U.S. patent application number 13/078184 was filed with the patent office on 2011-07-28 for method of administering therapeutic polypeptides, and polypeptides therefor.
This patent application is currently assigned to ABLYNX N.V.. Invention is credited to KAREN SILENCE, MARK VAECK, PAUL M. P. VAN BERGEN EN HENEGOUWEN.
Application Number | 20110184145 13/078184 |
Document ID | / |
Family ID | 32314976 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110184145 |
Kind Code |
A1 |
SILENCE; KAREN ; et
al. |
July 28, 2011 |
METHOD OF ADMINISTERING THERAPEUTIC POLYPEPTIDES, AND POLYPEPTIDES
THEREFOR
Abstract
The invention relates to a method suitable for administering
protein therapeutic molecules orally, sublingually, topically,
intravenously, subcutaneously, nasally, vaginally, rectally or by
inhalation so as to avoid inactivation, by using VHH polypeptides
derived from Camelidae antibodies. The invention further relates to
the said therapeutic molecules. The invention further a method for
delivering therapeutic molecules to the interior of cells. The
invention further relates to anti-IgE therapeutic molecules.
Inventors: |
SILENCE; KAREN; (OVERIJSE,
BE) ; VAECK; MARK; (HOFSTADE, BE) ; VAN BERGEN
EN HENEGOUWEN; PAUL M. P.; (UTRECHT, NL) |
Assignee: |
ABLYNX N.V.
ZWIJNAARDE
BE
|
Family ID: |
32314976 |
Appl. No.: |
13/078184 |
Filed: |
April 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12488077 |
Jun 19, 2009 |
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13078184 |
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10534292 |
May 9, 2005 |
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PCT/BE2003/000190 |
Nov 7, 2003 |
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12488077 |
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60425073 |
Nov 8, 2002 |
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60425063 |
Nov 8, 2002 |
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Current U.S.
Class: |
530/300 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 13/00 20180101; A61P 37/06 20180101; C07K 2319/31 20130101;
A61P 7/06 20180101; C07K 2317/22 20130101; A61P 35/00 20180101;
A61P 1/00 20180101; A61P 7/02 20180101; C07K 16/4291 20130101; A61P
1/04 20180101; A61P 5/00 20180101; A61P 43/00 20180101; C07K 16/18
20130101; A61P 9/08 20180101; A61P 17/06 20180101; A61P 37/08
20180101; C07K 2319/00 20130101; A61P 3/10 20180101; A61P 11/06
20180101; A61P 7/00 20180101; A61P 25/00 20180101; C07K 2317/31
20130101; A61P 1/14 20180101; C07K 16/40 20130101; A61P 21/04
20180101; A61P 19/02 20180101; C07K 16/241 20130101; C07K 2317/626
20130101; A61P 1/16 20180101; A61P 17/02 20180101; C07K 2317/76
20130101; A61P 27/02 20180101; A61P 31/06 20180101; A61P 37/00
20180101; A61P 25/02 20180101; A61P 13/12 20180101; A61P 31/16
20180101; A61P 37/02 20180101; C07K 2317/24 20130101; A61P 27/16
20180101; A61P 9/10 20180101; A61P 15/08 20180101; C07K 16/2875
20130101; C07K 2317/62 20130101; C07K 2317/77 20130101; A61P 11/00
20180101; C07K 16/2863 20130101; C07K 2317/565 20130101; C07K
2317/569 20130101; C07K 16/36 20130101; A61P 29/00 20180101; C07K
16/249 20130101 |
Class at
Publication: |
530/300 |
International
Class: |
C07K 2/00 20060101
C07K002/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 10, 2003 |
EP |
03447005.4 |
Jun 23, 2003 |
EP |
PCT/EP2003/006581 |
Jul 8, 2003 |
EP |
PCT/EP2003/007313 |
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 1.times.10.sup.-6 M or
better.
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, IgE, IFN-gamma, CEA, H. pylori, TB
(M. tuberculosis), influenza, Hepatitis E, MMP-12, epidermal growth
factor receptor (EGFR), ErBb2 receptor on tumor cells, LDL
receptor, FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF
receptor, VEGF receptor, PsmAr, PDK1, GSK1, Bad, caspase-9,
Forkhead, an antigen of Helicobacter pylori, an antigen of
Mycobacterium tuberculosis, an antigen of influenza virus, 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 1.times.10.sup.-6 M or
better.
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, IgE, IFN-gamma, CEA, H. pylori, TB
(M. tuberculosis), influenza, Hepatitis E, MMP-12, epidermal growth
factor receptor (EGFR), ErBb2 receptor on tumor cells, LDL
receptor, FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF
receptor, VEGF receptor, PsmAr, PDK1, GSK1, Bad, caspase-9,
Forkhead, an antigen of Helicobacter pylori, an antigen of
Mycobacterium tuberculosis, an antigen of influenza virus, 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, IgE, IFN-gamma, CEA, H.
pylori, TB (M. tuberculosis), influenza, Hepatitis E, MMP-12,
epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor
cells, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin
receptor, PDGF receptor, VEGF receptor, PsmAr, PDK1, GSK1, Bad,
caspase-9, Forkhead, an antigen of Helicobacter pylori, an antigen
of Mycobacterium tuberculosis, an antigen of influenza virus, 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 1.times.10.sup.-6
M or better.
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, IgE, IFN-gamma, CEA, H. pylori, TB
(M. tuberculosis), influenza, Hepatitis E, MMP-12, epidermal growth
factor receptor (EGFR), ErBb2 receptor on tumor cells, LDL
receptor, FGF2 receptor, ErbB2 receptor, transferrin receptor, PDGF
receptor, VEGF receptor, PsmAr, PDK1, GSK1, Bad, caspase-9,
Forkhead, an antigen of Helicobacter pylori, an antigen of
Mycobacterium tuberculosis, an antigen of influenza virus, 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, IgE, IFN-gamma, CEA, H.
pylori, TB (M. tuberculosis), influenza, Hepatitis E, MMP-12,
epidermal growth factor receptor (EGFR), ErBb2 receptor on tumor
cells, LDL receptor, FGF2 receptor, ErbB2 receptor, transferrin
receptor, PDGF receptor, VEGF receptor, PsmAr, PDK1, GSK1, Bad,
caspase-9, Forkhead, an antigen of Helicobacter pylori, an antigen
of Mycobacterium tuberculosis, an antigen of influenza virus, 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. application Ser.
No. 12/488,077, filed Jun. 19, 2009, which is a continuation of
U.S. application Ser. No. 10/534,292, filed May 9, 2005, which is a
national stage filing under 35 U.S.C. .sctn.371 of international
application PCT/BE03/00190, filed Nov. 7, 2003, which was published
under PCT Article 21(2) in English, which claims priority to
international application PCT/EP03/06581, filed Jun. 23, 2003, and
international application PCT/EP03/07313, filed Jul. 8, 2003, and
also claims the benefit under 35 U.S.C. 119(e) 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.
BACKGROUND
[0002] 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, they have one important drawback: these are complex, large
molecules and therefore relatively unstable, and they are sensitive
to breakdown by proteases. Because the degradation they undergo
during passage through, for instance, the gastrointestinal tract,
administration of conventional antibodies and their derived
fragments or single-chain formats (e.g. scFv's) is not very
effective. 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 is
therefore the most frequently used method of administration
although the method has many disadvantages, for example: (a) poor
tolerance by patients, especially when treating chronic disorder;
(b) a consequent risk of poor compliance with the dosage when the
drug is not a `life saver`; (c) difficulty of carrying out
self-administration by the patient; (d) possible non-availability
of suitable surroundings for carrying out the procedure in an
aseptic manner; (e) requires specialist training in order to use a
hypodermic syringe or needle correctly and safely. A method for the
delivery of therapeutic polypeptides which avoids the need for
injection has not only cost/time savings, but would also be more
convenient and more comfortable for the subject.
[0003] In most animal cells, a specialised pathway is present for
uptake of specific macromolecules from the extracellular fluid. The
macromolecules that bind to specific cell-surface receptors are
internalized, a process called receptor-mediated endocytosis.
Receptor internalization is based on the principle of regulation of
signal transduction by a process called sequestration, whereby
bound agonistic (i.e. receptor activation) ligands are recovered
from the cell surface in complex with the receptor. For many
applications it is necessary to deliver effector molecules across
the cell membrane and into the cytosol. This can be achieved by
taking advantage of such internalizing receptors. Antibodies have
been described that internalize upon binding to internalizing
receptors. However, they have important drawbacks: these antibodies
are complex, large molecules and therefore relatively unstable, and
they are sensitive to breakdown by proteases. Moreover, the domains
of such antibodies are held together by disulphide bonds that
dissociate in the reducing environment of the cytoplasm leading to
a substantial loss of binding activity. Therefore, they cannot be
used to target intracellular proteins.
[0004] Another process that relies on internalisation is the
efficient induction of an immune response. In particular, a T-cell
response depends heavily on efficient presentation of certain
epitopes to the T cells by antigen presenting cells (APCs). In the
case of a protein antigen this means that the APC has to take up
the protein, internally process it (this is cleaving it) and
express certain peptide fragments on its surface in association
with MHC (major histocompatibility complex) or HLA molecules. One
major and critical event in this process is the efficient uptake of
the protein antigen by its APC. Techniques which can enhance
antigen uptake by APCs enables an immune response to be elicited
against antigens which naturally elicit a weak or no immune
response. Therefore, a technique which can boost an immune response
against antigenic antigens, naturally weak or non-immunogenic
antigens has important implications for vaccination programs.
[0005] IgE plays a major role in allergic disease by causing the
release of histamine and other inflammatory mediatord from mast
cells. A mainstay of treatment of allergic disease, including
asthma, is allergen avoidance and treatment of symptoms. Presently,
the most effective treatments of allergic diseases are directed
towards a regulation of the inflammatory process with
corticosteroids. A more direct approach without the negative
effects of corticosteroids consists in regulating the allergic
process at the level of the initiatior of the allergic
inflammation, IgE, via an anti-IgE.
[0006] The concept of using anti-IgE antibodies as a treatment for
allergy has been widely disclosed in the scientific literature. A
few representative examples are as follows. Baniyash and Eshhar
(European Journal of Immunology 14:799-807 (1984)) demonstrated
that an anti-IgE monoclonal antibody could specifically block
passive cutaneous anaphylaxis reaction when injected intradermally
before challenging with the antigen; U.S. Pat. No. 4,714,759
discloses a product and process for treating allergy, using an
antibody specific for IgE; and Rup and Kahn (International Archives
Allergy and Applied Immunology, 89:387-393 (1989) discuss the
prevention of the development of allergic responses with monoclonal
antibodies which block mast cell-IgE sensitization.
[0007] Anti-IgE antibodies which block the binding of IgE to its
receptor on basophils and which fail to bind to IgE bound to the
receptor, thereby avoiding histamine release are disclosed, for
example, by Rup and Kahn (supra), by Baniyash et al. (Molecular
Immunology 25:705-711, 1988), and by Hook et al. (Federation of
American Societies for Experimental Biology, 71st Annual Meeting,
Abstract #6008, 1987).
[0008] Antagonists of IgE in the form of receptors, anti-IgE
antibodies, binding factors, or fragments thereof have been
disclosed in the art. For example, U.S. Pat. No. 4,962,035
discloses DNA encoding the alpha-subunit of the mast cell IgE
receptor or an IgE binding fragment thereof. Hook et al.
(Federation Proceedings Vol. 40, No. 3, Abstract #4177) disclose
monoclonal antibodies, of which one type is anti-idiotypic, a
second type binds to common IgE determinants, and a third type is
directed towards determinants hidden when IgE is on the basophil
surface.
[0009] U.S. Pat. No. 4,940,782 discloses monoclonal antibodies
which react with free IgE and thereby inhibit IgE binding to mast
cells, and react with IgE when it is bound to the B-cell FcE
receptor, but do not bind with IgE when it is bound to the mast
cell FcE receptor, nor block the binding of IgE to the B-cell
receptor.
[0010] U.S. Pat. No. 4,946,788 discloses a purified IgE binding
factor and fragments thereof, and monoclonal antibodies which react
with IgE binding factor and lymphocyte cellular receptors for IgE,
and derivatives thereof.
[0011] U.S. Pat. No. 5,091,313 discloses antigenic epitopes
associated with the extracellular segment of the domain which
anchors immunoglobulins to the B cell membrane. The epitopes
recognized are present on IgE-bearing B cells but not basophils or
in the secreted, soluble form of IgE. U.S. Pat. No. 5,252,467
discloses a method for producing antibodies specific for such
antigenic epitopes. U.S. Pat. No. 5,231,026 discloses DNA encoding
murine-human antibodies specific for such antigenic epitopes.
[0012] U.S. Pat. No. 4,714,759 discloses an immunotoxin in the form
of an antibody or an antibody fragment coupled to a toxin to treat
allergy.
[0013] Presta et al. (J. Immunol. 151:2623-2632 (1993)) disclose a
humanized anti-IgE antibody that prevents the binding of free IgE
to FceRl but does not bind to Fc.quadrature.RI-bound IgE.
[0014] Copending WO93/04173 discloses polypeptides which bind
differentially to the high- and low-affinity IgE receptors.
[0015] U.S. Pat. No. 5,428,133 discloses anti-IgE antibodies as a
therapy for allergy, especially antibodies which bind to IgE on B
cells, but not IgE on basophils. This publication mentions the
possibility of treating asthma with such antibodies. U.S. Pat. No.
5,422,258 discloses a method for making such antibodies.
[0016] EP0841946 discloses methods for treating allergic asthma
using IgE antagonists.
AIMS OF THE INVENTION
[0017] The aim of the invention is to provide a method of
administering protein therapeutic molecules orally, sublingually,
topically, nasally, vaginally, rectally, intraveneously,
subcutaneously or by inhalation which overcomes the problems of the
prior art. It is a further aim to provide said therapeutic
molecules.
[0018] Another aim of the invention is to provide a method for
delivering therapeutic substances to the interior of cells via
internalizing receptors without receptor activation.
[0019] It is further aim of the invention to provide a therapeutic
agent for the treatment of allergies.
[0020] It is a further aim of the invention to provide therapeutic
nanobodies.
SUMMARY OF THE INVENTION
[0021] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against IgE.
[0022] Another embodiment of the present invention is a polypeptide
construct as described above wherein at least one single domain
antibody is a Camelidae VHH.
[0023] Another embodiment of the present invention is a polypeptide
construct as described above wherein at least one single domain
antibody corresponds to a sequence represented by any of SEQ ID
NOs: 1 to 11.
[0024] Another embodiment of the present invention is a polypeptide
construct as described above, wherein the number of anti-IgE single
domain antibodies is at least two.
[0025] Another embodiment of the present invention is a polypeptide
construct as described above, wherein at least one single domain
antibody is a humanized Camelidae VHH.
[0026] Another embodiment of the present invention is a polypeptide
construct as described above, wherein a 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.
[0027] Another embodiment of the present invention is a polypeptide
construct as described above, wherein the polypeptide construct is
an homologous sequence, a functional portion, or a functional
portion of an homologous sequence of the full length polypeptide
construct.
[0028] Another embodiment of the present invention is a nucleic
acid encoding a polypeptide construct as described above.
[0029] Another embodiment of the present invention is a polypeptide
construct as described above for treating and/or preventing and/or
alleviating disorders relating to inflammatory processes.
[0030] Another embodiment of the present invention is a use of a
polypeptide construct as described above for the preparation of a
medicament for treating and/or preventing and/or alleviating
disorders relating to inflammatory reactions.
