that
are Camelidae VHHs. It further relates to methods of administering
said polypeptides. It further relates to protocols for screening
for agents that modulate the TNF-alpha receptor, and the agents
resulting from said screening.
Inventors: |
Silence; Karen; (Overijse,
BE) ; Lauwereys; Marc; (Haaltert, BE) ; De
Haard; Hans; (Oudelande, NL) |
Assignee: |
Ablynx N.V.
Zwijnaarde
BE
|
Family ID: |
35800205 |
Appl. No.: |
13/078241 |
Filed: |
April 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12905589 |
Oct 15, 2010 |
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13078241 |
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11804647 |
May 18, 2007 |
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12905589 |
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11788832 |
Apr 20, 2007 |
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11804647 |
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11636300 |
Dec 8, 2006 |
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11788832 |
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10534348 |
May 9, 2005 |
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PCT/BE03/00192 |
Nov 7, 2003 |
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11636300 |
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60425073 |
Nov 8, 2002 |
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60425063 |
Nov 8, 2002 |
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Current U.S.
Class: |
530/387.2 ;
530/387.1; 530/387.3; 530/389.1; 530/389.2; 530/389.3 |
Current CPC
Class: |
C07K 16/18 20130101;
A61P 31/00 20180101; A61P 37/06 20180101; C07K 2317/31 20130101;
C07K 2317/565 20130101; C07K 2317/567 20130101; A61P 31/04
20180101; C07K 16/2875 20130101; C07K 2317/76 20130101; C07K 16/241
20130101; A61P 19/02 20180101; C07K 2317/626 20130101; C07K 2317/92
20130101; A61P 35/00 20180101; C07K 2319/00 20130101; C07K 2317/34
20130101; C07K 16/40 20130101; C07K 16/4291 20130101; C07K 16/249
20130101; C07K 2317/22 20130101; C07K 16/2863 20130101; C07K
2317/569 20130101; C07K 16/36 20130101; A61K 2039/505 20130101;
C07K 2317/24 20130101 |
Class at
Publication: |
530/387.2 ;
530/387.1; 530/389.2; 530/389.3; 530/389.1; 530/387.3 |
International
Class: |
C07K 16/24 20060101
C07K016/24; C07K 16/00 20060101 C07K016/00; C07K 16/18 20060101
C07K016/18; C07K 16/42 20060101 C07K016/42; C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 10, 2003 |
EP |
EP 03447005.4 |
Jun 23, 2003 |
EP |
PCT/EP03/06581 |
Jul 8, 2003 |
EP |
PCT/EP03/07313 |
Claims
1. A ligand comprising a single variable domain, wherein the single
variable domain specifically binds to an antigen, and the variable
domain comprises a Kd for the antigen of better than 10.sup.-6
M.
2. The ligand of claim 1, wherein the antigen is selected from the
group consisting of human protein, animal protein, cytokine,
cytokine receptor, TNF-alpha, IFN-gamma, a serum protein, serum
albumin, serum immunoglobulins, thyroxine-binding protein,
transferrin, and fibrinogen.
3. A dual specific ligand comprising a first single variable domain
and a second a single variable domain where at least one of the
first single variable domain and the second single variable domain
comprises a Kd for the antigen of better than 10.sup.-6 M.
4. The dual specific ligand of claim 3, wherein said first single
variable domain specifically binds to an antigen selected from the
group consisting of, human, protein, animal protein, cytokine,
cytokine receptor, TNF-alpha, IFN-gamma, a serum protein, serum
albumin, serum immunoglobulins, thyroxine-binding protein,
transferrin, and fibrinogen; and wherein said second single
variable domain specifically binds to an antigen selected from the
group consisting of, human, protein, animal protein, cytokine,
cytokine receptor, TNF-alpha, IFN-gamma, a serum protein, serum
albumin, serum immunoglobulins, thyroxine-binding protein,
transferrin, and fibrinogen.
5. A dual specific ligand comprising a first single variable domain
and a second single variable domain, wherein the first single
variable domain specifically binds to an antigen and the second
single variable domain specifically binds to an antigen, wherein at
least one of the first single variable domain and the second single
variable domain comprises a Kd for the antigen of better than
10.sup.-6 M.
6. The dual specific ligand of claim 5, wherein said first single
variable domain specifically binds to an antigen selected from the
group consisting of human protein, animal protein, cytokine,
cytokine receptor, TNF-alpha, IFN-gamma, a serum protein, serum
albumin, serum immunoglobulins, thyroxine-binding protein,
transferrin, and fibrinogen; and wherein said second single
variable domain specifically binds to an antigen selected from the
group consisting of human protein, animal protein, cytokine,
cytokine receptor, TNF-alpha, IFN-gamma, a serum protein, serum
albumin, serum immunoglobulins, thyroxine-binding protein,
transferrin, and fibrinogen.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/905,589 filed Oct. 15, 2010, currently
pending, which is a continuation of U.S. patent application Ser.
No. 11/804,647 filed May 18, 2007, currently pending, which is a
continuation of U.S. patent application Ser. No. 11/788,832 filed
Apr. 20, 2007, currently pending, which is a continuation of U.S.
patent application Ser. No. 11/636,300 filed Dec. 8, 2006,
currently pending, which is a continuation of U.S. patent
application Ser. No. 10/534,348 filed May 9, 2005, currently
pending, which is a National Stage of PCT/BE03/00192, filed Nov. 7,
2003, which claims priority to PCT/EP03/06581, filed Jun. 23, 2003
and PCT/EP03/07313, filed Jul. 8, 2003; this application also
claims the benefit of U.S. provisional application Ser. No.
60/425,073, filed Nov. 8, 2002 and U.S. provisional application
Ser. No. 60/425,063, filed Nov. 8, 2002; all of the applications
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention provides polypeptides comprising one
or more single domain antibodies directed towards tumor necrosis
factor alpha (TNF-alpha). The present invention further relates to
their use in diagnosis and therapy. Such antibodies may have a
framework sequence with high homology to the human framework
sequences. Compositions comprising antibodies to tumor necrosis
factor alpha (TNF-alpha) alone or in combination with other drugs
are described.
BACKGROUND TO THE INVENTION
[0003] Tumor necrosis factor alpha (TNF-alpha) is believed to play
an important role in various disorders, for example in inflammatory
disorders such as rheumatoid arthritis, Crohn's disease, ulcerative
colitis and multiple sclerosis. Both TNF-alpha and the receptors
(CD120a, CD120b) have been studied in great detail. TNF-alpha in
its bioactive form is a trimer and the groove formed by neighboring
subunits is important for the cytokine-receptor interaction.
Several strategies to antagonize the action of the cytokine have
been developed and are currently used to treat various disease
states.
[0004] A TNF-alpha inhibitor which has sufficient specificity and
selectivity to TNF-alpha may be an efficient prophylactic or
therapeutic pharmaceutical compound for preventing or treating
disorders where TNF-alpha has been implicated as causative agent.
Methods of treating toxic shock (EP 486526), tumor regression,
inhibition of cytotoxicity (U.S. Pat. No. 6,448,380, U.S. Pat. No.
6,451,983, U.S. Pat. No. 6,498,237), autoimmune disease such as RA
and Crohn's disease (EP 663836, U.S. Pat. No. 5,672,347, U.S. Pat.
No. 5,656,272), graft versus host reaction (U.S. Pat. No.
5,672,347), bacterial meningitis (EP 585705) by means of an
antibody to TNF-alpha have been described.
[0005] Yet none of the presently available drugs are completely
effective for the treatment of autoimmune disease, and most are
limited by severe toxicity. In addition, it is extremely difficult
and a lengthy process to develop a new chemical entity (NCE) with
sufficient potency and selectivity to such target sequence.
Antibody-based therapeutics on the other hand have significant
potential as drugs because they have exquisite specificity to their
target and a low inherent toxicity. In addition, the development
time can be reduced considerably when compared to the development
of new chemical entities (NCE's). However, conventional antibodies
are difficult to raise against multimeric proteins where the
receptor-binding domain of the ligand is embedded in a groove, as
is the case with TNF-alpha. Heavy chain antibodies described in the
invention which are derived from Camelidae, are known to have
cavity-binding propensity (WO97/49805; Lauwereys et al, EMBO J. 17,
5312, 1998)). Therefore, such heavy chain antibodies are inherently
suited to bind to receptor binding domains of such ligands as TNF.
In addition, such antibodies are known to be stable over long
periods of time, therefore increasing their shelf-life (Perez et
al, Biochemistry, 40, 74, 2001). Furthermore, such heavy chain
antibody fragments can be produced `en-masse` in fermentors using
cheap expression systems compared to mammalian cell culture
fermentation, such as yeast or other microorganisms (EP 0 698
097).
[0006] The use of antibodies derived from sources such as mouse,
sheep, goat, rabbit etc., and humanised derivatives thereof as a
treatment for conditions which require a modulation of inflammation
is problematic for several reasons. Traditional antibodies are not
stable at room temperature, and have to be refrigerated for
preparation and storage, requiring necessary refrigerated
laboratory equipment, storage and transport, which contribute
towards time and expense. Refrigeration is sometimes not feasible
in developing countries. Furthermore, the manufacture or
small-scale production of said antibodies is expensive because the
mammalian cellular systems necessary for the expression of intact
and active antibodies require high levels of support in terms of
time and equipment, and yields are very low. Furthermore the large
size of conventional antibodies, would restrict tissue penetration,
for example, at the site of inflamed tissue. Furthermore,
traditional antibodies have a binding activity which depends upon
pH, and hence are unsuitable for use in environments outside the
usual physiological pH range such as, for example, in treating
gastric bleeding, gastric surgery. Furthermore, traditional
antibodies are unstable at low or high pH and hence are not
suitable for oral administration. However, it has been demonstrated
that camelidae antibodies resist harsh conditions, such as extreme
pH, denaturing reagents and high temperatures (Dumoulin et al,
Protein Science 11, 500, 2002), so making them suitable for
delivery by oral administration. Furthermore, traditional
antibodies have a binding activity, which depends upon temperature,
and hence are unsuitable for use in assays or kits performed at
temperatures outside biologically active-temperature ranges (e.g.
37.+-.20.degree. C.).
[0007] Polypeptide therapeutics and in particular antibody-based
therapeutics have significant potential as drugs because they have
exquisite specificity to their target and a low inherent toxicity.
However, it is known by the skilled addressee that an antibody
which has been obtained for a therapeutically useful target
requires additional modification in order to prepare it for human
therapy, so as to avoid an unwanted immunological reaction in a
human individual upon administration thereto. The modification
process is commonly termed "humanisation". It is known by the
skilled artisan that antibodies raised in species, other than in
humans, require humanisation to render the antibody therapeutically
useful in humans ((1) CDR grafting: Protein Design Labs: U.S. Pat.
No. 6,180,370, U.S. Pat. No. 5,693,761; Genentech U.S. Pat. No.
6,054,297; Celltech: 460167, EP 626390, U.S. Pat. No. 5,859,205;
(2) Veneering: Xoma: U.S. Pat. No. 5,869,619, U.S. Pat. No.
5,766,886, U.S. Pat. No. 5,821,123). There is a need for a method
for producing antibodies which avoids the requirement for
substantial humanisation, or which completely obviates the need for
humanisation. There is a need for a new class of antibodies which
have defined framework regions or amino acid residues and which can
be administered to a human subject without the requirement for
substantial humanisation, or the need for humanisation at all.
[0008] Another important drawback of conventional antibodies is
that they are complex, large molecules and therefore relatively
unstable, and they are sensitive to breakdown by proteases. This
means that conventional antibody drugs cannot be administered
orally, sublingually, topically, nasally, vaginally, rectally or by
inhalation because they are not resistant to the low pH at these
sites, the action of proteases at these sites and in the blood
and/or because of their large size. They have to be administered by
injection (intravenously, subcutaneously, etc.) to overcome some of
these problems. Administration by injection requires specialist
training in order to use a hypodermic syringe or needle correctly
and safely. It further requires sterile equipment, a liquid
formulation of the therapeutic polypeptide, vial packing of said
polypeptide in a sterile and stable form and, of the subject, a
suitable site for entry of the needle. Furthermore, subjects
commonly experience physical and psychological stress prior to and
upon receiving an injection. Therefore, there is need for a method
for the delivery of therapeutic polypeptides which avoids the need
for injection which is not only cost/time saving, but which would
also be more convenient and more comfortable for the subject.
[0009] Single domain antibody-based therapeutics have significant
potential as drugs because they have exquisite specificity to their
target and a low inherent toxicity. However, improving further
their intrinsic and functional affinity can lead to many benefits
for a patient such as reduced dose of therapeutic, faster therapy,
and reduced side effects.
THE AIMS OF THE PRESENT INVENTION
[0010] It is an aim of the present invention is to provide
polypeptides comprising one or more single domain antibodies which
bind to TNF-alpha, homologues of said polypeptides, functional
portions of homologues of said polypeptides. Said polypeptides
modify the biological activity of TNF-alpha upon binding. Such
polypeptides might bind into the receptor-binding groove of
TNF-alpha, or might not bind in the receptor binding groove. Such
polypeptides are single domain antibodies.
[0011] It is a further aim of the present invention to provide
single domain antibodies which may be any of the art, or any future
single domain antibodies. Examples include, but are not limited to,
heavy chain antibodies, antibodies naturally devoid of light
chains, single domain antibodies derived from conventional 4-chain
antibodies, engineered antibodies and single domain scaffolds other
than those derived from antibodies. According to one aspect of the
invention, a single domain antibody as used herein is a naturally
occurring single domain antibody known as heavy chain antibody
devoid of light chains (WO 9404678). For clarity reasons, this
variable domain derived from a heavy chain antibody devoid of light
chain will be called VHH or nanobody to distinguish it from the
conventional VH of four chain immunoglobulins. Such a VHH molecule
can be derived from antibodies raised in Camelidae species, for
example in camel, llama, dromedary, alpaca and guanaco.
[0012] It is a further aim of the invention to provide a method of
administering anti-TNF-alpha polypeptides intravenously,
subcutaneously, orally, sublingually, topically, nasally,
vaginally, rectally or by inhalation.
[0013] It is a further aim of the invention to enhance the binding
affinity of monovalent single domain antibodies.
SUMMARY OF THE INVENTION
[0014] One embodiment of the present invention is an anti-TNF-alpha
polypeptide comprising at least one anti-TNF-alpha single domain
antibody.
[0015] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above wherein a single
domain antibody corresponds to a sequence represented by any of SEQ
ID NOs: 1 to 16 and 79 to 84.
[0016] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above further comprising at
least one single domain antibody directed against a serum
protein.
[0017] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above wherein said serum
protein is any of serum albumin, serum immunoglobulins,
thyroxine-binding protein, transferring, or fibrinogen.
[0018] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above wherein a single
domain anti-serum protein single domain antibody correspond to a
sequence represented by any of SEQ ID NOs: 26 to 29 and 85 to
97.
[0019] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above corresponding to a
sequence represented by any of SEQ ID NOs: 30 to 43.
[0020] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above further comprising at
least one single domain antibody selected from the group consisting
of anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor
single domain antibody and anti-IFN-gamma receptor single domain
antibody.
[0021] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above, wherein the number
of single domain antibodies directed against TNF-alpha is at least
two.
[0022] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above corresponding to a
sequence represented by any of SEQ ID NOs: 73 to 76.
[0023] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above, wherein at least one
single domain antibody is a humanized Camelidae VHHs.
[0024] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above wherein a humanized
Camelidae VHH corresponds to a sequence represented by any of SEQ
ID NOs: 17 to 19 and 21 to 24.
[0025] Another embodiment of the present invention is a composition
comprising an anti-TNF-alpha polypeptide as described above and at
least one single domain antibody from the group consisting of
anti-IFN-gamma single domain antibody, anti-TNF-alpha receptor
single domain antibody and anti-IFN-gamma receptor single domain
antibody, for simultaneous, separate or sequential administration
to a subject.
[0026] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above, or a composition as
described above wherein at least one anti-IFN-gamma single domain
antibody correspond to a sequence represented by any of SEQ ID NOs:
44 to 72.
[0027] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above, or a composition as
described above, wherein said single domain antibody is an
homologous sequence, a functional portion, or a functional portion
of an homologous sequence of the full length single domain
antibody.
[0028] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above, or a composition as
described above, wherein the anti-TNF-alpha polypeptide is an
homologous sequence, a functional portion, or a functional portion
of an homologous sequence of the full length anti-TNF-alpha
polypeptide.
[0029] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above, or a composition as
described above wherein at least one single domain antibody is a
Camelidae VHH.
[0030] Another embodiment of the present invention is a nucleic
acid encoding an anti-TNF-alpha polypeptide as described above.
[0031] Another embodiment of the present invention is a method of
identifying an agent that modulates the binding of an
anti-TNF-alpha polypeptide as described above, to Tumor Necrosis
Factor-alpha comprising the steps of:
(a) contacting an anti-TNF-alpha polypeptide as described above
with a target that is Tumor Necrosis Factor alpha, in the presence
and absence of a candidate modulator under conditions permitting
binding between said polypeptide and target, and (b) measuring the
binding between the polypeptide and target of step (a), wherein a
decrease in binding in the presence of said candidate modulator,
relative to the binding in the absence of said candidate modulator
identified said candidate modulator as an agent that modulates the
binding of an anti-TNF-alpha polypeptide as described above and
Tumor Necrosis Factor-alpha.
[0032] Another embodiment of the present invention is a method of
identifying an agent that modulates Tumor Necrosis
Factor-alpha-mediated disorders through the binding of an
anti-TNF-alpha polypeptide as described above to Tumor Necrosis
Factor-alpha comprising:
(a) contacting an anti-TNF-alpha polypeptide as described above
with a target that is Tumor Necrosis Factor alpha, in the presence
and absence of a candidate modulator under conditions permitting
binding between said polypeptide and target, and (b) measuring the
binding between the polypeptide and target of step (a), wherein a
decrease in binding in the presence of said candidate modulator,
relative to the binding in the absence of said candidate modulator
identified, said candidate modulator as an agent that modulates
Tumor Necrosis Factor alpha-mediated disorders.
[0033] Another embodiment of the present invention is a method of
identifying an agent that modulates the binding of Tumor Necrosis
Factor alpha to its receptor through the binding of an
anti-TNF-alpha polypeptide as described above to Tumor Necrosis
Factor-alpha comprising:
(a) contacting an anti-TNF-alpha polypeptide as described above
with a target that is Tumor Necrosis Factor-alpha, in the presence
and absence of a candidate modulator under conditions permitting
binding between said polypeptide and target, and (b) measuring the
binding between the polypeptide and target of step (a), wherein a
decrease in binding in the presence of said candidate modulator,
relative to the binding in the absence of said candidate modulator
identified said candidate modulator as an agent that modulates the
binding of Tumor Necrosis Factor-alpha to its receptor.
[0034] Another embodiment of the present invention is a kit for
screening for agents that modulate Tumor Necrosis
Factor-alpha-mediated disorders comprising an anti-TNF-alpha
polypeptide as described above and Tumor Necrosis Factor-alpha.
[0035] Another embodiment of the present invention is an unknown
agent that modulates the binding of an anti-TNF-alpha polypeptide
as described above to Tumor Necrosis Factor-alpha, identified
according to the method as described above.
[0036] Another embodiment of the present invention is an unknown
agent that modulates Tumor Necrosis Factor-alpha-mediated
disorders, identified according to the methods as described
above.
[0037] Another embodiment of the present invention is an unknown
agent as described above wherein said disorders are one or more of
inflammation, rheumatoid arthritis, Crohn's disease, ulcerative
colitis, inflammatory bowel syndrome and multiple sclerosis.
[0038] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above, or a nucleic acid as
described above, or a composition as described above, or an agent
as described above for treating and/or preventing and/or
alleviating disorders relating to inflammatory processes.
[0039] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above or a nucleic acid as
described above, or a composition as described above, or an agent
as described above for the preparation of a medicament for treating
and/or preventing and/or alleviating disorders relating to
inflammatory reactions.
[0040] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for treating and/or preventing and/or alleviating
disorders susceptible to modulation by a TNF-alpha modulating
substance which is able pass through the gastric environment
without the substance being inactivated.
[0041] Another embodiment of the present invention is an use of an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for the preparation of a medicament for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance which is able
pass through the gastric environment without the substance being
inactivated.
[0042] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for treating and/or preventing and/or alleviating
disorders susceptible to modulation by a TNF-alpha modulating
substance delivered to the vaginal and/or rectal tract.
[0043] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for the preparation of a medicament for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance delivered to the
vaginal and/or rectal tract.
[0044] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for treating and/or preventing and/or alleviating
disorders susceptible to modulation by a TNF-alpha modulating
substance delivered to the nose, upper respiratory tract and/or
lung.
[0045] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for the preparation of a medicament for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance delivered to the
nose, upper respiratory tract and/or lung.
[0046] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for treating and/or preventing and/or alleviating
disorders susceptible to modulation by a TNF-alpha modulating
substance delivered to the intestinal mucosa, wherein said disorder
increases the permeability of the intestinal mucosa.
[0047] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for the preparation of a medicament for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance delivered to the
intestinal mucosa, wherein said disorder increases the permeability
of the intestinal mucosa.
[0048] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for treating and/or preventing and/or alleviating
disorders susceptible to modulation by a TNF-alpha modulating
substance which is able pass through the tissues beneath the tongue
effectively.
[0049] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for the preparation of a medicament for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance which is able
pass through the tissues beneath the tongue effectively.
[0050] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for treating and/or preventing and/or alleviating
disorders susceptible to modulation by a TNF-alpha modulating
substance which is able pass through the skin effectively.
[0051] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above or a composition as
described above, for the preparation of a medicament for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance which is able
pass through the skin effectively.
[0052] Another embodiment of the present invention is a method as
described above, a kit as described above, a nucleic acid or agent
as described above, use of a nucleic acid or agent as described
above, a composition as described above, use of a composition as
described above, an anti-TNF-alpha polypeptide as described above,
use of an anti-TNF-alpha polypeptide as described above wherein
said disorders are any of inflammation, rheumatoid arthritis,
Crohn's disease, ulcerative colitis, inflammatory bowel syndrome,
multiple sclerosis, Addison's disease, Autoimmune hepatitis,
Autoimmune parotitis, Diabetes Type I, Epididymitis,
Glomerulonephritis, Graves' disease, Guillain-Barre syndrome,
Hashimoto's disease, Hemolytic anemia, Systemic lupus
erythematosus, Male infertility, Multiple sclerosis, Myasthenia
Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid
arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome,
Spondyloarthropathies, Thyroiditis, and Vasculitis.
[0053] Another embodiment of the present invention is a composition
comprising a nucleic acid or agent as described above, an
anti-TNF-alpha polypeptide as described above, or a composition as
described above, and a suitable pharmaceutical vehicle.
[0054] Another embodiment of the present invention is a method of
diagnosing a disorder characterised by the dysfunction of Tumor
Necrosis Factor-alpha comprising:
(a) contacting a sample with an anti-TNF-alpha polypeptide as
described above, (b) detecting binding of said polypeptide to said
sample, and (c) comparing the binding detected in step (b) with a
standard, wherein a difference in binding relative to said sample
is diagnostic of a disorder characterised by dysfunction of Tumor
Necrosis Factor-alpha.
[0055] Another embodiment of the present invention is a kit for
screening for a disorder as cited above, using a method as
described above.
[0056] Another embodiment of the present invention is a kit for
screening for a disorder as cited above comprising an isolated
anti-TNF-alpha polypeptide as described above.
[0057] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above for the purification
of said Tumor Necrosis Factor-alpha.
[0058] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as described above for inhibiting the
interaction between Tumor Necrosis Factor-alpha and one or more
Tumor Necrosis Factor-alpha receptors.
[0059] Another embodiment of the present invention is a method for
producing an anti-TNF-alpha polypeptide as described above
comprising the steps of:
(a) obtaining double stranded DNA encoding a Camelidae VHH directed
to Tumor Necrosis Factor alpha, (b) cloning and expressing the DNA
selected in step (b).
[0060] Another embodiment of the present invention is a method of
producing an anti-TNF-alpha polypeptide as described above
comprising:
(a) culturing host cells comprising nucleic acid capable of
encoding an anti-TNF-alpha polypeptide as described above, under
conditions allowing the expression of the polypeptide, and, (b)
recovering the produced polypeptide from the culture.
[0061] Another embodiment of the present invention is a method as
described above, wherein said host cells are bacterial or
yeast.
[0062] Another embodiment of the present invention is a kit for
screening for any of inflammation, rheumatoid arthritis, Crohn's
disease, ulcerative colitis, inflammatory bowel syndrome or
multiple sclerosis comprising an anti-TNF-alpha polypeptide as
described above.
BRIEF DESCRIPTION OF FIGURES AND TABLES
[0063] FIG. 1 Alignment of anti-human TNF VHH's as described in
Example 1: VHH#3G (SEQ ID NO:121), VHH#3E (SEQ ID NO:122), VHH#1A
(SEQ ID NO:123), VHH#2B (SEQ ID NO:124), VHH#12B (SEQ ID NO:125),
VHH#7B (SEQ ID NO:126).
[0064] FIG. 2 Dilution series of anti-human TNF-alpha VHHs as
tested in ELISA according to Example 1.
[0065] FIG. 3 Antagonistic effect of VHH as determined in
cytotoxicity assay using human cell line KYM according to Example
1.
[0066] FIG. 4 In vitro receptor binding assay of wild type VHH#12B
and mutant A74S+Y76N+K83R+P84A.
[0067] FIG. 5 In vitro receptor binding assay of wild type VHH#12B
and mutant 1 E+Q5LA74S+Y76N+K83R+P84A.
[0068] FIG. 6 Binding in ELISA of wild type VHH#3E and mutant
VHH's.
[0069] FIG. 7 In vitro receptor binding assay of wild type VHH#3E
and mutant VHH's.
[0070] FIG. 8 Alignment of antagonistic anti-mouse TNF's as
described in Example 3: VHH#m3F (SEQ ID NO:127), VHH#m4B (SEQ ID
NO:128), VHH#m9A (SEQ ID NO:129), VHH#m9E (SEQ ID NO:130).
[0071] FIG. 9 Antagonistic effect of anti-mouse TNF VHH as
determined in cytotoxicity assay using murine cell line L929
according to Example 3.
[0072] FIG. 10 EcoRI-HindIII insert (SEQ ID NOs:131, 132) of vector
pAX11 (pUC119 backbone) for production of bi-valent or bispecific
VHH.
[0073] FIG. 11 Coomassie-stained PAGE (15%) of IMAC-purified
mono-(lane 8), bi- (lane 1), tri-(lanes 2, 3 and 5) and tetravalent
(lanes 4, 6 and 7) anti-TNFa VHH.
[0074] FIG. 12 Chromatogram of the analysis by gel filtration on
Superdex 75HR of the mono-, bi-, tri and tetravalent VHH.
[0075] FIG. 13 Comparison of the antagonistic characteristics of
the mono-, bi-, tri- and tetravalent form of the anti-human TNF VHH
with the clinically used products Remicade and Enbrel.
[0076] FIG. 14 Antagonistic behaviour of the mono- and bivalent
VHH's directed against mouse TNFalpha.
[0077] FIG. 15 Coomassie stained PAGE of VHH-Fc-fusion derived from
human IgG1 described in Example 4.
[0078] FIG. 16 Antagonistic efficacy of VHH-Fc fusion derived from
VHH#3E compared with bivalent format of VHH#3E as determined in
bioassay.
[0079] FIG. 17 ELISA of reference and pepsin-treated TNF3E at
pH2.2, pH3.2 and pH4.2 (100% is the signal measured at a 1/100
dilution).
[0080] FIG. 18 Experimental setting.
[0081] Table 1 Amino acid sequence listing of the peptides of
aspects of present invention directed against TNF-alpha.
[0082] Table 2 List of mutagenesis reactions, mutagenic primers and
templates used for mutagenesis of VHH#12B: mutation
A74S+Y76N+K83R+P84A (SEQ ID NOs: 107, 108); mutation
Q1E+Q5L+A74S+Y76N+K83R+P84A (SEQ ID NOs: 109, 110); mutation
Q1E+Q5L+A74S+Y76N+K83R+P84A+T93A (SEQ ID NOs:111, 112).
[0083] Table 3 List of mutagenesis reactions, mutagenic primers and
templates used for mutagenesis of VHH#3E: mutation F37V (SEQ ID
NOs: 113, 114); mutation E44G (SEQ ID NOs: 115, 116); mutation R45L
(SEQ ID NOs: 117, 118); mutation F47W (SEQ ID NOs: 119, 120).
[0084] Table 4 Overview of humanised and wild type VHH.
[0085] Table 5 Anti-mouse serum albumin/anti TNF-alpha
[0086] Table 6 Amino acid sequence listing of VHH's directed
against human IFN-gamma.