[0031] Another embodiment of the present invention is a method for
delivering an anti-target compound to a subject for the treatment
of a disorder without being inactivated by administering thereto a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0032] Another embodiment of the present invention is a method as
described above wherein said target is located in the gut system,
and said a polypeptide construct is delivered orally.
[0033] Another embodiment of the present invention is a method as
described above wherein said target is located in vaginal and/or
rectal tract, and said a polypeptide construct is delivered to the
vaginal and/or rectal tract.
[0034] Another embodiment of the present invention is a method as
described above wherein said target is located in nose, upper
respiratory tract and/or lung, and said a polypeptide construct is
delivered to nose, upper respiratory tract and/or lung.
[0035] Another embodiment of the present invention is a method as
described above wherein said target is located in intestinal
mucosa, and said a polypeptide construct is delivered orally.
[0036] Another embodiment of the present invention is a method as
described above wherein said target is located in the tissues
beneath the tongue, and said a polypeptide construct is delivered
to the tissues beneath the tongue.
[0037] Another embodiment of the present invention is a method as
described above wherein said target is located in the skin, and
said a polypeptide construct is delivered topically.
[0038] Another embodiment of the present invention is a method as
described above wherein said target is in, or accessible via the
blood, and said a polypeptide construct is delivered orally, to the
vaginal and/or rectal tract, nasally, by inhalation though the
mouth or nose, to the tissues beneath the tongue, or topically.
[0039] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target, for use in treating, preventing and/or
alleviating the symptoms of disorders which are susceptible to
modulation by an anti-target therapeutic compound that is able pass
through the gastric environment without being inactivated.
[0040] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders which are susceptible to modulation by an
anti-target therapeutic compound that is able pass through the wall
of the intestinal mucosa without being inactivated
[0041] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders which are susceptible to modulation by an
anti-target therapeutic compound that is able pass through the wall
of the nose, upper respiratory tract and/or lung without being
inactivated
[0042] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders which are susceptible to modulation by an
anti-target therapeutic compound that is able pass through the wall
of virginal and/or rectal tract without being inactivated
[0043] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders which are susceptible to modulation by a
therapeutic compound that is able pass through the tissues beneath
the tongue without being inactivated
[0044] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders which are susceptible to modulation by a
therapeutic compound that is able pass through the skin without
being inactivated
[0045] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is TNF-alpha and the disorder is
inflammation.
[0046] Another embodiment of the present invention is a method or
polypeptide as described above, wherein a single domain antibody
corresponds to a sequence represented by any of SEQ ID NOs: 12 to
14.
[0047] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is CEA and the disorder colon cancer.
[0048] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is EGFR and the disorder is any of head, neck,
lung and colon cancer.
[0049] Another embodiment of the present invention is a method or
polypeptide construct as described above, wherein a single domain
antibody corresponds to a sequence represented by any of SEQ ID
NOs: 23 to 44
[0050] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is antigen of Helicobacter pylori and the
disorder is any of indigestion, gastritis.
[0051] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is antigen of Mycobacterium tuberculosis and
the disorder is tuberculosis.
[0052] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is antigen of influenza virus and the disorder
is flu.
[0053] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is antigen of IgE and the disorder is allergic
response.
[0054] Another embodiment of the present invention is a method or
polypeptide construct as described above, wherein a single domain
antibody corresponds to a sequence represented by any of SEQ ID
NOs: 1 to 11
[0055] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is antigen of MMP and the disorder is
cancer.
[0056] Another embodiment of the present invention is a method or
polypeptide construct as described above, wherein a single domain
antibody corresponds to a sequence represented by any of SEQ ID
NOs: 15 to 22
[0057] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above,
wherein said target is antigen of IFN-gamma and the disorder is any
of cancer, transplant rejection, auto immune disorder.
[0058] Another embodiment of the present invention is a method or
polypeptide construct as described above, wherein a single domain
antibody corresponds to a sequence represented by any of SEQ ID
NOs: 45 to 70
[0059] Another embodiment of the present invention is a method as
described above or polypeptide construct as described above wherein
said target is any of antigen of Helicobacter pylori, antigen of
Mycobacterium tuberculosis, antigen of influenza virus.
[0060] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against an internalising cellular receptor, and at least one single
domain antibody directed against a therapeutic target.
[0061] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against an internalising cellular receptor, and at least one
therapeutic polypeptide or agent.
[0062] Another embodiment of the present invention is a polypeptide
construct as described above wherein said internalising cellular
receptor is Epidermal Growth Factor receptor.
[0063] Another embodiment of the present invention is a polypeptide
as described above wherein a single domain antibody directed
against an internalising cellular receptor corresponds to a
sequence represented by SEQ ID NO: 23 to 44.
[0064] Another embodiment of the present invention is a polypeptide
construct as described above wherein said internalising cellular
receptor is any of LDL receptor, FGF2r, ErbB2r, transferring
receptor, PDGr, VEGr, or PsmAr.
[0065] Another embodiment of the present invention is a polypeptide
construct as described above wherein a single domain antibody
directed against a therapeutic target, is directed against
PDK1.
[0066] Another embodiment of the present invention is a polypeptide
construct as described above use in treating cancer
[0067] Another embodiment of the present invention is a polypeptide
construct as described above wherein a single domain antibody
directed against a therapeutic target is directed against any of
GSK1, Bad, caspase and Forkhead.
[0068] Another embodiment of the present invention is a polypeptide
construct as described above use in treating cancer.
[0069] Another embodiment of the present invention is a method for
delivering an anti-target therapeutic compound to the interior of a
cell comprising administering to a subject a polypeptide construct
as described above.
[0070] Another embodiment of the present invention is a method for
delivering an anti-target therapeutic compound to the interior of a
cell without being inactivated comprising administering to a
subject a polypeptide construct as described above.
[0071] Another embodiment of the present invention is a method as
described above wherein said cell is located in the gut system, and
said a polypeptide construct is delivered orally.
[0072] Another embodiment of the present invention is a method as
described above wherein said cell is located in vaginal and/or
rectal tract, and said a polypeptide construct is delivered to the
vaginal and/or rectal tract.
[0073] Another embodiment of the present invention is a method as
described above wherein said cell is located in nose, upper
respiratory tract and/or lung, and said a polypeptide construct is
delivered to nose, upper respiratory tract and/or lung.
[0074] Another embodiment of the present invention is a method as
described above wherein said cell is located in intestinal mucosa,
and said a polypeptide construct is delivered orally.
[0075] Another embodiment of the present invention is a method as
described above wherein said cell is located in the tissues beneath
the tongue, and said a polypeptide construct is delivered to the
tissues beneath the tongue.
[0076] Another embodiment of the present invention is a method as
described above wherein said cell is located in the skin, and said
a polypeptide construct is delivered topically.
[0077] Another embodiment of the present invention is a method as
described above wherein said cell is in, or accessible via the
blood, and said a polypeptide construct is delivered orally, to the
vaginal and/or rectal tract, nasally, by inhalation though the
mouth or nose, to the tissues beneath the tongue, or topically.
[0078] Another embodiment of the present invention is a polypeptide
construct as described above, or a method as described above,
wherein the single domain antibodies are humanized Camelidae
VHHs.
[0079] Another embodiment of the present invention is a polypeptide
construct as described above, or a method 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.
[0080] Another embodiment of the present invention is a polypeptide
construct as described above or a method as described above,
wherein the polypeptide construct is an homologous sequence, a
functional portion, or a functional portion of an homologous
sequence of the full length polypeptide construct.
[0081] Another embodiment of the present invention is a polypeptide
construct as described above or a method as described above wherein
said single domain antibodies are Camelidae VHHs.
[0082] Another embodiment of the present invention is a nucleic
acid capable of encoding a polypeptide construct as described
above.
[0083] Another embodiment of the present invention is a composition
comprising a polypeptide construct as defined above, together with
a pharmaceutical carrier.
DETAILED DESCRIPTION OF THE INVENTION
[0084] The present invention relates to a polypeptide construct
comprising one or more single domain antibodies directed to one or
more target molecule(s), each in a suitable dosage form either
directly or as part of a composition containing an ingredient which
facilitates delivery.
[0085] The invention further relates to polypeptide constructs
comprising one or more single domain antibodies, for administration
to a subject by non-invasive methods, such as orally, sublingually,
topically, nasally, vaginally, rectally or by inhalation. Such
non-invasive routes of delivery unexpectly provide an effective
means to conveniently deliver therapeutic compounds
[0086] The present invention also relates to constructs comprising
one or more single domain antibodies, for administration to a
subject by normal invasive methods such as intravenously and
subcutaneously.
[0087] The invention further relates to a method for delivering
therapeutic peptides comprises the steps of administering a
polypeptide construct comprising one or more single domain
antibodies orally, sublingually, topically, intravenously,
subcutaneously, nasally, vaginally, rectally or by inhalation to a
subject.
[0088] The invention further relates to polypeptide constructs
comprising anti-IgE single domain antibodies.
[0089] Single domain antibodies are antibodies whose complementary
determining regions are part of a single domain polypetide.
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 antibody 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 9404678 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, llama,
dromedary, 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.
[0090] 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-albumin
VHH's may interact in a more efficient way with serum albumin 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.
[0091] The present invention further relates to a polypeptide
construct, wherein a single domain antibody is a VHH directed
against a target, 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 according to the Kabat
numbering. A VHH sequence represented by SEQ ID NO: 15 which binds
to MMP-12, belongs to this human-like class of VHH polypeptides. As
such, peptides belonging to this class 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.
[0092] Another human-like class of Camelidae single domain
antibodies represented by sequences 68 which binds to IFN gamma,
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 conventional
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.
[0093] 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 target 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.
[0094] Targets of the invention are any which are of pharmaceutical
interest. Examples are provided here of several targets, and are
not intended to limit the invention thereto. Examples of targets
include, TNF-alpha, IgE, IFN-gamma, MMP-12, EGFR, CEA, H. pylori,
TB, influenza. A single domain antibody directed against a target
means a single domain antibody that is capable of binding to said
target with an affinity of better than 10.sup.-6M.
[0095] Targets may also 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.
[0096] 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.sup.-6 M or better.
[0097] 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.
[0098] One embodiment of the present invention is a polypeptide
construct as disclosed herein, wherein the number of single domain
antibodies directed to a target is two or more. Such multivalent
polypeptide constructs have the advantage of unusually high
functional affinity for the target, displaying much higher than
expected inhibitory properties compared to their monovalent
counterparts.
[0099] Multivalent polypeptide constructs have functional
affinities that are several orders of magnitude higher than
polypeptide constructs which are monovalent. 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, the
multivalent polypeptide constructs of the present invention linked
to each other directly or via a short linker sequence show the high
functional affinities expected theoretically with multivalent
conventional four-chain antibodies.
[0100] 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. animal model of
chronic colitis.
[0101] A multivalent anti-target polypeptide as used herein refers
to a polypeptide comprising two or more anti-target polypeptides
which have been covalently linked. The anti-target polypeptides may
be identical in sequence or may be different in sequence, but are
directed against the same target or antigen. Depending on the
number of anti-target polypeptides linked, a multivalent
anti-target polypeptide may be bivalent (2 anti-target
polypeptides), trivalent (3 anti-target polypeptides), tetravalent
(4 anti-target polypeptides) or have a higher valency
molecules.
[0102] An example of a multivalent polypeptide construct of the
invention, comprising more than one anti-TNF-alpha VHHs is
described in Example 7.
[0103] The single domain antibodies may be joined to form any of
the polypeptide constructs disclosed herein comprising more than
one single domain antibody using methods known in the art or any
future method. They may be joined non-covalently (e.g. using
streptavidin/biotin combination, antibody/tag combination) or
covalently. 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. A method for producing
bivalent or multivalent VHH polypeptide constructs is disclosed in
PCT patent application WO 96/34103. One way of joining VHH
antibodies is via the genetic route by linking a VHH antibody
coding sequences either directly or via a peptide linker. For
example, the C-terminal end of the VHH antibody may be linked to
the N-terminal end of the next single domain antibody.
[0104] 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.
[0105] According to one 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
multivalent anti-target polypeptide is administered. The linker
sequence may provide sufficient flexibility to the multivalent
anti-target 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.
[0106] 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.
[0107] According to an aspect of the invention a polypeptide
construct may be a homologous sequence of a full-length polypeptide
construct. According to another aspect of the invention, a
polypeptide construct may be a functional portion of a full-length
polypeptide construct. According to another aspect of the
invention, a polypeptide construct may be a homologous sequence of
a full length polypeptide construct. According to another aspect of
the invention, a polypeptide construct may be a functional portion
of a homologous sequence of a full length polypeptide construct.
According to an aspect of the invention a polypeptide construct may
comprise a sequence of a polypeptide construct.
[0108] According to an aspect of the invention a single domain
antibody used to form a polypeptide construct 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.
[0109] As used herein, a 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.
[0110] A homologous sequence according to the present invention may
be a sequence of an anti-target polypeptide modified by the
addition, deletion or substitution of amino acids, said
modification not substantially altering the functional
characteristics compared with the unmodified polypeptide.
[0111] A homologous sequence of the present invention may be a
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.
[0112] A homologous sequence according to the present invention may
be a sequence which exists in other Camelidae species such as, for
example, camel, llama, dromedary, alpaca, guanaco etc.
[0113] 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.
[0114] Alternatively, a 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, H is, 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, H
is, 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; H is substituted by one of H
is, Glu, Lys, Gln, Thr, and Arg; Gln substituted by one of Gln,
Glu, Lys, Asn, H is, Thr, and Arg; Asn substituted by one of Asn,
Glu, Asp, Gln, and Ser; Lys substituted by one of Lys, Glu, Gln, H
is, and Arg; Asp substituted by one of Asp, Glu, and Asn; Glu
substituted by one of Glu, Asp, Lys, Asn, Gln, H is, and Arg; Met
substituted by one of Met, Phe, Ile, Val, Leu, and Tyr.
[0115] 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).
[0116] 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.
[0117] 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 its target.
[0118] 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 comprising 5 or more amino
acids or 15 or more nucleotides.
Anti-IgE Single Domain Antibodies
[0119] One aspect of the present invention relates to therapeutic
compounds which are suitable for alleviating the symptoms, for the
treatment and prevention of allergies. Said therapeutic compounds
interact with IgE, and modulate the cascade of immunological
responses that is responsible for an allergic response.
[0120] Another aspect of the present invention relates to the use
of anti-IgE single domain antibodies (e.g. VHHs) in the preparation
of topical ophthalmic compositions for the treatment of an ocular
allergic disorder (Example 2). Given the ease of production and the
low cost using bacterial or yeast expression systems for VHHs, for
example, compared to production of conventional antibodies in
mammalian cells, the economics of preparing such compositions using
VHHs of the invention are much more favourable then for
conventional antibodies.
[0121] Ocular penetration and consequently ocular efficacy is
highly unexpected with conventional antibodies and derived
fragments given their large size. The polypeptide constructs of the
invention however are expected to be highly efficient given their
high potency, stability combined with a low molecular weigth.
Therefore, applications for such indications other than topical can
be envisaged with polypeptide constructs of the invention.
[0122] One embodiment of the present invention is a polypeptide
construct comprising one or more single domain antibodies directed
against IgE.
[0123] Another embodiment of the present invention is a polypeptide
construct comprising one or more single domain antibodies directed
against IgE, wherein a single domain antibody corresponds to a
sequence represented by any of SEQ ID NOs: 1 to 11. Said sequences
are derived from Camelidae VHHs.
[0124] The present invention also relates to the finding that a
polypeptide construct comprising one or more single domain
antibodies directed against IgE and 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-IgE single domain antibody when not part of
said construct. Furthermore, such polypeptide constructs were found
to exhibit the same favourable properties of VHHs such as high
stability remaining intact in mice, extreme pH resistance, high
temperature stability and high target affinity.
[0125] Another embodiment of the present invention is a polypeptide
construct comprising one or more single domain antibodies directed
against IgE further comprising one or more single domain antibodies
directed against one or more serum proteins.
[0126] The serum protein may be any suitable protein found in the
serum of subject, or fragment thereof. 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.
[0127] One aspect of the invention, is a polypeptide construct
comprising one or more single domain antibodies directed against
IgE, further comprising an anti-serum albumin single domain
antibody corresponding to a sequence represented by any of SEQ ID
NO: 71 to 84.
Delivery of Polypeptide Constructs
[0128] The aspect of the invention relating to the delivery of
polypeptide constructs of the invention is not limited to a
polypeptide construct comprising anti-IgE single domain antibodies
disclosed herein, but, as shown below, is applicable to any target.
The polypeptide constructs may comprise single domain antibodies
directed against more than one target, optionally with the
variations described above.
[0129] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders susceptible to modulation by an
anti-target therapeutic compound that is able pass through the
gastric environment without being inactivated.
[0130] 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 VHHs 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
disorder whose targets that are located in the gut system.
[0131] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of a disorder
susceptible to modulation by a therapeutic compound that is able
pass through the gastric environment without being inactivated, by
orally administering to a subject a polypeptide construct
comprising one or more single domain antibodies specific for
antigen related to the disorder.
[0132] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the prepararion of a
medicament for treating, preventing and/or aleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound that is able pass through the gastric
environment without being inactivated.
[0133] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the gut system without being
inactivated, by orally administering to a subject a polypeptide
construct comprising one or more single domain antibodies directed
against said target.
[0134] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated, by orally administering to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0135] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target herein for use in treating, preventing and/or
alleviating the symptoms of disorders susceptible to modulation by
an anti-target therapeutic compound to the vaginal and/or rectal
tract.
[0136] In a non-limiting example, a formulation according to the
invention comprises a polypeptide construct as disclosed herein
comprising one or more VHHs directed against one or more targets 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).
[0137] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a therapeutic compound to the vaginal and/or
rectal tract, by vaginally and/or rectally administering to a
subject a polypeptide construct comprising one or more single
domain antibodies specific for antigen related to the disorder.
[0138] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the prepararion of a
medicament for treating, preventing and/or aleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound to the vaginal and/or rectal tract without
being inactivated.
[0139] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the vaginal and/or rectal tract
without being inactivated, by administering to the vaginal and/or
rectal tract of a subject a polypeptide construct comprising one or
more single domain antibodies directed against said target.
[0140] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated, by administering to the vaginal and/or
rectal tract of a subject a polypeptide construct comprising one or
more single domain antibodies directed against said target.
[0141] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target comprising at least one single domain antibody
directed against a target, for use in treating, preventing and/or
alleviating the symptoms of disorders susceptible to modulation by
an anti-target therapeutic compound to the nose, upper respiratory
tract and/or lung.
[0142] In a non-limiting example, a formulation according to the
invention, comprises a polypeptide construct as disclosed herein
directed against one or more targets in the form of a nasal spray
(e.g. an aerosol) or inhaler. Since the construct is small, it can
reach its target much more effectively than therapeutic IgG
molecules.
[0143] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a therapeutic compound to the upper respiratory
tract and lung, by administering to a subject a polypeptide
construct as disclosed herein wherein one or more single domain
antibodies are specific for an antigen related to the disorder, by
inhalation through the mouth or nose.
[0144] Another aspect of the invention is a dispersible VHH
composition, in particular dry powder dispersible VHH compositions,
such as those described in U.S. Pat. No. 6,514,496. These dry
powder compositions comprise a plurality of discrete dry particles
with an average particle size in the range of 0.4-10 .mu.m. Such
powders are capable of being readily dispersed in an inhalation
device. VHH's are particularly suited for such composition as
lyophilized material can be readily dissolved (in the lung
subsequent to being inhaled) due to its high solubilisation
capacity (Muyldermans, S., Reviews in Molecular Biotechnology, 74,
277-303, (2001)). Alternatively, such lyophilized VHH formulations
can be reconstituted with a diluent to generate a stable
reconstituted formulation suitable for subcutaneous administration.
For example, anti-IgE antibody formulations (Example 1; U.S. Pat.
No. 6,267,958, EP 841946) have been prepared which are useful for
treating allergic asthma.
[0145] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the prepararion of a
medicament for treating, preventing and/or aleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound to the nose, upper respiratory tract and/or
lung without being inactivated.
[0146] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the nose, upper respiratory
tract and lung, by administering to the nose, upper respiratory
tract and/or lung of a subject a polypeptide construct comprising
one or more single domain antibodies directed against said
target.
[0147] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the nose, upper respiratory
tract and/or lung without being inactivated, by administering to
the nose, upper respiratory tract and/or lung of a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0148] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated by administering to the nose, upper
respiratory tract and/or lung of a subject a polypeptide construct
comprising one or more single domain antibodies directed against
said target.
[0149] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders wherein the permeability of the
intestinal mucosa is increased. Because of their small size, a
polypeptide construct 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.
[0150] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders wherein the
permeability of the intestinal mucosa is increased, by orally
administering to a subject a polypeptide construct as disclosed
herein comprising one or more single domain antibodiesspecific for
an antigen related to the disorder.
[0151] 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 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.
[0152] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the prepararion of a
medicament for treating, preventing and/or aleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound to the intestinal mucosa, wherein said
disorder increases the permeability of the intestinal mucosa.
[0153] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the intestinal mucosa without
being inactivated, by administering orally to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0154] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated, by administering orally to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0155] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders susceptible to modulation by an
anti-target therapeutic compound that is able pass through the
tissues beneath the tongue effectively. 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.
[0156] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a therapeutic compound that is able pass through
the tissues beneath the tongue effectively, by sublingually
administering to a subject a VHH specific for an antigen related to
the disorder.
[0157] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the prepararion of a
medicament for treating, preventing and/or aleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound that is able to pass through the tissues
beneath the tongue.
[0158] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the tissues beneath the tongue
without being inactivated, by administering orally to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0159] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inecativated, by administering orally to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0160] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody for use in
treating, preventing and/or alleviating the symptoms of disorders
susceptible to modulation by an anti-target therapeutic compound
that is able pass through the skin effectively. A formulation of
said polypeptide construct, for example, a cream, film, spray,
drop, patch, is placed on the skin and passes through.
[0161] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a therapeutic compound that is able pass through
the skin effectively, by topically administering to a subject a
polypeptide construct as disclosed herein comprising one or more
single domain antibodies specific for an antigen related to the
disorder.
[0162] Another aspect of the invention is the use of a polypeptide
construct as disclosed herein as a topical ophthalmic composition
for the treatment of ocular disorder, such as allergic disorders,
which method comprises the topical administration of an ophthalmic
composition comprising polypeptide construct as disclosed herein,
said construct comprising one or more anti-IgE VHH (Example 1,
Example 2).
[0163] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the preparation of a
medicament for treating, preventing and/or aleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound that is able pass through the skin
effectively.
[0164] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the skin without being
inactivated, by administering topically to a subject a polypeptide
construct comprising one or more single domain antibodies directed
against said target.
[0165] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject,
by administering topically to a subject a polypeptide construct
comprising one or more single domain antibodies directed against
said target.
[0166] A non-limiting example of a therapeutic target against which
a polypeptide construct of the invention may be used is TNF, which
is involved in inflammatory processes. The blocking of TNF action
can have an anti-inflammatory effect, which is highly desirable in
certain disease states such as, for example, Crohn's disease.
Current therapy consists of intravenous administration of anti-TNF
antibodies. Our Examples (Example 4) demonstrate VHHs according to
the invention which bind TNF and moreover, block its binding to the
TNF receptor. Oral delivery of these anti-TNF polypeptide
constructs results in the delivery of such molecules in an active
form in the colon at sites that are affected by the disorder. These
sites are highly inflamed and contain TNF-producing cells. These
anti-TNF polypeptide constructs can neutralise the TNF 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 fragments (U.S.
Pat. No. 6,190,662, WO 0023471). Such modified microorganisms can
be used as vehicles for local production and delivery of antibody
fragments in the intestine. By using a strain which produces a TNF
neutralizing antibody fragment, inflammatory bowel disorder could
be treated. Another aspect of the invention is a polypeptide
construct comprising at least one single domain antibody specific
for TNF-alpha for use in the treatment, prevention and/or
alleviation of disorders relating to inflammatory processes,
wherein said polypeptide construct is administered orally,
sublingually, topically, intravenously, subcutaneously, nasally,
vaginally, rectally or by inhalation. Another aspect of the
invention is a method of treating, preventing and/or alleviating
disorders relating to inflammatory processes, comprising
administering to a subject a polypeptide construct comprising at
least one single domain antibody directed against for example
TNF-alpha orally, sublingually, topically, intravenously,
subcutaneously, nasally, vaginally, rectally or by inhalation.
[0167] According to one aspect of the invention, a polypeptide
construct of the invention comprises at least one single domain
antibody directed against TNF-alpha, said single domain antibody
corresponding to a sequence represented by any of SEQ ID NOs: 12 to
14. Said sequences are anti-TNF-alpha Camelidae VHHs.
[0168] Further non-limiting examples of therapeutic targets against
which a polypeptide construct of the invention may be used are
certain colon cancer specific antigens, such as, for example, CEA
or EGF receptors. In one aspect of the invention, therapeutic VHHs
against colon cancer antigens are linked to or provided with one
more tumor destroying reagents such as for example, a chemical
compound or a radioactive compound.
[0169] As stated above a colon cancer specific antigen according to
the invention is epidermal growth factor receptor (EGFR) which is
an essential mediator of cell division in mammalian cells and is a
recognised cellular oncogene. After the binding of EGF to its
receptor (EGFR), a signaling cascade is initiated resulting in cell
development. The EGFR is also involved in human tumorigenesis as it
is overexpressed on cells associated with epithelial malignancies
located in sites such as the head, neck, lung, colon. Another
aspect of the invention is a polypeptide construct comprising at
least one single domain antibody directed against EGFR for use in
the treatment, prevention and/or alleviation of disorders relating
to EGFR-mediated cancer, wherein said VHH is administered orally,
sublingually, topically, nasally, intravenously, subcutaneously,
vaginally, rectally or by inhalation (Examples 25 to 31). Another
aspect of the invention is a method of treating, preventing and/or
alleviating disorders relating to EGFR-mediated cancer, comprising
administering to a subject a polypeptide construct comprising at
least one single domain antibody directed against EGFR orally,
sublingually, topically, intravenously, subcutaneously, nasally,
vaginally, rectally or by inhalation.
[0170] According to one aspect of the invention, a polypeptide
construct of the invention comprises at least one single domain
antibody directed against EGFR, said single domain antibody
corresponding to a sequence represented by any of SEQ ID NOs: 23 to
44. Said sequences are anti-EGRF Camelidae VHHs.
[0171] As stated above another colon cancer specific antigen
according to the invention is carcinoembryonic antigen (CEA), a
recognized tumor marker. Another aspect of the invention is a
polypeptide construct comprising one or more single domain
antibodies specific for CEA for use in the treatment, prevention
and/or alleviation of disorders relating to CEA-mediated cancer,
wherein said polypeptide is administered orally, sublingually,
topically, intravenously, subcutaneously, nasally, vaginally,
rectally or by inhalation. Another aspect of the invention is a
method of treating, preventing and/or alleviating disorders
relating to CEA-mediated cancer, comprising administering to a
subject a polypeptide construct comprising at least one single
domain antibody directed against CEA, orally, sublingually,
topically, intravenously, subcutaneously, nasally, vaginally,
rectally or by inhalation. A few VHHs specific for this
glycoprotein have been isolated by selection on solid-phase coated
with CEA out of a dedicated library obtained after immunization of
a dromedary. By using FACS analysis it appeared that only two
fragments recognized the cell-bound antigen. One of the VHHs, that
recognised the native structure, has been used to construct a
fusion protein with .beta.-lactamase. The functionality of the
purified fusion protein was tested in vitro in a prodrug converting
cytotoxicity assay. In addition the immunoconjugate was tested in
vivo in a tumor-targeting biodistribution study.
[0172] A non-limiting example of a therapeutic target against which
a polypeptide construct of the invention may be used is
Helicobacter pylori, which is a bacterium that lives in the mucus
which coats the lining of the human stomach and duodenum. The
normal human stomach has a very thin layer of mucus that coats the
whole of its inside surface. This mucus has a protective role,
acting as a barrier between the acid in the stomach and the
sensitive stomach wall. H. pylori acts as an irritant to the lining
of the stomach, and this causes inflammation of the stomach
(gastritis). In one embodiment of the invention is a polypeptide
construct comprising at least one single domain antibody directed
against H. pylori, said construct and inhibits the enzymatic
function of urease. Since single domain antibodies, in particular
VHHs have the specific characteristic to occupy enzymatic sites,
selected VHHs would inhibit the enzymatic activity and neutralize
the virulence of a H. pylori infection. In another aspect of the
invention is a polypeptide construct comprising at least one single
domain antibody directed against H. pylori, said construct
inhibiting the adhesion of the bacteria to the stomach wall so
preventing irritation of the stomach wall and gastritis. One aspect
of the invention is a polypeptide construct comprising one or more
single domain antibodies directed against Helicobacter pylori for
use in the treatment, prevention and/or alleviation of disorders
relating to irritation of the stomach wall and gastritis, wherein
said polypeptide construct is administered orally, sublingually,
topically, intravenously, subcutaneously, nasally, vaginally,
rectally or by inhalation, but preferably orally. Another aspect of
the invention is a method of treating, preventing and/or
alleviating disorders relating to irritation of the stomach wall
and gastritis, comprising administering to a subject a polypeptide
construct comprising one or more single domain antibodies directed
against Helicobacter pylori, orally, sublingually, topically,
intravenously, subcutaneously, nasally, vaginally, rectally or by
inhalation, but preferably orally.
[0173] Another non-limiting example of a therapeutic target against
which the VHH of the invention may be used is Hepatitis E, which is
a viral disorder transmitted via the fecal/oral route. Symptoms
increase with age and include abdominal pain, anorexia, dark urine,
fever, hepatomegaly, jaundice, malaise, nausea, and vomiting. The
overall fatality rate is 1-3%, but 15-25% in pregnant women. Once
encountered, most patients develop a neutralizing IgG response
which gives life-long protection Neutralizing VHH molecules have
the advantage over conventional IgG molecules because they may be
administered orally. Since most infections with hepatitis E occur
in North-Africa, Central-Africa, Asia and Central-America, oral
administration is a significant advantage, since medical logistics
are less developed in those countries. One aspect of the invention
is one or more VHHs specific for HEV capsid protein (56 kDa) for
use in the treatment, prevention and/or alleviation of disorders
relating hepatitis E, wherein said VHH is administered orally,
sublingually, topically, intravenously, subcutaneously, nasally,
vaginally, rectally or by inhalation. Another aspect of the
invention is a method of treating, preventing and/or alleviating
disorders relating to hepatitis E, comprising administering to a
subject said VHH orally, sublingually, topically, intravenously,
subcutaneously, nasally, vaginally, rectally or by inhalation."