[0087] Table 7 Sequences of bivalent (BIV 3E, BIV#m3F), trivalent
(TRI3E) or tetravalent (TETRA 3E) VHH directed against
TNF-alpha.
[0088] Table 8 Fractional homologies between the amino acid
sequences of anti-mouse serum albumin VHHs of the invention.
[0089] Table 9 Fractional homologies between anti-TNF-alpha VHHs of
the invention.
[0090] Table 10 Percentage homologies between anti-IFN-gamma VHHs
of the invention.
[0091] Table 11 Treatment schedule.
DETAILED DESCRIPTION
[0092] The present invention relates to an anti-tumour necrosis
factor-alpha (TNF-alpha) polypeptide, comprising one or more single
domain antibodies which are directed against TNF-alpha. The
invention also relates to nucleic acids capable of encoding said
polypeptides.
[0093] Single domain antibodies are antibodies whose complementary
determining regions are part of a single domain polypeptide.
Examples include, but are not limited to, heavy chain antibodies,
antibodies naturally devoid of light chains, single domain
antibodies derived from conventional 4-chain antibodies, engineered
antibodies and single domain scaffolds other than those derived
from antibodies. Single domain antibodies may be any of the art, or
any future single domain antibodies. Single domain antibodies may
be derived from any species including, but not limited to mouse,
human, camel, llama, goat, rabbit, bovine. According to one aspect
of the invention, a single domain antibodies as used herein is a
naturally occurring single domain antibody known as heavy chain
antibody devoid of light chains. Such single domain antibodies are
disclosed in WO 94/04678 for example. For clarity reasons, this
variable domain derived from a heavy chain antibody naturally
devoid of light chain is known herein as a VHH or nanobody to
distinguish it from the conventional VH of four chain
immunoglobulins. Such a VHH molecule can be derived from antibodies
raised in Camelidae species, for example in camel, dromedary,
llama, alpaca and guanaco. Other species besides Camelidae may
produce heavy chain antibodies naturally devoid of light chain;
such VHHs are within the scope of the invention.
[0094] VHHs, according to the present invention, and as known to
the skilled addressee are heavy chain variable domains derived from
immunoglobulins naturally devoid of light chains such as those
derived from Camelidae as described in WO 94/04678 (and referred to
hereinafter as VHH domains or nanobodies). VHH molecules are about
10.times. smaller than IgG molecules. They are single polypeptides
and very stable, resisting extreme pH and temperature conditions.
Moreover, they are resistant to the action of proteases which is
not the case for conventional antibodies. Furthermore, in vitro
expression of VHHs produces high yield, properly folded functional
VHHs. In addition, antibodies generated in Camelids will recognize
epitopes other than those recognised by antibodies generated in
vitro through the use of antibody libraries or via immunisation of
mammals other than Camelids (WO 9749805). As such, anti-TNF-alpha
VHH's may interact more efficiently with TNF-alpha than
conventional antibodies, thereby blocking its interaction with the
TNF-alpha receptor more efficiently.
[0095] According to the invention, TNF-alpha is derived from any
species. Examples of species relevant to the invention include as
rabbits, goats, mice, rats, cows, calves, camels, llamas, monkeys,
donkeys, guinea pigs, chickens, sheep, dogs, cats, horses, and
preferably humans.
[0096] TNF-alpha is also a fragment of TNF-alpha, capable of
eliciting an immune response. TNF-alpha is also a fragment of
TNF-alpha, capable of binding to a single domain antibody raised
against the full length TNF-alpha.
[0097] A single domain antibody directed against TNF-alpha means
single domain antibody that it is capable of binding to TNF-alpha
with an affinity of better than 10.sup.-6 M.
[0098] One embodiment of the present invention is an anti-TNF
polypeptide, wherein the single domain antibodies comprise
Camelidae VHH directed against TNF-alpha.
[0099] The one or more single domain antibodies of the anti-TNF
polypeptide which are directed against a TNF-alpha may be of the
same sequence. Alternatively they may not all have the same
sequence. It is within the scope of the invention that an anti-TNF
polypeptide comprises anti-TNF-alpha single domain antibodies which
do not all share the same sequence, but which are directed against
the same target, one or more antigens thereof.
[0100] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide, wherein a single domain antibody
corresponds to a sequence represented by any of SEQ ID NOs: 1 to 16
and 79 to 84 as shown in Table 1. Said sequences are derived from
Camelidae heavy chain antibodies (VHHs) which are directed against
TNF-alpha.
[0101] The present invention further relates to an anti-TNF-alpha
polypeptide, wherein said single domain antibody is a VHH directed
against TNF-alpha, wherein the VHH belongs to a class having
human-like sequences. The class is characterised in that the VHHs
carry an amino acid from the group consisting of glycine, alanine,
valine, leucine, isoleucine, proline, phenylalanine, tyrosine,
tryptophan, methionine, serine, threonine, asparagine, or glutamine
at position 45, such as, for example, L45 and a tryptophan at
position 103, according to the Kabat numbering. The new class of
Camelidae single-domain antibodies described in this invention
(Table 1, Example 1) is represented by VHH#2B (SEQ ID NO: 3) and
VHH#12B (SEQ ID No. 14) containing the hydrophobic residues in FR2
in combination with the hydrophobic residue tryptophan at position
103.
[0102] Another human-like class of Camelidae single domain
antibodies represented by sequences VHH#1A (SEQ ID NO. 1), VHH#4B
(SEQ ID NO. 12), VHH#8-29 (SEQ ID NO. 81), VHH#8-41 (SEQ ID NO.
82), VHH#8-42 (SEQ ID NO. 83) and VHH#8-44 (SEQ ID NO. 84) (Table
1, Example 1) have been described in WO03035694 and contain the
hydrophobic FR2 residues typically found in conventional antibodies
of human origin or from other species, but compensating this loss
in hydrophilicity by the charged arginine residue on position 103
that substitutes the conserved tryptophan residue present in VH
from double-chain antibodies. As such, peptides belonging to these
two classes show a high amino acid sequence homology to human VH
framework regions and said peptides might be administered to a
human directly without expectation of an unwanted immune response
therefrom, and without the burden of further humanisation. The
invention also relates to nucleic acids capable of encoding said
polypeptides.
[0103] Therefore, one aspect of the present invention allows for
the direct administration of an anti-TNF-alpha polypeptide, wherein
the single domain antibodies belong to the humanized class of VHH,
and comprise a sequence represented by any of SEQ ID NO:1, 3, 12,
14, 81, 82, 83, and 84 to a patient in need of the same.
[0104] Any of the VHHs as used by the invention may be of the
traditional class or of the classes of human-like Camelidae
antibodies. Said antibodies may be directed against whole TNF-alpha
or a fragment thereof, or a fragment of a homologous sequence
thereof. These polypeptides include the full length Camelidae
antibodies, namely Fc and VHH domains, chimeric versions of heavy
chain Camelidae antibodies with a human Fc domain or VHH's by
themselves or derived fragments.
[0105] Anti-serum albumin VHH's may interact in a more efficient
way with serum albumin than conventional antibodies which is known
to be a carrier protein. As a carrier protein some of the epitopes
of serum albumin may be inaccessible by bound proteins, peptides
and small chemical compounds. Since VHH's are known to bind into
`unusual` or non-conventional epitopes such as cavities (WO
97/49805), the affinity of such VHH's to circulating albumin may be
increased.
[0106] The present invention also relates to the finding that an
anti-TNF polypeptide as described herein further comprising one or
more single domain antibodies directed against one or more serum
proteins of a subject, surprisingly has significantly prolonged
half-life in the circulation of said subject compared with the
half-life of the anti-TNF-alpha single domain antibody when not
part of said construct. Examples of such polypeptides are
represented in Table 5 by SEQ ID NOs: 30 to 43. Furthermore, the
said polypeptides were found to exhibit the same favourable
properties of single domain antibodies such as high stability
remaining intact in mice, extreme pH resistance, high temperature
stability and high target affinity.
[0107] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide further comprising one or more single
domain antibodies directed against one or more serum proteins, said
anti-TNF alpha polypeptide comprising a sequence corresponding to
any represented by SEQ ID NOs: 30 to 43 (Table 5).
[0108] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide, wherein an anti-serum protein single
domain antibody corresponds to a sequence represented by any of SEQ
ID NOs: 26 to 29 and 85 to 97 as shown in Table 5.
[0109] The serum protein may be any suitable protein found in the
serum of subject. In one aspect of the invention, the serum protein
is serum albumin, serum immunoglobulins, thyroxine-binding protein,
transferrin, or fibrinogen. Depending on the intended use such as
the required half-life for effective treatment and/or
compartimentalisation of the target antigen, the VHH-partner can be
directed to one of the above serum proteins.
[0110] Another aspect of the invention is an anti-TNF-alpha
polypeptide as disclosed herein further comprising at least one
polypeptide selected from the group consisting of an anti-IFN-gamma
polypeptide, an anti-TNF-alpha receptor polypeptide and
anti-IFN-gamma receptor polypeptide.
[0111] It is an embodiment of the invention that a single domain
antibody directed against IFN-gamma corresponds to a sequence
represented by any of SEQ ID NOs: 44 to 72 as shown in Table 6.
[0112] According to one aspect of the invention, a single domain
antibody is directed against TNF-alpha receptor. Said single domain
antibody may be a Camelidae VHH.
[0113] According to one aspect of the invention, a single domain
antibody is directed against IFN-gamma receptor. Said single domain
antibody may be a Camelidae VHH.
[0114] Another aspect of the invention is a method of treating an
autoimmune disease or condition as cited herein, comprising
administering to a patient an effective amount of an anti-TNF-alpha
polypeptide further comprising a least one polypeptide selected
from the group consisting of anti-IFN-gamma polypeptide,
anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor
polypeptide, such polypeptides joined to each other as described
below.
[0115] Such multi-specific constructs may have improved potency as
inflammatory therapeutic compound over mono-specific
constructs.
[0116] One aspect of the invention is a composition comprising an
anti-TNF-alpha polypeptide as disclosed herein and at least one
polypeptide selected from the group consisting of anti-IFN-gamma
polypeptide, anti-TNF-alpha receptor polypeptide and anti-IFN-gamma
receptor polypeptide, for simultaneous, separate or sequential
administration to a subject.
[0117] One aspect of the invention is a method for treating
autoimmune disease comprising administering to an individual an
effective amount of an anti-TNF-alpha polypeptide and a least one
polypeptide selected from the group consisting of anti-IFN-gamma
polypeptide, anti-TNF-alpha receptor polypeptide and anti-IFN-gamma
receptor polypeptide, simultaneously, separately or
sequentially.
[0118] Another aspect of the invention is a kit containing an
anti-TNF-alpha polypeptide and a least one polypeptide selected
from the group consisting of anti-IFN-gamma polypeptide,
anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor
polypeptide for simultaneous, separate or sequential administration
to a subject. It is an aspect of the invention that the kit may be
used according to the invention. It is an aspect of the invention
that the kit may be used to treat the diseases as cited herein.
[0119] By simultaneous administration means the polypeptides are
administered to a subject at the same time. For example, as a
mixture of the polypeptides or a composition comprising said
polypeptides. Examples include, but are not limited to a solution
administered intraveneously, a tablet, liquid, topical cream, etc.,
wherein each preparation comprises the polypeptides of
interest.
[0120] By separate administration means the polypeptides are
administered to a subject at the same time or substantially the
same time. The polypeptides are present in the kit as separate,
unmixed preparations. For example, the different polypeptides may
be present in the kit as individual tablets. The tablets may be
administered to the subject by swallowing both tablets at the same
time, or one tablet directly following the other.
[0121] By sequential administration means the polypeptides are
administered to a subject sequentially. The polypeptides are
present in the kit as separate, unmixed preparations. There is a
time interval between doses. For example, one polypeptide might be
administered up to 336, 312, 288, 264, 240, 216, 192, 168, 144,
120, 96, 72, 48, 24, 20, 16, 12, 8, 4, 2, 1, or 0.5 hours after the
other component.
[0122] In sequential administration, one polypeptide may be
administered once, or any number of times and in various doses
before and/or after administration of another polypeptide.
Sequential administration may be combined with simultaneous or
sequential administration.
[0123] The medical uses of the anti-TNF-alpha polypeptide described
below, also apply to the composition comprising an anti-TNF-alpha
polypeptide as disclosed herein and at least one polypeptide
selected from the group consisting of anti-IFN-gamma polypeptide,
anti-TNF-alpha receptor polypeptide and anti-IFN-gamma receptor
polypeptide, for simultaneous, separate or sequential
administration to a subject as disclosed here above.
[0124] According to one aspect of the invention, an anti-IFN-gamma
polypeptide anti-TNF-alpha a single domain antibody directed
against IFN-gamma. Said single domain antibody may be a Camelidae
VHH.
[0125] It is an embodiment of the invention that a single domain
antibody directed against IFN-gamma corresponds to a sequence
represented by any of SEQ ID NOs: 44 to 72 as shown in Table 6.
[0126] According to one aspect of the invention, anti-TNF-alpha a
single domain antibody directed against TNF-alpha receptor. Said
single domain antibody may be a Camelidae VHH.
[0127] According to one aspect of the invention, an anti-IFN-gamma
receptor polypeptide anti-TNF-alpha a single domain antibody
directed against IFN-gamma receptor. Said single domain antibody
may be a Camelidae VHH.
[0128] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as disclosed herein, wherein the number
of single domain antibodies directed against TNF-alpha is two or
more. Such multivalent anti-TNF-alpha polypeptides have the
advantage of unusually high functional affinity for the target,
displaying much higher than expected inhibitory properties compared
to their monovalent counterparts.
[0129] The multivalent anti-TNF-alpha polypeptides have functional
affinities that are several orders of magnitude higher than the
monovalent parent anti-TNF-alpha polypeptides. The inventors have
found that the functional affinities of these multivalent
polypeptides are much higher than those reported in the prior art
for bivalent and multivalent antibodies. Surprisingly,
anti-TNF-alpha polypeptides of the present invention linked to each
other directly (SEQ ID No. 77 and 78) or via a short linker
sequence show the high functional affinities expected theoretically
with multivalent conventional four-chain antibodies.
[0130] The inventors have found that such large increased
functional activities can be detected preferably with antigens
composed of multidomain and multimeric proteins, either in straight
binding assays or in functional assays, e.g. cytotoxicity
assays.
[0131] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as disclosed herein, wherein the number
of single domain antibodies directed against TNF-alpha is two or
more, said anti-TNF-alpha polypeptide comprising a sequence
corresponding to any represented by SEQ ID NOs: 73 to 76.
[0132] The single domain antibodies may be joined to form any of
the polypeptides disclosed herein comprising more than one single
domain antibody using methods known in the art or any future
method. For example, they may be fused by chemical cross-linking by
reacting amino acid residues with an organic derivatising agent
such as described by Blattler et al, Biochemistry 24, 1517-1524;
EP294703. Alternatively, the single domain antibody may be fused
genetically at the DNA level i.e. a polynucleotide construct formed
which encodes the complete polypeptide construct comprising one or
more anti-target single domain antibodies and one or more
anti-serum protein single domain antibodies. A method for producing
bivalent or multivalent VHH polypeptide constructs is disclosed in
PCT patent application WO 96/34103. One way of joining multiple
single domain antibodies is via the genetic route by linking single
domain antibody coding sequences either directly or via a peptide
linker. For example, the C-terminal end of the first single domain
antibody may be linked to the N-terminal end of the next single
domain antibody. This linking mode can be extended in order to link
additional single domain antibodies for the construction and
production of tri-, tetra-, etc. functional constructs.
[0133] According to one aspect of the present invention, the single
domain antibodies are linked to each other directly, without use of
a linker. Contrary to joining bulky conventional antibodies where a
linker sequence is needed to retain binding activity in the two
subunits, polypeptides of the invention can be linked directly (SEQ
ID No. 77 and 78) thereby avoiding potential problems of the linker
sequence, such as antigenicity when administered to a human
subject, instability of the linker sequence leading to dissociation
of the subunits.
[0134] According to another aspect of the present invention, the
single domain antibodies are linked to each other via a peptide
linker sequence. Such linker sequence may be a naturally occurring
sequence or a non-naturally occurring sequence. The linker sequence
is expected to be non-immunogenic in the subject to which the
anti-TNF-alpha polypeptide is administered. The linker sequence may
provide sufficient flexibility to the multivalent anti-TNF-alpha
polypeptide, at the same time being resistant to proteolytic
degradation. A non-limiting example of a linker sequences is one
that can be derived from the hinge region of VHHs described in WO
96/34103.
[0135] According to another aspect of the invention, multivalent
single domain antibodies comprising more than two single domain
antibodies can be linked to each other either directly or via a
linker sequence. Such constructs are difficult to produce with
conventional antibodies and due to steric hindrance of the bulky
subunits, functionality will be lost or greatly diminished rather
than increased considerably as seen with VHH's of the invention
compared to the monovalent construct (see FIG. 12 for gel
filtration analyses of such multivalent VHH constructs).
[0136] The polypeptide constructs disclosed herein may be made by
the skilled artisan according to methods known in the art or any
future method. For example, VHHs may be obtained using methods
known in the art such as by immunising a camel and obtaining
hybridomas therefrom, or by cloning a library of single domain
antibodies using molecular biology techniques known in the art and
subsequent selection by using phage display.
[0137] According to an aspect of the invention an anti-TNF-alpha
polypeptide may be a homologous sequence of a full-length
anti-TNF-alpha polypeptide. According to another aspect of the
invention, an anti-TNF-alpha polypeptide may be a functional
portion of a full-length anti-TNF-alpha polypeptide. According to
another aspect of the invention, an anti-TNF-alpha polypeptide may
be a homologous sequence of a full-length anti-TNF-alpha
polypeptide. According to another aspect of the invention, an
anti-TNF-alpha polypeptide may be a functional portion of a
homologous sequence of a full-length anti-TNF-alpha polypeptide.
According to an aspect of the invention an anti-TNF-alpha
polypeptide may comprise a sequence of an anti-TNF-alpha
polypeptide.
[0138] According to an aspect of the invention a single domain
antibody used to form an anti-TNF-alpha polypeptide may be a
complete single domain antibody (e.g. a VHH) or a homologous
sequence thereof. According to another aspect of the invention, a
single domain antibody used to form the polypeptide construct may
be a functional portion of a complete single domain antibody.
According to another aspect of the invention, a single domain
antibody used to form the polypeptide construct may be a homologous
sequence of a complete single domain antibody. According to another
aspect of the invention, a single domain antibody used to form the
polypeptide construct may be a functional portion of a homologous
sequence of a complete single domain antibody.
[0139] As used herein, an homologous sequence of the present
invention may comprise additions, deletions or substitutions of one
or more amino acids, which do not substantially alter the
functional characteristics of the polypeptides of the invention.
The number of amino acid deletions or substitutions is preferably
up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69 or 70 amino acids.
[0140] A homologous sequence according to the present invention may
a polypeptide modified by the addition, deletion or substitution of
amino acids, said modification not substantially altering the
functional characteristics compared with the unmodified
polypeptide.
[0141] A homologous sequence according to the present invention may
be a polypeptide modified by the addition, deletion or substitution
of amino acids, said modification not substantially altering the
functional characteristics compared with the unmodified
polypeptide.
[0142] A homologous sequence according to the present invention may
be a sequence which exists in other Camelidae species such as, for
example, camel, dromedary, llama, alpaca, guanaco etc.
[0143] Where homologous sequence indicates sequence identity, it
means a sequence which presents a high sequence identity (more than
70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity) with the
parent sequence and is preferably characterised by similar
properties of the parent sequence, namely affinity, said identity
calculated using known methods.
[0144] Alternatively, an homologous sequence may also be any amino
acid sequence resulting from allowed substitutions at any number of
positions of the parent sequence according to the formula
below:
Ser substituted by Ser, Thr, Gly, and Asn; Arg substituted by one
of Arg, His, Gln, Lys, and Glu; Leu substituted by one of Leu, Ile,
Phe, Tyr, Met, and Val; Pro substituted by one of Pro, Gly, Ala,
and Thr; Thr substituted by one of Thr, Pro, Ser, Ala, Gly, His,
and Gln; Ala substituted by one of Ala, Gly, Thr, and Pro; Val
substituted by one of Val, Met, Tyr, Phe, Ile, and Leu; Gly
substituted by one of Gly, Ala, Thr, Pro, and Ser; Ile substituted
by one of Ile, Met, Tyr, Phe, Val, and Leu; Phe substituted by one
of Phe, Trp, Met, Tyr, Ile, Val, and Leu; Tyr substituted by one of
Tyr, Trp, Met, Phe, Ile, Val, and Leu; His substituted by one of
His, Glu, Lys, Gln, Thr, and Arg; Gln substituted by one of Gln,
Glu, Lys, Asn, His, Thr, and Arg; Asn substituted by one of Asn,
Glu, Asp, Gln, and Ser; Lys substituted by one of Lys, Glu, Gln,
His, and Arg; Asp substituted by one of Asp, Glu, and Asn; Glu
substituted by one of Glu, Asp, Lys, Asn, Gln, His, and Arg; Met
substituted by one of Met, Phe, Ile, Val, Leu, and Tyr.
[0145] A homologous nucleotide sequence according to the present
invention may refer to nucleotide sequences of more than 50, 100,
200, 300, 400, 500, 600, 800 or 1000 nucleotides able to hybridize
to the reverse-complement of the nucleotide sequence capable of
encoding the patent sequence, under stringent hybridisation
conditions (such as the ones described by Sambrook et al.,
Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor
Laboratory press, New York).
[0146] As used herein, a functional portion refers to a sequence of
a single domain antibody that is of sufficient size such that the
interaction of interest is maintained with affinity of
1.times.10.sup.-6 M or better.
[0147] Alternatively, a functional portion comprises a partial
deletion of the complete amino acid sequence and still maintains
the binding site(s) and protein domain(s) necessary for the binding
of and interaction with the target.
[0148] As used herein, a functional portion refers to less than
100% of the complete sequence (e.g., 99%, 90%, 80%, 70%, 60% 50%,
40%, 30%, 20%, 10%, 5%, 1% etc.), but comprises 5 or more amino
acids or 15 or more nucleotides.
[0149] Targets as mentioned herein such as TNF-alpha, TNF-alpha
receptor, serum proteins (e.g. serum albumin, serum
immunoglobulins, thyroxine-binding protein, transferrin,
fibrinogen) and IFN-gamma, IFN-gamma receptor may be fragments of
said targets. Thus a target is also a fragment of said target,
capable of eliciting an immune response. A target is also a
fragment of said target, capable of binding to a single domain
antibody raised against the full length target.
[0150] A fragment as used herein refers to less than 100% of the
sequence (e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%
etc.), but comprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25 or more amino acids. A fragment is
of sufficient length such that the interaction of interest is
maintained with affinity of 1.times.10-6 M or better.
[0151] A fragment as used herein also refers to optional
insertions, deletions and substitutions of one or more amino acids
which do not substantially alter the ability of the target to bind
to a single domain antibody raised against the wild-type target.
The number of amino acid insertions deletions or substitutions is
preferably up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69 or 70 amino acids.
[0152] A homologous sequence of the present invention may include
an anti-TNF-alpha polypeptide which has been humanised. The
humanisation of antibodies of the new class of VHHs would further
reduce the possibility of unwanted immunological reaction in a
human individual upon administration.
[0153] One embodiment of the present invention relates to a method
for preparing modified polypeptides based upon llama antibodies by
determining the amino acid residues of the antibody variable domain
(VHH) which may be modified without diminishing the native affinity
of the domain for antigen and while reducing its immunogenicity
with respect to a heterologous species; the use of VHHs having
modifications at the identified residues which are useful for
administration to heterologous species; and to the VHH so
modified.
[0154] More specifically, the invention relates to the preparation
of modified VHHs, which are modified for administration to humans,
the resulting VHH themselves, and the use of such "humanized" VHHs
in the treatment of diseases in humans. By humanised is meant
mutated so that immunogenicity upon administration in human
patients is minor or nonexistent. Humanising a polypeptide,
according to the present invention, comprises a step of replacing
one or more of the Camelidae amino acids by their human counterpart
as found in the human consensus sequence, without that polypeptide
losing its typical character, i.e. the humanisation does not
significantly affect the antigen binding capacity of the resulting
polypeptide. Such methods are known by the skilled addressee.
[0155] Humanization of Camelidae single domain antibodies requires
the introduction and mutagenesis of a limited amount of amino acids
in a single polypeptide chain. This is in contrast to humanization
of scFv, Fab, (Fab).sub.2 and IgG, which requires the introduction
of amino acid changes in two chains, the light and the heavy chain
and the preservation of the assembly of both chains.
[0156] As a non-limited example, the polypeptide of VHH#12B
containing human-like residues in FR2 was humanized. Humanization
required mutagenesis of residues in FR1 at position 1 and 5 which
were introduced by the primer used for repertoire cloning and do
not occur naturally in the llama sequence. Mutagenesis of those
residues did not result in loss of binding and/or inhibition
activity. Humanization also required mutagenesis of residues in FR3
at position 74, 76, 83, 84, 93. Mutagenesis of those residues did
not result in a dramatic loss of binding and/or inhibition activity
(see FIG. 4). Combining the mutations of FR1 and FR3 therefore did
not affect the binding and/or inhibition activity (FIG. 5).
[0157] Humanization also required mutagenesis of residues in FR4 at
position 108. Mutagenesis of Q108L resulted in lower production
level in Escherichia coli. Position 108 is solvent exposed in
camelid VHH, while in human antibodies this position is buried at
the VH-VL interface (Spinelli, 1996; Nieba, 1997). In isolated VHs
position 108 is solvent exposed. The introduction of a non-polar
hydrophobic Leu instead of polar uncharged Gln can have a drastic
effect on the intrinsic folding/stability of the molecule.
[0158] As a non-limited example, the polypeptide represented in the
VHH#3E containing camelid hallmark residues at position 37, 44, 45
and 47 with hydrophilic characteristics, was humanized. Replacement
of the hydrophilic residues by human hydrophobic residues at
positions 44 and 45 (E44G and R45L), did not have an effect on
binding and/or inhibition. However, loss of binding and/or
inhibition activity was observed when F37V and F47W were
introduced. Modeling data confirmed the critical residue 37 to
preserve the integrity of the CDR3 loop conformation and hence on
activity (see FIG. 6) (all numbering according to the Kabat).
[0159] SEQ ID NO: 3 and 14 display more than 90% amino acid
sequence homology to human VH framework regions and therefore said
VHH might be administered to patients directly without expectation
of an immune response therefrom, and without the additional burden
of humanisation. Therefore, one aspect of the present invention
allows for the direct administration of the polypeptide comprising
SEQ ID NO: 3 and 14, homologous sequence thereof, or a functional
portion of an homologous sequence thereof to a patient in need of
the same.
[0160] One embodiment of the present invention is a method for
humanizing a VHH comprising the steps of replacing of any of the
following residues either alone or in combination: [0161] FR1
position 1, 5, 28 and 30, [0162] the hallmark amino acid at
position 44 and 45 in FR2, [0163] FR3 residues 74, 75, 76, 83, 84,
93 and 94, [0164] and positions 103, 104, 108 and 111 in FR4;
numbering according to the Kabat numbering.
[0165] One embodiment of the present invention is an anti-TNF-alpha
polypeptide, or a nucleic acid capable of encoding said polypeptide
for use in treating, preventing and/or alleviating the symptoms of
disorders relating to inflammatory processes. TNF-alpha is involved
in inflammatory processes, and the blocking of TNF-alpha action can
have an anti-inflammatory effect, which is highly desirable in
certain disease states such as, for example, Crohn's disease. Our
Examples demonstrate VHHs according to the invention which bind
TNF-alpha and moreover, block its binding to the TNF-alpha
receptor.
[0166] The anti-TNF-alpha polypeptides of the present invention are
applicable to autoimmune diseases, such as Addison's disease
(adrenal), Autoimmune diseases of the ear (ear), Autoimmune
diseases of the eye (eye), Autoimmune hepatitis (liver), Autoimmune
parotitis (parotid glands), Crohn's disease (intestine), Diabetes
Type I (pancreas), Epididymitis (epididymis), Glomerulonephritis
(kidneys), Graves' disease (thyroid), Guillain-Barre syndrome
(nerve cells), Hashimoto's disease (thyroid), Hemolytic anemia (red
blood cells), Systemic lupus erythematosus (multiple tissues), Male
infertility (sperm), Multiple sclerosis (nerve cells), Myasthenia
Gravis (neuromuscular junction), Pemphigus (primarily skin),
Psoriasis (skin), Rheumatic fever (heart and joints), Rheumatoid
arthritis (joint lining), Sarcoidosis (multiple tissues and
organs), Scleroderma (skin and connective tissues), Sjogren's
syndrome (exocrine glands, and other tissues),
Spondyloarthropathies (axial skeleton, and other tissues),
Thyroiditis (thyroid), Vasculitis (blood vessels). Within
parenthesis is the tissue affected by the disease. This listing of
autoimmune diseases is intended to be exemplary rather than
inclusive.