[0174] Other non-limiting examples of therapeutic targets against
which a polypeptide construct of the invention may be used are
micro-organisms induce respiratory disorders such as the TB
bacterium and influenza virus. TB or tuberculosis, is a disorder
caused by bacteria called Mycobacterium tuberculosis. The bacteria
can attack any part of the body, but they usually attack the lungs.
Influenza is a viral disorder that causes `flu`. Influenza viruses
are also present in the lung. One aspect of the invention is a
polypeptide construct comprising at least one single domain
antibody directed against Mycobacterium tuberculosis epitope for
use in the treatment, prevention and/or alleviation of disorders
relating TB, wherein said polypeptide construct is administered
orally, sublingually, topically, intravenously, subcutaneously,
nasally, vaginally, rectally or by inhalation. Another aspect of
the invention is a method of treating, preventing and/or
alleviating disorders relating to TB, comprising administering to a
subject said polypeptide construct orally, sublingually, topically,
intravenously, subcutaneously, nasally, vaginally, rectally or by
inhalation. Another aspect of the invention is a polypeptide
construct comprising at least one single domain antibody directed
against an influenza virus epitope for use in the treatment,
prevention and/or alleviation of disorders relating flu, wherein
said polypeptide construct is administered orally, sublingually,
topically, intravenously, subcutaneously, nasally, vaginally,
rectally or by inhalation. Another aspect of the invention is a
method of treating, preventing and/or alleviating disorders
relating to flu, comprising administering to a subject said
polypeptide construct orally, sublingually, topically,
intravenously, subcutaneously, nasally, vaginally, rectally or by
inhalation.
[0175] Another non-limiting example of a therapeutic target against
which a polypeptide of the invention may be used is IgE in relation
to allergies. During their lifetime, subjects may develop an
allergic response to harmless parasites (e.g. Dermatophagoides
pteronyssinus, house dust mite) or substances (clumps, plastics,
metals). This results in the induction of IgE molecules that
initiate a cascade of immunological responses. One aspect of the
present invention is a polypeptide construct comprising at least
one single domain antibody directed against IgE, said polypeptide
preventing the interaction of IgE with their receptor(s) on mast
cells and basophils. As such they prevent the initiation of the
immunological cascade, an allergic reaction. Since IgE molecules
are present in the bloodstream, it is within the scope of the
invention to fuse the VHH one or more active transport carriers in
order to reach their target. Another aspect of the invention is a
polypeptide construct comprising at least one single domain
antibody directed against an IgE epitope for use in the treatment,
prevention and/or alleviation of disorders relating to allergies,
wherein said polypeptide construct is administered orally,
sublingually, topically, nasally, vaginally, rectally or by
inhalation. Another aspect of the invention is a method of
treating, preventing and/or alleviating disorders relating to
allergies, comprising administering to a subject said polypeptide
construct orally, sublingually, topically, intravenously,
subcutaneously, nasally, vaginally, rectally or by inhalation.
[0176] According to one aspect of the invention, a polypeptide
construct of the invention comprises at least one single domain
antibody directed against IgE, said single domain antibody
corresponding to a sequence represented by any of SEQ ID NOs: 1 to
11. Said sequences are anti-IgE Camelidae VHHs.
[0177] Another non-limiting example of a therapeutic target against
which a polypeptide construct of the invention may be used is human
macrophage elastase (MMP-12), which is a member of the family of
matrix metalloproteases (MMPs). These enzymes play an important
role in normal and inflammatory processes contributing to tissue
remodeling and destruction. MMPs play besides proper extracellular
matrix remodeling also an important role in diverse disease states
such as cancer and inflammation. Macrophage elastase or MMP-12 has
a large specificity pocket and broad substrate specificity. It
plays a role in several disorders owing to excessive protein
degradation of extracellular proteins (e.g. lung damage in smoke
induced emphysema, Churg et al, A. 2003) or increased matrix
degradation (e.g. higher MMP-12 enzymatic activity in obesity,
Chavey et al, 2003). Other clinical indications include coeliac
disorder and dermatitis herpetiformis (Salmela et al, 2001),
glomerulo nephritis (Kaneko et al, 2003), esophageal squamous cell
carcinoma (Ding et al, 2002) and skin cancer (Kerkela et al,
2000).
[0178] MMP-12 is secreted into the extracellular space by lung
alveolar macrophages and dysregulation of MMP-12 is a possible
reason for degradation of the alveolar membrane leading to lung
emphysema. Target substrates of MMP-12 include extracellular matrix
proteins such as elastin, fibronectin and laminin, but also
.alpha.1-antitrypsin and tissue factor protease inhibitor. One
aspect of the invention is a polypeptide construct comprising at
least one single domain antibody directed against MMP-12 for use in
the treatment, prevention and/or alleviation of disorders relating
to inflammatory processes, wherein said polypeptide construct is
administered orally, sublingually, topically, nasally, vaginally,
rectally or by inhalation. Another aspect of the invention is a
method of treating, preventing and/or alleviating disorders
relating to inflammatory processes, comprising administering to a
subject said polypeptide construct orally, sublingually, topically,
intravenously, subcutaneously, nasally, vaginally, rectally or by
inhalation.
[0179] Another aspect of this invention consists of (1) VHH's that
specifically bind to a metalloproteinase and are not degraded by a
metalloproteinase, (2) VHH's which inhibit the proteolytic activity
of one or more metalloproteinase and (3) inhibitory VHH's which are
highly specific for one MMP (e.g. MMP-12 specific antagonist),
unlike none-specific chemical inhibitors (e.g. batimastat,
merimastat . . . )
[0180] According to one aspect of the invention, a polypeptide
construct of the invention comprises at least one single domain
antibody directed against human MMP-12, said single domain antibody
corresponding to a sequence represented by any of SEQ ID NOs: 15 to
22. Said sequences are anti-MMP-12 Camelidae VHHs.
[0181] Another non-limiting example of a therapeutic target against
which a polypeptide construct of the invention may be used is
IFN-gamma, which is secreted by some T cells. In addition to its
anti-viral activity, IFN gamma stimulates natural killer (NK) cells
and T helper 1 (Th1) cells, and activates macrophages and
stimulates the expression of MHC molecules on the surface of cells.
Hence, IFN gamma generally serves to enhance many aspects of immune
function, and is a candidate for treatment of disease states where
the immune system is over-active (e.g. Crohn's disease), e.g.,
autoimmune disorders and organ plant rejection. One aspect of the
invention is a polypeptide construct comprising at least one single
domain antibody directed against IFN-gamma for use in the
treatment, prevention and/or alleviation of disorders relating to
the immune response, wherein said polypeptide construct is
administered orally, sublingually, topically, intravenously,
subcutaneously, nasally, vaginally, rectally or by inhalation.
Another aspect of the invention is a method of treating, preventing
and/or alleviating disorders relating to the immune response,
comprising administering to a subject said polypeptide construct
orally, sublingually, topically, intravenously, subcutaneously,
nasally, vaginally, rectally or by inhalation. In other embodiments
of the present invention polypeptide constructs that neutralize IFN
gamma are used to treat patients with psoriasis.
[0182] According to one aspect of the invention, a polypeptide
construct of the invention comprises at least one single domain
antibody directed against IFN-gamma, said single domain antibody
corresponding to a sequence represented by any of SEQ ID NOs: 45 to
70. Said sequences are anti-IFN-gamma Camelidae VHHs.
[0183] The invention also relates to a method of identifying single
domain antibodies (e.g. VHHs) harbouring specific sequences which
facilitates the delivery or transport of the anti-target single
domain antibodies across human or animal tissues (as described in
U.S. Pat. No. 6,361,938), including without limitation GIT
epithelial layers, alveolar cells, endothelial of the blood-brain
barrier, vascular smooth muscle cells, vascular endothelial cells,
renal epithelial cells, M cells of the Peyers Patch, and
hepatocytes. Furthermore, delivery systems could be used in
conjunction with the VHH's of the invention, comprising
nanoparticles, microparticles, liposomes, micelles, cyclodextrines.
Only small (<600 daltons) and hydrophobic (Partridge et al, Adv.
Drug Delivery Reviews, 15, 5-36 (1995)) molecules can easily pass
the blood-brain barrier, severely limiting the development of novel
brain drugs which can be used without the use of invasive
neurosurgical procedures.
Delivering Polypeptide Constructs to the Interior of Cells
[0184] Another aspect of the present invention is a method and
molecules for delivering therapeutic polypeptides and/or agents to
the inside of cells. A further aspect of the invention is a method
and molecules for delivering antigens to the inside of antigen
presenting cells, and thereby eliciting a powerful immune response
thereto. A still further aspect of the invention is to provide a
method and molecules for delivery of therapeutic polypeptides
and/or agents across natural barriers such as the blood-brain
barrier, lung-blood barrier.
[0185] One aspect of the invention is a polypeptide construct
comprising one or more single domain antibodies directed against a
target and comprising one or more single domain antibodies directed
against an internalising cellular receptor, wherein said
polypeptide construct internalises upon binding to said
receptor.
[0186] The targets inside cells may affect the functioning of said
cell, or binding thereto may lead to a change in the phenotype of
the cell itself by itself. This can be for example, cell death,
effects on cell cycling or cell growth or interference with
intracellular signaling pathways (see, for example, Poul M A et al,
J Mol Biol, 2000, 301, 1149-1161).
[0187] One embodiment of the present invention is a polypeptide
construct comprising one or more single domain antibodies specific
for an internalising cellular receptor, wherein said construct
internalises upon binding to said receptor, wherein the polypeptide
construct comprises a therapeutic polypeptide or agent which is
covalently or non-covalently linked thereto. Said therapeutic
polypeptide or agent has one or more targets which acts
intracellularly. See, for example, FIG. 12. Said therapeutic
polypeptides may harbour specific sequences which target the
polypeptide to specific compartiments in the cell, comprising
vesicles, organelles and other cytoplasmic structures,
membrane-bound structures, the nucleus.
[0188] An internalising receptor according to the invention is a
receptor displayed on the surface of a cell which upon binding to a
ligand, mediates the internalisation of said ligand into the
cytoplasm of the cell. Internalising receptors according to the
invention include, but are not limited to, LDL receptors, EGFr,
FGF2r, ErbB2r, transferrin receptor, PDGFr, VEGFr, PsmAr or antigen
presenting cell internalising receptors.
[0189] One embodiment of the present invention is a polypeptide
construct comprising one or more single domain antibodies specific
for an internalising cellular receptor as disclosed herein, further
comprising an antigen.
[0190] One embodiment of the present invention is a polypeptide
construct comprising one or more single domain antibodies specific
for an internalising cellular receptor as disclosed herein, wherein
said receptor is an internalising receptor on an antigen presenting
cell (APC). Preferably the receptor is highly specific for APCs and
not present or is present in lower amounts on other cell types.
[0191] Another embodiment of the invention is a polypeptide
construct comprising one or more anti-receptor single domain
antibodies and an antigen. Thus by linking an antigen to a VHH
directed towards an internalising receptor on an APC, antigen
uptake by APC is not determined by the passive interaction between
APC and antigen, but by the "active" binding between VHH and said
receptor. This not only makes the process more efficient, but also
more reproducible and not dependent on the antigen structure which
causes great variability in the T-cell activation from antigen to
antigen.
[0192] After internalization, the complex is digested by the APC
and pieces of the antigen can be exposed on the surface in
association with MHC/HLA and elicit a more powerful immune
response.
[0193] Another embodiment of the present invention is a method for
immunising a subject against an antigen comprising administering to
a subject in need thereof a polypeptide construct comprising at
least one single domain antibody directed against an antigen
present on an APC, wherein said single domain antibody further
comprises the antigen of interest.
[0194] One embodiment of the present invention is a polypeptide
construct comprising one or more single domain antibodies specific
for an internalising cellular receptor as disclosed herein, wherein
said receptor is EGFR. In general internalization of receptors
occurs upon binding of the agonistic ligand in a proces called
sequestration. In order to ensure that extracellular signals are
translated into intracellular signals of appropriate magnitude and
specificity, the signalling cascades are tightly regulated via the
process of sequestration, whereby receptors are physically removed
from the cell surface by internalization to a cytosolic compartment
(Carman, C. V. and Benovic, J. L. Current Opinion in Neurobiology
1998, 8: 335-344). This implies that only agonistic ligands or
antibodies indeed are expected to internalize via such receptors.
In terms of therapeutic use it is not a desired effect that the
antibody first triggers proliferation of the tumorcells, before it
can deliver a toxic payload to the interiour of the cell.
[0195] Some of internalising receptors are over-expressed on
certain cells, such as the epidermal growth factor receptor (EGFR)
or ErBb2 receptor on tumor cells. Epidermal growth factor (EGF) is
an essential mediator of cell division in mammalian cells and is a
recognized cellular oncogene and is therefore an appropriate target
for anti-receptor therapy. After the binding of EGF to its receptor
(EGFR), a signaling cascade is initiated resulting in cell
development. The EGFR is involved in human tumorigenesis as it is
overexpressed on cells of many epithelial malignancies such as
head, neck, lung, colon. VHH that are internalised upon binding to
one of these receptors can be used to deliver molecules inside the
cell.
[0196] One embodiment of the present invention a polypeptide
construct comprising one or more single domain antibodies directed
against EGFR, wherein a single domain antibody corresponds to a
sequence represented by any of SEQ ID NOs: 23 to 44. Surprisingly,
one of the single domain antibodies, did not activate the EGFR,
despite the fact that it was internalized efficiently. Such types
of antibodies are preferred for therapeutic applications, since
these can deliver toxic payloads into cells without stimulating its
proliferation.
[0197] Another embodiment of the present invention is a polypeptide
construct construct comprising one or more single domain antibodies
directed against for EGFR, wherein said anti-EGFR single domain
antibody does not activate the EGFR. Said polypeptide construct may
be used for the delivery of a therapeutic agents and/or
polypeptides into a cell, as mentioned herein, without stimulating
the EGFR.
[0198] Another embodiment of the present is a polypeptide construct
construct comprising one or more single domain antibodies directed
against for EGFR, wherein said anti-EGFR single domain antibody
does not activate the EGFR and corresponds to a sequence
represented by SEQ ID NO: 31.
[0199] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against an internalising cellular receptor, wherein said construct
internalises upon binding to said receptor, and further comprising
one or more single domain antibodies directed against an
intracellular target, said single domain antibodies covalently or
non-covalently linked. This multispecific polypeptide construct may
be used in the treatment, prevention and/or alleviation of
disorders, according to the target of the non-receptor specific
single domain antibody. This target can be, for example, a kinase
such as PDK1. PDK1 is over-expressed in breast tumor cells. It
activates Akt by phosphorylating T308 in the activation loop. A
number of downstream substrates of Akt play a direct role in
promoting cell survival. These include GSK3, Bad, caspase-9 and
Forkhead.
[0200] One embodiment of the present invention is a polypeptide
construct comprising a single domain antibody directed against an
internalising cellular receptor, wherein said construct
internalises upon binding to said receptor, and further comprising
one or more single domain antibodies directed against any of PDK1,
GSK1, Bad, caspase-9 and Forkhead. Another aspect of the invention
the use of said construct for treating cancer. Another aspect of
the invention is said construct for the preparation of a medicament
for treating cancer.
[0201] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against an internalising cellular receptor, wherein said construct
internalises upon binding to said receptor, wherein the construct
further comprises a drug or a toxic compound covalently or
non-covalently linked thereto. One example of a toxic compound is a
compound that is only active intracellularly due to reducing
environment (e.g. an enzyme recombinantly modified with additional
cysteins resulting in inactive enzyme, but active in reducing
environment). Another example of a toxic compound is a one that is
specifically toxic only to a particular cell-type. An example of a
toxic compound or a drug is a compound activated by a ligand
present inside the cell and leading to the phenotype of interest.
Other examples include prodrugs, small organic molecules. One
aspect of the invention the use of said construct in the treatment
of disorder requiring administration of the same. Another aspect of
the invention is said construct for the preparation of a medicament
for the treatment of disorder requiring administration of the
same.
[0202] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against an internalising cellular receptor, wherein said construct
internalises upon binding to said receptor, and wherein a
filamentous phage expresses said construct on its surface. Said
construct may be attached to the tip of the phage. In one aspect of
the invention, construct-phage assembly can be used to package and
deliver DNA to the cell for use as a gene therapy vector. According
to the invention, the phage may carry DNA in additional to that
encoding said construct, for use therapeutically. According to the
invention, the phage may carry a gene encoding a therapeutic
polypeptide controlled by a promoter for the expression of said
gene inside the cell. An example of said promoter includes, but is
not limited to, the CMV promoter (Kassner et al, Biochem Biophys
Res Commun, 1999, 264: 921-928). Phage have distinct advantages
over existing gene therapy vectors because they are simple,
economical to produce at high titer, have no intrinsic tropism for
mammalian cells, and are relatively simple to genetically modify
and evolve (Larocca D et al, Curr. Pharm. Biotechnol,
2002:3:45-57).
[0203] Another embodiment of the present invention is a polypeptide
construct as disclosed herein, wherein said single domain antibody
is a peptide derived from a VHH specific for an internalising
cellular receptor. Said VHH peptide may bind their antigen almost
only through the peptide. Internalising VHHs may be prepared from a
peptide library which is screened for internalising properties. It
is an aspect of the invention that these VHH peptides can be added
as a tag to therapeutic polypeptides or agents, for intracellular
uptake. The VHH peptide, may, for example, be used to transport a
therapeutic VHH into a cell. In one embodiment of the invention,
the VHH peptide is the CDR3. In another one embodiment of the
invention, the VHH peptide is any other CDR.
[0204] Another embodiment of the present invention is a method of
selecting for VHHs specific for an internalising cellular receptor,
wherein said VHH internalise upon binding to said receptor,
comprising panning receptor-displaying cells with a phage library
(naive or immune) of VHH, and selecting for internalising VHH by
recovering the endocytosed phage from within the cell. The
invention includes a selection method which uses cell lines that
overexpress a receptor or cell lines transfected with a receptor
gene to allow the easy selection of phage antibodies binding to the
receptor. This avoids the need for protein expression and
purification, speeding up significantly the generation of
internalizing VHH.
[0205] Another embodiment of the present invention is a method for
delivering a therapeutic polypeptide, agent or antigen for uptake
by cellular internalisation by covalently or non-covalently
attaching thereto a polypeptide construct comprising at least one
single domain antibody specific for an internalising cellular
receptor, wherein said construct internalises upon binding to said
receptor.
[0206] The VHHs according to the invention may be used to treat,
prevent and/or alleviate symptoms of disorders requiring the
administration of the same.
[0207] Another embodiment of the present invention is a method for
delivering a therapeutic polypeptide or agent that interacts with
intracellular targets molecules comprising administering to a
subject in need thereof one or more VHHs specific for an
internalising cellular receptor, wherein said VHH internalise upon
binding to said receptor, wherein said VHH is fused to said
polypeptide or agent.
[0208] Another embodiment of the present invention is a method for
delivering a therapeutic polypeptide, agent or antigen across a
natural barrier by covalently or non-covalently attaching thereto a
polypeptide construct comprising at least one single domain
antibody directed against an internalising cellular receptor,
wherein said construct internalises upon binding to said receptor.
According to the invention, a natural barrier includes, but is not
limited to, the blood-brain, lung-blood, gut-blood, vaginal-blood,
rectal-blood and nasal-blood barriers.
[0209] For example, a peptide construct delivered via the upper
respiratory tract and lung can be used for transport of therapeutic
polypeptides or agents from the lung lumen to the blood. The
construct binds specifically to a receptor present on the mucosal
surface (bronchial epithelial cells) resulting in transport, via
cellular internalisation, of the therapeutic polypeptides or agents
specific for bloodstream targets from the lung lumen to the blood.
In another example, a therapeutic polypeptide or agent is linked to
a polypeptide construct comprising at least one single domain
antibody directed against an internalising cellular receptor
present on the intestinal wall into the bloodstream. Said construct
induces a transfer through the wall, via cellular internalization,
of said therapeutic polypeptide or agent.
[0210] Another embodiment of the present invention is a VHH
specific for an internalising cellular receptor, wherein said VHH
internalises upon binding to said receptor, said VHH is covalently
or non-covalently attached to a therapeutic polypeptide or agent,
and said VHH crosses a natural barrier.
[0211] Another embodiment of the present invention is a method for
delivering a therapeutic polypeptide, agent or antigen for uptake
at a local by covalently or non-covalently attaching it to a VHH
specific for an internalising cellular receptor, wherein said VHH
internalises upon binding to said receptor. A local area, according
to the invention, includes, but is not limited to, the brain, lung,
gut, vaginal, rectal and nasal areas.
[0212] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders susceptible to modulation by an
anti-target therapeutic compound that is able pass through the
gastric environment without being inactivated.
[0213] 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 VHHs 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
disorder whose targets that are located in the gut system.
[0214] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of a disorder
susceptible to modulation by a therapeutic compound that is able
pass through the gastric environment without being inactivated, by
orally administering to a subject a polypeptide construct
comprising one or more single domain antibodies specific for
antigen related to the disorder.
[0215] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the prepararion of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound that is able pass through the gastric
environment without being inactivated.
[0216] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the gut system without being
inactivated, by orally administering to a subject a polypeptide
construct comprising one or more single domain antibodies directed
against said target.
[0217] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated, by orally administering to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0218] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders susceptible to modulation by an
anti-target therapeutic compound delivered to the vaginal and/or
rectal tract.
[0219] In a non-limiting example, a formulation according to the
invention comprises a polypeptide construct as disclosed herein
comprising one or more VHHs directed against one or more targets 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).
[0220] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by an anti-target therapeutic compound delivered to
the vaginal and/or rectal tract, by vaginally and/or rectally
administering to a subject a polypeptide construct comprising one
or more single domain antibodies directed against said target.
[0221] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound delivered to the vaginal and/or rectal tract
without being inactivated.
[0222] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the vaginal and/or rectal tract
without being inactivated, by administering to the vaginal and/or
rectal tract of a subject a polypeptide construct comprising one or
more single domain antibodies directed against said target.
[0223] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated, by administering to the vaginal and/or
rectal tract of a subject a polypeptide construct comprising one or
more single domain antibodies directed against said target.
[0224] Another embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target comprising at least one single domain antibody
directed against a target, for use in treating, preventing and/or
alleviating the symptoms of disorders susceptible to modulation by
an anti-target therapeutic compound delivered to the nose, upper
respiratory tract and/or lung.
[0225] In a non-limiting example, a formulation according to the
invention, comprises a polypeptide construct as disclosed herein
directed against one or more targets in the form of a nasal spray
(e.g. an aerosol) or inhaler. Since the construct is small, it can
reach its target much more effectively than therapeutic IgG
molecules.
[0226] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by an anti-target therapeutic delivered to the nose,
upper respiratory tract and lung, by administering to a subject a
polypeptide construct as disclosed herein wherein one or more
single domain antibodies are specific for an antigen related to the
disorder, by inhalation through the mouth or nose.
[0227] Another aspect of the invention is a dispersible VHH
composition, in particular dry powder dispersible VHH compositions,
such as those described in U.S. Pat. No. 6,514,496. These dry
powder compositions comprise a plurality of discrete dry particles
with an average particle size in the range of 0.4-10 mm. Such
powders are capable of being readily dispersed in an inhalation
device. VHH's are particularly suited for such composition as
lyophilized material can be readily dissolved (in the lung
subsequent to being inhaled) due to its high solubilisation
capacity (Muyldermans, S., Reviews in Molecular Biotechnology, 74,
277-303, (2001)). Alternatively, such lyophilized VHH formulations
can be reconstituted with a diluent to generate a stable
reconstituted formulation suitable for subcutaneous administration.
For example, anti-IgE antibody formulations (Example 1; U.S. Pat.
No. 6,267,958, EP 841946) have been prepared which are useful for
treating allergic asthma.
[0228] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound delivered to the nose, upper respiratory tract
and/or lung without being inactivated.
[0229] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the nose, upper respiratory
tract and lung, by administering to the nose, upper respiratory
tract and/or lung of a subject a polypeptide construct comprising
one or more single domain antibodies directed against said
target.
[0230] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the nose, upper respiratory
tract and/or lung without being inactivated, by administering to
the nose, upper respiratory tract and/or lung of a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0231] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated by administering to the nose, upper
respiratory tract and/or lung of a subject a polypeptide construct
comprising one or more single domain antibodies directed against
said target.
[0232] One embodiment of the present invention is a polypeptide
construct as disclosed herein for use in treating, preventing
and/or alleviating the symptoms of disorders susceptible to
modulation by an anti-target therapeutic compound delivered to the
intestinal mucosa, wherein said disorder increases the permeability
of the intestinal mucosa. Because of their small size, a
polypeptide construct 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.
[0233] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by an anti-target therapeutic compound delivered to
the intestinal mucosa, wherein said disorder increases the
permeability of the intestinal mucosa, by orally administering to a
subject a polypeptide construct as disclosed herein.
[0234] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound delivered to the intestinal mucosa, wherein
said disorder increases the permeability of the intestinal
mucosa.
[0235] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the intestinal mucosa without
being inactivated, by administering orally to a subject a
polypeptide construct of the invention.
[0236] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated, by administering orally to a subject a
polypeptide construct of the invention.
[0237] 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, a polypeptide construct
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.
[0238] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody directed
against a target for use in treating, preventing and/or alleviating
the symptoms of disorders susceptible to modulation by an
anti-target therapeutic compound that is able pass through the
tissues beneath the tongue effectively. 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.
[0239] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a therapeutic compound that is able pass through
the tissues beneath the tongue effectively, by sublingually
administering to a subject a VHH specific for an antigen related to
the disorder.
[0240] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound that is able to pass through the tissues
beneath the tongue.
[0241] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the tissues beneath the tongue
without being inactivated, by administering orally to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0242] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject
without being inactivated, by administering orally to a subject a
polypeptide construct comprising one or more single domain
antibodies directed against said target.
[0243] One embodiment of the present invention is a polypeptide
construct comprising at least one single domain antibody for use in
treating, preventing and/or alleviating the symptoms of disorders
susceptible to modulation by an anti-target therapeutic compound
that is able pass through the skin effectively. A formulation of
said polypeptide construct, for example, a cream, film, spray,
drop, patch, is placed on the skin and passes through.
[0244] An aspect of the invention is a method for treating,
preventing and/or alleviating the symptoms of disorders susceptible
to modulation by a therapeutic compound that is able pass through
the skin effectively, by topically administering to a subject a
polypeptide construct as disclosed herein comprising one or more
single domain antibodies specific for an antigen related to the
disorder.
[0245] Another aspect of the invention is the use of a polypeptide
construct as disclosed herein as a topical ophthalmic composition
for the treatment of ocular disorder, such as allergic disorders,
which method comprises the topical administration of an ophthalmic
composition comprising polypeptide construct as disclosed herein,
said construct comprising one or more anti-IgE VHH (Example 1,
Example 2).
[0246] Another embodiment of the present invention is a use of a
polypeptide construct as disclosed herein for the preparation of a
medicament for treating, preventing and/or alleviating the symptoms
of disorders susceptible to modulation by an anti-target
therapeutic compound that is able pass through the skin
effectively.
[0247] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the skin without being
inactivated, by administering topically to a subject a polypeptide
construct comprising one or more single domain antibodies directed
against said target.
[0248] An aspect of the invention is a method for delivering an
anti-target therapeutic compound to the bloodstream of a subject,
by administering topically to a subject a polypeptide construct
comprising one or more single domain antibodies directed against
said target.
[0249] Another aspect of the present invention is a method to
determine which single domain antibodies (e.g. VHHs) molecules
cross a natural barrier into the bloodstream upon administration
using, for example, oral, nasal, lung, skin. In a non-limiting
example, the method comprises administering a naive, synthetic or
immune single domain antibody phage library to a small animal such
as a mouse. At different time points after administration, blood is
retrieved to rescue phages that have been actively transferred to
the bloodstream. Additionally, after administration, organs can be
isolated and bound phages can be stripped off. A non-limiting
example of a receptor for active transport from the lung lumen to
the bloodstream is the Fc receptor N (FcRn). The method of the
invention thus identifies single domain antibodies which are not
only actively transported to the blood, but are also able to target
specific organs. The method may identify which VHH are transported
across the gut and into the blood; across the tongue (or beneath)
and into the blood; across the skin and into the blood etc.
[0250] One aspect of the invention are the single domain antibodies
obtained by using said method. According to the invention, said
single domain antibody may be used as a single domain antibody in a
polypeptide construct of the invention. Said construct, further
comprising another single domain antibody, a therapeutic agent, or
polypeptide carrier directed against a target accessible via or in
the blood may be administered by the route most efficient for said
single domain antibody.
[0251] In general, "therapeutically effective amount",
"therapeutically effective dose" and "effective amount" means the
amount needed to achieve the desired result or results (such as for
instance modulating IFN-gamma binding; treating or preventing
inflammation). One of ordinary skills in the art will recognize
that the potency and, therefore, an "effective amount" can vary for
the various compounds that modulate ligand-target binding, such as
for instance IFN-gamma binding used in the invention. One skilled
in the art can readily assess the potency of the compound.
[0252] As used herein, the term "compound" refers to a polypeptide
construct of the present invention, or a nucleic acid capable of
encoding said polypeptide construct.
[0253] 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.
[0254] The polypeptide constructs of the present invention are
useful for treating or preventing conditions in a subject and
comprises administering a pharmaceutically effective amount of a
compound or composition.
[0255] The polypeptide constructs 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, doxorubicin.
[0256] 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.
[0257] 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.,
parenterally, intravenously, intramuscularly, subcutaneously, to
the vaginal and/or rectal tract, nasally, by inhalation though the
mouth or nose, to the tissues beneath the tongue, or topically.
[0258] 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.
[0259] Thus, the present compound may be administered 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.
[0260] 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.
[0261] 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.
[0262] 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.
[0263] 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.
[0264] 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.
[0265] 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. 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.
[0266] 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.
[0267] 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).
[0268] 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.
[0269] 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-%.
[0270] 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.
[0271] 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.
[0272] 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.
BRIEF DESCRIPTION OF FIGURES
[0273] FIG. 1: Schematic illustrating the regions of IgE
[0274] FIG. 2: 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)
[0275] FIG. 3: Experimental setting
[0276] FIG. 4: Capacity of VHH clones to inhibit the proteolytic
activity of human catalytic domain of MMP12
[0277] FIG. 5: ELISA to detect A431 specific antibody titers in
llama serum.
[0278] FIG. 6: Detection of EGFR specific antibody titers in llama
serum.
[0279] FIG. 7: Detection of EGFR specific antibody titers in serum
of llama 024 and 025 and of llama 026 and 027.