[0167] Autoimmune conditions for which the anti-TNF-alpha
polypeptides of the present invention is applicable include, for
example, AIDS, atopic allergy, bronchial asthma, eczema, leprosy,
schizophrenia, inherited depression, transplantation of tissues and
organs, chronic fatigue syndrome, Alzheimer's disease, Parkinson's
disease, myocardial infarction, stroke, autism, epilepsy, Arthus's
phenomenon, anaphylaxis, and alcohol and drug addiction. In the
above-identified autoimmune conditions, the tissue affected is the
primary target, in other cases it is the secondary target. These
conditions are partly or mostly autoimmune syndromes. Therefore, in
treating them, it is possible to use the same methods, or aspects
of the same methods that are herein disclosed, sometimes in
combination with other methods.
[0168] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide according to the invention, or a nucleic
acid capable of encoding said polypeptide for the preparation of a
medicament for treating a disorder relating to inflammatory
processes. Examples of disorders include rheumatoid arthritis,
Crohn's disease, ulcerative colitis, inflammatory bowel syndrome
and multiple sclerosis
[0169] Polypeptides and nucleic acids according to the present
invention may be administered to a subject by conventional routes,
such as intravenously. However, a special property of the
anti-TNF-alpha polypeptides of the invention is that they penetrate
barriers such as tissue membranes and/or tumours and act locally
and act locally thereon, and they are sufficiently stable to
withstand extreme environments such as in the stomach. Therefore,
another aspect of the present invention relates to the delivery of
anti-TNF-alpha polypeptides.
[0170] A subject according to the invention can be any mammal
susceptible to treatment by therapeutic polypeptides.
[0171] Oral delivery of anti-TNF-alpha polypeptides of the
invention results in the provision of such molecules in an active
form in the colon at local sites that are affected by the disorder.
These sites may be highly inflamed and contain TNF-alpha-producing
cells. The anti-TNF-alpha polypeptides of the invention which bind
to TNF-alpha can neutralise the TNF-alpha locally, avoiding
distribution throughout the whole body and thus limiting negative
side-effects. Genetically modified microorganisms such as
Micrococcus lactis are able to secrete antibody or functional
portions thereof. Such modified microorganisms can be used as
vehicles for local production and delivery of antibodies or
functional portions thereof in the intestine. By using a strain
which produces an anti-TNF-alpha polypeptide, inflammatory bowel
syndrome could be treated.
[0172] Another aspect of the invention involves delivering anti-TNF
polypeptides by using surface expression on or secretion from
non-invasive bacteria, such as Gram-positive host organisms like
Lactococcus spec. using a vector such as described in
WO00/23471.
[0173] One embodiment of the present invention is an anti-TNF-alpha
polypeptide as disclosed herein for use in treating, preventing
and/or alleviating the symptoms of disorders susceptible to
modulation by a TNF-alpha modulating substance which is able pass
through the gastric environment without the substance being
inactivated.
[0174] Examples of disorders are any that cause inflammation,
including, but not limited to rheumatoid arthritis, Crohn's
disease, ulcerative colitis, inflammatory bowl syndrome, and
multiple sclerosis. As known by persons skilled in the art, once in
possession of said polypeptide construct, formulation technology
may be applied to release a maximum amount of polypeptide in the
right location (in the stomach, in the colon, etc.). This method of
delivery is important for treating, prevent and/or alleviate the
symptoms of disorders whose targets are located in the gut
system.
[0175] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of a disorder
susceptible to modulation by a TNF-alpha modulating substance which
is able pass through the gastric environment without being
inactivated, by orally administering to a subject an anti-TNF-alpha
polypeptide as disclosed herein.
[0176] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as disclosed herein for the preparation
of a medicament for treating, preventing and/or alleviating the
symptoms of disorders susceptible to modulation by a TNF-alpha
modulating substance which is able pass through the gastric
environment without being inactivated.
[0177] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the gut system without said
substance being inactivated, by orally administering to a subject
an anti-TNF-alpha polypeptide as disclosed herein.
[0178] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the bloodstream of a subject
without the substance being inactivated, by orally administering to
a subject an anti-TNF-alpha polypeptide as disclosed herein.
[0179] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as disclosed herein for use in treating,
preventing and/or alleviating the symptoms or disorders susceptible
to modulation by a TNF-alpha modulating substance delivered to the
vaginal and/or rectal tract.
[0180] Examples of disorders are any that cause inflammation,
including, but not limited to rheumatoid arthritis, Crohn's
disease, ulcerative colitis, inflammatory bowl syndrome, and
multiple sclerosis. In a non-limiting example, a formulation
according to the invention comprises an anti-TNF-alpha polypeptide
as disclosed herein, in the form of a gel, cream, suppository,
film, or in the form of a sponge or as a vaginal ring that slowly
releases the active ingredient over time (such formulations are
described in EP 707473, EP 684814, U.S. Pat. No. 5,629,001).
[0181] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance delivered to the
vaginal and/or rectal tract, by vaginally and/or rectally
administering to a subject an anti-TNF-alpha polypeptide as
disclosed herein.
[0182] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as disclosed herein for the preparation
of a medicament for treating, preventing and/or alleviating the
symptoms of disorders susceptible to modulation by a TNF-alpha
modulating substance delivered to the vaginal and/or rectal
tract.
[0183] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the vaginal and/or rectal tract
without being said substance being inactivated, by administering to
the vaginal and/or rectal tract of a subject an anti-TNF-alpha
polypeptide as disclosed herein.
[0184] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the bloodstream of a subject
without said substance being inactivated, by administering to the
vaginal and/or rectal tract of a subject an anti-TNF-alpha
polypeptide as disclosed herein.
[0185] Another embodiment of the present invention is an
anti-TNF-alpha polypeptide as disclosed herein, for use in
treating, preventing and/or alleviating the symptoms of disorders
susceptible to modulation by a TNF-alpha modulating substance
delivered to the nose, upper respiratory tract and/or lung.
[0186] Examples of disorders are any that cause inflammation,
including, but not limited to rheumatoid arthritis, Crohn's
disease, ulcerative colitis, inflammatory bowl syndrome, and
multiple sclerosis. In a non-limiting example, a formulation
according to the invention, comprises an anti-TNF-alpha polypeptide
as disclosed herein in the form of a nasal spray (e.g. an aerosol)
or inhaler. Since the polypeptide construct is small, it can reach
its target much more effectively than therapeutic IgG
molecules.
[0187] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance delivered to the
upper respiratory tract and lung, by administering to a subject an
anti-TNF-alpha polypeptide as disclosed herein, by inhalation
through the mouth or nose.
[0188] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as disclosed herein for the preparation
of a medicament for treating, preventing and/or alleviating the
symptoms of disorders susceptible to modulation by a TNF-alpha
modulating substance delivered to the nose, upper respiratory tract
and/or lung, without said polypeptide being inactivated.
[0189] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the nose, upper respiratory tract
and lung without inactivation, by administering to the nose, upper
respiratory tract and/or lung of a subject an anti-TNF-alpha
polypeptide as disclosed herein.
[0190] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the bloodstream of a subject
without inactivation by administering to the nose, upper
respiratory tract and/or lung of a subject an anti-TNF-alpha
polypeptide as disclosed herein.
[0191] One embodiment of the present invention is an anti-TNF-alpha
polypeptide as disclosed herein for use in treating, preventing
and/or alleviating the symptoms of disorders susceptible to
modulation by a TNF-alpha modulating substance delivered to the
intestinal mucosa, wherein said disorder increases the permeability
of the intestinal mucosa. Because of their small size, an
anti-TNF-alpha polypeptide as disclosed herein can pass through the
intestinal mucosa and reach the bloodstream more efficiently in
subjects suffering from disorders which cause an increase in the
permeability of the intestinal mucosa, for example Crohn's
disease.
[0192] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance delivered to the
intestinal mucosa, wherein said disorder increases the permeability
of the intestinal mucosa, by orally administering to a subject an
anti-TNF-alpha polypeptide as disclosed herein.
[0193] This process can be even further enhanced by an additional
aspect of the present invention--the use of active transport
carriers. In this aspect of the invention, VHH is fused to a
carrier that enhances the transfer through the intestinal wall into
the bloodstream. In a non-limiting example, this "carrier" is a
second VHH which is fused to the therapeutic VHH. Such fusion
constructs are made using methods known in the art. The "carrier"
VHH binds specifically to a receptor on the intestinal wall which
induces an active transfer through the wall.
[0194] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as disclosed herein for the preparation
of a medicament for treating, preventing and/or alleviating the
symptoms of disorders susceptible to modulation by a TNF-alpha
modulating substance delivered to the intestinal mucosa, wherein
said disorder increases the permeability of the intestinal
mucosa.
[0195] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the intestinal mucosa without
being inactivated, by administering orally to a subject an
anti-TNF-alpha polypeptide of the invention.
[0196] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the bloodstream of a subject
without being inactivated, by administering orally to a subject an
anti-TNF-alpha polypeptide of the invention.
[0197] This process can be even further enhanced by an additional
aspect of the present invention--the use of active transport
carriers. In this aspect of the invention, an anti-TNF-alpha
polypeptide as described herein is fused to a carrier that enhances
the transfer through the intestinal wall into the bloodstream. In a
non-limiting example, this "carrier" is a VHH which is fused to
said polypeptide. Such fusion constructs made using methods known
in the art. The "carrier" VHH binds specifically to a receptor on
the intestinal wall which induces an active transfer through the
wall.
[0198] One embodiment of the present invention is an anti-TNF-alpha
polypeptide as disclosed herein for use in treating, preventing
and/or alleviating the symptoms of disorders susceptible to
modulation by a TNF-alpha modulating substance which is able pass
through the tissues beneath the tongue effectively.
[0199] Examples of disorders are any that cause inflammation,
including, but not limited to rheumatoid arthritis, Crohn's
disease, ulcerative colitis, inflammatory bowl syndrome, and
multiple sclerosis. A formulation of said polypeptide construct as
disclosed herein, for example, a tablet, spray, drop is placed
under the tongue and adsorbed through the mucus membranes into the
capillary network under the tongue.
[0200] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance which is able
pass through the tissues beneath the tongue effectively, by
sublingually administering to a subject an anti-TNF-alpha
polypeptide as disclosed herein.
[0201] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as disclosed herein for the preparation
of a medicament for treating, preventing and/or alleviating the
symptoms of disorders susceptible to modulation by a TNF-alpha
modulating substance which is able to pass through the tissues
beneath the tongue.
[0202] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the tissues beneath the tongue
without being inactivated, by administering sublingually to a
subject an anti-TNF-alpha polypeptide as disclosed herein.
[0203] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the bloodstream of a subject
without being inactivated, by administering orally to a subject an
anti-TNF-alpha polypeptide as disclosed herein.
[0204] One embodiment of the present invention is an anti-TNF-alpha
polypeptide as disclosed herein for use in treating, preventing
and/or alleviating the symptoms of disorders susceptible to
modulation by a TNF-alpha modulating substance which is able pass
through the skin effectively.
[0205] Examples of disorders are any that cause inflammation,
including, but not limited to rheumatoid arthritis, Crohn's
disease, ulcerative colitis, inflammatory bowl syndrome, and
multiple sclerosis. A formulation of said polypeptide construct,
for example, a cream, film, spray, drop, patch, is placed on the
skin and passes through.
[0206] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a TNF-alpha modulating substance which is able
pass through the skin effectively, by topically administering to a
subject an anti-TNF-alpha polypeptide as disclosed herein.
[0207] Another embodiment of the present invention is a use of an
anti-TNF-alpha polypeptide as disclosed herein for the preparation
of a medicament for treating, preventing and/or alleviating the
symptoms of disorders susceptible to modulation by a TNF-alpha
modulating substance which is able pass through the skin
effectively.
[0208] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the skin without being
inactivated, by administering topically to a subject an
anti-TNF-alpha polypeptide as disclosed herein.
[0209] An aspect of the invention is a method for delivering a
TNF-alpha modulating substance to the bloodstream of a subject, by
administering topically to a subject an anti-TNF-alpha polypeptide
as disclosed herein.
[0210] In another embodiment of the present invention, an
anti-TNF-alpha polypeptide further comprises a carrier single
domain antibody (e.g. VHH) which acts as an active transport
carrier for transport said anti-TNF-alpha polypeptide, from the
lung lumen to the blood.
[0211] An anti-TNF-alpha polypeptide further comprising a carrier
binds specifically to a receptor present on the mucosal surface
(bronchial epithelial cells) resulting in the active transport of
the polypeptide from the lung lumen to the blood. The carrier
single domain antibody may be fused to the polypeptide construct.
Such fusion constructs may be made using methods known in the art
and are describe herein. The "carrier" single domain antibody binds
specifically to a receptor on the mucosal surface which induces an
active transfer through the surface.
[0212] Another aspect of the present invention is a method to
determine which single domain antibodies (e.g. VHHs) are actively
transported into the bloodstream upon nasal administration.
Similarly, a naive or immune VHH phage library can be administered
nasally, and after different time points after administration,
blood or organs can be isolated to rescue phages that have been
actively transported to the bloodstream. A non-limiting example of
a receptor for active transport from the lung lumen to the
bloodstream is the Fc receptor N (FcRn). One aspect of the
invention includes the VHH molecules identified by the method. Such
VHH can then be used as a carrier VHH for the delivery of a
therapeutic VHH to the corresponding target in the bloodstream upon
nasal administration.
[0213] In one aspect of the invention, one can use an
anti-TNF-alpha polypeptide as disclosed herein, in order to screen
for agents that modulate the binding of the polypeptide to
TNF-alpha. When identified in an assay that measures binding or
said polypeptide displacement alone, agents will have to be
subjected to functional testing to determine whether they would
modulate the action of the antigen in vivo. Examples of screening
assays are given below primarily in respect of SEQ ID NO: 3, though
any anti-TNF-alpha polypeptide as disclosed herein as disclosed
herein may be appropriate.
[0214] In an example of a displacement experiment, phage or cells
expressing TNF-alpha or a fragment thereof are incubated in binding
buffer with, for example, a polypeptide represented by SEQ ID NO: 3
which has been labeled, in the presence or absence of increasing
concentrations of a candidate modulator. To validate and calibrate
the assay, control competition reactions using increasing
concentrations of said polypeptide and which is unlabeled, can be
performed. After incubation, cells are washed extensively, and
bound, labeled polypeptide is measured as appropriate for the given
label (e.g., scintillation counting, fluorescence, etc.). A
decrease of at least 10% in the amount of labeled polypeptide bound
in the presence of candidate modulator indicates displacement of
binding by the candidate modulator. Candidate modulators are
considered to bind specifically in this or other assays described
herein if they displace 50% of labeled polypeptide (sub-saturating
polypeptide dose) at a concentration of 1 .mu.M or less.
[0215] Alternatively, binding or displacement of binding can be
monitored by surface plasmon resonance (SPR). Surface plasmon
resonance assays can be used as a quantitative method to measure
binding between two molecules by the change in mass near an
immobilized sensor caused by the binding or loss of binding of, for
example, the polypeptide represented by SEQ ID NO: 3 from the
aqueous phase to TNF-alpha immobilized in a membrane on the sensor.
This change in mass is measured as resonance units versus time
after injection or removal of the said polypeptide or candidate
modulator and is measured using a Biacore Biosensor (Biacore AB).
TNF-alpha can be for example immobilized on a sensor chip (for
example, research grade CM5 chip; Biacore AB) in a thin film lipid
membrane according to methods described by Salamon et al. (Salamon
et al., 1996, Biophys J. 71: 283-294; Salamon et al., 2001,
Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends Biochem.
Sci. 24: 213-219, each of which is incorporated herein by
reference). Sarrio et al. demonstrated that SPR can be used to
detect ligand binding to the GPCR A(1) adenosine receptor
immobilized in a lipid layer on the chip (Sarrio et al., 2000, Mol.
Cell. Biol. 20: 5164-5174, incorporated herein by reference).
Conditions for the binding of SEQ ID NO:3 to TNF-alpha in an SPR
assay can be fine-tuned by one of skill in the art using the
conditions reported by Sarrio et al. as a starting point.
[0216] SPR can assay for modulators of binding in at least two
ways. First, a polypeptide represented by SEQ ID NO: 3, for
example, can be pre-bound to immobilized TNF-alpha followed by
injection of candidate modulator at a concentration ranging from
0.1 nM to 1 .mu.M. Displacement of the bound polypeptide can be
quantitated, permitting detection of modulator binding.
Alternatively, the membrane-bound TNF-alpha can be pre-incubated
with a candidate modulator and challenged with, for example, a
polypeptide represented by SEQ ID NO: 3. A difference in binding
affinity between said polypeptide and TNF-alpha pre-incubated with
the modulator, compared with that between said polypeptide and
TNF-alpha in absence of the modulator will demonstrate binding or
displacement of said polypeptide in the presence of modulator. In
either assay, a decrease of 10% or more in the amount of said
polypeptide bound in the presence of candidate modulator, relative
to the amount of said polypeptide bound in the absence of candidate
modulator indicates that the candidate modulator inhibits the
interaction of TNF-alpha and said polypeptide.
[0217] Another method of detecting inhibition of binding of, for
example, a polypeptide represented by SEQ ID NO: 3, to TNF-alpha
uses fluorescence resonance energy transfer (FRET). FRET is a
quantum mechanical phenomenon that occurs between a fluorescence
donor (D) and a fluorescence acceptor (A) in close proximity to
each other (usually <100 .ANG. of separation) if the emission
spectrum of D overlaps with the excitation spectrum of A. The
molecules to be tested, e.g. a polypeptide represented by SEQ ID
NO: 3 and a TNF-alpha are labelled with a complementary pair of
donor and acceptor fluorophores. While bound closely together by
the TNF-alpha: polypeptide interaction, the fluorescence emitted
upon excitation of the donor fluorophore will have a different
wavelength from that emitted in response to that excitation
wavelength when the said polypeptide and TNF-alpha are not bound,
providing for quantitation of bound versus unbound molecules by
measurement of emission intensity at each wavelength. Donor
fluorophores with which to label the TNF-alpha are well known in
the art. Of particular interest are variants of the A. Victoria GFP
known as Cyan FP (CFP, Donor (D)) and Yellow FP (YFP, Acceptor
(A)). As an example, the YFP variant can be made as a fusion
protein with TNF-alpha. Vectors for the expression of GFP variants
as fusions (Clontech) as well as fluorophore-labeled reagents
(Molecular Probes) are known in the art. The addition of a
candidate modulator to the mixture of fluorescently-labelled
polypeptide and YFP-TNF-alpha will result in an inhibition of
energy transfer evidenced by, for example, a decrease in YFP
fluorescence relative to a sample without the candidate modulator.
In an assay using FRET for the detection of TNF-alpha: polypeptide
interaction, a 10% or greater decrease in the intensity of
fluorescent emission at the acceptor wavelength in samples
containing a candidate modulator, relative to samples without the
candidate modulator, indicates that the candidate modulator
inhibits the TNF-alpha:polypeptide interaction.
[0218] A sample as used herein may be any biological sample
containing TNF-alpha such as clinical (e.g. cell fractions, whole
blood, plasma, serum, tissue, cells, etc.), derived from clinical,
agricultural, forensic, research, or other possible samples. The
clinical samples may be from human or animal origin. The sample
analysed can be both solid or liquid in nature. It is evident when
solid materials are used, these are first dissolved in a suitable
solution.
[0219] A variation on FRET uses fluorescence quenching to monitor
molecular interactions. One molecule in the interacting pair can be
labelled with a fluorophore, and the other with a molecule that
quenches the fluorescence of the fluorophore when brought into
close apposition with it. A change in fluorescence upon excitation
is indicative of a change in the association of the molecules
tagged with the fluorophore:quencher pair. Generally, an increase
in fluorescence of the labelled TNF-alpha is indicative that
anti-TNF-alpha polypeptide bearing the quencher has been displaced.
For quenching assays, a 10% or greater increase in the intensity of
fluorescent emission in samples containing a candidate modulator,
relative to samples without the candidate modulator, indicates that
the candidate modulator inhibits TNF-alpha: anti-TNF-alpha
polypeptide interaction.
[0220] In addition to the surface plasmon resonance and FRET
methods, fluorescence polarization measurement is useful to
quantitate binding. The fluorescence polarization value for a
fluorescently-tagged molecule depends on the rotational correlation
time or tumbling rate. Complexes, such as those formed by TNF-alpha
associating with a fluorescently labelled anti-TNF-alpha
polypeptide, have higher polarization values than uncomplexed,
labelled polypeptide. The inclusion of a candidate inhibitor of the
TNF-alpha:anti-TNF-alpha polypeptide interaction results in a
decrease in fluorescence polarization, relative to a mixture
without the candidate inhibitor, if the candidate inhibitor
disrupts or inhibits the interaction of TNF-alpha with said
polypeptide. Fluorescence polarization is well suited for the
identification of small molecules that disrupt the formation of
TNF-alpha:anti-TNF-alpha polypeptide complexes. A decrease of 10%
or more in fluorescence polarization in samples containing a
candidate modulator, relative to fluorescence polarization in a
sample lacking the candidate modulator, indicates that the
candidate modulator inhibits the TNF-alpha:anti-TNF-alpha
polypeptide interaction.
[0221] Another alternative for monitoring TNF-alpha:anti-TNF-alpha
polypeptide interactions uses a biosensor assay. ICS biosensors
have been described in the art (Australian Membrane Biotechnology
Research Institute; Cornell B, Braach-Maksvytis V, King L, Osman P,
Raguse B, Wieczorek L, and Pace R. "A biosensor that uses
ion-channel switches" Nature 1997, 387, 580). In this technology,
the association of TNF-alpha and a anti-TNF-alpha polypeptide is
coupled to the closing of gramacidin-facilitated ion channels in
suspended membrane bilayers and thus to a measurable change in the
admittance (similar to impedence) of the biosensor. This approach
is linear over six orders of magnitude of admittance change and is
ideally suited for large scale, high throughput screening of small
molecule combinatorial libraries. A 10% or greater change (increase
or decrease) in admittance in a sample containing a candidate
modulator, relative to the admittance of a sample lacking the
candidate modulator, indicates that the candidate modulator
inhibits the interaction of TNF-alpha and said polypeptide. It is
important to note that in assays testing the interaction of
TNF-alpha with an anti-TNF-alpha polypeptide, it is possible that a
modulator of the interaction need not necessarily interact directly
with the domain(s) of the proteins that physically interact with
said polypeptide. It is also possible that a modulator will
interact at a location removed from the site of interaction and
cause, for example, a conformational change in the TNF-alpha.
Modulators (inhibitors or agonists) that act in this manner are
nonetheless of interest as agents to modulate the binding of
TNF-alpha to its receptor.
[0222] Any of the binding assays described can be used to determine
the presence of an agent in a sample, e.g., a tissue sample, that
binds to TNF-alpha, or that affects the binding of, for example, a
polypeptide represented by SEQ ID NO: 3 to the TNF-alpha. To do so
a TNF-alpha is reacted with said polypeptide in the presence or
absence of the sample, and polypeptide binding is measured as
appropriate for the binding assay being used. A decrease of 10% or
more in the binding of said polypeptide indicates that the sample
contains an agent that modulates the binding of said polypeptide to
the TNF-alpha. Of course, the above-generalized method might easily
be applied to screening for candidate modulators which alter the
binding between any anti-TNF-alpha polypeptide of the invention, an
homologous sequence thereof, a functional portion thereof or a
functional portion of an homologous sequence thereof, and TNF-alpha
or a fragment thereof.
[0223] One embodiment of the present invention is an unknown agent
identified by the method disclosed herein.
[0224] One embodiment of the present invention is an unknown agent
identified by the method disclosed herein for use in treating,
preventing and/or alleviating the symptoms of disorders relating to
inflammatory processes.
[0225] Another embodiment of the present invention is a use of an
unknown agent identified by the method disclosed herein for use in
treating, preventing and/or alleviating the symptoms of disorders
relating to inflammatory processes.
[0226] Examples of disorders include rheumatoid arthritis, Crohn's
disease, ulcerative colitis, inflammatory bowel syndrome and
multiple sclerosis
[0227] A cell that is useful according to the invention is
preferably selected from the group consisting of bacterial cells
such as, for example, E. coli, yeast cells such as, for example, S.
cerevisiae, P. pastoris, insect cells or mammal cells.
[0228] A cell that is useful according to the invention can be any
cell into which a nucleic acid sequence encoding a polypeptide
comprising an anti-TNF-alpha of the invention, an homologous
sequence thereof, a functional portion thereof or a functional
portion of an homologous sequence thereof according to the
invention can be introduced such that the polypeptide is expressed
at natural levels or above natural levels, as defined herein.
Preferably a polypeptide of the invention that is expressed in a
cell exhibits normal or near normal pharmacology, as defined
herein. Most preferably a polypeptide of the invention that is
expressed in a cell comprises the nucleotide sequence capable of
encoding any one of the amino acid sequences presented in Table 1
or capable of encoding an amino acid sequence that is at least 70%
identical to the amino acid sequence presented in Table 1.
[0229] According to a preferred embodiment of the present
invention, a cell is selected from the group consisting of
COS7-cells, a CHO cell, a LM (TK-) cell, a NIH-3T3 cell, HEK-293
cell, K-562 cell or a 1321N1 astrocytoma cell but also other
transfectable cell lines.
[0230] In general, "therapeutically effective amount",
"therapeutically effective dose" and "effective amount" means the
amount needed to achieve the desired result or results (modulating
TNF-alpha binding; treating or preventing inflammation). One of
ordinary skill in the art will recognize that the potency and,
therefore, an "effective amount" can vary for the various compounds
that modulate TNF-alpha binding used in the invention. One skilled
in the art can readily assess the potency of the compound.
[0231] As used herein, the term "compound" refers to an
anti-TNF-alpha polypeptide of the present invention, a composition,
or a nucleic acid capable of encoding said polypeptide or an agent
identified according to the screening method described herein or
said polypeptide comprising one or more derivatised amino
acids.
[0232] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be administered to an individual along with the compound without
causing any undesirable biological effects or interacting in a
deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained.
[0233] Anti-TNF-alpha polypeptides as disclosed herein is useful
for treating or preventing conditions in a subject and comprises
administering a pharmaceutically effective amount of a compound or
composition.
[0234] Anti-TNF polypeptides of the present invention are useful
for treating or preventing conditions relating to rheumatoid
arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel
syndrome and multiple sclerosis in a subject and comprises
administering a pharmaceutically effective amount of a compound or
composition that binds TNF-alpha.
[0235] Anti-TNF-alpha polypeptides as disclosed here in are useful
for treating or preventing conditions in a subject and comprises
administering a pharmaceutically effective amount of a compound
combination with another, such as, for example, aspirin.
[0236] The anti-TNF-alpha polypeptides as disclosed here in are
useful for treating or preventing conditions relating to rheumatoid
arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel
syndrome and multiple sclerosis in a subject and comprises
administering a pharmaceutically effective amount of a compound
combination with another, such as, for example, aspirin.
[0237] The present invention is not limited to the administration
of formulations comprising a single compound of the invention. It
is within the scope of the invention to provide combination
treatments wherein a formulation is administered to a patient in
need thereof that comprises more than one compound of the
invention.
[0238] Conditions mediated by TNF-alpha include, but are not
limited rheumatoid arthritis, Crohn's disease, ulcerative colitis,
inflammatory bowel syndrome and multiple sclerosis.
[0239] A compound useful in the present invention can be formulated
as pharmaceutical compositions and administered to a mammalian
host, such as a human patient or a domestic animal in a variety of
forms adapted to the chosen route of administration, i.e., orally
or parenterally, by intranassally by inhalation, intravenous,
intramuscular, topical or subcutaneous routes.
[0240] A compound of the present invention can also be administered
using gene therapy methods of delivery. See, e.g., U.S. Pat. No.
5,399,346, which is incorporated by reference in its entirety.
Using a gene therapy method of delivery, primary cells transfected
with the gene for the compound of the present invention can
additionally be transfected with tissue specific promoters to
target specific organs, tissue, grafts, tumors, or cells.
[0241] Thus, the present compound may be systemically administered,
e.g., orally, in combination with a pharmaceutically acceptable
vehicle such as an inert diluent or an assimilable edible carrier.
They may be enclosed in hard or soft shell gelatin capsules, may be
compressed into tablets, or may be incorporated directly with the
food of the patient's diet. For oral therapeutic administration,
the active compound may be combined with one or more excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 0.1% of
active compound. The percentage of the compositions and
preparations may, of course, be varied and may conveniently be
between about 2 to about 60% of the weight of a given unit dosage
form. The amount of active compound in such therapeutically useful
compositions is such that an effective dosage level will be
obtained.