[0280] FIG. 8: Phage response to EGFR
[0281] FIG. 9: Amino acid alignment of 31 clones identified by the
epitope specific elution selection procedure
[0282] FIG. 10: Phage ELISA on cells (panel A) or on solid-phase
immobilized EGFR (panel B) of the 20 unique EGFR specific clones
identified via the epitope specific elution selection procedure
[0283] FIG. 11: Effect of nanobody EGFR-IIIa42 on receptor
internalization and signalling. Fluorescence microscopy
visualization of EGFR-IIIa42 under conditions that allow
internalization, with Her-14 (panel A) or 3T3 (panel B). A Western
blot that shows the effect of EGFR-IIIa42 on receptor tyrosin
kinase activity is represented in panel C.
[0284] FIG. 12: Schematic illustrating a use of VHHs directed
towards internalising receptors to deliver therapeutic protein,
toxic compound, drug or polynucleotide.
BRIEF DESCRIPTION OF TABLES
[0285] Table 1: Immunization scheme as described in Example 1
[0286] Table 2: Presence of insert by PCR with vector specific
primers as described in Example 1
[0287] Table 3: First selection as described in Example 1
[0288] Table 4: Second selection using the rescued phages from the
first selection as described in Example 1
[0289] Table 5: Second round selection using neutravidine coated
tubes as described in Example 1
[0290] Table 6: Number of clones that score positive for binding to
both human IgE and chimeric IgE versus the number of clones tested
in ELISA as described in Example 1
[0291] Table 7: Treatment schedule
[0292] Table 8: Overview of the libraries, their diversity and %
insert derived from different llama's and tissues as described in
Example 7 and 8
[0293] Table 9: Immunization schedule and tissue collections
[0294] Table 10: Overview of constructed libraries
[0295] Table 11: Overview of epitope specific elution selection
procedure
[0296] Table 12: Overview of `internalization` selection
procedure
[0297] Table 13: Primer sequences
[0298] Table 14: Sequence listing
EXAMPLES
Legend of Examples
IgE
[0299] Example 1: VHH directed against IgE Example 2: Formulation
of VHH anti-IgE Example 3: Anti-IgE formulation
TNF-alpha
[0300] Example 4: Selection of anti-TNF-alpha Example 5: Stability
testing of antibody fragments specific for human TNF.alpha. Example
6: Oral administration of an anti-human TNF.alpha. specific VHH in
mice Example 7: Efficacy in an animal model for IBD
MMP12
Example 8: Immunization
Example 9: Repertoire Cloning
[0301] Example 10: Rescue of the library and phage preparation
Example 11: Selection of human MMP-12 specific VHH Example 12:
Specificity of selected VHH's Example 13: Diversity of selected
VHH's Example 14: Expression and purification of VHH Example 15:
Functional characterization of selected VHH's: inhibition of MMP-12
proteolytic activity by a VHH in a colorimetric assay. Example 16:
Formulation of anti-MMP12 VHH for pulmonary delivery Interferon
gamma
Example 17: Immunization
Example 18: Repertoire Cloning
[0302] Example 19: Rescue of the library and phage preperation
Example 20: Selection of human-IFN gamma VHH Example 21: Diversity
of selected VHH's Example 22: Expression and purification of VHH
Example 23: Topical applications of anti-IFN gamma VHH's
Therapeutic VHH-fraoments
[0303] Example 24: Expression of VHH-CDR3 of anti-TNF alpha VHH#3E
EGFR
Example 25: Immunization
Example 26: Evaluation of Immune Response
[0304] Example 27: Cloning of the heavy-chain antibody fragment
(VHH) repertoire
Example 28: Evaluation of the Cloned Repertoire
[0305] Example 29: Multiple selection strategies to identify EGFR
specific nanobodies Example 30: Characterization of EGFR specific
nanobodies Example 31: EGF receptor mediated internalization of
nanobodies
PDK1
Example 32: Immunisation of Llamas
Example 33: Repertoire Cloning
[0306] Example 34: Rescue of the library, phage preparation
Example 35: Selection
Example 36: Screening
[0307] Example 37: Screen for internalised VHH Example 38: Screen
for VHH inhibiting PDK1-Akt interaction
Example 39: Making a Bispecific Construct
[0308] Example 40: Endocytosis and lysis of tumor cells Example 41:
Calculation of homologies between anti-target-single domain
antibodies of the invention Example 42: Construction of a
bispecific constructs containing a VHH-CDR3 fragment fused to an
anti-serum albumin VHH
EXAMPLES
IgE
Example 1
VHH Directed Against IgE
[0309] Two llama's were immunized with human IgE, Scripps
laboratories, Cat nr. 10224. The following immunization schemes
were used according to Table 1.
[0310] Different sources for RNA extraction were used: [0311] 150
ml immune blood, between 4 and 10 days after the last antigen
injection [0312] lymph node biopsy 4 days after the last antigen
injection
[0313] Peripheral blood lymphocytes (PBLs) were isolated by
centrifugation on a density gradient (Ficoll-Paque Plus Amersham
Biosciences). PBLs and lymph node were used to extract total RNA
(Chomczynski and Sacchi 1987). cDNA was prepared on 200 .mu.g total
RNA with MMLV Reverse Transcriptase (Gibco BRL) using oligo d(T)
oligonucleotides (de Heard et al., 1999). The cDNA was purified
with a phenol/chloroform extraction, followed by an ethanol
precipitation and subsequently used as template to amplify the VHH
repertoire.
[0314] In a first PCR, the repertoire of both conventional (1.6 kb)
and heavy-chain (1.3 kb) antibody gene segments were amplified
using a leader specific primer (5'-GGCTGAGCTCGGTGGTCCTGGCT-3'; SEQ
ID NO:85) and the oligo d(T) primer
(5'-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT-3'; SEQ ID
NO:86). The resulting DNA fragments were separated by agarose gel
electrophoresis and the 1.3 kb fragment encoding heavy-chain
antibody segments was purified from the agarose gel. A second PCR
was performed using a mixture of FR1 reverse primers (WO03/054016
sequences ABL037 to ABL043) and the same oligo d(T) forward primer.
The PCR products were digested with SfiI (introduced in the FR1
primer) and BstEII (naturally occurring in framework 4). Following
gel electrophoresis, the DNA fragments of approximately 400
basepairs were purified from gel and ligated into the corresponding
restriction sites of phagemid pAX004 to obtain a library of cloned
VHHs after electroporation of Escherichia coli TG1. pAX004 allows
the production of phage particles, expressing the individual VHHs
as a fusion protein with a c-myc tag, a hexahistidine tag and the
geneIII product. The percentage insert was determined in PCR using
a combination of vector based primers.
[0315] Results are summarized in Table 2.
[0316] Selections were done using chimaeric IgE instead of human
IgE, used for immunization, in order to select for VHH molecules
directed against the constant region of IgE. The region interacting
with the Fc.epsilon.-receptor is located in the constant part of
IgE, more in particular in the region covered by
C.epsilon.2-C.epsilon.3 as shown in FIG. 1.
[0317] A first selection was performed using the pool of PBL day4,
PBL day10 and lymph node day4 libraries for each of the two
llama's. Chimaeric IgE was solid phase coated at 5 .mu.g/ml and 0.5
.mu.g/ml and specific phages were eluted using 0.1 M glycine
pH=2.5.
[0318] The results obtained are shown in Table 3.
[0319] A second selection was performed using the rescued phages
from the first selection using 5 .mu.g/ml. Chimaeric IgE was solid
phase coated at 1 .mu.g/ml and specific phages were eluted using
buffy coat cells or lysozyme for 1 hr. Buffy coat cells contain
cells expressing the Fc.epsilon.receptor, while lysozyme is an
irrelevant protein and serves as a control. The results obtained
are shown in Table 4.
[0320] Another second round selection was performed using
neutravidine coated tubes and 2 nM biotinylated IgE. Specific
phages were eluted using buffy coat cells or lysozyme for 1 hr.
Buffy coat cells contain cells expressing the Fc.epsilon.receptor,
while lysozyme is an irrelevant protein and serves as a control.
The results obtained are shown in Table 5.
[0321] Individual clones obtained from the first round of selection
were screened in an ELISA using solid phase coated human IgE or
chimaeric IgE. The number of clones that score positive for binding
to both human IgE and chimeric IgE versus the number of clones
tested in ELISA are summarized in Table 6.
[0322] Clones were picked which were positive for human and
chimaeric IgE binding, amplified by PCR and digested with Hinfl.
Hinfl profiles were determined on agarose gel and representative
clones for different profiles were sequenced. The sequences
obtained are shown in Table 14 SEQ ID NOs: 1 to 11.
Example 2
Topical Applications of Anti-IgE VHH's
[0323] To obtain anti-allergic pharmaceutical compositions for
ophthalmic topical applications, a solution of anti-IgE VHH was
prepared as follows: [0324] eye drops containing a therapeutic dose
of anti-IgE VHH dissolved in 100 ml of sterilized water containing
0.9 g sodium chloride, 0.02 g sodium citrate, 0.02 g methyl
parahydroxybenzoate, 0.1 g chlorobutanol and acetic acid suitabe to
obtain a pH of 6.5. [0325] eye ointment containing a therapeutic
dose of anti-IgE VHH was prepared according to the conventional
method containing 1.0 g of liquid paraffin and a suitable amount of
soft paraffin to obtain a total mixture of 100 g.
Example 3
Anti-IgE Formulation
[0326] Anti-IgE VHH's that block binding of IgE to its
high-affinity receptor are of potential therapeutic value in the
treatment of allergy.
[0327] Highly purified VHH#2H11 was dialysed into formulation
buffer, followed by addition of lyoprotectant at an isotonic
concentration. Isotonic formulation was performed as follows:
VHH#2H11 at 25 mg/ml was formulated in 5 mM histidine buffer at pH
6 with 500 moles of sugar per mole antibody. This formulation is
reconstututed with BWFI (0.9% benzyl alcohol) at a volume which
results in a 100 mg/ml of antibody in 20 mM histidine at pH 6 with
an isotonic sugar concentration of 340 nM. The binding activity of
the anti-IgE VHH in the isotonic formulations was measured in an
IgE receptor inhibition assay. It was found that binding activity
was essentially unchanged following storage at 4.degree. C. for up
to 3 months.
TNF-Alpha
Example 4
Selection of Anti-TNF-Alpha
[0328] Two llamas were immunized with 100 .mu.g human TNF-alpha
Doer injection according to the schedule described in Example 1.
The libraries (short and long immunization procedure) were
constructed and selected with in vitro biotinylated TNF-alpha. The
biotinylation was carried out as described by Magni et al (Anal
Biochem 2001, 298, 181-188).
[0329] The incorporation of biotin in TNF was evaluated by SDS-PAGE
analysis and detection with Extravidin-alkaline phosphatase
conjugate (Sigma).
[0330] The functionality of the modified protein was evaluated for
its ability to bind to the solid phase coated recombinant a p75
receptor. {biotinylation} In the first round of selection 400 ng
and 50 ng of biotinylated TNF-alpha was captured on neutravidin
(Pierce; 10 .mu.g/ml in PBS) coated on the wells of a microtiter
plate (NUNC maxisorb). Phage (1.2.times.10.sup.10 TU-s) were added
to the wells and incubated for two hours at room temperature. After
washing (20 times with PBS-tween and two times with PBS) bound
phage was eluted by adding an excess of receptor (extracellular
domain of CD120b or p75; 10 .mu.M) or with cells expressing the
intact TNF receptor. Between 30,000 and 100,000 phage clones were
eluted with TNF from the library derived from the llama immunized
using the rapid scheme, while about 10% of these numbers were
obtained when eluted with BSA (3 .mu.M; negative control).
[0331] From the other library (long immunization scheme) 10-fold
high numbers were eluted with receptor and BSA, yielding the same
enrichment factor (10) as observed before. New phage was prepared
from the elution of 50 ng TNF (rapid immunization scheme) and 400
ng TNF (slow scheme) and used for another round of selection on
400, 50 and 10 ng of captured TNF (input: 1.2.times.10.sup.10 phage
per well). Approx. 2.5.times.10.sup.7 phage were eluted with
receptor (10 .mu.M) from the well containing 400 ng and 50 ng of
captured TNF and about 2.times.10.sup.6 from the well with 10 ng of
TNF, while the negative control (elution with 10 .mu.M of BSA) gave
only 5 to 10% of those numbers. The observed numbers of eluted
phage suggest that the elution with receptor is specific and that
those VHH fragments should be eluted that bind to the receptor
binding site of TNF.
[0332] Individual clones were picked and grown in microtiter plate
for the production of VHH in culture supernatants. ELISA screening
with TNF captured on Extravidin coated plates revealed about 50%
positive clones. HinFl-fingerprint analysis showed that 14
different clones were selected, which were grown and induced on 50
ml scale.
[0333] Periplasmic fractions were prepared, the VHH fragments
purified with IMAC and used in an assay to analyze their
antagonistic characteristics, i.e. preventing the interaction of
TNF with its receptor. For this purpose the VHH (1 .mu.M and 0.3
.mu.M) was incubated with TNF-alpha (3 and 0.7 nM) for 1.5 hours at
room temperature (in 0.2% casein/PBS). 100 .mu.l of this mixture
was transferred to a well of a microtiter plate, in which the
extracellular domain of the receptor was immobilized. After an
incubation of one hour the plate was washed and bound TNF was
detected with alkaline phosphatase conjugated streptavidin. Two VHH
fragments gave antagonistic profiles similar as obtained with 3 and
0.3 .mu.M intact mAB Remicade (Infliximab; Centercor) in spite of
the fact that the VHH is truly monomeric, whereas the dimeric
appearance of the mAB probably favors the binding of the trimeric
TNF-molecule. Similar experiments showing the efficacy of the VHH
were performed using the murine sarcoma cell line WEHI and a human
cell line expressing the TNF receptor. The sequences obtained are
shown in Table 14 SEQ ID NOs: 12 to 13.
Example 5
Stability Testing of Antibody Fragments Specific for Human
Tnf.alpha.
[0334] 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
TNF3E to pepsin which are supposed to mimic the gastric
environment. TNF3E a VHH specific to human TNF.alpha. was produced
as recombinant protein in E. coli and purified to homogeneity by
IMAC and gelfiltration chromatography. The protein concentration
after purification was determined spectrophotometrically by using
the calculated molar exctinction coefficient at 280 nm. Diluted
solutions at 100 microgram/ml were prepared in Mcllvaine 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 para nitrophenyl phosphate. The data plotted in FIG.
2 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 6
Oral Administration of an Anti-Human TNF.alpha. Specific VHH in
Mice
[0335] An antibody solution containing the anti-human TNF.alpha.
specific VHH#TNF3E (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 plater 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 phosphate. The ELISA signals
obtained after 10 minutes clearly demonstrated the presence of
functional VHH TNF3E 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 7
Efficacy in an Animal Model for IBD
1) Animal Model of Chronic Collitis
[0336] 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. 3.
2) VHH Treatment Schedule
[0337] During the VHH treatment period the mice (8 animals per
group) were treated daily for 14 consecutive days with bivalent
VHH#3F (VHH#3F-VHH#3F; SEQ ID No. 14) 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 7.
3) Results
[0338] 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.
MMP12
Example 8
Immunization
[0339] One llama's (llama 5) was immunized intramuscularly with
recombinant human catalytic domain of MMP12 using an appropriate
animal-friendly adjuvant Stimune (Cedi Diagnostics BV, The
Netherlands). The recombinant catalytic domain was acquired from
Prof. H. Tschesche Universitat Bielefeld and was supplied as a 56
.mu.g/ml solution in 5 mM Tris/HCl pH=7.5, 100 mM NaCl, 5 mM
CaCl.sub.2 (Lang, R. et al. (2001). The llama received 6 injections
at weekly intervals, the first two injections containing each 10
.mu.g of MMP-12, the last four injections containing each 5 .mu.g
of MMP-12. Four days after the last immunization a lymph node
biopsy (LN) and a blood sample (PBL1) of 150 ml was collected from
the animal and serum was prepared. Ten days after the last
immunization a second blood sample (PBL2) of 150 ml was taken and
serum was prepared. Peripheral blood lymphocytes (PBLs), as the
genetic source of the llama heavy chain immunoglobulins (HcAbs),
were isolated from the 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 was isolated from PBLs and
lymph nodes according to the method of Chomczynski and Sacchi
(1987).