[0242] The tablets, troches, pills, capsules, and the like may also
contain the following: binders such as gum tragacanth, acacia, corn
starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound may be incorporated into sustained-release
preparations and devices.
[0243] The active compound may also be administered intravenously
or intraperitoneally by infusion or injection. Solutions of the
active compound or its salts can be prepared in water, optionally
mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0244] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form must be sterile,
fluid and stable under the conditions of manufacture and storage.
The liquid carrier or vehicle can be a solvent or liquid dispersion
medium comprising, for example, water, ethanol, a polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycols,
and the like), vegetable oils, nontoxic glyceryl esters, and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the formation of liposomes, by the maintenance of
the required particle size in the case of dispersions or by the use
of surfactants. The prevention of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. In many cases, it will be preferable to
include isotonic agents, for example, sugars, buffers or sodium
chloride. Prolonged absorption of the injectable compositions can
be brought about by the use in the compositions of agents delaying
absorption, for example, aluminum monostearate and gelatin.
[0245] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze
drying techniques, which yield a powder of the active ingredient
plus any additional desired ingredient present in the previously
sterile-filtered solutions.
[0246] For topical administration, the present compound may be
applied in pure form, i.e., when they are liquids. However, it will
generally be desirable to administer them to the skin as
compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0247] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, hydroxyalkyls or
glycols or water-alcohol/glycol blends, in which the present
compound can be dissolved or dispersed at effective levels,
optionally with the aid of non-toxic surfactants. Adjuvants such as
fragrances and additional antimicrobial agents can be added to
optimize the properties for a given use.
[0248] The resultant liquid compositions can be applied from
absorbent pads, used to impregnate bandages and other dressings, or
sprayed onto the affected area using pump-type or aerosol
sprayers.
[0249] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0250] Examples of useful dermatological compositions which can be
used to deliver the compound to the skin are known to the art; for
example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.
Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and
Wortzman (U.S. Pat. No. 4,820,508).
[0251] Useful dosages of the compound can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art; for example, see
U.S. Pat. No. 4,938,949.
[0252] Generally, the concentration of the compound(s) in a liquid
composition, such as a lotion, will be from about 0.1-25 wt-%,
preferably from about 0.5-10 wt-%. The concentration in a
semi-solid or solid composition such as a gel or a powder will be
about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
[0253] The amount of the compound, or an active salt or derivative
thereof, required for use in treatment will vary not only with the
particular salt selected but also with the route of administration,
the nature of the condition being treated and the age and condition
of the patient and will be ultimately at the discretion of the
attendant physician or clinician. Also the dosage of the compound
varies depending on the target cell, tumor, tissue, graft, or
organ.
[0254] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0255] An administration regimen could include long-term, daily
treatment. By "long-term" is meant at least two weeks and
preferably, several weeks, months, or years of duration. Necessary
modifications in this dosage range may be determined by one of
ordinary skill in the art using only routine experimentation given
the teachings herein. See Remington's Pharmaceutical Sciences
(Martin, E. W., ed. 4), Mack Publishing Co., Easton, Pa. The dosage
can also be adjusted by the individual physician in the event of
any complication. The invention provides for an agent that is a
modulator of TNF-alpha/TNF-alpha-receptor interactions.
[0256] The candidate agent may be a synthetic agent, or a mixture
of agents, or may be a natural product (e.g. a plant extract or
culture supernatant). A candidate agent according to the invention
includes a small molecule that can be synthesized, a natural
extract, peptides, proteins, carbohydrates, lipids etc.
[0257] Candidate modulator agents from large libraries of synthetic
or natural agents can be screened. Numerous means are currently
used for random and directed synthesis of saccharide, peptide, and
nucleic acid based agents. Synthetic agent libraries are
commercially available from a number of companies including
Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex
(Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and
Microsource (New Milford, Conn.). A rare chemical library is
available from Aldrich (Milwaukee, Wis.). Combinatorial libraries
are available and can be prepared. Alternatively, libraries of
natural agents in the form of bacterial, fungal, plant and animal
extracts are available from e.g., Pan Laboratories (Bothell, Wash.)
or MycoSearch (NC), or are readily producible by methods well known
in the art. Additionally, natural and synthetically produced
libraries and agents are readily modified through conventional
chemical, physical, and biochemical means.
[0258] Useful agents may be found within numerous chemical classes.
Useful agents may be organic agents, or small organic agents. Small
organic agents have a molecular weight of more than 50 yet less
than about 2,500 daltons, preferably less than about 750, more
preferably less than about 350 daltons. Exemplary classes include
heterocycles, peptides, saccharides, steroids, and the like. The
agents may be modified to enhance efficacy, stability,
pharmaceutical compatibility, and the like. Structural
identification of an agent may be used to identify, generate, or
screen additional agents. For example, where peptide agents are
identified, they may be modified in a variety of ways to enhance
their stability, such as using an unnatural amino acid, such as a
D-amino acid, particularly D-alanine, by functionalizing the amino
or carboxylic terminus, e.g. for the amino group, acylation or
alkylation, and for the carboxyl group, esterification or
amidification, or the like.
[0259] For primary screening, a useful concentration of a candidate
agent according to the invention is from about 10 mM to about 100
.mu.M or more (i.e. 1 mM, 10 mM, 100 mM, 1 M etc.). The primary
screening concentration will be used as an upper limit, along with
nine additional concentrations, wherein the additional
concentrations are determined by reducing the primary screening
concentration at half-log intervals (e.g. for 9 more
concentrations) for secondary screens or for generating
concentration curves.
High Throughput Screening Kit
[0260] A high throughput screening kit according to the invention
comprises all the necessary means and media for performing the
detection of an agent that modulates TNF-alpha/TNF-alpha receptor
interactions by interacting with TNF-alpha in the presence of a
polypeptide, preferably at a concentration in the range of 1 .mu.M
to 1 mM.
[0261] The kit comprises the following. Recombinant cells of the
invention, comprising and expressing the nucleotide sequence
encoding TNF-alpha, which are grown according to the kit on a solid
support, such as a microtiter plate, more preferably a 96 well
microtiter plate, according to methods well known to the person
skilled in the art especially as described in WO 00/02045.
Alternatively TNF-alpha is supplied in a purified form to be
immobilized on, for example, a 96 well microtiter plate by the
person skilled in the art. Alternatively TNF-alpha is supplied in
the kit pre-immobilized on, for example, a 96 well microtiter
plate. The TNF-alpha may be whole TNF-alpha or a fragment
thereof.
[0262] Modulator agents according to the invention, at
concentrations from about 1 .mu.M to 1 mM or more, are added to
defined wells in the presence of an appropriate concentration of
anti-TNF-alpha polypeptide, an homologous sequence thereof, a
functional portion thereof or a functional portion of an homologous
sequence thereof, said concentration of said polypeptide preferably
in the range of 1 .mu.M to 1 mM. Kits may contain one or more
anti-TNF-alpha polypeptide (e.g. one or more of a polypeptide
represented by any of the SEQ ID NOs: 1 to 15 or other
anti-TNF-alpha polypeptides, an homologous sequence thereof, a
functional portion thereof or a functional portion of an homologous
sequence thereof).
[0263] Binding assays are performed as according to the methods
already disclosed herein and the results are compared to the
baseline level of, for example TNF-alpha binding to an
anti-TNF-alpha polypeptide, an homologous sequence thereof, a
functional portion thereof or a functional portion of an homologous
sequence thereof, but in the absence of added modulator agent.
Wells showing at least 2 fold, preferably 5 fold, more preferably
10 fold and most preferably a 100 fold or more increase or decrease
in TNF-alpha-polypeptide binding (for example) as compared to the
level of activity in the absence of modulator, are selected for
further analysis.
Other Kits Useful According to the Invention
[0264] The invention provides for kits useful for screening for
modulators of TNF-alpha/TNF-alpha receptor binding, as well as kits
useful for diagnosis of disorders characterised by dysfunction of
TNF-alpha. The invention also provides for kits useful for
screening for modulators of disorders as well as kits for their
diagnosis, said disorders characterised by one or more process
involving TNF-alpha. Kits useful according to the invention can
include an isolated TNF-alpha. Alternatively, or in addition, a kit
can comprise cells transformed to express TNF-alpha. In a further
embodiment, a kit according to the invention can comprise a
polynucleotide encoding TNF-alpha. In a still further embodiment, a
kit according to the invention may comprise the specific primers
useful for amplification of TNF-alpha. Kits useful according to the
invention can comprise an isolated TNF-alpha polypeptide, a
homologue thereof, or a functional portion thereof. A kit according
to the invention can comprise cells transformed to express said
polypeptide. Kits may contain more than one polypeptide. In a
further embodiment, a kit according to the invention can comprise a
polynucleotide encoding TNF-alpha. In a still further embodiment, a
kit according to the invention may comprise the specific primers
useful for amplification of a macromolecule such as, for example,
TNF-alpha. All kits according to the invention will comprise the
stated items or combinations of items and packaging materials
therefore. Kits will also include instructions for use.
EXAMPLES
[0265] The invention is illustrated by the following non-limiting
examples.
Example 1
Example of Camelidae Antibodies Against Human Tumor Necrosis Factor
Alpha
1) Immunization and Library Constructions
[0266] A llama (Llama glama) was immunized with human TNF-alpha.
For immunization, the cytokine was formulated as an emulsion with
an appropriate, animal-friendly adjuvant (Specoll, CEDI Diagnostics
B.V.). The antigen cocktail was administered by double-spot
injections intramuscularly in the neck. The animal received 6
injections of the emulsion, containing 100 .mu.g of TNF-alpha at
weekly intervals. At different time points during immunization,
10-ml blood samples were collected from the animal and sera were
prepared. The induction of an antigen specific humoral immune
response was verified using the serum samples in an ELISA
experiment with TNF (data not shown). Five days after the last
immunization, a blood sample of 150 ml was collected. From this
sample, conventional and heavy-chain antibodies (HcAbs) were
fractionated (Lauwereys et al. 1998) and used in an ELISA, which
revealed that the HcAbs were responsible for the antigen specific
humoral immune response (data not shown). Peripheral blood
lymphocytes (PBLs), as the genetic source of the llama heavy chain
immunoglobulins (HcAbs), were isolated from the 150-ml blood sample
using a Ficoll-Paque gradient (Amersham Biosciences) yielding
5.times.10.sup.8 PBLs. The maximal diversity of antibodies is
expected to be equal to the number of sampled B-lymphocytes, which
is about 10% of the number of PBLs (5.times.10.sup.7). The fraction
of heavy-chain antibodies in llama is up to 20% of the number of
B-lymphocytes. Therefore, the maximal diversity of HcAbs in the 150
ml blood sample is calculated as 10.sup.7 different molecules.
Total RNA (around 400 .mu.g) was isolated from these cells using an
acid guanidinium thiocyanate extraction (Chomczynski and Sacchi,
1987).
[0267] cDNA was prepared on 100 .mu.g total RNA with M-MLV Reverse
Transcriptase (Gibco BRL) and oligo-dT-primer or hexanucleotide
random primers (Amersham Biosciences) as described before (de Haard
et al., 1999). The cDNA was purified with a phenol/chloroform
extraction combined with an ethanol precipitation and subsequently
used as template to specifically amplify the VHH repertoire.
[0268] The VHH repertoire was amplified using oligo-dT primed cDNA
as template with a single degenerated framework1 (FR1) primer
ABL013 (5'-GAGGTBCARCTGCAGGASTCYGG-3') (SEQ ID NO:98), introducing
a PstI restriction site (in bold), in combination with the oligo-dT
primer as is described in EP01205100.9. This amplification yields
two fragments of 1650 bp and 1300 bp, the latter being the product
derived from the CH1-deleted HcAb genes. The smaller PCR-product
was gel purified and subsequently digested with PstI and BstEII.
The BstEII-site frequently occurs within the FR4 of heavy-chain
derived VHH encoding DNA-fragments.
[0269] Alternatively, the VHH-repertoire was amplified in a
hinge-dependent approach using two IgG specific oligonucleotide
primers. In a single PCR reaction a short
(5'-AACAGTTAAGCTTCCGCTTGCGGCCGCGGAGCTGGGGTCTTCGCTGTGGTGCG-3') (SEQ
ID NO:99) or long (5'-AACAGTTAAGCTTCCGCTTGCGGCCGCTGGTTGTGG
TTTTGGTGTCTTGGGTT-3') (SEQ ID NO:100) hinge primer known to be
specific for HcAbs was combined with the FR1-primer ABL013 (see
above). A PstI and NotI (bold underlined) restriction site was
introduced within the FR1 and hinge primers respectively, to allow
cloning. Subsequently, the DNA fragments were ligated into
PstI-BstEII or PstI-NotI digested phagemid vector pAX004, which is
identical to pHEN1 (Hoogenboom et al., 1991), but encodes a
carboxyterminal (His).sub.6- and c-myc-tag for purification and
detection, respectively. The ligation mixture was desalted on a
Microcon filter (YM-50, Millipore) and electroporated into E. coli
TG1 cells to obtain a library containing 1.8.times.10.sup.7 clones.
The transformed cells were grown overnight at 37.degree. C. on a
single 20.times.20 cm plate with LB containing 100 .mu.g/ml
ampicillin and 2% glucose. The colonies were scraped from plates
using 2.times.TY medium and stored at -80.degree. C. in 20%
glycerol.
[0270] As quality control the percentage of insert containing
clones was verified on 24 clones for each library by PCR using a
combination of vector based primers. This analysis revealed that
95% of the clones contained a VHH encoding insert. The variability
was examined by HinfI fingerprint analysis of the amplified VHH
fragment of these 24 clones, thereby showing that all clones were
indeed different (data not shown).
2) Selection of Antagonistic Anti-TNF VHH's
[0271] From both libraries phage was prepared. To rescue the
polyclonal phage repertoire, libraries were grown to logarithmic
phase (OD600=0.5) at 37.degree. C. in 2.times.TY containing 100
.mu.g/ml ampicillin and 2% glucose and subsequently superinfected
with M13K07 helper phage for 30 minutes at 37.degree. C. Infected
cells were pelleted for 5 minutes at 4000 rpm and resuspended in
2.times.TY containing 100 .mu.g/ml ampicillin and 25 .mu.g/ml
kanamycin. Bacteriophage was propagated by overnight growth at
37.degree. C. and 250 rpm. Overnight cultures were centrifuged for
15 minutes at 4500 rpm and phage was precipitated with one fifth
volume of a [20% polyethyleneglycol 6000, 1.5 M NaCl]-solution by
incubation for 30 minutes on ice. Phage was pelleted by
centrifugation for 15 minutes at 4000.times.g and 4.degree. C.
After resuspension of the phages in PBS, cell debris was pelleted
by centrifugation for 1 minute at maximal speed (15000.times.g) in
microcentrifuge tubes. The supernatant containing the phage
particles was transferred to a new tube and again phage was
precipitated as described above. Phage was dissolved in PBS and
separated from remaining cell debris as mentioned above. The titer
of phage was determined by infection of logarithmic TG1 cells
followed by plating on selective medium.
[0272] The library was selected using in vitro biotinylated
TNF-alpha. The biotinylation was carried out as described by Magni
et al (Anal Biochem 2001, 298, 181-188). The incorporation of
biotin in TNF was evaluated by SDS-PAGE analysis and detection with
Extravidin-alkaline phosphatase conjugate (Sigma). The
functionality of the modified protein was evaluated for its ability
to bind to the solid phase coated recombinant a p75 receptor.
[0273] VHH were selected by capturing biotinylated TNF-alpha (10 to
400 ng per well during 2 hours at room temperature) on streptavidin
coated microtiter plates (coated with 100 .mu.l of 10 .mu.g/ml
streptavidin during 16 hours at +4.degree. C.). Antagonistic VHH
were obtained by elution with an excess of receptor, either the
extracellular ligand binding domain or with cells expressing the
receptor. After 2 hours incubation of phage with captured cytokine,
the non-specific phage was washed away, while specific phage
displaying antagonistic VHH was eluted for 30 minutes with receptor
(extracellular domain of CD120b or p75; 10 .mu.M) or with receptor
displaying cells (>10.sup.5 KYM cells per well). High
enrichments, i.e. the ratio of the number of phage eluted with
receptor and those eluted by serum albumin (50 .mu.g per well), of
more than a factor of 20 suggested the successful selection of
TNF-alpha specific clones. Alternatively, instead of elution with
receptor a standard procedure was applied, in which a low pH causes
the denaturation of VHH and/or antigen (0.1 M glycine buffer pH
2.5). Log phase growing E. coli cells were infected with the eluted
and neutralized phage and plated on selective medium.
[0274] Individual clones were picked and grown in microtiter plate
for the production of VHH in culture supernatant. ELISA screening
with TNF-alpha captured on Extravidin coated plates revealed about
50% positive clones. HinfI-fingerprint analysis showed that 13
different clones were selected, which were grown and induced on 50
ml scale. The sequences of said clones are shown in Table 1.
[0275] Five clones, coded VHH#1A, #2B, #3E, #3G, #7B and #12B, with
different sequences (FIG. 1) were characterized in more detail.
VHH#3E, #3G and #7B are single-domain antibody fragments carrying
the typical hydrophilic residue at position 45 (arginine) and the
phenylalanine to tryptophan substitution in position 47 in FR2
thereby conferring the advantageous characteristics in terms of
solubility. VHH#1A contains the hydrophobic FR2 residues typically
found in double-chain antibodies of human origin or from other
species, but compensating this loss in hydrophilicity by the
charged arginine residue on position 103 that substitutes the
conserved tryptophan residue present in VH from double-chain
antibodies (PCT/EP02/07804). A new class of humanised Camelidae
single-domain antibodies described in this invention is represented
by VHH#2B and VHH#12B, which contains the hydrophobic residues in
FR2 in combination with the hydrophobic residue tryptophan at
position 103. Larger amounts of antibody fragments were expressed
by cultivation on 50 ml scale and purified by IMAC using TALON
resin (Clontech). After dialysis against PBS to remove the eluent
imidazol the amount of VHH was determined by OD280; approximately
300 .mu.g of VHH was obtained from each clone.
[0276] This material was used for determining the sensitivity of
detection of (biotinylated) TNF in ELISA. For this purpose a
streptavidin (10 .mu.g/ml) coated microtiterplate was employed for
capture of biotinylated TNF (1 .mu.g/ml), VHH was diluted in 0.2%
casein/PBS and incubated for 2 hours at room temperature. Bound VHH
was detected with anti-MYC mAB 9E10 (0.5 .mu.g/ml) and anti-mouse
AP conjugate (1000-fold diluted, Sigma). The results are shown in
FIG. 2.
3) Determination of Antagonistic Effect in Cytotoxicity Assay with
KYM Cell Line
[0277] TNF-alpha-induced cytostasis/cytotoxicity was determined by
the colorimetric MTT assay as described by Vandenabeele and
colleagues (Vandenabeele, P., Declercq, W., Vercammen, D., Van de
Craen, M., Grooten, J., Loetscher, H., Brockhaus, M., Lesslauer,
W., Fiers, W. (1992) Functional characterization of the human tumor
necrosis factor receptor p75 in a transfected rat/mouse T cell
hybridoma. J. Exp. Med. 176, 1015-1024). MTT
(3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) is
a pale yellow substrate that is cleaved by living cells to yield a
dark blue formazan product. This process requires active
mitochondria, and even freshly dead cells do not cleave significant
amounts of MTT. KYM cells (Sekiguchi M, Shiroko Y, Suzuki T, Imada
M, Miyahara M, Fujii G. (1985) Characterization of a human
rhabdomyosarcoma cell strain in tissue culture. Biomed.
Pharmacother. 39, 372-380) were seeded in 96 well microtiterplates
and cultured in the presence or absence of TNF-alpha (0.216 ng/ml
or approx. 5 pM of trimer). In addition to TNF variable amounts of
antibody (VHH or Remicade) were included during cultivation. For
the assay MTT was added to the culture medium at a final
concentration of 500 .mu.g/ml and the plates were incubated at
37.degree. C. to achieve cleavage of MTT by mitochondrial enzymes.
The formed formazon product, which appear as black, fuzzy crystals
on the bottom of the well were dissolved by addition of acid
isopropanol (40 nM HCl in isopropanol) or DMSO. The absorbance is
measured at 570 nm.
[0278] The MTT assay (FIG. 3) shows that VHH#1A, which has arginine
on position 103 in combination with the human-like hydrophobic
residues in FR2, has a moderate antagonistic effect (IC50.about.100
nM). VHH#7B with the characteristic hydrophilic residues in FR2
does not prevent binding of TNF-.alpha. to its ligand in spite of
its sensitive detection of the cytokine in ELISA (curve not shown).
In contrast, VHH#3E and #3G with hydrophilic FR2 hallmark residues
are very potent antagonistic VHH's (IC50 of 20 nM). VHH#3E and #3G
have a high degree of homology and are clonally related (Harmsen et
al., Mol. Immunol. 37, 579-590), but VHH#3E is more potent,
probably due to the fact that it has a higher affinity than VHH#3G
(FIG. 2). The (chimeric) monoclonal antibody Remicade is very
potent (IC50 of 80 pM), but its derived Fab fragment lost most of
this efficacy (IC50 is 3 nM, 30 fold less than the intact mAB).
This clearly shows the avidity effect of the interaction between
the antibody and the cytokine: the mAB with two binding sites
interacts more efficiently with the trimeric TNF molecule via
cooperative binding. VHH fragments are strictly monovalent and
therefore it was speculated that increasing the avidity by
genetically fusing VHH genes might increase their antagonistic
efficacy (see Example 4).
[0279] These experiments show that a new class of human-like VHH
has bona fide binding and functional characteristics, thereby
enabling their application for therapeutic purposes.
Example 2
Humanization of VHH#12B and VHH#3E by Site Directed Mutagenesis
1) Homology Between VHH#3E/VHH#12B and Human Germline Heavy Chain
V-Region DP-47
[0280] Alignment of VHH#12B and a human VH3 germline sequence
(DP-47) revealed a high degree of homology: [0281] 4 AA changes in
FR1 on position 1, 5, 28 and 30 [0282] 5 AA changes in FR3 on
position 74, 76, 83, 84 and 93 [0283] 1 AA change in FR4 on
position 108 as represented in the following sequence alignment in
which DP-47 is SEQ ID NO:101 and VHH#12B is SEQ ID NO:102:
TABLE-US-00001 [0283] DP-47 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS
WVRQAPGKGLEWVS AISGSGGSTYY VHH#12B QVQLQESGGGLVQPGGSLRLSCAASGFEFE
NHWMY WVRQAPGKGLEWVS TVNTNGLITRY DP-47 ADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK ------------- ----------- VHH#12B
ADSVKG RFTISRDNAKYTLYLQMNSLKSEDTAVYYCTK VLPPYSDDSRTNAD
WGQGTQVTVSS
[0284] A specific inhibitor for the TNF-alpha cytokine, with high
homology to the human germline gene DP-47 was therefore an ideal
candidate to further humanize and evaluate the influence of
mutagenesis on inhibition capacity in ELISA.
[0285] Alignment of VHH#3E and a human VH3 germline (DP-47)
revealed the presence of hydrophilic amino acid residues in FR2 of
VHH#3E compared to hydrophobic residues in DP-47.
TABLE-US-00002 DP-47 EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS-----
WVRQAPGKGLEWVS AISGSGGSTYY VHH#3E QVQLQESGGGLVQPGGSLRLSCAASGRTFS
DHSGYTYTIG WFRQAPGKEREFVA RIYWSSGNTYY DP-47 ADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK ------------- ----------- VHH#3E
ADSVKG RFAISRDIAKNTVDLTMNNLEPEDTAVYYCAA RDGIPTSRSVESYNY
WGQGTQVTVSS
[0286] Evaluation of the effect of substituting the hydrophilic by
hydrophobic residues as present in human VH is important, since the
majority of camelid VHH sequences contain hydrophilic residues.
2) Mutagenesis of VHH#12B
[0287] VHH#12B was mutated by using a non-PCR based site-directed
mutagenesis method as described by Chen and Ruffner and
commercialized by Stratagene (Quickchange site-directed
mutagenesis). Plasmid DNA was used as template in combination with
2 mutagenic primers introducing the desired mutation(s). The 2
primers are each complementary to opposite strands of the template
plasmid DNA. In a polymerase reaction using the Pfu DNA polymerase
each strand is extended from the primer sequence during a cycling
program using a limited number of cycles. This results in a mixture
of wild type and mutated strands.
[0288] Digestion with DpnI results in selection of the mutated in
vitro synthesized DNA strand, since only the template strand is
sensitive for digestion. The DNA was precipitated and transformed
to E. coli and analyzed for the required mutation by sequence
analysis. The generated mutant VHH's and the mutagenic primers are
listed in Table 2.
[0289] Plasmid was prepared from mutant clones and was transformed
into WK6 electrocompetent cells. A single colony was used to start
an overnight culture in LB containing 2% glucose and 100 .mu.g/ml
ampicillin. This overnight culture was diluted 100-fold in 300 ml
TB medium containing 100 .mu.g/ml ampicillin, and incubated at
37.degree. C. until OD600 nm=2, when 1 mM IPTG and 5 mM MgSO.sub.4
(final concentrations) was added and the culture was incubated for
3 more hours at 37.degree. C.
[0290] Cultures were centrifuged for 20 minutes at 4,500 rpm at
4.degree. C. The pellet was frozen overnight or for 1 hour at
-20.degree. C. Next, the pellet was thawed at room temperature for
40 minutes, re-suspended in 20 ml PBS/1 mM EDTA/1M NaCl and shaken
on ice for 1 hour. Periplasmic fraction was isolated by
centrifugation for 20 minutes at 4.degree. C. at 4,500 rpm. The
supernatant containing the VHH was loaded on TALON (ClonTech) and
purified to homogeneity. The yield of VHH was determined using the
calculated extinction coefficient.
[0291] All mutant VHH's expressed comparable to the wild type. The
mutants were analyzed for their inhibition capacity in an in vitro
receptor binding assay.
[0292] A microtiter plate was coated overnight at 4.degree. C. with
Enbrel (Wyeth) at 2 .mu.g/ml in PBS. The plate was washed five
times with PBS-Tween and blocked for 1 hour at room temperature
with PBS containing 1% casein. The plate was washed five times with
PBS-Tween. Biotinylated human TNF-alpha (80 .mu.g/ml) was
pre-incubated with a dilution series of mutant or wild type VHH#12B
for 1 hr at RT and the mixture was incubated for 1 hr at room
temperature in the wells of the microtiterplate. The plate was
washed five times with PBS-Tween. Bound human TNF-.alpha. was
detected using Extravidin-AP (1/1,000 dilution) and
paranitrophenylphosphate (pNPP). Signals were measured after 30
minutes at 405 nm. The results are presented in FIGS. 4 and 5. The
IC50 increased 3-fold from 66 nM (wild type) to 200 nM (mutant
Q1E+Q5L+A74S+Y76N+K83R+P84A). Mutation of position T93A resulted in
loss of inhibition (data not shown). The positions that still need
to be humanized are: E28, E30 and Q108. However, E28 and E30 are
part of the H1 canonical structure and thus part of the CDR1
according to Chothia numbering system.
[0293] The amino acid sequences of mutant VHHs are presented in
Table 4 SEQ ID NOs: 17 to 19.
3) Mutagenesis of VHH#3E
[0294] VHH#3E was mutated by using a non-PCR based site-directed
mutagenesis method as described above. The obtained mutant VHH's
and the mutagenic primers are listed in Table 3.
[0295] All mutant VHH's expressed comparable to the wild type. The
purified mutant VHH's were analyzed for binding in ELISA and
inhibition capacity in receptor binding assay identical to the
method described above.
[0296] The results of the ELISA are shown in FIG. 6, those from the
receptor binding assay in FIG. 7.
[0297] The amino acid sequences of mutant VHHs are presented in
Table 4 SEQ ID NOs 21 to 24.
Example 3
Isolation of Antagonistic VHH Against Mouse TNF-Alpha
1) Selection of Anti-Mouse TNF-Alpha VHH
[0298] In order to perform efficacy studies in mouse models for IBD
or Crohn's disease mouse TNF specific VHH were selected. Therefore
a llama was immunized with mouse TNF-alpha as described in Example
1. RNA was extracted from PBL's sampled 4 and 10 days after the
last immunization, as well as from a biopsy taken from a lymph node
after day 4. Total RNA was converted in either random primed or
oligo-dT primed cDNA and used as template for the amplification of
the VHH encoding gene segments using Ig derived primers or a
combination of oligo-dT primer and a single Ig primer (see example
1). With the Ig primers a library containing 8.5.times.10.sup.7
clones was generated from the first PBL's, and a library with
7.times.10.sup.6 clones for the second PBL sample and
5.8.times.10.sup.8 clones for the lymph node. Using the combination
of the oligo-dT primer and the Ig primer libraries from the first
PBL sample were made containing 1.2.times.10.sup.8 clones, from the
second sample of PBL's a library of 5.7.times.10.sup.7 clones and
the lymph node derived library contained 2.times.10.sup.8 clones.