Example 9
Repertoire Cloning
[0340] cDNA was prepared on 200 .mu.g total RNA with MMLV Reverse
Transcriptase (Gibco BRL) using oligo d(T) oligonucleotides (de
Heard et al., 1999). The cDNA was purified with a phenol/chloroform
extraction, followed by an ethanol precipitation and subsequently
used as template to amplify the VHH repertoire.
[0341] In a first PCR, the repertoire of both conventional (1.6 kb)
and heavy-chain (1.3 kb) antibody gene segments were amplified
using a leader specific primer (5'-GGCTGAGCTCGGTGGTCCTGGCT-3'; SEQ
ID NO:87) and the oligo d(T) primer
(5'-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT-3'; SEQ ID
NO:88). The resulting DNA fragments were separated by agarose gel
electrophoresis and the 1.3 kb fragment encoding heavy-chain
antibody segments was purified from the agarose gel. A second PCR
was performed using a mixture of FR1 reverse primers (WO03/054016
sequences ABL037 to ABL043) and the same oligo d(T) forward primer.
The PCR products were digested with SfiI (introduced in the FR1
primer) and BstEII (naturally occurring in framework 4). Following
gel electrophoresis, the DNA fragments of approximately 400
basepairs were purified from gel and ligated into the corresponding
restriction sites of phagemid pAX004 to obtain a library of cloned
VHHs after electroporation of Escherichia coli TG1. pAX004 allows
the production of phage particles, expressing the individual VHHs
as a fusion protein with a c-myc tag, a hexahistidine tag and the
geneIII product. The diversity obtained after electroporation of
TG1 cells is presented in Table 8. The percentage insert was
determined in PCR using a combination of vector based primers.
Example 10
Rescue of the Library and Phage Preparation
[0342] The library was grown at 37.degree. C. in 10 ml 2.times.TY
medium containing 2% glucose, and 100 .mu.g/ml ampicillin, until
the OD.sub.600nm reached 0.5. M13KO7 phages (10.sup.12) were added
and the mixture was incubated at 37.degree. C. for 2.times.30
minutes, first without shaking, then with shaking at 100 rpm. Cells
were centrifuged for 5 minutes at 4,500 rpm at room temperature.
The bacterial pellet was resuspended in 50 ml of 2.times.TY medium
containing 100 .mu.g/ml ampicillin and 25 .mu.g/ml kanamycin, and
incubated overnight at 37.degree. C. with vigorously shaking at 250
rpm. The overnight cultures were centrifuged for 15 minutes at
4,500 rpm at 4.degree. C. Phages were PEG precipitated (20%
poly-ethylene-glycol and 1.5 M NaCl) for 30 minutes on ice and
centrifuged for 20 minutes at 4,500 rpm. The pellet was resuspended
in 1 ml PBS. Phages were again PEG precipitated for 10 minutes on
ice and centrifuged for 10 minutes at 14,000 rpm and 4.degree. C.
The pellet was dissolved in 1 ml 0.5% skimmed milk or PBS-BSA [1
mg/ml] (Sigma, Cat Nr A3059).
Example 11
Selection of Human MMP-12 Specific VHH
[0343] Phages were rescued and prepared as described above in
Example 10.
[0344] Two approaches were followed to obtain MMP-12 specific
binders: [0345] a. Inactive MMP-12 coated on PVDF Membrane [0346]
100 ng human MMP-12 catalytic domain (diluted in 33 .mu.l PBS) was
spotted on small pieces (1 cm.sup.2) of PVDF (Immobilon-P,
Millipore, Cat Nr IPVH 15150) following the manufacturers
guidelines, resulting in an inactive MMP due to the MeOH fixation.
As controls an equal amount of lysozyme (Sigma, Cat Nr L-6876) and
33 .mu.l PBS were also spotted and immobilized. The membrane pieces
were blocked overnight in 5% skimmed milk at 4.degree. C. and were
washed 3 times with PBS before the phage preparation was applied
(4.times.10.sup.9 phages in 1 ml [5% skimmed milk]). Phages and
membrane pieces (in 1.5 ml tubes) were incubated for 3 hrs at room
temperature with rotation. Then the membranes were transferred to
15 ml tubes and were washed 6 times with 10 ml [PBS+0.05%
Tween-20]. Phages were eluted by exposing the membranes to 500
.mu.l TEA [70 .mu.l in 5 ml H.sub.2O] for 10 min while rotating.
The solution containing the eluted phages was removed and the pH
was neutralized with 1M Tris pH=7.5. [0347] Log phase growing TG1
cells were infected with the eluted phages and serial dilutions
were plated on selective medium. Enrichment was determined by the
number of transfected TG1 colonies after selection obtained from
the MMP-12 coated membrane as compared with the negative control
where lysozyme was immobilized. Bacteria from MMP selections
showing enrichment were scraped and used for a second round of
selection. [0348] The bacteria were superinfected with helperphage
to produce recombinant phages to do a second selection against
MMP-12 (as described in Example 9). MMP-12 was immobilized as above
and the membrane was blocked overnight at 4.degree. C. in 5% skim
milk. Phages (2.5.times.10.sup.9 in 1 ml) were prepared and exposed
to the membranes and further selected for MMP binding as during the
first round of selection. Log phase growing TG1 cells were infected
with the eluted and pH neutralized phages and plated on selective
medium. Enrichment was determined by the number of transfected TG1
colonies from the MMP-12 coated membrane as compared with the
negative control (immobilized lysozyme). [0349] b. Active MMP-12
coated on Nitrocellulose Membrane [0350] 250 ng human MMP-12
catalytic domain (Biomol Research laboratories Inc, SE 138-9090)
was spotted directly on a piece of Hybond-C extra (Amersham
Biosciences, Cat Nr RPN 303E) following the suppliers guidelines.
As control an equal volume of PBS was spotted. A 5 mm diameter
disk, containing the spotted area was cut out from each membrane
and was transferred to a 1.5 ml tube and blocked overnight at
4.degree. C. in 1 ml BSA-PBS [1 mg/ml]. The disks were washed three
times in 15 ml PBS and subsequently transferred and exposed to the
200 .mu.l phage preparation in a microtiterplate well. The phages
were prepared as in Example 9 but were preincubated in BSA-PBS for
15 min at room temperature. The disks were washed 5 times with
PBS/0.05% Tween-20 and were blocked with PBS-BSA for 2 hrs at room
temperature. Phages were eluted by exposing the membranes to 100
.mu.l TEA [70 .mu.l in 5 ml H.sub.2O] for 10 min while rotating.
The solution containing the eluted phages was removed and the pH
was neutralized with 1M Tris pH=7.5. [0351] Log phase growing TG1
cells were infected with the eluted phages and plated on selective
medium. Enrichment was determined by the number of transfected TG1
colonies after selection on the MMP-12 membrane disk as compared
with the negative control (PBS). Bacteria from selections with
MMP-12 were scraped and used for a second round of selection.
[0352] The bacteria were superinfected with helperphage to produce
recombinant phages to do a second selection against MMP-12 (as
described in Example 9). MMP-12 was immobilized as above and the
membrane was blocked overnight at 4.degree. C. in PBS-BSA [1
mg/m1]. Phages (2.5.times.10.sup.9 in 1 ml) were prepared and
exposed to the membranes and further selected for MMP binding as
during the first round of selection. Log phase growing TG1 cells
were infected with the eluted and neutralized phages and plated on
selective medium. Enrichment was determined by the number of
transfected TG1 colonies from the MMP-12 coated membrane as
compared with the negative control.
Example 12
Specificity of Selected VHH's
[0353] Individual clones were picked, grown in 150 .mu.l 2.times.TY
containing 0.1% glucose and 100 .mu.g/ml ampicillin in a microtiter
plate at 37.degree. C. until OD.sub.600nm=0.6. Then 1 mM IPTG and 5
mM MgSO.sub.4 was added and the culture was incubated 4 hours at
37.degree. C. ELISA was performed on the periplasmic extracts (PE,
preparation see Example 13) of the cells to examine specificity of
the selected clones.
[0354] To examine the clones selected using solid phase coated
human MMP-12, plates were coated with human MMP-12 catalytic domain
at a concentration of 1 .mu.g/ml overnight at 4.degree. C. Plates
were washed 5 times with PBS/0.05% Tween-20. Wells were blocked
with 1% skimmed milk for 2 hrs at room temperature. Periplasmic
extracts (100 .mu.l) were applied to the wells and incubated for 1
hour at room temperature. Plates were washed 5 times with PBS/0.05%
Tween-20. Detection was performed using anti-c-myc antibody,
followed by anti-mouse-HRP and ABTS/H.sub.2O.sub.2 as substrate.
Plates were read at 405 nm after 30 minutes incubation at room
temperature.
[0355] To examine the clones selected using membrane immobilized
human MMP-12, 50 ng human MMP-12 catalytic domain samples were
spotted on PVDF membrane as described in the manufacturers
guidelines. 50 ng lysozyme was spotted as a negative control. The
membranes were blocked with skimmed milk overnight at 4.degree. C.,
washed 5 times with PBS and transferred to 1,5 ml tubes.
Periplasmic extracts (100 .mu.l) were tenfold diluted in 1% skimmed
milk and 1 ml was applied per membrane (2 cm.sup.2) and rotated for
1 hour at room temperature. Membranes were washed 5 times with
PBS/0.05% Tween-20. Detection was performed using anti-c-myc
antibody, followed by anti-mouse-HRP and DAP as substrate.
Membranes were incubated with substrate at room temperature until
clear spots were visible. Seven clones which were found to be
MMP-12 specific binders are shown in Table 14 SEQ ID NOs 15 to
21.
[0356] In order to check for non specific binding to other MMPs a
similar approach was followed in which 50 ng of active catalytic
domain of MMP 1, 2, 3, 7, 9 and 13 (all from Biomol Research
laboratories Inc) was immobilized on Hybond C-extra. The membranes
were blocked with skimmed milk overnight at 4.degree. C., washed 5
times with PBS and transferred to 1.5 ml tubes. Periplasmic
extracts (100 .mu.l) were tenfold diluted in 1% skimmed milk and 1
ml was applied per membrane (2 cm.sup.2) and rotated for 1 hour at
room temperature. Membranes were washed times with PBS/0.05%
Tween-20. Detection was performed using anti-c-myc antibody,
followed by anti-mouse-HRP and DAP as substrate. Membranes were
incubated with substrate at room temperature until clear spots were
visible. No significant detection of the seven selected VHH clones
was observed on any of the MMPs other than MMP-12.
[0357] Results on binders selected against PVDF membrane
immobilized human MMP-12 catalytic domain are presented in Table 14
SEQ ID NOs 15 to 21.
[0358] Results on MMP-12 inhibitors selected via Hybond membrane
immobilization are presented in Table 14 SEQ ID NO 22.
Example 13
Diversity of Selected VHH's
[0359] PCR was performed using M13 reverse and genIII forward
primers. The clones were analyzed using Hinf1 fingerprinting and
representative clones were sequenced. Sequence analysis was
performed resulting in the sequences which are presented in Table
14 SEQ ID NOs 15 to 21 for Immobilon-P selections and in Table 14
SEQ ID NO 22 for Hybond-C.
Example 14
Expression and Purification of VHH
[0360] Clones were grown in 50 ml 2.times.TY containing 0.1%
glucose and 100 .mu.g/ml ampicillin in a shaking flask at
37.degree. C. until OD.sub.600nm=2. 1 mM IPTG and 5 mM MgSO.sub.4
was added and the culture was incubated for 3 more hours at
37.degree. C. Cultures were centrifuged for 10 minutes at 4,500 rpm
at 4.degree. C. The pellet was frozen overnight at -20.degree. C.
Next, the pellet was thawed at room temperature for 40 minutes,
re-suspended in 1 ml PBS/1 mM EDTA/1M NaCl and shaken on ice for 1
hour. Periplasmic fraction was isolated by centrifugation for 10
minutes at 4.degree. C. at 4,500 rpm. The supernatant containing
the VHH was loaded on Ni-NTA (Qiagen) and purified to homogeneity
on an Akta FPLC chromatography system (Amersham Biosciences). The
VHH were eluted from the Ni-NTA using 25 mM citric acid pH=4.0 and
directly applied on a cation exchange column equilibrated in 25 mM
citric acid pH=4.0 (Source 30S in a HR5/5 column, Amersham
Biosciences). The VHH were eluted with 1M NaCl in PBS and further
purified on a size exclusion column (Superdex 75 HR10/30, Amersham
Biosciences) equilibrated in MMP-12 assay buffer [50 mM HEPES, 100
mM NaCl, 0.05% Brij-35]. The yield of VHH was calculated according
to the extinction coefficient and peak surface area.
Example 15
Functional Characterization of Selected VHH's: Inhibition of MMP-12
Proteolytic Activity by a VHH in a Colorimetric Assay
[0361] VHHs were expressed and purified as described in Example 13.
Purified VHH was analyzed for the ability to inhibit human MMP-12
catalytic domain using the MMP-12 Colorimetric Assay Kit for Drug
Discovery (AK-402) from BIOMOL Research Laboratories. The
experimental method conditions described in the Kit were
followed.
[0362] The inhibitor supplied with the Kit (PI115-9090) was used as
positive control at the recommended concentration. VHH were applied
at a concentration of 7 .mu.M. The assay was performed in the
microtiterplate supplied with the BIOMOL Kit and MMP-12 proteolytic
activity was followed in a plate reader (405 nm) at 37.degree.
C.
[0363] The results of one inhibitory VHH and an inactive VHH are
presented in FIG. 4 together with a positive control.
[0364] Only one VHH molecule (clone P5-29) from selections using
active MMP-12 coated on nitrocellulose (Example 12) showed
inhibition of human MMP-12 catalytic domain. All other MMP-12
binders (only clone P5-5 is shown), although they bind MMP-12, did
not inhibit MMP-12.
Example 16
Formulation of Anti-MMP12 VHH for Pulmonary Delivery
[0365] A 100% formulation of antibody was prepared by dissolving 5
mg of VHH in 1.0 ml of deionized water. The pH of the solution was
6.5. A 90% formulation of antibody was prepared by dissolving 4.5
mg of VHH in 1.0 ml of 2 mM citrate buffer. A 70% formulation of
antibody was prepared by dissolving 3.5 mg of VHH in 1 mg/ml of
excipient in 1 ml of citrate buffer at pH 6.5. The various classes
of excipients used were as follows: Sugar excipients: sucrose,
lactose, mannitol, raffinose and trehalose. Polymeric excipients:
ficoll and PVP. Protein excipients: HSA.
[0366] Dry powders of the above formulations were produced by spray
drying using a Buchi Spray Dryer.
[0367] The particle size distribution was measure by centrifugal
sedimentation.