The libraries were pooled dependent on the used combination of
primers and the resulting two libraries were grown for propagation
of phage as was described before. Selections were performed on
biotinylated mouse TNF-alpha captured on coated streptavidin, bound
phage was eluted by competition with the human receptor p75, which
is known to cross-react with mouse TNF-alpha. Two distinct mouse
TNF-alpha specific VHH (VHH#m3F and VHH#m9E) were selected from the
library obtained by amplification with Ig derived primers, while
two closely related VHH's were retrieved from the library
constructed by PCR with oligo-dT primer and Ig-primer (FIG. 8).
2) Determination Antagonistic Efficacy in Cytotoxicity Assay with
L929 Cell Line (FIG. 9)
[0299] The same type of assay was applied as described in Example
1, but with the murine cell line L929. VHH#m3F and VHH#m4B (FIG. 9)
turned out to be 10-fold more potent then the other two VHH's.
Example 4
Enhancing the Antagonistic Efficacy by Increasing the Avidity Using
Multivalent Camelidae Antibodies
1) Antagonistic Efficacy of Bi-, Tri- and Tetravalent VHH Against
Human and Mouse TNF-Alpha
[0300] The E. coli production vector pAX11 (FIG. 10) was designed,
which allows the two-step cloning of bivalent or bispecific VHH.
The carboxy terminal VHH is cloned first with PstI and BstEII,
while in the second step the other VHH is inserted by SfiI and
NotI, which do not cut within the first gene fragment. The
procedure avoids the enforcement of new sites by amplification and
thus the risk of introducing PCR errors.
[0301] With this vector the bivalent derivative of the antagonistic
anti-human TNF-alpha VHH#3E was generated. The plasmid vector
encoding the bivalent VHH was used to generate a tri- and
tetrameric derivative, which was accomplished by partial digestion
of the plasmid with BstEII, which occurs in both VHH gene segments.
The linearized vector was purified from gel, subsequently
de-phosphorylated and used as acceptor for cloning of the BstEII
fragment of approx. 350 bp that was obtained by complete digestion
of the same plasmid. Ligation of the BstEII fragment alone prior to
addition to the vector enhances the insertion of multimeric VHH
encoding gene segments. After transformation in E. coli TG1 the
resulting clones were screened by PCR with M13Rev and M13Fwd
primers; since BstEII is an a-symmetric cutter (5 nt overhang) only
correctly oriented inserts were obtained as was confirmed by
digesting the plasmids with PstI alone (350 bp) or double digesting
with EcoRI and HindIII (1000 bp for bivalent (BIV 3E, SEQ ID NO:
73), 1350 bp for trivalent (TRI 3E, SEQ ID NO: 74) and 1700 bp for
tetravalent (TETRA 3E, SEQ ID NO: 75), data not shown). The
sequences are listed in Table 7.
[0302] The clones were grown and induced on 50 ml scale,
periplasmic fractions prepared and used for IMAC purification with
TALON resin. Analysis of the purified products on Coomassie stained
PAGE revealed good production levels (between 2 and 10 mg per liter
cell culture) of intact multivalent VHH (see FIG. 11). The
molecular appearance of the IMAC purified VHH was determined by gel
filtration on a Superdex 75HR column and as expected the molecules
with higher avidities came earlier from the column (see FIG.
12).
[0303] The antagonistic efficacy was analyzed with the cell based
assay using KYM cells. The cells were seeded in microtiterplates
and cultured in the presence or absence of TNF-alpha .quadrature.
(1.29 ng/ml or approx. 25 pM of trimer). The assays (FIG. 13)
revealed that the monovalent molecules used in this study had the
poorest antagonistic characteristics, what is reflected by their
IC50 values: the Fab derived from the chimeric antibody Remicade
has an IC50 of 2 nM and for VHH#3E it is 12 nM (see also FIG. 3).
The avidity of the used molecules turned out to have a dramatic
influence on the antagonistic efficacy as was observed with the
bivalent IgG molecule Remicade, which is 40-fold more effective
(IC50 50 pM) than the Fab. TNF-alpha is a trimeric molecule, which
interacts to a dimeric receptor and therefore it can be expected
that the avidity of the IgG permits the mutual binding to two
epitopes on the cytokine and supports the formation of large
complexes as has been described before (Santora et al, Anal.
Biochem. 299, 119-129). Surprisingly, increasing the avidity of the
VHH from monomer to dimer has a far more spectacular effect than
observed with Remicade, since the IC50 of the dimer (30 pM) is 400
fold lower than of the monomer. Increasing the avidity even more
leads to a still better antagonistic behaviour: the trimeric VHH
has an IC50 of 20 pM and the tetravalent format 6 pM. All higher
avidity formats of the VHH are more efficient than Remicade, while
the tetravalent format is even better than Enbrel, which consists
of the extracellular domain of the receptor p75 fused to the Fc of
an IgG and therefore has a bivalent binding mode.
[0304] The same unexpected effect of avidity on antagonistic
behaviour was observed with VHH generated against mouse TNF (FIG.
14). The same type of cytotoxicity assay was performed using MTT as
substrate and mouse TNF-alpha (65 pg/ml or 1.3 pM), but with the
murine cell line L929, which expresses the mouse specific receptor.
Three different antagonistic (monovalent) VHH were identified coded
9E and 3F, of which the first two have IC50's of 25 nM and the
latter 2 nM (see also Example 3). Conversion of 3F into the
bivalent format (BIV#m3F, SEQ ID NO: 76) yielded a 1000 fold
increase in IC50 (2 pM), thereby demonstrating once more that the
increased avidity of the antibody leads to an unexpected
improvement of the antagonistic characteristics.
2) Comparison with VHH-Fc Fusion
[0305] VHH#3E, directed against human TNF, was cloned via PstI and
BstEII in an adapted vector derived from pcDNA3, thereby generating
a genetic fusion to the CH1 deleted Fc portion of human IgG1. After
confirmation by sequencing, the plasmid construct was transfected
to the myeloma cell line NS0. The obtained cell line was grown and
the VHH-Fc fusion was secreted into the culture supernatant. The
product was purified with an anti-human Fc VHH resin and analyzed
on a Coomassie stained gel (FIG. 15). In the presence of DTT the
fusion was visible as a 45 kDa protein, in the absence of DTT the
dimeric molecule with a molecular weight of 90 kDa could be
observed. This dimeric product results from the linkage of two
chains by two disulfide bridges, which originate from cysteine
residues located in the hinge region.
[0306] The VHH-fusion was tested in the bioassay with the human
cell line KYM and turned out to be 5-fold less effective than the
bivalent VHH in spite of the fact that both molecules have the same
avidity and that they both originate from VHH#3E (FIG. 16).
Probably steric hindrance by the bulky Fc tail might cause this
discrepancy.
Example 5
Calculation of Homologies Between Anti-Target-Single Domain
Antibodies of the Invention
[0307] The degree of amino acid sequence homology between
anti-target single domain antibodies of the invention was
calculated using the Bioedit Sequence Alignment Editor. The
calculations indicate the proportion of identical residues between
all of the sequences as they are aligned by ClustalW. (Thompson, J.
D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through
sequence weighting, position specific gap penalties and weight
matrix choice. Nucleic Acids Research, submitted, June 1994). Table
8 indicates the fraction homology between anti-serum albumin VHHs
of the invention. Table 9 indicates the fraction homology between
anti-TNF-alpha VHHs of the invention. Table 10 indicates the
percentage homology between anti-IFN-gamma VHHs of the
invention.
Example 6
Expression of a VHH-CDR3 Fragment of VHH#3E
[0308] The CDR3 region of VHH#3E was amplified by using a sense
primer located in the framework 4 region (Forward:
CCCCTGGCCCCAGTAGTTATACG) (SEQ ID NO:103) and an anti-sense primer
located in the framework 3 region (Reverse: TGTGCAGCAAGAGACGG) (SEQ
ID NO:104).
[0309] In order to clone the CDR-3 fragment in pAX10, a second
round PCR amplification was performed with following primers
introducing the required restriction sites:
TABLE-US-00003 Reverse primer Sfi1: (SEQ ID NO: 105)
GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCAGCAAGAGACGG Forward primer Not1:
(SEQ ID NO: 106) GTCCTCGCAACTGCGCGGCCGCCCCCTGGCCCCAGTAGTTATACG
[0310] The PCR reactions were performed in 50 ml reaction volume
using 50 pmol of each primer. The reaction conditions for the
primary PCR were 11 min at 94.degree. C., followed by 30/60/120 sec
at 94/55/72.degree. C. for 30 cycles, and 5 min at 72.degree. C.
All reaction were performed with 2.5 mM MgCl2, 200 mM dNTP and 1.25
U AmpliTaq God DNA Polymerase (Roche Diagnostics, Brussels,
Belgium).
[0311] After cleavage with Sfi1 and Not1 the PCR product was cloned
in pAX10.
Example 7
Stability Testing of Antibody Fragments Specific for Human
TNF.alpha.
[0312] Orally administered proteins are subject to denaturation at
the acidic pH of the stomach and as well to degradation by pepsin.
We have selected conditions to study the resistance of the VHH#3E
to pepsin which are supposed to mimic the gastric environment.
VHH#3E a VHH specific to human TNF.alpha. was produced as
recombinant protein in E. coli and purified to homogeneity by IMAC
and gel filtration chromatography. The protein concentration after
purification was determined spectrophotometrically by using the
calculated molar extinction coefficient at 280 nm. Diluted
solutions at 100 microgram/ml were prepared in McIlvaine buffer (J.
Biol. Chem. 49, 1921, 183) at pH 2, pH3 and 4 respectively. These
solutions were subsequently incubated for 15 minutes at 37.degree.
C., prior the addition of porcine gastric mucosa pepsin at a 1/30
w/w ratio. Sixty minutes after adding the protease a sample was
collected and immediately diluted 100-fold in PBS pH7.4 containing
0.1% casein to inactivate the pepsin. Seven additional 3-fold
dilutions were prepared from this sample for assessing the presence
of functional antibody fragment by ELISA. Identical dilutions
prepared from an aliquot collected prior the addition of the
protease served as a reference. In the ELISA assay biotinylated
TNF.alpha. was captured in wells of a microtiter plate coated with
neutravidin. For both the pepsin-treated and reference samples
similar serial dilutions of the samples were prepared and 100
microliter of those dilutions were added to the wells. After
incubation for 1 hour the plates were washed. For the detection of
VHH binding to of the captured TNF.alpha. a polyclonal rabbit
anti-VHH antiserum (R42) and an anti-rabbit IgG alkaline
phosphatase conjugate was used. After washing, the plates were
developed with paranitrophenyl phosphate. The data plotted in FIG.
17 shows similar curves for all of the samples exposed to digestive
conditions as well as for the reference samples. This indicates
that the VHH#3E essentially retains its functional activity under
all of the chosen conditions.
Example 8
Oral Administration of an Anti-Human TNF.alpha. Specific VHH in
Mice
[0313] An antibody solution containing the anti-human TNF.alpha.
specific VHH#3E (100 microgram per milliliter in 100-fold diluted
PBS) was prepared. Three mice which were first deprived from
drinking water for 12 hours and subsequently allowed to freely
access the antibody solution during the next two hours. Afterwards
the mice were sacrificed and their stomachs were dissected.
Immediately the content of the stomachs was collected by flushing
the stomach with 500 microliter PBS containing 1% BSA. This flushed
material was subsequently used to prepare serial three-fold
dilutions, starting at a 1/5 dilution from the undiluted material.
One hundred microliter of these samples was transferred to
individual wells of a microtiter plate coated with human
TNF.alpha.. After incubation for 1 hour and following extensive
washing the presence of immuno-reactive material was assessed with
a polyclonal rabbit anti-VHH antiserum (R42) followed by incubation
with an anti-rabbit alkaline-phosphatase conjugate. The ELISA was
developed with paranitrophenyl acetate. The ELISA signals obtained
after 10 minutes clearly demonstrated the presence of functional
VHH#3E in the gastric flushings of these mice. By comparing to the
standard curve we determined the concentration of the functional
antibody fragment in the gastric flushing fluid to be 1.5, 12.6 and
8.6 microgram/ml for the three mice tested.
Example 9
Efficacy in an Animal Model for IBD
1) Animal Model of Chronic Colitis
[0314] The efficacy of bivalent VHH constructs applied via various
routes of administration was assessed in a DSS (dextran sodium
sulfate) induced model of chronic colitis in BALB/c mice. This
model was originally described by Okayasu et al. [Okayasu et al.
Gastroenterology 1990; 98: 694-702] and modified by Kojouharoff et.
al. [G. Kojouharoff et al. Clin. Exp. Immunol. 1997; 107: 353-8].
The animals were obtained from Charles River Laboratories, Germany,
at an age of 11 weeks and kept in the animal facility until they
reached a body weight between 21 and 22 g. Chronic colitis was
induced in the animals by four DSS treatment cycles. Each cycle
consisted of a DSS treatment interval (7 days) where DSS was
provided with the drinking water at a concentration of 5% (w/v) and
a recovery interval (12 days) with no DSS present in the drinking
water. The last recovery period was prolonged from 12 to 21 days to
provide for an inflammation status rather representing a chronic
than an acute inflammation at the time of the treatment. Subsequent
to the last recovery interval the mice were randomly assigned to
groups of 8 mice and treatment with the VHH-constructs was started.
The treatment interval was 2 weeks. One week after the end of the
treatment interval the animals were sacrificed, the intestine was
dissected and histologically examined. The experimental setting is
shown schematically in FIG. 18.
2) VHH Treatment Schedule
[0315] During the VHH treatment period the mice (8 animals per
group) were treated daily for 14 consecutive days with bivalent
VHH#3F (VHH#m3F-VHH#m3F; SEQ ID No. 76) by intra-gastric or
intra-venous application of 100 .mu.g bivalent VHH 3F. An
additional group of animals was treated rectally with the bivalent
VHH#3F every other day for a period of 14 days. In all treatment
groups a dose of 100 .mu.g of the bivalent VHH#3F was applied at a
concentration of 1 mg/ml in a buffered solution. The negative
control groups received 100 .mu.l of PBS under otherwise identical
conditions. The treatment schedule is shown in Table 11.
3) Results
[0316] After the mice were sacrificed the body weight was
determined and the colon was dissected. The length of the dissected
colon was determined and the histology of the colon was assessed by
Haematoxilin-Eosin (HE) stain (standard conditions). As compared to
the negative controls (PBS treatment) the groups treated with
bivalent nanobody 3F showed a prorogued colon length as well as an
improved histological score [G. Kojouharoff et al. Clin. Exp.
Immunol. 1997; 107: 353-8] thereby demonstrating efficacy of the
treatment.
TABLE-US-00004 TABLE 1 Amino acid sequence listing of the peptides
of aspects of present invention directed against TNF-alpha. SEQ ID
NAME NO SEQUENCE VHH#1A 1 QVQLQESGGGLVQPGGSLRLSCATSGFDFSVSWMYW
VRQAPGKGLEWVSEINTNGLITKYVDSVKGRFTISR
DNAKNTLYLQMDSLIPEDTALYYCARSPSGSFRGQG TQVTVSS VHH#7B 2
QVQLQESGGGLVQPGGSLRLSCAASGSIFRVNAMGW
YRQVPGNQREFVAIITSGDNLNYADAVKGRFTISTD
NVKKTVYLQMNVLKPEDTAVYYCNAILQTSRWSIPS NYWGQGTQVTVSS VHH#2B 3
QVQLQESGGGLVQPGGSLRLSCATSGFTFSDYWMYW
VRQAPGKGLEWVSTVNTNGLITRYADSVKGRFTISR
DNAKYTLYLQMNSLKSEDTAVYYCTKVVPPYSDDSR TNADWGQGTQVTVSS VHH#3E 4
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYT
YTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGR
FAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIP TSRSVESYNYWGQGTQVTVSS VHH#3G 5
QVQLQDSGGGLVQAGGSLRLSCAVSGRTFSAHSVYT
MGWFRQAPGKEREFVARIYWSSANTYYADSVKGRFT
ISRDNAKNTVDLLMNSLKPEDTAVYYCAARDGIPTS RTVGSYNYWGQGTQVTVSS VHH#10A 6
QVQLQESGGGLVQPGGSLRLSCAASGSIFRVNAMGW
YRQVPGNQREFVAIITSSDTNDTTNYADAVKGRFTI
STDNVKKTVYLQMNVLKPEDTAVYYCNAVLQTSRWS IPSNYWGQGTQVTVSS VHH#2G 7
QVQLQDSGGGLVQAGGSLRLSCTTSGRTISVYAMGW
FRQAPGKEREFVASISGSGAITPYADSVKGRFTISR
DNAKNTVYLQMNSLNPEDTAVYYCAASRYARYRDVH AYDYWGQGTQVTVSS VHH#1F 8
QVQLQDSGGGLVQAGGSLRLSCAASTRTFSRYVVGW
FRQAPGKEREFVATISWNGEHTYYADSVKGRYTISR
DNAKNTVYLQMGSLKPEDTAVYYCAARSFWGYNVEQ RDFGSWGQGTPVTVSS VHH#9C 9
QVQLQESGGGLVQPGGSLRLSCAASGSIFRVNAMGW
YRQVPGNQREFVAIITNDTTNYADAVKGRFTISTDN
VKKTVYLQMNVLKPEDTAVYYCNTVLQTSRWNIPTN YWGQGTQVTVSS VHH#11E 10
QVQLQESGGGLVQPGGSLRLSCAASGSIFRVNAMGW
YRQVPGNQREFVAIISGDTTNYADAVKGRFTISTDN
VKKTVYLQMNVLESEDTAVYYCNAVLQTSRWSIPSN YWGQGTQVTVSS VHH#10C 11
QVQLQDSGGGLVQPGGSLRLACVASGSIFSIDVMGW
YRQAPGQQRELVATITNSWTTNYADSVKGRFTISRD
NAKNVVYLQMNSLKLEDTAVYYCNARRWYQPEAWGQ GTQVTVSS VHH#4B 12
QVQLQDSGGGLVQPGGSLRLSCAASGFTFSTHWMYW
VRQAPGKGLEWVSTINTNGLITDYIHSVKGRFTISR
DNAKNTLYLQMNSLKSEDTAVYYCALNQAGLSRGQG TQVTVSS VHH#10D 13
QVQLQESGGGLVQAGGSLRLSCAASRRTFSGYAMGW
FRQAPGKEREFVAVVSGTGTIAYYADSVKGRFTISR
DNAENTVYLQMNSLKPEDTGLYYCAVGPSSSRWYYR GASLVDYWGKGTLVTVSS VHH#12B 14
QVQLQESGGGLVQPGGSLRLSCAASGFEFENHWMYW
VRQAPGKGLEWVSTVNTNGLITRYADSVKGRFTISR
DNAKYTLYLQMNSLKSEDTAVYYCTKVLPPYSDDSR TNADWGQGTQVTVSS VHH#m9A 79
EVQLVESGGGLVQAGGSLRLSCAASGGTLSSYITGW
FRQAPGKEREFVGAVSWSSSTIVYADSVEGRFTISR
DNHQNTVYLQMDSLKPEDTAVYYCAARPYQKYNWAS ASYNVWGQGTQVTVSS VHH#m9E 15
EVQLVESGGGLVQAGGSLRLSCAASGGTLSSYITGW
FRQAPGKEREFVGAVSWSSSTIVYADSVEGRFTISR
DNHQNTVYLQMDSLKPEDTAVYYCAARPYQKYNWAS ASYNVWGQGTQVTVSS VHH#m3F 16
QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAW
FRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISR
DSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWAS ASYNVWGQGTQVTVSS VHH#m4B 80
QVQLQDSGGGLVQAGGSLRLSCGVSGLSFSGYTMGW
FRQAPGKEREFAAAIGWNSGTTEYRNSVKGRFTISR
DNAKNTVYLQMNSLKPEDTAVYYCAASPKYMTAYER SYDFWGQGTQVTVSS VHH#8-29 81
QVQLVESGGGLVQPGGSLRLSCAASGFAFGDSWMYW
VRQAPGKGLEWVSEINTNGLITKYKDSVTGRFTISR
DNAKNTLHLEMNRLKPEDTALYYCARDPSGKLRGPG TQVTVSS VHH#8-41 82
QVQLVESGGGLVQPGGPLRLSCAASGFAFGDSWMYW
VRQAPGKGLEWVSEINTNGLITKYKDSVTGRFTISR
DNAKNTLHLEMNRLKPEDTALYYCARDPSGKLRGPG TQVTVSS VHH#8-42 83
QVQLVESGGGLVQPGGSLRLSCAASGFAFGDSWMYW
VRQAPGKGLEWVSEINTNGLITKYKDSVTGRFTIS
RDNAKNTLHLEMNRLKPEDTALYYCARDPSGKLRG PGTQVTVSS VHH#8-44 84
QVQLVESGGGLVQPGGSLRLSCAASGFTFSDHWMY
WVRQAPGKGLEWVSTINTNGLITNYIHSVKGRFTI
SRDNAKNTLYLQMNSLKSEDTAVYYCALNQAGLSR GQGTQVTVSS
TABLE-US-00005 TABLE 2 List of mutagenesis reactions, mutagenic
primers and templates used for mutagenesis of VHH#12B Mutation
Template Primer sequence A74S + Y76N + Wild type 5'-AGA GAC AAC TCC
AAG AAC ACG CTG TAT K83R + P84A CTG CAA ATG AAC AGC CTG AGA GCT GAG
GAC ACG-3' Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Q1E + Q5L + A74S + Y76N + 5'-C ATG GCT GAG
GTG CAG CTG CTC GAG TCT A74S + Y76N + K83R + P84A GG-3' K83R + P84A
Met Ala Glu Val Gln Leu Leu Glu Ser Q1E + Q5L + Q1E + Q5L + 5'-G
GAC ACG GCC GTC TAT TAC TGT GCA AAA A74S + Y76N + A74S + Y76N + GTA
CTT C-3' K83R + P84A + K83R + P84A Asp Thr Ala Val Tyr Tyr Cys Ala
Lys T93A Val Leu
TABLE-US-00006 TABLE 3 List of mutagenesis reactions, mutagenic
primers and templates used for mutagenesis of VHH#3E Mutation
Template Primer sequence F37V Wild type 5'-ACC TAT ACC ATT GGC TGG
GTC CGC CAG GCT-3' Thr Tyr Thr Ile Gly Trp Val Arg Gln Ala E44G
Wild type 5'-CGC CAG GCT CCA GGG AAG GGG CGT GAG TTT-3' Arg Gln Ala
Pro Gly Lys Gly Arg Glu Phe R45L Wild type 5'-A GGG AAG GAG CTT GAG
TTT GTA GCG CGT AT-3' Gly Lys Glu Leu Glu Phe Val Ala Arg F47W Wild
type 5'-A GGG AAG GAG CGT GAG TGG GTA GCG CGT AT-3' Gly Lys Glu Arg
Glu Trp Val Ala Arg
TABLE-US-00007 TABLE 4 Overview of humanized and wild type
anti-TNF-alpha VHH SEQ ID Name Sequence 17 VHH#12B
QVQLQESGGGLVQPGGSLRLSCAASGFEFENHWMYWVRQAPGKGLEW A74S + Y76N +
VSTVNTNGLITRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY K83R + P84A
YCTKVLPPYSDDSRTNADWGQGTQVTVSS 18 VHH#12B
EVQLLESGGGLVQPGGSLRLSCAASGFEFENHWMYWVRQAPGKGLEW Q1E + Q5L +
VSTVNTNGLITRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY A74S + Y76N +
YCTKVLPPYSDDSRTNADWGQGTQVTVSS K83R + P84A 19 VHH#12B
EVQLLESGGGLVQPGGSLRLSCAASGFEFENHWMYWVRQAPGKGLEW Q1E + Q5L +
VSTVNTNGLITRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY A74S + Y76N +
YCAKVLPPYSDDSRTNADWGQGTQVTVSS K83R + P84A + T93A 20 VHH#12B
QVQLQESGGGLVQPGGSLRLSCAASGFEFENHWMYWVRQAPGKGLEW Wild type
VSTVNTNGLITRYADSVKGRFTISRDNAKYTLYLQMNSLKSEDTAVY
YCTKVLPPYSDDSRTNADWGQGTQVTVSS 21 VHH#3E
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWVRQAPG F37V
KEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPE
DTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS 22 VHH#3E
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPG E44G
KGREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPE
DTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS 23 VHH#3E
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPG R45L
KELEFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPE
DTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS 24 VHH#3E
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPG F47W
KEREWVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPE
DTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS 25 VHH#3E
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPG Wild type
KEREFVARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPE
DTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS
TABLE-US-00008 TABLE 5 Anti-mouse serum albumin, and anti-mouse
serum albumin + anti TNF-alpha VHH Name SEQ ID Sequence Anti-mouse
serum albumin MSA21 26
QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWV
TIGGSLNPGGQGTQVTVSS MSA24 27
QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSWVRQAPGKEPEWV
SSISGSGSNTIYADSVKDRFTISRDNAKSTLYLQMNSLKPEDTAVYYC
TIGGSLSRSSQGTQVTVSS MSA210 28
QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC VIGRGSPSSQGTQVTVSS
MSA212 29 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKGLEWV
SAISADGSDKRYADSVKGRFTISRDNGKKMLTLDMNSLKPEDTAVYYC VIGRGSPASQGTQVTVSS
MSAc16 85 AVQLVESGGGLVQAGDSLRLSCVVSGTTFSSAAMGWFRQAPGKEREFV
GAIKWSGTSTYYTDSVKGRFTISRDNVKNTVYLQMNNLKPEDTGVYTC
AADRDRYRDRMGPMTTTDFRFWGQGTQVTVSS MSAc112 86
QVKLEESGGGLVQTGGSLRLSCAASGRTFSSFAMGWFRQAPGREREFV
ASIGSSGITTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGLCYC
AVNRYGIPYRSGTQYQNWGQGTQVTVSS MSAc110 87
EVQLEESGGGLVQPGGSLRLSCAASGLTFNDYAMGWYRQAPGKERDMV
ATISIGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCV
AHRQTVVRGPYLLWGQGTQVTVSS MSAc114 88
QVQLVESGGKLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFV
AGSGRSNSYNYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC
AASTNLWPRDRNLYAYWGQGTQVTVSS MSAc116 89
EVQLVESGGGLVQAGDSLRLSCAASGRSLGIYRMGWFRQVPGKEREFV
AAISWSGGTTRYLDSVKGRFTISRDSTKNAVYLQMNSLKPEDTAVYYC
AVDSSGRLYWTLSTSYDYWGQGTQVTVSS MSAc119 90
QVQLVEFGGGLVQAGDSLRLSCAASGRSLGIYKMAWFRQVPGKEREFV
AAISWSGGTTRYIDSVKGRFTLSRDNTKNMVYLQMNSLKPDDTAVYYC
AVDSSGRLYWTLSTSYDYWGQGTQVTVSS MSAc15 91
EVQLVESGGGLVQAGGSLSLSCAASGRTFSPYTMGWFRQAPGKEREFL
AGVTWSGSSTFYGDSVKGRFTASRDSAKNTVTLEMNSLNPEDTAVYYC
AAAYGGGLYRDPRSYDYWGRGTQVTVSS MSc111 92
AVQLVESGGGLVQAGGSLRLSCAASGFTLDAWPIAWFRQAPGKEREGV
SCIRDGTTYYADSVKGRFTISSDNANNTVYLQTNSLKPEDTAVYYCAA
PSGPATGSSHTFGIYWNLRDDYDNWGQGTQVTVSS MSAc115 93
EVQLVESGGGLVQAGGSLRLSCAASGFTFDHYTIGWFRQVPGKEREGV
SCISSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNTLEPDDTAVYYC
AAGGLLLRVEELQASDYDYWGQGIQVTVSS MSAc18 94
AVQLVDSGGGLVQPGGSLRLSCTASGFTLDYYAIGWFRQAPGKEREGV
ACISNSDGSTYYGDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYC
ATADRHYSASHHPFADFAFNSWGQGTQVTVSS MSAc17 95
EVQLVESGGGLVQAGGSLRLSCAAYGLTFWRAAMAWFRRAPGKERELV
VARNWGDGSTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC
AAVRTYGSATYDIWGQGTQVTVSS MSAc120 96
EVQLVESGGGLVQDGGSLRLSCIFSGRTFANYAMGWFRQAPGKEREFV
AAINRNGGTTNYADALKGRFTISRDNTKNTAFLQMNSLKPDDTAVYYC
AAREWPFSTIPSGWRYWGQGTQVTVSS MSAc14 97
DVQLVESGGGWVQPGGSLRLSCAASGPTASSHAIGWFRQAPGKEREFV
VGINRGGVTRDYADSVKGRFAVSRDNVKNTVYLQMNRLKPEDSAIYIC
AARPEYSFTAMSKGDMDYWGKGTLVTVSS Anti-mouse serum albumin/anti
TNF-alpha MSA21/ 30
QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWV VHH#3E
SGISSLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC
TIGGSLNPGGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
LRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYY
ADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSV ESYNYWGQGTQVTVSS
MSA24/ 31 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSWVRQAPGKEPEWV VHH#3E
SSISGSGSNTIYADSVKDRFTISRDNAKSTLYLQMNSLKPEDTAVYYC
TIGGSLSRSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
LRLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYY
ADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSV ESYNYWGQGTQVTVSS
MSA210/ 32 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV VHH#3E
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC
VIGRGSPSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYA
DSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVE SYNYWGQGTQVTVSS
MSA212/ 33 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKGLEWV VHH#3E
SAISADGSDKRYADSVKGRFTISRDNGKKMLTLDMNSLKPEDTAVYYC
VIGRGSPASQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYA
DSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVE SYNYWGQGTQVTVSS
MSA21/ 34 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWV MSA21/
SGISSLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC VHH#3E
TIGGSLNPGGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
LRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGISSLGDSTLYADSVK
GRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSLNPGGQGTQVTV
SSEPKTPKPQPAAAQVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSG
YTYTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRDIAKN
TVDLTMNNLEPEDTAVYYCAARDGIPTSRSVESYNYWGQGTQVTVSS MSA210/ 35
QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV VHH#1A
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC
VIGRGSPSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCATSGFDFSVSWMYWVRQAPGKGLEWVSEINTNGLITKYVDSVKG
RFTISRDNAKNTLYLQMDSLIPEDTALYYCARSPSGSFRGQGTQVTVS S MSA210/ 36
QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV VHH#7B
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC
VIGRGSPSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCAASGSIFRVNAMGWYRQVPGNQREFVAIITSGDNLNYADAVKGR
FTISTDNVKKTVYLQMNVLKPEDTAVYYCNAILQTSRWSIPSNYWGQG TQVTVSS MSA210/ 37
QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV VHH#2B
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC
VIGRGSPSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCATSGFTFSDYWMYWVRQAPGKGLEWVSTVNTNGLITRYADSVKG
RFTISRDNAKYTLYLQMNSLKSEDTAVYYCTKVVPPYSDDSRTNADWG QGTQVTVSS MSA210/
38 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV VHH#3E
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC
VIGRGSPSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCAASGRTFSDHSGYTYTIGWFRQAPGKEREFVARIYWSSGNTYYA
DSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVE SYNYWGQGTQVTVSS
MSA210/ 39 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV VHH#3G
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC
VIGRGSPSSQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQAGGSL
RLSCAVSGRTFSAHSVYTMGWFRQAPGKEREFVARIYWSSANTYYADS
VKGRFTISRDNAKNTVDLLMNSLKPEDTAVYYCAARDGIPTSRTVGSY NYWGQGTQVTVSS
MSA21/ 40 QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWV VHH#12B
SGISSLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC
TIGGSLNPGGQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
LRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNTNGLITRYADSVK
GRFTISRDNAKYTLYLQMNSLKSEDTAVYYCTKVLPPYSDDSRTNADW GQGTQVTVSS MSA24/
41 QVQLQESGGGLVQPGNSLRLSCAASGFTFRNFGMSWVRQAPGKEPEWV VHH#12B
SSISGSGSNTIYADSVKDRFTISRDNAKSTLYLQMNSLKPEDTAVYYC
TIGGSLSRSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGS
LRLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNTNGLITRYADSVK
GRFTISRDNAKYTLYLQMNSLKSEDTAVYYCTKVLPPYSDDSRTNADW GQGTQVTVSS MSA210/
42 QVQLQESGGGLVQPGGSLRLTCTASGFTFSSFGMSWVRQAPGKGLEWV VHH#12B
SAISSDSGTKNYADSVKGRFTISRDNAKKMLFLQMNSLRPEDTAVYYC
VIGRGSPSSQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNTNGLITRYADSVKG
RFTISRDNAKYTLYLQMNSLKSEDTAVYYCTKVLPPYSDDSRTNADWG QGTQVTVSS MSA212/
43 QVQLQESGGGLVQPGGSLRLTCTASGFTFRSFGMSWVRQAPGKGLEWV VHH#12B
SAISADGSDKRYADSVKGRFTISRDNGKKMLTLDMNSLKPEDTAVYYC
VIGRGSPASQGTQVTVSSEPKTPKPQPAAAQVQLQESGGGLVQPGGSL
RLSCAASGFEFENHWMYWVRQAPGKGLEWVSTVNTNGLITRYADSVKG
RFTISRDNAKYTLYLQMNSLKSEDTAVYYCTKVLPPYSDDSRTNADWG QGTQVTVSS
TABLE-US-00009 TABLE 6 Amino acid sequence listing of VHH's
directed against human IFN-gamma. Seq. Seq. Family Name Id Sequence
1 MP3D2SRA 44 QVQLQDSGGGTVQAGGSLRLSCAASGRTFSDYAVGWFRQA
PGKEREFVARILWTGASRSYANSVDGRFTVSTDNAKNTVY
LQMNSLKPEDTAIYYCAALPSNIITTDYLRVYYWGQGTQV TVSS 1 MP3A3SR 45
QVQLQDSGGGTVQAGGSLRLSCAASGRTFSNYAVGWFRQA
PGKEREFVARIKWSGGSRSYANSVDGRFTVSTDNAKNTVY
LQMNSLKPEDTAIYYCA?LPSNIITTDYLRVYYWGQGTQV TVSS 2 MP3C5SR 46
QVQLQESGGGLVQAGGSLRLSCAAAGISGSVFSRTPMGWY
RQAPGKQRELVAGILTSGATSYAESVKGRFTISRDNAKNT
VYLQMNSLSPEDTAEYYCNTYPTWVLSWGQGTQVTVSS 2 MP3C1SR 47
QVQLQDSGGGLVQAGGSLRLSCAAAGISGSVFSRTPMGWY
RQAPGKQRELVAGILSSGATVYAESVKGRFTISRDNAKNT
VYLQMNSLSPEDTAEYYCNTYPTWVLSWGQGTQVTVSS 2 MP3G8SR 48
QVQLQESGGGLVQAGGSLRLSCAAAGISGSVFSRTPMGWY
RQAPGKQRELVAGILSSGATAYAESVKGRFTISRDNAKNT
VYLQMNSLSPEDTAEYYCNTYPTWVLSWGQGTQVTVSS 3 MP3D2BR 49
QVQLQESGGGLVQPGESLRLSCAASRGIFRFNAGGWYRQA
PGKQRELVAFIGVDNTTRYIDSVKGRFTISRDNAKTTVYL
QMNSLQPEDTAVYYCNKVPYIDWGQGTQVTVSS 4 MP3H6SRA 50
QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYNMGWFRQA
PGKEREFVAGISWNGGSIYYTSSVEGRFTISRDNAENTVY
LQMNSLKPEDTGVYYCASKGRPYGVPSPRQGDYDYWGQGT QVTVSS 4 MP3B4SRA 51
QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYNMGWFRQA
PGKEREFVAGISWNGGSIYYTSSVEGRFTISRDNAENTVY
LQMNSLKPEDTGVYYCASKGRPYGVPSPRQGDYDYWGQGT QVTVSS 4 MP4E4BR 52
QVQLQESGGGLVQAGGSLRLSCAASGRTFSIYNMGWFRQA
PGKEREFVAAISWNGGSIYYTSSVEGRFTISRDNAINTVY
LQMNSLKPEDTGVYYCASKGRPYGVPSPRQGEYDYWGQGT QVTVSS 4 MP4H8SR 53
QVQLQESGGGLVQAGGSLRLSCAASGRTFNIYNMGWFRQA
PGKERDFVAAISWNGGSIYYTSSVEGRFTISRDNAENTVY
LQMNSLKPEDTGVYYCASKGRPYGVPSPRQGDYDYWGQGT QVTVSS 5 MP2F6SR 54
QVKLEESGGGLVQAGGSLRLSCAASGRTFNNYNMGWFRQA
PGKEREFVAAISWNGGSTYYDDSVKGRFTISRDNANNLVY
LQMNSLNFEDTAVYYCACAANPYGIPQYRENRYDFWGQGT QVTVSS 5 MP3D1BR 55
QVQLQESGGGLVQAGGSLRLSCAASGRTFDNYNMGWFRQA
PGKEREFVAAISWNGGSTYYDDSVKGRFTISRDNFQKLVY
LQMNSLKLEDTAVYYCACAANPYGIPQYRENRYDFWGQGT QVTVSS 6 MP2B5BR 56
QVQLVESGGRLVQAGGSLRLSCIASGRTISDYAAGWFRQA
PGKEREFLASVTWGFGSTSYADSVKGRFTISRDKAKDTVY
LQMNTLEPDDTSVYYCASSPRYCAGYRCYVTASEFDSWGQ GTQVTVSS 6 MP2C1BR 57
QVKLEESGGRLVQAGGSLRLSCIASGRTISDYAAGWFRQA
PGKEREFLASVSWGFGSTYYADSVKGRFTISRDTAKDTVY
LQMNTLEPDDTSVYYCASSPRYCAGYRCYATASEFDSWGQ GTQVTVSS 6 MP4A12SR 58
QVQLQESGGRLVQAGGSLRLSCIASGRTISDYAAGWFRQA
PGKEREFLASVTWGFGSTYYADSVKGRFTISRDKAKDTVY
LQMNTLEPDDTSAYYCASSPRYCAGYRCYVTASEFDSWGP GTQVTVSS 7 MP3F4SRA 59
QVQLQDSGGGLVQAGDSLRLSCAASGRSFSSYGMGWFRQA
PGKEHEFVAGIWRSGVSLYYTDSVKGRFTISRDDAKMTVS
LQMNSLKPEDTAVYYCAAEATFPTWSRGRFADYDYRGQGT QVTVSS 7 MP3D3BR 60
QVQLQESGGGLVQAGDSLRLSCTASGRSFSSYGMGWFRQA
PGKDHEFVAGIWRSGVSLYYADSVKGRFTISRDDAKMTVS
LQMNGLKPEDTAVYYCAAEATFPTWNRGTFADYDYRGQGT QVTVSS 7 MP3E5BR 61
QVQLQESGGGLVQAGDSLRLSCAASGRSFSSYGMGWFRQA
PGKEHEFVAGIWRSGVSLYYADSVKGRFTISRDDAKMTVS
LQMNGLKPEDTAVYYCAAEATFPTWNRGSFADYDYRGQGT QVTVSS 7 MP3C7SRA 62
QVQLQESGGGLVQAGDSLRLSCAASGRSFSSYGMGWFRQA
PGKEHEFVAGIWRSGVSLYYADSVKGRFTISRDDAKMTVS
LQMNSLKPEDTAVYYCAAEATFPTWNRGRFADYDYSGQGT QVTVSS 8 MP2F1BR 63
AVQLVESGGGLVQTGDSLRLSCVASGGTFSRYAMGWFRQA
PGKEREFVARIGYSGRSISYATSVEGRFAISRDNAKNTVY
LQMNSLKPEDTAVYYCASLVSGTLYQADYWGQGTQVTVSS 8 MP2C5BR 64
QVQLVESGGGLVQTGDSLRLSCVASGGTFSRYAMGWFRQP
PGKERDFVARIGYSGQSISYATSVEGRFAISRDNAKNTVY
LQMNSLKPEDTAVYYCASLVSGTLYKPNYWGQGTQVTVSS 9 MP2C10BR 65
QVKLEESGGGLVQAGGSLRLSCAASGLTYTVGWFRQAPGK
EREFVAAISWSGGSALYADSVKGRFTISRDNAKNTVYLQM
GSLEPEDTAYYSCAAPGTRYYGSNQVNYNYWGQGTQVTVS S 9 MP2G5SR 66
QVKLEESGGGLVQAGDSLRLSCAASGLTYTVGWFRQAPGK
EREFVAAIDWSGGSALYADSVKGRFTISRDNTKNTVYLQM
GSLEPEDTAVYWCAAPGTRYHGRNQVNYNYWGQGTQVTVS S 10 MP3B1SRA 67
QVQLQESGGGLVQPGGSLRLSCAASGFTSSNYAMSWVRQA
PGKGLEWVSSINSRTGSITYADSVKGRFTITLDNAKNTLY
LQMNSLKPEDTAVYYCASRVDDRVSRGQGTQVTVSS 11 MP2F10SR 68
QVQLVESGGGLVQAGGSLRLSCAASGRTISSFRMGWFRRA
PGEEREFVAFVRSNGTSTYYADSVEGRFTITRDNAKNTVY
LRMDSLKPEDTAVYYCAAATRDYGGSFDYWGQGTQVTVSS 11 MP3A7SRA 69
QVQLQDSGGGLVQAGGSLRLSCAASGRTFSSFRMGWFRRA
PGEEREFVAFVRSNGTSTYYADSVEGRFTITRDNAKNTVY
LRMDSLKPEDTAVYYCAAATRDYGGSFDYWGQGTQVIVSS 12 MP4C10SR 70
QVQLQESGGGLVQPGGSLRLSCAASGFTVSNYAMSWVRQP
PGKGIEWVSSINNRNDHITYADSVKGRFTIARDNANNILY
LQMNSLKPEDTAVYYCASRVDDRVSRGQGTQVTVSS 13 MP4D5BR 71
QVQLQDSGGGLVQPGGSLRLSCAASGRTFSSYGMAWFRQA
PGKERELVVAINRSGGATSYATSVRGRFTISRDNAKNTMY
LQMNSLNPEDTAVYYCAARDPTRTYSSYFEYTYWGQGTQV TVSS 14 MP3F1SRA 72
QVQLQESGGGLVQAGGSLTLSCVASGRTISDYAVGWFRQA
PGKEREFVASISWGGGFTAFADSMKGRFTISRDNAKNTVY
LQTHTLEPDDTSVYYCASSRRYCTGYRCYATASEFDSWGQ GTQVTVSS
TABLE-US-00010 TABLE 7 Sequences of bivalent (BIV 3E, BIV#m3F),
trivalent (TRI3E) or tetravalent (TETRA 3E) VHH directed against
TNF-alpha. SEQ Name ID Sequence With linker sequence (underlined)
BIV 3E 73 QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAW
FRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISR
DSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWAS
ASYNVWGQGTQVTVSSEPKTPKPQPAAAQVQLQDSG
GGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKE
REFVGAVSWSGGTTVYADSVLGRFEISRDSARKSVY
LQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQ GTQVTVSS TRI 3E 74
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYT
YTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGR
FAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIP
TSRSVESYNYWGQGTQVTVSSEPKTPKPQPAAAQVQ
LQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTI
GWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAI
SRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSR
SVESYNYWGQGTQVTVSSEPKTPKPQPAAAQVQLQE
SGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWF
RQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRD
IAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVE SYNYWGQGTQVTVSS TETRA 75
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYT 3E
YTIGWFRQAPGKEREFVARIYWSSGNTYYADSVKGR
FAISRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIP
TSRSVESYNYWGQGTQVTVSSEPKTPKPQPAAAQVQ
LQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTI
GWFRQAPGKEREFVARIYWSSGNTYYADSVKGRFAI
SRDIAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSR
SVESYNYWGQGTQVTVSSEPKTPKPQPAAAQVQLQE
SGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWF
RQAPGKEREFVARIYWSSGNTYYADSVKGRFAISRD
IAKNTVDLTMNNLEPEDTAVYYCAARDGIPTSRSVE SYNYWGQGTQVTVSS BIV#m 76
QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAW 3F
FRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISR
DSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWAS
ASYNVWGQGTQVTVSSEPKTPKPQPAAAQVQLQDSG
GGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKE
REFVGAVSWSGGTTVYADSVLGRFEISRDSARKSVY
LQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQ GTQVTVSS Without linker
sequence BIV#3 77 QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAW Edir
FRQAPGKEREFVGAVSWSGGTTVYADSVLGRFEISR
DSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWAS
ASYNVWGQGTQVTVSSQVQLQDSGGGLVQAGGSLRL
SCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGG
TTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTA
VYYCAARPYQKYNWASASYNVWGQGTQVTVSS BIV#12 78
QVQLQESGGGLVQPGGSLRLSCAASGFEFENHWMYW Bdir
VRQAPGKGLEWVSTVNTNGLITRYADSVKGRFTISR
DNAKYTLYLQMNSLKSEDTAVYYCTKVLPPYSDDSR
TNADWGQGTQVTVSSQVQLQESGGGLVQPGGSLRLS
CAASGFEFENHWMYWVRQAPGKGLEWVSTVNTNGLI
TRYADSVKGRFTISRDNAKYTLYLQMNSLKSEDTAV
YYCTKVLPPYSDDSRTNADWGQGTQVTVSS
TABLE-US-00011 TABLE 8 Fractional homologies between the amino acid
sequences of anti-mouse serum albumin VHHs of the invention. SEQ
MSA21 MSA24 MSA210 MSA212 MSA21 1.000 0.834 0.800 0.782 MSA24 --
1.000 0.782 0.791 MSA210 -- -- 1.000 0.903 MSA212 -- -- --
1.000
TABLE-US-00012 TABLE 9 Fractional homologies between anti-TNF-alpha
VHHs of the invention SEQ VHH#1A VHH#7B VHH#2B VHH#3E VHH#3G
VHH#10A VHH#2G VHH#1F VHH#1A 1.000 0.601 0.764 0.596 0.622 0.600
0.682 0.629 VHH#7B -- 1.000 0.604 0.635 0.645 0.943 0.653 0.616
VHH#2B -- -- 1.000 0.620 0.645 0.611 0.682 0.661 VHH#3E -- -- --
1.000 0.875 0.641 0.713 0.689 VHH#3G -- -- -- -- 1.000 0.651 0.779
0.740 VHH#10A -- -- -- -- -- 1.000 0.658 0.614 VHH#2G -- -- -- --
-- -- 1.000 0.741 VHH#1F -- -- -- -- -- -- -- 1.000 VHH#9C -- -- --
-- -- -- -- -- VHH#11E -- -- -- -- -- -- -- -- VHH#10C -- -- -- --
-- -- -- -- VHH#4B -- -- -- -- -- -- -- -- VHH#10D -- -- -- -- --
-- -- -- VHH#12B -- -- -- -- -- -- -- -- VHH#9E -- -- -- -- -- --
-- -- VHH#3F SEQ VHH#9C VHH#11E VHH#10C VHH#4B VHH#10D VHH#12B
VHH#9E VHH#3F VHH#1A 0.609 0.601 0.614 0.818 0.642 0.747 0.596
0.604 VHH#7B 0.933 0.933 0.719 0.593 0.614 0.620 0.616 0.624 VHH#2B
0.629 0.620 0.637 0.796 0.634 0.951 0.620 0.645 VHH#3E 0.620 0.643
0.612 0.604 0.648 0.596 0.674 0.682 VHH#3G 0.637 0.637 0.653 0.645
0.689 0.622 0.708 0.716 VHH#10A 0.935 0.935 0.725 0.592 0.612 0.626
0.622 0.637 VHH#2G 0.653 0.669 0.685 0.666 0.746 0.650 0.701 0.717
VHH#1F 0.616 0.616 0.664 0.661 0.714 0.645 0.709 0.717 VHH#9C 1.000
0.941 0.743 0.601 0.622 0.645 0.600 0.616 VHH#11E -- 1.000 0.719
0.601 0.622 0.637 0.608 0.624 VHH#10C -- -- 1.000 0.650 0.606 0.637
0.600 0.632 VHH#4B -- -- -- 1.000 0.611 0.796 0.588 0.629 VHH#10D
-- -- -- -- 1.000 0.619 0.674 0.674 VHH#12B -- -- -- -- -- 1.000
0.604 0.637 VHH#9E -- -- -- -- -- -- 1.000 0.854 VHH#3F 1.000
TABLE-US-00013 TABLE 10 Percentage homologies between
anti-IFN-gamma VHHs of the invention % Homology MP3D2SRA MP3A3SR
MP3C5SR MP3C1SR MP3G8SR MP3D2BR MP3H6SRA MP3B4SRA MP4E4BR MP4H8SR
MP2F6SR MP3D1BR MP2B5BR MP2C1BR MP3D2SRA X 96 66 66 66 62 71 71 71
70 68 69 65 63 MP3A3SR -- X 66 66 66 62 72 72 72 71 70 71 65 63
MP3C5SR -- -- X 97 98 73 65 65 64 63 63 63 60 58 MP3C1SR -- -- -- X
98 72 64 64 64 62 62 62 58 57 MP3G8SR -- -- -- -- X 73 65 65 64 63
63 63 59 58 MP3D2BR -- -- -- -- -- X 63 63 63 62 63 64 59 58
MP3H6SRA -- -- -- -- -- -- X 100 97 97 80 81 67 68 MP3B4SRA -- --
-- -- -- -- -- X 97 97 80 81 67 68 MP4E4BR -- -- -- -- -- -- -- --
X 97 81 82 68 69 MP4H8SR -- -- -- -- -- -- -- -- -- X 81 81 66 66
MP2F6SR -- -- -- -- -- -- -- -- -- -- X 94 65 68 MP3D1BR -- -- --
-- -- -- -- -- -- -- -- X 65 66 MP2B5BR -- -- -- -- -- -- -- -- --
-- -- -- X 95 MP2C1BR -- -- -- -- -- -- -- -- -- -- -- -- -- X
MP4A12SR -- -- -- -- -- -- -- -- -- -- -- -- -- -- MP3F4SRA -- --
-- -- -- -- -- -- -- -- -- -- -- -- MP3D3BR -- -- -- -- -- -- -- --
-- -- -- -- -- -- MP3E5BR -- -- -- -- -- -- -- -- -- -- -- -- -- --
MP3C7SRA -- -- -- -- -- -- -- -- -- -- -- -- -- -- MP2F1BR -- -- --
-- -- -- -- -- -- -- -- -- -- -- MP2C5BR -- -- -- -- -- -- -- -- --
-- -- -- -- -- MP2C10BR -- -- -- -- -- -- -- -- -- -- -- -- -- --
MP2G5SR -- -- -- -- -- -- -- -- -- -- -- -- -- -- MP3B1SRA -- -- --
-- -- -- -- -- -- -- -- -- -- -- MP2F10SR -- -- -- -- -- -- -- --
-- -- -- -- -- -- MP3A7SRA -- -- -- -- -- -- -- -- -- -- -- -- --
-- MP4C10SR -- -- -- -- -- -- -- -- -- -- -- -- -- -- MP4D5BR -- --
-- -- -- -- -- -- -- -- -- -- -- -- MP3F1SRA -- -- -- -- -- -- --
-- -- -- -- -- -- -- MP6D6BR -- -- -- -- -- -- -- -- -- -- -- -- --
-- MP6B1BR -- -- -- -- -- -- -- -- -- -- -- -- -- -- MP6A8BR -- --
-- -- -- -- -- -- -- -- -- -- -- -- MP6B12BR -- -- -- -- -- -- --
-- -- -- -- -- -- -- MP6C11BR MP6B10BR % Homology MP4A12SR MP3F4SRA
MP3D3BR MP3E5BR MP3C7SRA MP2F1BR MP2C5BR MP2C10BR MP2G5SR MP3B1SRA
MP2F10SR MP3A7SRA MP4C10SR MP3D2SRA 64 68 66 67 68 71 70 68 67 63
67 68 60 MP3A3SR 64 68 66 67 68 72 72 69 67 64 66 67 60 MP3C5SR 59
64 64 65 66 65 65 65 63 63 64 64 61 MP3C1SR 58 65 64 64 65 64 63 64
62 63 64 65 60 MP3G8SR 59 64 64 65 66 65 64 65 63 63 65 65 61
MP3D2BR 58 62 61 62 63 64 63 63 63 64 63 63 63 MP3H6SRA 67 75 71 73
75 73 71 73 71 66 75 75 63 MP3B4SRA 67 75 71 73 75 73 71 73 71 66
75 75 63 MP4E4BR 68 73 70 71 73 73 71 73 71 66 75 75 63 MP4H8SR 66
72 69 71 72 71 71 72 71 64 73 73 62 MP2F6SR 64 70 67 69 71 67 65 73
71 63 71 70 62 MP3D1BR 65 71 69 71 72 67 65 70 69 63 71 71 62
MP2B5BR 97 63 64 64 64 65 63 64 63 60 66 63 57 MP2C1BR 95 63 64 64
64 63 61 66 65 59 66 63 56 MP4A12SR X 63 64 64 64 62 60 63 62 59 65
63 56 MP3F4SRA -- X 94 96 97 69 67 68 68 62 67 69 60 MP3D3BR -- --
X 98 96 70 68 67 67 62 67 67 70 MP3E5BR -- -- -- X 98 70 68 68 69
63 68 68 60 MP3C7SRA -- -- -- -- X 71 69 69 70 63 69 69 61 MP2F1BR
-- -- -- -- -- X 94 66 67 63 68 67 61 MP2C5BR -- -- -- -- -- -- X
66 67 63 67 65 62 MP2C10BR -- -- -- -- -- -- -- X 94 62 68 66 59
MP2G5SR -- -- -- -- -- -- -- -- X 62 67 65 59 MP3B1SRA -- -- -- --
-- -- -- -- -- X 66 65 91 MP2F10SR -- -- -- -- -- -- -- -- -- -- X
97 61 MP3A7SRA -- -- -- -- -- -- -- -- -- -- -- X 61 MP4C10SR -- --
-- -- -- -- -- -- -- -- -- -- X MP4D5BR -- -- -- -- -- -- -- -- --
-- -- -- -- MP3F1SRA -- -- -- -- -- -- -- -- -- -- -- -- -- MP6D6BR
-- -- -- -- -- -- -- -- -- -- -- -- -- MP6B1BR -- -- -- -- -- -- --
-- -- -- -- -- -- MP6A8BR -- -- -- -- -- -- -- -- -- -- -- -- --
MP6B12BR -- -- -- -- -- -- -- -- -- -- -- -- -- MP6C11BR MP6B10BR %
Homology MP4D5BR MP3F1SRA MP6D6BR MP6B1BR MP6A8BR MP6B12BR MP6C11BR
MP6B10BR MP3D2SRA 72 65 68 67 66 67 76 70 MP3A3SR 73 65 67 67 65 66
77 71 MP3C5SR 67 60 74 63 60 63 70 64 MP3C1SR 67 59 73 63 60 62 70
65 MP3G8SR 66 60 73 63 61 63 71 64 MP3D2BR 65 58 73 64 60 63 68 67
MP3H6SRA 71 69 71 71 68 70 82 70 MP3B4SRA 71 69 71 71 68 70 82 70
MP4E4BR 72 70 71 71 68 70 80 71 MP4H8SR 70 67 69 70 67 70 79 71
MP2F6SR 69 66 67 69 68 67 78 69 MP3D1BR 68 66 67 71 69 69 79 70
MP2B5BR 63 84 65 63 63 62 70 65 MP2C1BR 61 85 65 64 63 62 70 65
MP4A12SR 61 84 64 63 63 62 70 65 MP3F4SRA 72 63 67 68 65 65 76 71
MP3D3BR 70 64 66 66 64 64 75 69 MP3E5BR 72 64 67 68 65 66 77 71
MP3C7SRA 72 64 68 68 66 66 78 71 MP2F1BR 70 64 68 65 64 64 74 67
MP2C5BR 69 63 67 64 62 63 73 67 MP2C10BR 67 66 69 68 64 68 74 73
MP2G5SR 67 65 67 66 64 66 73 73 MP3B1SRA 67 60 67 69 68 69 69 65
MP2F10SR 67 65 71 66 65 67 77 68 MP3A7SRA 68 63 71 65 65 67 77 69
MP4C10SR 64 58 65 64 63 66 66 63 MP4D5BR X 64 69 68 65 67 76 73
MP3F1SRA -- X 65 64 64 63 71 68 MP6D6BR -- -- X 70 65 70 77 73
MP6B1BR -- -- -- X 78 81 76 71 MP6A8BR -- -- -- -- X 75 74 66
MP6B12BR -- -- -- -- -- X 73 68 MP6C11BR X 77 MP6B10BR X
TABLE-US-00014 TABLE 11 Treatment schedule Group Animals
Description Schedule 1 8 negative control 1 daily 100 .mu.l PBS
i.p. + ip 2 8 negative control 2 every other day 100 .mu.l PBS
rectal for 2 rectal weeks 3 8 negative control 3 daily 100 .mu.l
PBS intragastric for 14 intragastric consecutive days 4 8 positive
control 1 5 .mu.g i.p. for 7 consecutive days dexamethasone 5 8
positive control 2 applied orally once per day for 14 IL10
expressing I. lactis consecutive days 6 8 bivalent VHH 3F daily 100
.mu.g bivalent VHH 3F.sub.2 intra-gastric intragastric on 14
consecutive days 7 8 bivalent VHH 3F daily 100 .mu.g bivalent VHH
3F i.p. for 14 i.p. consecutive days 8 8 bivalent VHH 3F 100 .mu.g
bivalent VHH 3F rectally in 100 .mu.l rectally PBS every other day
for two weeks
Sequence CWU 1
1
1321115PRTLama glama 1Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly
Phe Asp Phe Ser Val Ser 20 25 30Trp Met Tyr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile Asn Thr Asn Gly Leu
Ile Thr Lys Tyr Val Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asp Ser
Leu Ile Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Ala Arg Ser Pro
Ser Gly Ser Phe Arg Gly Gln Gly Thr Gln Val Thr 100 105 110Val Ser
Ser 1152121PRTLama glama 2Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ser Ile Phe Arg Val Asn 20 25 30Ala Met Gly Trp Tyr Arg Gln Val
Pro Gly Asn Gln Arg Glu Phe Val 35 40 45Ala Ile Ile Thr Ser Gly Asp
Asn Leu Asn Tyr Ala Asp Ala Val Lys 50 55 60Gly Arg Phe Thr Ile Ser
Thr Asp Asn Val Lys Lys Thr Val Tyr Leu65 70 75 80Gln Met Asn Val
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Ala Ile Leu
Gln Thr Ser Arg Trp Ser Ile Pro Ser Asn Tyr Trp Gly 100 105 110Gln
Gly Thr Gln Val Thr Val Ser Ser 115 1203123PRTLama glama 3Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Thr Lys Val Val Pro Pro Tyr Ser Asp Asp Ser
Arg Thr Asn Ala Asp 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 1204129PRTLama glama 4Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly Tyr Thr Tyr Thr
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45Glu Arg Glu Phe Val
Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55 60Tyr Ala Asp Ser
Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65 70 75 80Lys Asn
Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr 85 90 95Ala
Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser 100 105
110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125Ser5127PRTLama glama 5Gln Val Gln Leu Gln Asp Ser Gly
Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Val Ser Gly Arg Thr Phe Ser Ala His 20 25 30Ser Val Tyr Thr Met Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg 35 40 45Glu Phe Val Ala Arg
Ile Tyr Trp Ser Ser Ala Asn Thr Tyr Tyr Ala 50 55 60Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn65 70 75 80Thr Val
Asp Leu Leu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 85 90 95Tyr
Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Thr Val Gly 100 105
110Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 1256124PRTLama glama 6Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ser Ile Phe Arg Val Asn 20 25 30Ala Met Gly Trp Tyr Arg Gln Val
Pro Gly Asn Gln Arg Glu Phe Val 35 40 45Ala Ile Ile Thr Ser Ser Asp
Thr Asn Asp Thr Thr Asn Tyr Ala Asp 50 55 60Ala Val Lys Gly Arg Phe
Thr Ile Ser Thr Asp Asn Val Lys Lys Thr65 70 75 80Val Tyr Leu Gln
Met Asn Val Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Asn
Ala Val Leu Gln Thr Ser Arg Trp Ser Ile Pro Ser Asn 100 105 110Tyr
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 1207123PRTLama
glama 7Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Thr Ser Gly Arg Thr Ile Ser
Val Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Ala Ser Ile Ser Gly Ser Gly Ala Ile Thr Pro Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Asn Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ser Arg Tyr Ala Arg Tyr
Arg Asp Val His Ala Tyr Asp Tyr 100 105 110Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 1208124PRTLama glama 8Gln Val Gln Leu Gln
Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Thr Arg Thr Phe Ser Arg Tyr 20 25 30Val Val Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Thr
Ile Ser Trp Asn Gly Glu His Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75
80Leu Gln Met Gly Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Ala Arg Ser Phe Trp Gly Tyr Asn Val Glu Gln Arg Asp Phe
Gly 100 105 110Ser Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser 115
1209120PRTLama glama 9Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Ser Ile Phe Arg Val Asn 20 25 30Ala Met Gly Trp Tyr Arg Gln Val Pro
Gly Asn Gln Arg Glu Phe Val 35 40 45Ala Ile Ile Thr Asn Asp Thr Thr
Asn Tyr Ala Asp Ala Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Thr Asp
Asn Val Lys Lys Thr Val Tyr Leu Gln65 70 75 80Met Asn Val Leu Lys
Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Thr 85 90 95Val Leu Gln Thr
Ser Arg Trp Asn Ile Pro Thr Asn Tyr Trp Gly Gln 100 105 110Gly Thr
Gln Val Thr Val Ser Ser 115 12010120PRTLama glama 10Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Arg Val Asn 20 25 30Ala Met
Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg Glu Phe Val 35 40 45Ala
Ile Ile Ser Gly Asp Thr Thr Asn Tyr Ala Asp Ala Val Lys Gly 50 55
60Arg Phe Thr Ile Ser Thr Asp Asn Val Lys Lys Thr Val Tyr Leu Gln65
70 75 80Met Asn Val Leu Glu Ser Glu Asp Thr Ala Val Tyr Tyr Cys Asn
Ala 85 90 95Val Leu Gln Thr Ser Arg Trp Ser Ile Pro Ser Asn Tyr Trp
Gly Gln 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115
12011116PRTLama glama 11Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ala Cys Val Ala Ser Gly
Ser Ile Phe Ser Ile Asp 20 25 30Val Met Gly Trp Tyr Arg Gln Ala Pro
Gly Gln Gln Arg Glu Leu Val 35 40 45Ala Thr Ile Thr Asn Ser Trp Thr
Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Val Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu
Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Ala Arg Arg Trp
Tyr Gln Pro Glu Ala Trp Gly Gln Gly Thr Gln Val 100 105 110Thr Val
Ser Ser 11512115PRTLama glama 12Gln Val Gln Leu Gln Asp Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Thr His 20 25 30Trp Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Ile Asn Thr Asn
Gly Leu Ile Thr Asp Tyr Ile His Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Leu
Asn Gln Ala Gly Leu Ser Arg Gly Gln Gly Thr Gln Val Thr 100 105
110Val Ser Ser 11513126PRTLama glama 13Gln Val Gln Leu Gln Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Arg Arg Thr Phe Ser Gly Tyr 20 25 30Ala Met Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Val Val Ser
Gly Thr Gly Thr Ile Ala Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Leu Tyr Tyr Cys 85 90
95Ala Val Gly Pro Ser Ser Ser Arg Trp Tyr Tyr Arg Gly Ala Ser Leu
100 105 110Val Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 12514123PRTLama glama 14Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Glu Phe Glu Asn His 20 25 30Trp Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Val Asn Thr Asn
Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Lys
Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp 100 105
110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12015124PRTLama
glama 15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Leu Ser
Ser Tyr 20 25 30Ile Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Gly Ala Val Ser Trp Ser Ser Ser Thr Ile Val Tyr
Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn His
Gln Asn Thr Val Tyr65 70 75 80Leu Gln Met Asp Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Pro Tyr Gln Lys Tyr
Asn Trp Ala Ser Ala Ser Tyr Asn 100 105 110Val Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 12016124PRTLama glama 16Gln Val Gln Leu
Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Ile 20 25 30Ile Met
Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly
Ala Val Ser Trp Ser Gly Gly Thr Thr Val Tyr Ala Asp Ser Val 50 55
60Leu Gly Arg Phe Glu Ile Ser Arg Asp Ser Ala Arg Lys Ser Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Arg Pro Tyr Gln Lys Tyr Asn Trp Ala Ser Ala Ser
Tyr Asn 100 105 110Val Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 12017123PRTLama glama 17Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Glu Phe Glu Asn His 20 25 30Trp Met Tyr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Val Asn Thr Asn Gly
Leu Ile Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Lys Val
Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp 100 105 110Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12018123PRTLama glama
18Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn
His 20 25 30Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Lys Val Leu Pro Pro Tyr Ser Asp
Asp Ser Arg Thr Asn Ala Asp 100 105 110Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 12019123PRTLama glama 19Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His 20 25 30Trp Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Val
Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala
Asp 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
12020123PRTLama glama 20Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Glu Phe Glu Asn His 20 25 30Trp Met Tyr Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Val Asn Thr Asn Gly Leu
Ile Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Tyr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Lys Val Leu
Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp 100 105 110Trp Gly
Gln Gly Thr Gln
Val Thr Val Ser Ser 115 12021129PRTLama glama 21Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly Tyr
Thr Tyr Thr Ile Gly Trp Val Arg Gln Ala Pro Gly Lys 35 40 45Glu Arg
Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55 60Tyr
Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65 70 75
80Lys Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr
85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg
Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser 115 120 125Ser22129PRTLama glama 22Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly Tyr
Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45Gly Arg
Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55 60Tyr
Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65 70 75
80Lys Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr
85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg
Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser 115 120 125Ser23129PRTLama glama 23Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly Tyr
Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45Glu Leu
Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55 60Tyr
Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65 70 75
80Lys Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr
85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg
Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser 115 120 125Ser24129PRTLama glama 24Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly Tyr
Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45Glu Arg
Glu Trp Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55 60Tyr
Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65 70 75
80Lys Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr
85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg
Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser 115 120 125Ser25129PRTLama glama 25Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly Tyr
Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45Glu Arg
Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55 60Tyr
Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65 70 75
80Lys Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr
85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg
Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser 115 120 125Ser26115PRTLama glama 26Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30Gly Met Thr
Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40 45Ser Gly
Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val
Thr 100 105 110Val Ser Ser 11527115PRTLama glama 27Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45Ser
Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr Ala Asp Ser Val 50 55
60Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln
Val Thr 100 105 110Val Ser Ser 11528114PRTLama glama 28Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu
Phe65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr
Gln Val Thr Val 100 105 110Ser Ser 29114PRTLama glama 29Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30Gly
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ala Ile Ser Ala Asp Gly Ser Asp Lys Arg Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Lys Met Leu
Thr65 70 75 80Leu Asp Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Val Ile Gly Arg Gly Ser Pro Ala Ser Gln Gly Thr
Gln Val Thr Val 100 105 110Ser Ser 30256PRTLama glama 30Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe 20 25 30Gly
Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val 35 40
45Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Asn Pro Gly Gly Gln Gly
Thr Gln Val Thr 100 105 110Val Ser Ser Glu Pro Lys Thr Pro Lys Pro
Gln Pro Ala Ala Ala Gln 115 120 125Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser 130 135 140Leu Arg Leu Ser Cys Ala
Ala Ser Gly Arg Thr Phe Ser Asp His Ser145 150 155 160Gly Tyr Thr
Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 165 170 175Arg
Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr 180 185
190Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys
195 200 205Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp
Thr Ala 210 215 220Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr
Ser Arg Ser Val225 230 235 240Glu Ser Tyr Asn Tyr Trp Gly Gln Gly
Thr Gln Val Thr Val Ser Ser 245 250 25531256PRTLama glama 31Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Phe 20 25
30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val
35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr Ala Asp Ser
Val 50 55 60Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln
Gly Thr Gln Val Thr 100 105 110Val Ser Ser Glu Pro Lys Thr Pro Lys
Pro Gln Pro Ala Ala Ala Gln 115 120 125Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140Leu Arg Leu Ser Cys
Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser145 150 155 160Gly Tyr
Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 165 170
175Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr
180 185 190Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile
Ala Lys 195 200 205Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro
Glu Asp Thr Ala 210 215 220Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile
Pro Thr Ser Arg Ser Val225 230 235 240Glu Ser Tyr Asn Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 25532255PRTLama glama
32Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser
Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Lys Met Leu Phe65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Val Ile Gly Arg Gly Ser Pro Ser Ser
Gln Gly Thr Gln Val Thr Val 100 105 110Ser Ser Glu Pro Lys Thr Pro
Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125Gln Leu Gln Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140Arg Leu Ser
Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser Gly145 150 155
160Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
165 170 175Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr
Tyr Ala 180 185 190Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp
Ile Ala Lys Asn 195 200 205Thr Val Asp Leu Thr Met Asn Asn Leu Glu
Pro Glu Asp Thr Ala Val 210 215 220Tyr Tyr Cys Ala Ala Arg Asp Gly
Ile Pro Thr Ser Arg Ser Val Glu225 230 235 240Ser Tyr Asn Tyr Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 25533255PRTLama
glama 33Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg
Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Ala Ile Ser Ala Asp Gly Ser Asp Lys Arg Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly
Lys Lys Met Leu Thr65 70 75 80Leu Asp Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Ile Gly Arg Gly Ser Pro Ala
Ser Gln Gly Thr Gln Val Thr Val 100 105 110Ser Ser Glu Pro Lys Thr
Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120 125Gln Leu Gln Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser Gly145 150 155
160Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
165 170 175Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr
Tyr Ala 180 185 190Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp
Ile Ala Lys Asn 195 200 205Thr Val Asp Leu Thr Met Asn Asn Leu Glu
Pro Glu Asp Thr Ala Val 210 215 220Tyr Tyr Cys Ala Ala Arg Asp Gly
Ile Pro Thr Ser Arg Ser Val Glu225 230 235 240Ser Tyr Asn Tyr Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 25534383PRTLama
glama 34Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser
Arg Phe 20 25 30Gly Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val
Glu Trp Val 35 40 45Ser Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Asn Pro
Gly Gly Gln Gly Thr Gln Val Thr 100 105 110Val Ser Ser Glu Pro Lys
Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140Leu Arg
Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Arg Phe Gly145 150 155
160Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Val Glu Trp Val Ser
165 170 175Gly Ile Ser Ser Leu Gly Asp Ser Thr Leu Tyr Ala Asp Ser
Val Lys 180 185 190Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr Leu 195 200 205Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Thr 210 215 220Ile Gly Gly Ser Leu Asn Pro Gly
Gly Gln Gly Thr Gln Val Thr Val225 230 235 240Ser Ser Glu Pro Lys
Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 245 250 255Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 260 265 270Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His Ser Gly 275 280
285Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
290 295 300Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly
Asn Thr Tyr Tyr Ala305 310 315 320Asp Ser Val Lys Gly Arg Phe Ala
Ile Ser Arg Asp Ile Ala Lys Asn 325 330 335Thr Val Asp Leu Thr Met
Asn Asn Leu Glu Pro Glu Asp Thr Ala Val 340 345 350Tyr Tyr Cys Ala
Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val Glu 355 360 365Ser Tyr
Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 370 375
38035241PRTLama glama 35Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly
Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Ser Asp Ser Gly
Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Lys Met Leu Phe65 70 75 80Leu Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Ile Gly Arg
Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110Ser Ser
Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120
125Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
130 135 140Arg Leu Ser Cys Ala Thr Ser Gly Phe Asp Phe Ser Val Ser
Trp Met145 150 155 160Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser Glu 165 170 175Ile Asn Thr Asn Gly Leu Ile Thr Lys
Tyr Val Asp Ser Val Lys Gly 180 185 190Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Thr Leu Tyr Leu Gln 195 200 205Met Asp Ser Leu Ile
Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg 210 215 220Ser Pro Ser
Gly Ser Phe Arg Gly Gln Gly Thr Gln Val Thr Val Ser225 230 235
240Ser36247PRTLama glama 36Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Thr Cys Thr Ala Ser
Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Ser Asp Ser
Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Lys Met Leu Phe65 70 75 80Leu Gln Met Asn
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Ile Gly
Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110Ser
Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120
125Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
130 135 140Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Arg Val Asn
Ala Met145 150 155 160Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln Arg
Glu Phe Val Ala Ile 165 170 175Ile Thr Ser Gly Asp Asn Leu Asn Tyr
Ala Asp Ala Val Lys Gly Arg 180 185 190Phe Thr Ile Ser Thr Asp Asn
Val Lys Lys Thr Val Tyr Leu Gln Met 195 200 205Asn Val Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala Ile 210 215 220Leu Gln Thr
Ser Arg Trp Ser Ile Pro Ser Asn Tyr Trp Gly Gln Gly225 230 235
240Thr Gln Val Thr Val Ser Ser 24537249PRTLama glama 37Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Ala Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu
Phe65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr
Gln Val Thr Val 100 105 110Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln
Pro Ala Ala Ala Gln Val 115 120 125Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu 130 135 140Arg Leu Ser Cys Ala Thr
Ser Gly Phe Thr Phe Ser Asp Tyr Trp Met145 150 155 160Tyr Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr 165 170 175Val
Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys Gly 180 185
190Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu Gln
195 200 205Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
Thr Lys 210 215 220Val Val Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn
Ala Asp Trp Gly225 230 235 240Gln Gly Thr Gln Val Thr Val Ser Ser
24538255PRTLama glama 38Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly
Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Ser Asp Ser Gly
Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Lys Met Leu Phe65 70 75 80Leu Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Ile Gly Arg
Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110Ser Ser
Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120
125Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
130 135 140Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His
Ser Gly145 150 155 160Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg 165 170 175Glu Phe Val Ala Arg Ile Tyr Trp Ser
Ser Gly Asn Thr Tyr Tyr Ala 180 185 190Asp Ser Val Lys Gly Arg Phe
Ala Ile Ser Arg Asp Ile Ala Lys Asn 195 200 205Thr Val Asp Leu Thr
Met Asn Asn Leu Glu Pro Glu Asp Thr Ala Val 210 215 220Tyr Tyr Cys
Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser Val Glu225 230 235
240Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245
250 25539253PRTLama glama 39Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Thr Cys Thr Ala Ser
Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Ser Asp Ser
Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Lys Met Leu Phe65 70 75 80Leu Gln Met Asn
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Ile Gly
Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr Val 100 105 110Ser
Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120
125Gln Leu Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu
130 135 140Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Phe Ser Ala His
Ser Val145 150 155 160Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Glu Arg Glu Phe 165 170 175Val Ala Arg Ile Tyr Trp Ser Ser Ala
Asn Thr Tyr Tyr Ala Asp Ser 180 185 190Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val 195 200 205Asp Leu Leu Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 210 215 220Cys Ala Ala
Arg Asp Gly Ile Pro Thr Ser Arg Thr Val Gly Ser Tyr225 230 235
240Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245
25040250PRTLama glama 40Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly
Phe Thr Phe Ser Arg Phe 20 25 30Gly Met Thr Trp Val Arg Gln Ala Pro
Gly Lys Gly Val Glu Trp Val 35 40 45Ser Gly Ile Ser Ser Leu Gly Asp
Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly
Ser Leu Asn Pro Gly Gly Gln Gly Thr Gln Val Thr 100 105 110Val Ser
Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120
125Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
130 135 140Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn
His Trp145 150 155 160Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ser 165 170 175Thr Val Asn Thr Asn Gly Leu Ile Thr
Arg Tyr Ala Asp Ser Val Lys 180 185 190Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Tyr Thr Leu Tyr Leu 195 200 205Gln Met Asn Ser Leu
Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr 210 215 220Lys Val Leu
Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp Trp225 230 235
240Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 25041250PRTLama
glama 41Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg
Asn Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro
Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asn Thr Ile Tyr
Ala Asp Ser Val 50 55 60Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Ser Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg
Ser Ser Gln Gly Thr Gln Val Thr 100 105 110Val Ser Ser Glu Pro Lys
Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln 115 120 125Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His Trp145 150 155
160Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
165 170 175Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser
Val Lys 180 185 190Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr
Thr Leu Tyr Leu 195 200 205Gln Met Asn Ser Leu Lys Ser Glu Asp Thr
Ala Val Tyr Tyr Cys Thr 210 215 220Lys Val Leu Pro Pro Tyr Ser Asp
Asp Ser Arg Thr Asn Ala Asp Trp225 230 235 240Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 245 25042249PRTLama glama 42Gln Val Gln Leu Gln
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Thr Cys Thr Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Ser Asp Ser Gly Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Phe65 70 75
80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Val Ile Gly Arg Gly Ser Pro Ser Ser Gln Gly Thr Gln Val Thr
Val 100 105 110Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala
Ala Gln Val 115 120 125Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu 130 135 140Arg Leu Ser Cys Ala Ala Ser Gly Phe
Glu Phe Glu Asn His Trp Met145 150 155 160Tyr Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ser Thr 165 170 175Val Asn Thr Asn
Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys Gly 180 185 190Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu Gln 195 200
205Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Lys
210 215 220Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp
Trp Gly225 230 235 240Gln Gly Thr Gln Val Thr Val Ser Ser
24543249PRTLama glama 43Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly
Phe Thr Phe Arg Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Ala Asp Gly Ser
Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Gly Lys Lys Met Leu Thr65 70 75 80Leu Asp Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Val Ile Gly Arg
Gly Ser Pro Ala Ser Gln Gly Thr Gln Val Thr Val 100 105 110Ser Ser
Glu Pro Lys Thr Pro Lys Pro Gln Pro Ala Ala Ala Gln Val 115 120
125Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
130 135 140Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His
Trp Met145 150 155 160Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser Thr 165 170 175Val Asn Thr Asn Gly Leu Ile Thr Arg
Tyr Ala Asp Ser Val Lys Gly 180 185 190Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Tyr Thr Leu Tyr Leu Gln 195 200 205Met Asn Ser Leu Lys
Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Lys 210 215 220Val Leu Pro
Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp Trp Gly225 230 235
240Gln Gly Thr Gln Val Thr Val Ser Ser 24544124PRTLama glama 44Gln
Val Gln Leu Gln Asp Ser Gly Gly Gly Thr Val Gln Ala Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr
20 25 30Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Arg Ile Leu Trp Thr Gly Ala Ser Arg Ser Tyr Ala Asn
Ser Val 50 55 60Asp Gly Arg Phe Thr Val Ser Thr Asp Asn Ala Lys Asn
Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Leu Pro Ser Asn Ile Ile Thr Thr
Asp Tyr Leu Arg Val Tyr 100 105 110Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 12045123PRTLama glama 45Gln Val Gln Leu Gln Asp
Ser Gly Gly Gly Thr Val Gln Ala Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr
20 25 30Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Arg Ile Lys Trp Ser Gly Gly Ser Arg Ser Tyr Ala Asn
Ser Val 50 55 60Asp Gly Arg Phe Thr Val Ser Thr Asp Asn Ala Lys Asn
Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Ile Tyr Tyr Cys 85 90 95Ala Leu Pro Ser Asn Ile Ile Thr Thr Asp
Tyr Leu Arg Val Tyr Tyr 100 105 110Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 12046118PRTLama glama 46Gln Val Gln Leu Gln Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ala Gly Ile Ser Gly Ser Val Phe 20 25 30Ser Arg Thr Pro Met
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg 35 40 45Glu Leu Val Ala
Gly Ile Leu Thr Ser Gly Ala Thr Ser Tyr Ala Glu 50 55 60Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr65 70 75 80Val
Tyr Leu Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Glu Tyr 85 90
95Tyr Cys Asn Thr Tyr Pro Thr Trp Val Leu Ser Trp Gly Gln Gly Thr
100 105 110Gln Val Thr Val Ser Ser 11547118PRTLama glama 47Gln Val
Gln Leu Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ala Gly Ile Ser Gly Ser Val Phe 20 25
30Ser Arg Thr Pro Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg
35 40 45Glu Leu Val Ala Gly Ile Leu Ser Ser Gly Ala Thr Val Tyr Ala
Glu 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr65 70 75 80Val Tyr Leu Gln Met Asn Ser Leu Ser Pro Glu Asp
Thr Ala Glu Tyr 85 90 95Tyr Cys Asn Thr Tyr Pro Thr Trp Val Leu Ser
Trp Gly Gln Gly Thr 100 105 110Gln Val Thr Val Ser Ser
11548118PRTLama glama 48Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ala Gly
Ile Ser Gly Ser Val Phe 20 25 30Ser Arg Thr Pro Met Gly Trp Tyr Arg
Gln Ala Pro Gly Lys Gln Arg 35 40 45Glu Leu Val Ala Gly Ile Leu Ser
Ser Gly Ala Thr Ala Tyr Ala Glu 50 55 60Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr65 70 75 80Val Tyr Leu Gln Met
Asn Ser Leu Ser Pro Glu Asp Thr Ala Glu Tyr 85 90 95Tyr Cys Asn Thr
Tyr Pro Thr Trp Val Leu Ser Trp Gly Gln Gly Thr 100 105 110Gln Val
Thr Val Ser Ser 11549113PRTLama glama 49Gln Val Gln Leu Gln Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Glu1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Arg Gly Ile Phe Arg Phe Asn 20 25 30Ala Gly Gly Trp Tyr
Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Phe Ile Gly
Val Asp Asn Thr Thr Arg Tyr Ile Asp Ser Val Lys 50 55 60Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr Leu65 70 75 80Gln
Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90
95Lys Val Pro Tyr Ile Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser
100 105 110Ser50126PRTLama glama 50Gln Val Gln Leu Gln Glu Ser Gly
Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Arg Thr Phe Ser Thr Tyr 20 25 30Asn Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Gly Ile Ser Trp
Asn Gly Gly Ser Ile Tyr Tyr Thr Ser Ser Val 50 55 60Glu Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala
Ser Lys Gly Arg Pro Tyr Gly Val Pro Ser Pro Arg Gln Gly Asp 100 105
110Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12551126PRTLama glama 51Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Ser Thr Tyr 20 25 30Asn Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Gly Ile Ser Trp Asn Gly Gly
Ser Ile Tyr Tyr Thr Ser Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Glu Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Ser Lys Gly
Arg Pro Tyr Gly Val Pro Ser Pro Arg Gln Gly Asp 100 105 110Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12552126PRTLama glama 52Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Ser Ile Tyr 20 25 30Asn Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Asn Gly Gly
Ser Ile Tyr Tyr Thr Ser Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Ile Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Ser Lys Gly
Arg Pro Tyr Gly Val Pro Ser Pro Arg Gln Gly Glu 100 105 110Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12553126PRTLama glama 53Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Asn Ile Tyr 20 25 30Asn Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Asp Phe Val 35 40 45Ala Ala Ile Ser Trp Asn Gly Gly
Ser Ile Tyr Tyr Thr Ser Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Glu Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95Ala Ser Lys Gly
Arg Pro Tyr Gly Val Pro Ser Pro Arg Gln Gly Asp 100 105 110Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12554126PRTLama glama 54Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Asn Asn Tyr 20 25 30Asn Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Asn Gly Gly
Ser Thr Tyr Tyr Asp Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Asn Asn Leu Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Asn Phe Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Cys Ala Ala
Asn Pro Tyr Gly Ile Pro Gln Tyr Arg Glu Asn Arg 100 105 110Tyr Asp
Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12555126PRTLama glama 55Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Asp Asn Tyr 20 25 30Asn Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Asn Gly Gly
Ser Thr Tyr Tyr Asp Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Phe Gln Lys Leu Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Cys Ala Ala
Asn Pro Tyr Gly Ile Pro Gln Tyr Arg Glu Asn Arg 100 105 110Tyr Asp
Phe Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12556128PRTLama glama 56Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly
Arg Thr Ile Ser Asp Tyr 20 25 30Ala Ala Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Leu 35 40 45Ala Ser Val Thr Trp Gly Phe Gly
Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Lys Ala Lys Asp Thr Val Tyr65 70 75 80Leu Gln Met Asn Thr
Leu Glu Pro Asp Asp Thr Ser Val Tyr Tyr Cys 85 90 95Ala Ser Ser Pro
Arg Tyr Cys Ala Gly Tyr Arg Cys Tyr Val Thr Ala 100 105 110Ser Glu
Phe Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12557128PRTLama glama 57Gln Val Lys Leu Glu Glu Ser Gly Gly Arg Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly
Arg Thr Ile Ser Asp Tyr 20 25 30Ala Ala Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Leu 35 40 45Ala Ser Val Ser Trp Gly Phe Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Thr Ala Lys Asp Thr Val Tyr65 70 75 80Leu Gln Met Asn Thr
Leu Glu Pro Asp Asp Thr Ser Val Tyr Tyr Cys 85 90 95Ala Ser Ser Pro
Arg Tyr Cys Ala Gly Tyr Arg Cys Tyr Ala Thr Ala 100 105 110Ser Glu
Phe Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12558128PRTLama glama 58Gln Val Gln Leu Gln Glu Ser Gly Gly Arg Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Ala Ser Gly
Arg Thr Ile Ser Asp Tyr 20 25 30Ala Ala Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Leu 35 40 45Ala Ser Val Thr Trp Gly Phe Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Lys Ala Lys Asp Thr Val Tyr65 70 75 80Leu Gln Met Asn Thr
Leu Glu Pro Asp Asp Thr Ser Ala Tyr Tyr Cys 85 90 95Ala Ser Ser Pro
Arg Tyr Cys Ala Gly Tyr Arg Cys Tyr Val Thr Ala 100 105 110Ser Glu
Phe Asp Ser Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser 115 120
12559126PRTLama glama 59Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu
Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Ser Phe Ser Ser Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu His Glu Phe Val 35 40 45Ala Gly Ile Trp Arg Ser Gly Val
Ser Leu Tyr Tyr Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ala Lys Met Thr Val Ser65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Glu Ala
Thr Phe Pro Thr Trp Ser Arg Gly Arg Phe Ala Asp 100 105 110Tyr Asp
Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12560126PRTLama glama 60Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly
Arg Ser Phe Ser Ser Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Asp His Glu Phe Val 35 40 45Ala Gly Ile Trp Arg Ser Gly Val
Ser Leu Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ala Lys Met Thr Val Ser65 70 75 80Leu Gln Met Asn Gly
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Glu Ala
Thr Phe Pro Thr Trp Asn Arg Gly Thr Phe Ala Asp 100 105 110Tyr Asp
Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12561126PRTLama glama 61Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Ser Phe Ser Ser Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu His Glu Phe Val 35 40 45Ala Gly Ile Trp Arg Ser Gly Val
Ser Leu Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ala Lys Met Thr Val Ser65 70 75 80Leu Gln Met Asn Gly
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Glu Ala
Thr Phe Pro Thr Trp Asn Arg Gly Ser Phe Ala Asp 100 105 110Tyr Asp
Tyr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12562126PRTLama glama 62Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Ser Phe Ser Ser Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu His Glu Phe Val 35 40 45Ala Gly Ile Trp Arg Ser Gly Val
Ser Leu Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ala Lys Met Thr Val Ser65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Glu Ala
Thr Phe Pro Thr Trp Asn Arg Gly Arg Phe Ala Asp 100 105 110Tyr Asp
Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12563120PRTLama glama 63Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Thr Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly
Gly Thr Phe Ser Arg Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Arg Ile Gly Tyr Ser Gly Arg
Ser Ile Ser Tyr Ala Thr Ser Val 50 55 60Glu Gly Arg Phe Ala Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Leu Val
Ser Gly Thr Leu Tyr Gln Ala Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Gln Val Thr Val Ser Ser 115 12064120PRTLama glama 64Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Asp1 5 10 15Ser Leu Arg
Leu Ser Cys Val Ala Ser Gly Gly Thr Phe Ser Arg Tyr 20 25 30Ala Met
Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Asp Phe Val 35 40 45Ala
Arg Ile Gly Tyr Ser Gly Gln Ser Ile Ser Tyr Ala Thr Ser Val 50 55
60Glu Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85
90 95Ala Ser Leu Val Ser Gly Thr Leu Tyr Lys Pro Asn Tyr Trp Gly
Gln 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115 12065121PRTLama
glama 65Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Tyr Thr
Val Gly 20 25 30Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
Ala Ala Ile 35 40 45Ser Trp Ser Gly Gly Ser Ala Leu Tyr Ala Asp Ser
Val Lys Gly Arg 50 55 60Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val Tyr Leu Gln Met65 70 75 80Gly Ser Leu Glu Pro Glu Asp Thr Ala
Tyr Tyr Ser Cys Ala Ala Pro 85 90 95Gly Thr Arg Tyr Tyr Gly Ser Asn
Gln Val Asn Tyr Asn Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr
Val Ser Ser 115 12066121PRTLama glama 66Gln Val Lys Leu Glu Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Leu Thr Tyr Thr Val Gly 20 25 30Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile 35 40 45Asp Trp Ser Gly
Gly Ser Ala Leu Tyr Ala Asp Ser Val Lys Gly Arg 50 55 60Phe Thr Ile
Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr Leu Gln Met65 70 75 80Gly
Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Trp Cys Ala Ala Pro 85 90
95Gly Thr Arg Tyr His Gly Arg Asn Gln Val Asn Tyr Asn Tyr Trp Gly
100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12067116PRTLama
glama 67Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Ser
Asn Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45Ser Ser Ile Asn Ser Arg Thr Gly Ser Ile Thr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Thr Leu Asp Asn Ala
Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Val Asp Asp Arg Val
Ser Arg Gly Gln Gly Thr Gln Val 100 105 110Thr Val Ser Ser
11568120PRTLama glama 68Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Ile Ser Ser Phe 20 25 30Arg Met Gly Trp Phe Arg Arg Ala Pro
Gly Glu Glu Arg Glu Phe Val 35 40 45Ala Phe Val Arg Ser Asn Gly Thr
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Thr
Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Arg Met Asp Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ala Thr
Arg Asp Tyr Gly Gly Ser Phe Asp Tyr Trp Gly Gln 100 105 110Gly Thr
Gln Val Thr Val Ser Ser 115 12069120PRTLama glama 69Gln Val Gln Leu
Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Phe 20 25 30Arg Met
Gly Trp Phe Arg Arg Ala Pro Gly Glu Glu Arg Glu Phe Val 35 40 45Ala
Phe Val Arg Ser Asn Gly Thr Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Glu Gly Arg Phe Thr Ile Thr Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Arg Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Ala Thr Arg Asp Tyr Gly Gly Ser Phe Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Gln Val Ile Val Ser Ser 115
12070116PRTLama glama 70Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Val Ser Asn Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Pro Pro
Gly Lys Gly Ile Glu Trp Val 35 40 45Ser Ser Ile Asn Asn Arg Asn Asp
His Ile Thr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ala
Arg Asp Asn Ala Asn Asn Ile Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ser Arg Val
Asp Asp Arg Val Ser Arg Gly Gln Gly Thr Gln Val 100 105 110Thr Val
Ser Ser 11571124PRTLama glama 71Gln Val Gln Leu Gln Asp Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Gly Met Ala Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45Val Ala Ile Asn Arg Ser
Gly Gly Ala Thr Ser Tyr Ala Thr Ser Val 50 55 60Arg Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Arg Asp Pro Thr Arg Thr Tyr Ser Ser Tyr Phe Glu Tyr Thr 100 105
110Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
12072128PRTLama glama 72Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Thr Leu Ser Cys Val Ala Ser Gly
Arg Thr Ile Ser Asp Tyr 20 25 30Ala Val Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ser Ile Ser Trp Gly Gly Gly
Phe Thr Ala Phe Ala Asp Ser Met 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Thr His Thr
Leu Glu Pro Asp Asp Thr Ser Val Tyr Tyr Cys 85 90 95Ala Ser Ser Arg
Arg Tyr Cys Thr Gly Tyr Arg Cys Tyr Ala Thr Ala 100 105 110Ser Glu
Phe Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12573260PRTLama glama 73Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Gly Thr Phe Ser Ser Ile 20 25 30Ile Met Ala Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala Val Ser Trp Ser Gly Gly
Thr Thr Val Tyr Ala Asp Ser Val 50 55 60Leu Gly Arg Phe Glu Ile Ser
Arg Asp Ser Ala Arg Lys Ser Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Pro
Tyr Gln Lys Tyr Asn Trp Ala Ser Ala Ser Tyr Asn 100 105 110Val Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr 115 120
125Pro Lys Pro Gln Pro Ala Ala Ala Gln Val Gln Leu Gln Asp Ser Gly
130 135 140Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala145 150 155 160Ser Gly Gly Thr Phe Ser Ser Ile Ile Met Ala
Trp Phe Arg Gln Ala 165 170 175Pro Gly Lys Glu Arg Glu Phe Val Gly
Ala Val Ser Trp Ser Gly Gly 180 185 190Thr Thr Val Tyr Ala Asp Ser
Val Leu Gly Arg Phe Glu Ile Ser Arg 195 200 205Asp Ser Ala Arg Lys
Ser Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 210 215 220Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Ala Arg Pro Tyr Gln Lys Tyr225 230 235
240Asn Trp Ala Ser Ala Ser Tyr Asn Val Trp Gly Gln Gly Thr Gln Val
245 250 255Thr Val Ser Ser 26074411PRTLama glama 74Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly
Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45Glu
Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55
60Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65
70 75 80Lys Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp
Thr 85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser
Arg Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser 115 120 125Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro
Ala Ala Ala Gln Val Gln 130 135 140Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg145 150 155 160Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Asp His Ser Gly Tyr 165 170 175Thr Tyr Thr
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 180 185 190Phe
Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala Asp 195 200
205Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn Thr
210 215 220Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala
Val Tyr225 230 235 240Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser
Arg Ser Val Glu Ser 245 250 255Tyr Asn Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser Glu Pro 260 265 270Lys Thr Pro Lys Pro Gln Pro
Ala Ala Ala Gln Val Gln Leu Gln Glu 275 280 285Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 290 295 300Ala Ala Ser
Gly Arg Thr Phe Ser Asp His Ser Gly Tyr Thr Tyr Thr305 310 315
320Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala
325 330 335Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala Asp Ser
Val Lys 340 345 350Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn
Thr Val Asp Leu 355 360 365Thr Met Asn Asn Leu Glu Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 370 375 380Ala Arg Asp Gly Ile Pro Thr Ser
Arg Ser Val Glu Ser Tyr Asn Tyr385 390 395 400Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 405 41075552PRTLama glama 75Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp His 20 25 30Ser Gly
Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45Glu
Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr 50 55
60Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala65
70 75 80Lys Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp
Thr 85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser
Arg Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser 115 120 125Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro
Ala Ala Ala Gln Val Gln 130 135 140Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg145 150 155 160Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Asp His Ser Gly Tyr 165 170 175Thr Tyr Thr
Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 180 185 190Phe
Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala Asp 195 200
205Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn Thr
210 215 220Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala
Val Tyr225 230 235 240Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser
Arg Ser Val Glu Ser 245 250 255Tyr Asn Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser Glu Pro 260 265 270Lys Thr Pro Lys Pro Gln Pro
Ala Ala Ala Gln Val Gln Leu Gln Glu 275 280 285Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 290 295 300Ala Ala Ser
Gly Arg Thr Phe Ser Asp His Ser Gly Tyr Thr Tyr Thr305 310 315
320Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala
325 330 335Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr Ala Asp Ser
Val Lys 340 345 350Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys Asn
Thr Val Asp Leu 355 360 365Thr Met Asn Asn Leu Glu Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 370 375 380Ala Arg Asp Gly Ile Pro Thr Ser
Arg Ser Val Glu Ser Tyr Asn Tyr385 390 395 400Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro 405 410 415Lys Pro Gln
Pro Ala Ala Ala Gln Val Gln Leu Gln Glu Ser Gly Gly 420 425 430Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 435 440
445Gly Arg Thr Phe Ser Asp His Ser Gly Tyr Thr Tyr Thr Ile Gly Trp
450 455 460Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Arg
Ile Tyr465 470 475 480Trp Ser Ser Gly Asn Thr Tyr Tyr Ala Asp Ser
Val Lys Gly Arg Phe 485 490 495Ala Ile Ser Arg Asp Ile Ala Lys Asn
Thr Val Asp Leu Thr Met Asn 500 505 510Asn Leu Glu Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Ala Arg Asp 515 520 525Gly Ile Pro Thr Ser
Arg Ser Val Glu Ser Tyr Asn Tyr Trp Gly Gln 530 535 540Gly Thr Gln
Val Thr Val Ser Ser545 55076260PRTLama glama 76Gln Val Gln Leu Gln
Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Ile 20 25 30Ile Met Ala
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala
Val Ser Trp Ser Gly Gly Thr Thr Val Tyr Ala Asp Ser Val 50 55 60Leu
Gly Arg Phe Glu Ile Ser Arg Asp Ser Ala Arg Lys Ser Val Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Ala Arg Pro Tyr Gln Lys Tyr Asn Trp Ala Ser Ala Ser Tyr
Asn 100 105 110Val Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
Pro Lys Thr 115 120 125Pro Lys Pro Gln Pro Ala Ala Ala Gln Val Gln
Leu Gln Asp Ser Gly 130 135 140Gly Gly Leu Val Gln Ala Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala145 150 155 160Ser Gly Gly Thr Phe Ser
Ser Ile Ile Met Ala Trp Phe Arg Gln Ala 165 170 175Pro Gly Lys Glu
Arg Glu Phe Val Gly Ala Val Ser Trp Ser Gly Gly 180 185 190Thr Thr
Val Tyr Ala Asp Ser Val Leu Gly Arg Phe Glu Ile Ser Arg 195 200
205Asp Ser Ala Arg Lys Ser Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
210 215 220Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg
Pro Tyr Gln Lys Tyr225 230 235 240Asn Trp Ala Ser Ala Ser Tyr Asn
Val Trp Gly Gln Gly Thr Gln Val 245 250 255Thr Val Ser Ser
26077248PRTLama glama 77Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Gly Thr Phe Ser Ser Ile 20 25 30Ile Met Ala Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala Val Ser Trp Ser Gly Gly
Thr Thr Val Tyr Ala Asp Ser Val 50 55 60Leu Gly Arg Phe Glu Ile Ser
Arg Asp Ser Ala Arg Lys Ser Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Pro
Tyr Gln Lys Tyr Asn Trp Ala Ser Ala Ser Tyr Asn 100 105 110Val Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gln Val Gln Leu 115 120
125Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu
130 135 140Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Ile Ile Met
Ala Trp145 150 155 160Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val Gly Ala Val Ser 165 170 175Trp Ser Gly Gly Thr Thr Val Tyr Ala
Asp Ser Val Leu Gly Arg Phe 180 185 190Glu Ile Ser Arg Asp Ser Ala
Arg Lys Ser Val Tyr Leu Gln Met Asn 195 200 205Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg Pro 210 215 220Tyr Gln Lys
Tyr Asn Trp Ala Ser Ala Ser Tyr Asn Val Trp Gly Gln225 230 235
240Gly Thr Gln Val Thr Val Ser Ser 24578246PRTLama glama 78Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His 20 25
30Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Thr Lys Val Leu Pro Pro Tyr Ser Asp Asp Ser
Arg Thr Asn Ala Asp 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser Gln Val Gln Leu Gln 115 120 125Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140Cys Ala Ala Ser Gly
Phe Glu Phe Glu Asn His Trp Met Tyr Trp Val145 150 155 160Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Thr Val Asn Thr 165 170
175Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
180 185 190Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr Leu Gln Met
Asn Ser 195 200 205Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr
Lys Val Leu Pro 210 215 220Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala
Asp Trp Gly Gln Gly Thr225 230 235 240Gln Val Thr Val Ser Ser
24579124PRTLama glama 79Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Gly Thr Leu Ser Ser Tyr 20 25 30Ile Thr Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala Val Ser Trp Ser Ser Ser
Thr Ile Val Tyr Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser
Arg Asp Asn His Gln Asn Thr Val Tyr65 70 75 80Leu Gln Met Asp Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Pro
Tyr Gln Lys Tyr Asn Trp Ala Ser Ala Ser Tyr Asn 100 105 110Val Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12080123PRTLama glama
80Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Gly Val Ser Gly Leu Ser Phe Ser Gly
Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Ala 35 40 45Ala Ala Ile Gly Trp Asn Ser Gly Thr Thr Glu Tyr Arg
Asn Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ser Pro Lys Tyr Met Thr Ala
Tyr Glu Arg Ser Tyr Asp Phe 100 105 110Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 12081115PRTLama glama 81Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Ala Phe Gly Asp Ser 20 25 30Trp Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Glu Ile
Asn Thr Asn Gly Leu Ile Thr Lys Tyr Lys Asp Ser Val 50 55 60Thr Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu His65 70 75
80Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95Ala Arg Asp Pro Ser Gly Lys Leu Arg Gly Pro Gly Thr Gln Val
Thr 100 105 110Val Ser Ser 11582115PRTLama glama 82Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Pro Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Asp Ser 20 25 30Trp Met
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Lys Asp Ser Val 50 55
60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu His65
70 75 80Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Ala Arg Asp Pro Ser Gly Lys Leu Arg Gly Pro Gly Thr Gln
Val Thr 100 105 110Val Ser Ser 11583115PRTLama glama 83Gln Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Gly Asp Ser 20 25 30Trp
Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Lys Asp Ser Val
50 55 60Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
His65 70 75 80Leu Glu Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Leu
Tyr Tyr Cys 85 90 95Ala Arg Asp Pro Ser Gly Lys Leu Arg Gly Pro Gly
Thr Gln Val Thr 100 105 110Val Ser Ser 11584115PRTLama glama 84Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His
20 25 30Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Thr Ile Asn Thr Asn Gly Leu Ile Thr Asn Tyr Ile His
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Leu Asn Gln Ala Gly Leu Ser Arg Gly
Gln Gly Thr Gln Val Thr 100 105 110Val Ser Ser 11585128PRTLama
glama 85Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
Asp1 5 10 15Ser Leu Arg Leu Ser Cys Val Val Ser Gly Thr Thr Phe Ser
Ser Ala 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Gly Ala Ile Lys Trp Ser Gly Thr Ser Thr Tyr Tyr
Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val
Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Asn Leu Lys Pro Glu
Asp Thr Gly Val Tyr Thr Cys 85 90 95Ala Ala Asp Arg Asp Arg Tyr Arg
Asp Arg Met Gly Pro Met Thr Thr 100 105 110Thr Asp Phe Arg Phe Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 12586124PRTLama
glama 86Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Thr Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser
Ser Phe 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Arg Glu Arg
Glu Phe Val 35 40 45Ala Ser Ile Gly Ser Ser Gly Ile Thr Thr Asn Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Gly Leu Cys Tyr Cys 85 90 95Ala Val Asn Arg Tyr Gly Ile Pro
Tyr Arg Ser Gly Thr Gln Tyr Gln 100 105 110Asn Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 12087120PRTLama glama 87Glu Val Gln Leu
Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Asn Asp Tyr 20 25 30Ala Met
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Asp Met Val 35 40 45Ala
Thr Ile Ser Ile Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
Val 85 90 95Ala His Arg Gln Thr Val Val Arg Gly Pro Tyr Leu Leu Trp
Gly Gln 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115
12088123PRTLama glama 88Gln Val Gln Leu Val Glu Ser Gly Gly Lys Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Ser Asn Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Gly Ser Gly Arg Ser Asn Ser
Tyr Asn Tyr Tyr Ser Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ser Thr
Asn Leu Trp Pro Arg Asp Arg Asn Leu Tyr Ala Tyr 100 105 110Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 12089125PRTLama glama 89Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Leu Gly Ile Tyr
20 25 30Arg Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Thr Thr Arg Tyr Leu Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Thr Lys Asn
Ala Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Val Asp Ser Ser Gly Arg Leu Tyr Trp
Thr Leu Ser Thr Ser Tyr 100 105 110Asp Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 12590125PRTLama glama 90Gln Val Gln Leu
Val Glu Phe Gly Gly Gly Leu Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Ser Leu Gly Ile Tyr 20 25 30Lys Met
Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Ala Ile Ser Trp Ser Gly Gly Thr Thr Arg Tyr Ile Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Thr Lys Asn Met Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Val Asp Ser Ser Gly Arg Leu Tyr Trp Thr Leu Ser Thr
Ser Tyr 100 105 110Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 12591124PRTLama glama 91Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Ser Leu Ser Cys Ala
Ala Ser Gly Arg Thr Phe Ser Pro Tyr 20 25 30Thr Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Phe Leu 35 40 45Ala Gly Val Thr Trp
Ser Gly Ser Ser Thr Phe Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe
Thr Ala Ser Arg Asp Ser Ala Lys Asn Thr Val Thr65 70 75 80Leu Glu
Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Ala Ala Tyr Gly Gly Gly Leu Tyr Arg Asp Pro Arg Ser Tyr Asp 100 105
110Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 115
12092131PRTLama glama 92Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Leu Asp Ala Trp 20 25 30Pro Ile Ala Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Gly Val 35 40 45Ser Cys Ile Arg Asp Gly Thr Thr
Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg Phe Thr Ile Ser Ser Asp
Asn Ala Asn Asn Thr Val Tyr Leu Gln65 70 75 80Thr Asn Ser Leu Lys
Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 85 90 95Pro Ser Gly Pro
Ala Thr Gly Ser Ser His Thr Phe Gly Ile Tyr Trp 100 105 110Asn Leu
Arg Asp Asp Tyr Asp Asn Trp Gly Gln Gly Thr Gln Val Thr 115 120
125Val Ser Ser 13093126PRTLama glama 93Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Asp His Tyr 20 25 30Thr Ile Gly Trp Phe
Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ser Cys Ile Ser
Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu
Gln Met Asn Thr Leu Glu Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Ala Gly Gly Leu Leu Leu Arg Val Glu Glu Leu Gln Ala Ser Asp
100 105 110Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr Val Ser Ser
115 120 12594128PRTLama glama 94Ala Val Gln Leu Val Asp Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala
Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30Ala Ile Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ala Cys Ile Ser Asn Ser
Asp Gly Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Thr Thr Val Tyr65 70
75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Thr Ala Asp Arg His Tyr Ser Ala Ser His His Pro Phe
Ala Asp 100 105 110Phe Ala Phe Asn Ser Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 12595120PRTLama glama 95Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Tyr Gly Leu Thr Phe Trp Arg Ala 20 25 30Ala Met Ala
Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45Val Ala
Arg Asn Trp Gly Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75
80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Ala Val Arg Thr Tyr Gly Ser Ala Thr Tyr Asp Ile Trp Gly
Gln 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115 12096123PRTLama
glama 96Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Asp Gly
Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Phe Ser Gly Arg Thr Phe Ala
Asn Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Ala Ala Ile Asn Arg Asn Gly Gly Thr Thr Asn Tyr
Ala Asp Ala Leu 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr
Lys Asn Thr Ala Phe65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Glu Trp Pro Phe Ser
Thr Ile Pro Ser Gly Trp Arg Tyr 100 105 110Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 12097125PRTLama glama 97Asp Val Gln Leu Val
Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Pro Thr Ala Ser Ser His 20 25 30Ala Ile Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Val Gly
Ile Asn Arg Gly Gly Val Thr Arg Asp Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Ala Val Ser Arg Asp Asn Val Lys Asn Thr Val Tyr65 70 75
80Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Ser Ala Ile Tyr Ile Cys
85 90 95Ala Ala Arg Pro Glu Tyr Ser Phe Thr Ala Met Ser Lys Gly Asp
Met 100 105 110Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser
115 120 1259823DNALama glama 98gaggtbcarc tgcaggastc ygg
239953DNALama glama 99aacagttaag cttccgcttg cggccgcgga gctggggtct
tcgctgtggt gcg 5310053DNALama glama 100aacagttaag cttccgcttg
cggccgctgg ttgtggtttt ggtgtcttgg gtt 5310198PRTHomo sapiens 101Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys102123PRTLama glama 102Gln Val
Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His 20 25
30Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Thr Lys Val Leu Pro Pro Tyr Ser Asp Asp Ser
Arg Thr Asn Ala Asp 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 12010323DNALama glama 103cccctggccc cagtagttat acg
2310417DNALama glama 104tgtgcagcaa gagacgg 1710544DNALama glama
105gtcctcgcaa ctgcggccca gccggcctgt gcagcaagag acgg 4410645DNALama
glama 106gtcctcgcaa ctgcgcggcc gccccctggc cccagtagtt atacg
4510760DNALama glama 107agagacaact ccaagaacac gctgtatctg caaatgaaca
gcctgagagc tgaggacacg 6010820PRTLama glama 108Arg Asp Asn Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg1 5 10 15Ala Glu Asp Thr
2010930DNALama glama 109catggctgag gtgcagctgc tcgagtctgg
301109PRTLama glama 110Met Ala Glu Val Gln Leu Leu Glu Ser1
511135DNALama glama 111ggacacggcc gtctattact gtgcaaaagt acttc
3511211PRTLama glama 112Asp Thr Ala Val Tyr Tyr Cys Ala Lys Val
Leu1 5 1011330DNALama glama 113acctatacca ttggctgggt ccgccaggct
3011410PRTLama glama 114Thr Tyr Thr Ile Gly Trp Val Arg Gln Ala1 5
1011530DNALama glama 115cgccaggctc cagggaaggg gcgtgagttt
3011610PRTLama glama 116Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe1 5
1011730DNALama glama 117agggaaggag cttgagtttg tagcgcgtat
301189PRTLama glama 118Gly Lys Glu Leu Glu Phe Val Ala Arg1
511929DNALama glama 119gggaaggagc gtgagtgggt agcgcgtat
291209PRTLama glama 120Gly Lys Glu Arg Glu Trp Val Ala Arg1
5121136PRTLama glama 121Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly
Arg Thr Phe Ser Ala His 20 25 30Ser Val Tyr Thr Met Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg 35 40 45Glu Phe Val Ala Arg Ile Tyr Trp
Ser Ser Ala Asn Thr Tyr Tyr Ala 50 55 60Asp Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn65 70 75 80Thr Val Asp Leu Leu
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val 85 90 95Tyr Tyr Cys Ala
Ala Arg Asp Gly Ile Pro Thr Ser Arg Thr Val Gly 100 105 110Ser Tyr
Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu 115 120
125Pro Lys Thr Pro Lys Pro Gln Pro 130 135122138PRTLama glama
122Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp
His 20 25 30Ser Gly Tyr Thr Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro
Gly Lys 35 40 45Glu Arg Glu Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly
Asn Thr Tyr 50 55 60Tyr Ala Asp Ser Val Lys Gly Arg Phe Ala Ile Ser
Arg Asp Ile Ala65 70 75 80Lys Asn Thr Val Asp Leu Thr Met Asn Asn
Leu Glu Pro Glu Asp Thr 85 90 95Ala Val Tyr Tyr Cys Ala Ala Arg Asp
Gly Ile Pro Thr Ser Arg Ser 100 105 110Val Glu Ser Tyr Asn Tyr Trp
Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125Ser Glu Pro Lys Thr
Pro Lys Pro Gln Pro 130 135123124PRTLama glama 123Gln Val Gln Leu
Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Thr Ser Gly Phe Asp Phe Ser Val Ser 20 25 30Trp Met
Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Glu Ile Asn Thr Asn Gly Leu Ile Thr Lys Tyr Val Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asp Ser Leu Ile Pro Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Ala Arg Ser Pro Ser Gly Ser Phe Arg Gly Gln Gly Thr Gln
Val Thr 100 105 110Val Ser Ser Glu Pro Lys Thr Pro Lys Pro Gln Pro
115 120124132PRTLama glama 124Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr
Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30Trp Met Tyr Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Thr Val Asn Thr Asn
Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Lys
Val Val Pro Pro Tyr Ser Asp Asp Ser Arg Thr Asn Ala Asp 100 105
110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro
115 120 125Lys Pro Gln Pro 130125132PRTLama glama 125Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Glu Phe Glu Asn His 20 25 30Trp
Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Thr Val Asn Thr Asn Gly Leu Ile Thr Arg Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Thr Lys Val Leu Pro Pro Tyr Ser Asp Asp Ser Arg
Thr Asn Ala Asp 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser Glu Pro Lys Thr Pro 115 120 125Lys Pro Gln Pro 130126130PRTLama
glama 126Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe
Arg Val Asn 20 25 30Ala Met Gly Trp Tyr Arg Gln Val Pro Gly Asn Gln
Arg Glu Phe Val 35 40 45Ala Ile Ile Thr Ser Gly Asp Asn Leu Asn Tyr
Ala Asp Ala Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Thr Asp Asn Val
Lys Lys Thr Val Tyr Leu65 70 75 80Gln Met Asn Val Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95Ala Ile Leu Gln Thr Ser Arg
Trp Ser Ile Pro Ser Asn Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val
Thr Val Ser Ser Glu Pro Lys Thr Pro Lys Pro 115 120 125Gln Pro
130127133PRTLama glama 127Gln Val Gln Leu Gln Asp Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Gly Thr Phe Ser Ser Ile 20 25 30Ile Met Ala Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala Val Ser Trp Ser Gly
Gly Thr Thr Val Tyr Ala Asp Ser Val 50 55 60Leu Gly Arg Phe Glu Ile
Ser Arg Asp Ser Ala Arg Lys Ser Val Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg
Pro Tyr Gln Lys Tyr Asn Trp Ala Ser Ala Ser Tyr Asn 100 105 110Val
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr 115 120
125Pro Lys Pro Gln Pro 130128132PRTLama glama 128Gln Val Gln Leu
Gln Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Gly Val Ser Gly Leu Ser Phe Ser Gly Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45Ala
Ala Ile Gly Trp Asn Ser Gly Thr Thr Glu Tyr Arg Asn Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Ser Pro Lys Tyr Met Thr Ala Tyr Glu Arg Ser Tyr
Asp Phe 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu
Pro Lys Thr Pro 115 120 125Lys Pro Gln Pro 130129124PRTLama glama
129Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Leu Ser Ser
Tyr 20 25 30Ile Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Gly Ala Val Ser Trp Ser Ser Ser Thr Ile Val Tyr Ala
Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn His Gln
Asn Thr Val Tyr65 70 75 80Leu Gln Met Asp Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Arg Pro Tyr Gln Lys Tyr Asn
Trp Ala Ser Ala Ser Tyr Asn 100 105 110Val Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120130124PRTLama glama 130Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Glu Gly Thr Leu Ser Gly Tyr 20 25 30Ile Leu
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly
Ala Val Ser Trp Ser Gly Gly Thr Ile Val Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Glu Ile Ser Arg Asp Asn Ala Arg Asn Thr Val Tyr65
70 75 80Leu Gln Met Asp Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Arg Pro Tyr Gln Arg Phe Asn Trp Ala Ser Ala Ser
Tyr Asn 100 105 110Val Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
115 120131253DNALama glama 131aagcttgcat gcaaattcta tttcaaggag
acagtcataa tgaaatacct attgcctacg 60gcagccgctg gattgttatt actcgcggcc
cagccggcca tggggcctaa taggcggccg 120cacaggtgca gctgcaggag
tcataatgag ggacccaggt caccgtctcc tcagaacaaa 180aactcatctc
agaagaggat ctgaatgggg ccgcacatca tcatcatcat cattaatgag
240aattcactgg ccg 25313261PRTLama glama 132Met Lys Tyr Leu Leu Pro
Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met
Gly Pro Ala Ala Ala Gln Val Gln Leu Gln Glu 20 25 30Ser Gly Thr Gln
Val Thr Val Ser Ser Glu Gln Lys Leu Ile Ser Glu 35 40 45Glu Asp Leu
Asn Gly Ala Ala His His His His His His 50 55 60
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