Interferon-damma
Example 17
Immunization
[0368] Four llama's (llama 5, 6, 22 and 23) were immunized
intramuscularly with human IFN-.gamma. (PeproTech Inc, USA, Cat Nr:
300-02) using an appropriate animal-friendly adjuvant Stimune (Cedi
Diagnostics BV, The Netherlands). Two llama's (llama 29 and 31)
were immunized intramuscularly with mouse IFN-.gamma. (Protein
Expression & Purification core facility, VIB-RUG, Belgium)
using an appropriate animal-friendly adjuvant Stimune (Cedi
Diagnostics BV, The Netherlands). The llama's received 6 injections
at weekly intervals, the first two injections containing each 100
.mu.g of IFN-.gamma., the last four injections containing each 50
.mu.g of IFN-.gamma.. Four days after the last immunization a blood
sample (PBL1) of 150 ml and a lymph node biopsy (LN) was collected
from each animal and sera were prepared. Ten days after the last
immunization a second blood sample (PBL2) of 150 ml was taken from
each animal and sera were prepared. Peripheral blood lymphocytes
(PBLs), as the genetic source of the llama heavy chain
immunoglobulins (HcAbs), were isolated from the 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 was isolated from PBLs and lymph nodes according to the
method of Chomczynski and Sacchi (1987).
Example 18
Repertoire Cloning
[0369] cDNA was prepared on 200 .mu.g total RNA with MMLV Reverse
Transcriptase (Gibco BRL) using oligo d(T) oligonucleotides (de
Heard et al., 1999). The cDNA was purified with a phenol/chloroform
extraction, followed by an ethanol precipitation and subsequently
used as template to amplify the VHH repertoire.
[0370] In a first PCR, the repertoire of both conventional (1.6 kb)
and heavy-chain (1.3 kb) antibody gene segments were amplified
using a leader specific primer (5'-GGCTGAGCTCGGTGGTCCTGGCT-3'; SEQ
ID NO:89) and the oligo d(T) primer
(5'-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT-3'; SEQ ID
NO:90). The resulting DNA fragments were separated by agarose gel
electrophoresis and the 1.3 kb fragment encoding heavy-chain
antibody segments was purified from the agarose gel. A second PCR
was performed using a mixture of FR1 reverse primers (WO03/054016
sequences ABL037 to ABL043) and the same oligo d(T) forward
primer.
[0371] The PCR products were digested with SfiI (introduced in the
FR1 primer) and BstEII (naturally occurring in framework 4).
Following gel electrophoresis, the DNA fragments of approximately
400 basepairs were purified from gel and ligated into the
corresponding restriction sites of phagemid pAX004 to obtain a
library of cloned VHHs after electroporation of Escherichia coli
TG1. pAX004 allows the production of phage particles, expressing
the individual VHHs as a fusion protein with a c-myc tag, a
hexahistidine tag and the geneIII product. The diversity obtained
after electroporation of TG1 cells is presented in Table 1. The
percentage insert was determined in PCR using a combination of
vector based primers.
Example 19
Rescue of the Library and Phage Preparation
[0372] The library was grown at 37.degree. C. in 10 ml 2.times.TY
medium containing 2% glucose, and 100 .mu.g/ml ampicillin, until
the OD.sub.600nm reached 0.5. M13KO7 phages (10.sup.12) were added
and the mixture was incubated at 37.degree. C. for 2.times.30
minutes, first without shaking, then with shaking at 100 rpm. Cells
were centrifuged for 5 minutes at 4,500 rpm at room temperature.
The bacterial pellet was resuspended in 50 ml of 2.times.TY medium
containing 100 .mu.g/ml ampicillin and 25 .mu.g/ml kanamycin, and
incubated overnight at 37.degree. C. with vigorously shaking at 250
rpm. The overnight cultures were centrifuged for 15 minutes at
4,500 rpm at 4.degree. C. Phages were PEG precipitated (20%
poly-ethylene-glycol and 1.5 M NaCl) for 30 minutes on ice and
centrifuged for 20 minutes at 4,500 rpm. The pellet was resuspended
in 1 ml PBS. Phages were again PEG precipitated for 10 minutes on
ice and centrifuged for 10 minutes at 14,000 rpm and 4.degree. C.
The pellet was dissolved in 1 ml PBS-0.1% casein.
Example 20
Selection of Human IFN-.gamma. Specific VHH
[0373] Phages were rescued and prepared as described above in
example 17
[0374] Two approaches were followed to obtain IFN-.gamma. specific
binders: [0375] a. Solid phase coated IFN-.gamma. [0376] Microtiter
wells were coated with human IFN-.gamma. at different
concentrations of 10-0.4 .mu.g/well overnight at 4.degree. C.
Plates were washed 5 times with PBS/0.05% Tween-20. Wells were
blocked with PBS+1% caseine for 2 hrs at room temperature. Phages
were incubated for 2 hrs at room temperature. Wells were washed 20
times with PBS+0.05% Tween-20. The two final washes were performed
using PBS. Specific phages were eluted using 1 to 2 .mu.g of
IFN-.gamma. R1 (R&D Systems, Cat Nr: 673-IR/CF) for 1 hr. As
negative control elutions were performed using 10 .mu.g Ovalbumine
(Sigma, A2512) as irrelevant protein. Log phase growing TG1 cells
were infected with the eluted phages and plated on selective
medium. Enrichment was determined by the number of transfected TG1
colonies after selection using the receptor for elution as compared
with negative control using ovalbumine for elution. Bacteria from
selections showing enrichment were scraped and used for a second
round of selection. [0377] The bacteria were superinfected with
helperphage to produce recombinant phages as described in example
3. Microtiter wells were coated with IFN-.gamma. at different
concentrations of 2-0.1 .mu.g/well overnight at 4.degree. C. Plates
were washed 5 times with PBS/0.05% Tween-20. Wells were blocked
with PBS+1% caseine for 2 hrs at room temperature. Phages were
incubated for 2 hrs at room temperature. Wells were washed 20 times
with PBS+0.05% Tween-20. The two final washes were performed using
PBS. Specific phages were eluted using 1 to 2 .mu.g of IFN-.gamma.
R1 or 10 .mu.g Ovalbumine as irrelevant protein for 1 hr,
subsequently overnight at 4.degree. C. and subsequently, phages
were eluted using 0.1 M glycine pH 2.5 for 15 minutes at room
temperature and neutralized with 1M Tris-HCl pH=7.5. Log phase
growing TG1 cells were infected with the eluted and neutralized
phages and plated on selective medium. Enrichment was determined by
the number of transfected TG1 colonies after selection using the
receptor for elution as compared with negative control using
ovalbumine for elution. [0378] b. Biotinylated IFN-v [0379]
Microtiter wells were coated with neutravidine at a concentration
of 2 .mu.g/ml overnight at 4.degree. C. Plates were washed 5 times
with PBS/0.05% Tween-20. Wells were blocked with PBS+1% caseine for
2 hrs at room temperature. Biotinylated human IFN-.gamma. at a
concentration of 100-10 ng/well was captured overnight at 4.degree.
C. Plates were washed 5 times with PBS/0.05% Tween-20. Phages were
incubated for 2 hrs at room temperature. Wells were washed with
PBS+0.05% Tween-20. The two final washes were performed using PBS.
Specific phages were eluted using 1 to 2 .mu.g of IFN-.gamma. R1
(R&D Systems, Cat Nr: 673-IR/CF) for 1 hr. As negative control
elutions were performed using 10 .mu.g Ovalbumine (Sigma, A2512) as
irrelevant protein. Log phase growing TG1 cells were infected with
the eluted phages and plated on selective medium. Enrichment was
determined by the number of transfected TG1 colonies after
selection using the receptor for elution as compared with negative
control using ovalbumine for elution. Bacteria from selections
showing enrichment were scraped and used for a second round of
selection. Bacteria were superinfected with helperphage to produce
recombinant phages. Microtiter wells were coated with neutravidine
at a concentration of 2 .mu.g/ml overnight at 4.degree. C. Plates
were washed 5 times with PBS/0.05% Tween-20. Wells were blocked
with PBS+1% caseine for 2 hrs at room temperature. Biotinylated
human IFN-.gamma. at a concentration of 20-2.5 ng/100 .mu.l was
captured overnight at 4.degree. C. Plates were washed 5 times with
PBS/0.05% Tween-20. Phages were incubated for 2 hrs at room
temperature. Wells were washed 20 times with PBS+0.05% Tween-20.
The two final washes were performed using PBS. Specific phages were
eluted using 1 to 2 .mu.g of IFN-.gamma. R1 or 10 .mu.g Ovalbumine
as irrelevant protein for 1 hr, subsequently overnight at 4.degree.
C. and subsequently, phages were eluted using 0.1 M glycine pH 2.5
for 15 minutes at room temperature and neutralized with 1M Tris-HCl
pH=7.5. Log phase growing TG1 cells were infected with the eluted
and neutralized phages and plated on selective medium. Enrichment
was determined by the number of transfected TG1 colonies after
selection using the receptor for elution as compared with negative
control using ovalbumine for elution.
Example 21
Diversity of Selected VHH's
[0380] PCR was performed using M13 reverse and genIII forward
primers. The clones were analyzed using Hinf1 fingerprinting and
representative clones were sequenced. Sequence analysis was
performed resulting in the sequences presented in Table 4 for human
IFN-.gamma. SEQ ID No. 45-70).
Example 22
Expression and Purification of VHH
[0381] Small scale expressions were started after transformation of
DNA into WK6 Escherichia coli cells.
[0382] Clones were grown in 50 ml 2.times.TY containing 0.1%
glucose and 100 .mu.g/ml ampicillin in a shaking flask at
37.degree. C. until OD.sub.600nm=2. 1 mM IPTG and 5 mM MgSO.sub.4
was added and the culture was incubated for 3 more hours at
37.degree. C. Cultures were centrifuged for 10 minutes at 4,500 rpm
at 4.degree. C. The pellet was frozen overnight at -20.degree. C.
Next, the pellet was thawed at room temperature for 40 minutes,
re-suspended in 1 ml PBS/1 mM EDTA/1M NaCl and shaken on ice for 1
hour. Periplasmic fraction was isolated by centrifugation for 10
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 calculated according to the extinction
coefficient.
Example 23
Topical Applications of Anti-IFN Gamma VHH's
[0383] 1: To obtain anti-allergic pharmaceutical compositions for
ophthalmic topical applications, a solution of at least one
anti-IFN gamma VHH was prepared as follows: [0384] eye drops
containing a therapeutic dose of anti-IFN gamma VHH dissolved in
100 ml of sterilized water containing 0.9 g sodium chloride, 0.02 g
sodium citrate, 0.02 g methyl parahydroxybenzoate, 0.1 g
chlorobutanol and acetic acid suitable to obtain a pH of 6.5.
[0385] eye ointment containing a therapeutic dose of anti-IFN gamma
VHH was prepared according to the conventional method containing
1.0 g of liquid paraffin and a suitable amount of soft paraffin to
obtain a total mixture of 100 g.
[0386] 2: To obtain anti-inflammatory pharmaceutical applications,
topical preparations of the present invention contained at least
one anti-IFN gamma VHH and a pharmaceutically acceptable carrier.
They were prepared as follows: [0387] Preparation of Base Cream
[0388] The reagents for preparing the base cream are as follows
(contents for 100 kg base cream): Dimethyl silicon oil (17 kg),
Liquid paraffin (9 kg), Stearic acid (7.5 kg), Cetyl alcohol (1
kg), Stearyl alcohol (3 kg), Glycerol (20 kg), Ethylparaben (0.1
kg), Peregal A-20 (0.45 kg), Softener SG (0.85 kg), 0.01 M
Phosphate Buffer (pH 7.2)(41.1 kg)
[0389] The stainless steel tank was placed into a thermostat water
bath and heated to 80.degree. C., which took approximately 10
minutes. The liquid was thoroughly mixed. Then, emulsifying and
homogenizing equipment was placed into the open stainless steel
tank, the mixture was stirred for 20 minutes at 3500 rpm until
fully emulsified. The temperature of the thermostat water bath was
cooled naturally to room temperature, until the mixture became a
semi-solid cream. The mixture was being continually stirred. [0390]
Preparation of Liquid Antibody Mixture [0391] VHH#MP3B1SRA was
prepared in accordance with Example 22. The lyophilized antibodies
were reconstituted with 0.01 M phosphate buffer (pH 7.2) to a
concentration of 2 mg/ml. For 1000 gm of base cream, 45 mg of
VHH#MP3B1SRA antibody was added.
Therapeutic VHH-fragments
Example 24
Expression of a VHH-CDR3 Fragment of Anti-TNF.alpha. VHH#3E
[0392] The CDR3 region of VHH#3E was amplified by using a sense
primer located in the framework 4 region (Forward:
CCCCTGGCCCCAGTAGTTATACG; SEQ ID NO:91) and an anti-sense primer
located in the framework 3 region (Reverse: TGTGCAGCAAGAGACGG; SEQ
ID NO:92).
[0393] 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-00001 Reverse primer Sfi1: (SEQ ID NO: 93)
GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCAGCAAGAGACGG Forward primer Not1:
(SEQ ID NO: 94) GTCCTCGCAACTGCGCGGCCGCCCCCTGGCCCCAGTAGTTATACG
[0394] The PCR reactions were performed in 50 .mu.l 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).
[0395] After cleavage with Sfi1 and Not1 the PCR product was cloned
in pAX10.
EGFR
Example 25
Immunization
[0396] After approval of the Ethical Committee of the Faculty of
Veterinary Medicine (University Ghent, Belgium), 4 llamas (024,
025, 026 and 027) were immunized with the tumor antigen epidermal
growth factor receptor (EGFR) according to all current animal
welfare regulations. To generate an antibody dependent immune
response (Table 9), two animals were injected with intact human
vulvar squamous carcinoma cells (A431, ATCC CRL 1555), expressing
EGFR on its cell surface, while A431 derived membrane extracts were
administered to two other llamas (026 and 027). Each animal
received seven doses of subcutaneously administered antigens at
weekly intervals (Table 9). When immunizing with intact cells, each
dose consisted of 10.sup.8 freshly harvested A431 cells. The dose
for immunization with membrane extracts consisted of vesicles
prepared from 10.sup.8 A431 cells. Vesicles were prepared according
to Cohen and colleagues (Cohen S, Ushiro H, Stoscheck C, Chinkers
M, 1982. A native 170,000 epidermal growth factor receptor-kinase
complex from shed plasma membrane vesicles. J. Biol. Chem.
257:1523-31). Vesicles were stored at -80.degree. C. before
administration. Two extra injections of eight microgram purified
EGFR (Sigma) in an emulsion with the adjuvant Stimune (CEDI
Diagnostics B.V., Lelystad, The Netherlands) were administered
intramuscularly to llama 025 (Table 9).
Example 26
Evaluation of Immune Response
[0397] At day 0, 28 and 42, 10 ml of (pre-)immune blood was
collected and serum was used to evaluate the induction of the
immune responses in the 4 animals. A first ELISA was performed to
verify whether the animals generated antibodies that recognized
A431 epitopes. After coating a tissue-culture treated 96-well plate
with gelatin (0.5% in PBS for 10 minutes), the excess of gelatin
was removed and A431 cells were grown overnight in the microwells
to confluency. Cells were fixed with 4% paraformaldehyde in PBS for
30 minutes at room temperature. Subsequently, the fixative was
blocked with 100 mM glycine in PBS for 10 minutes, followed by
blocking of the wells with a 4% skim milk-PBS solution, again for
10 minutes. Serum dilutions of immunized animals were applied and
A431 specific antibodies were detected with a polyclonal anti-llama
antiserum developed in rabbit, followed by a secondary goat
anti-rabbit horse radish peroxidase (HRP) conjugate (Dako,
Denmark). For all four animals, immunization with intact cells or
membrane vesicles resulted in the induction of a significant
A431-specific antibody titer (FIG. 5).
[0398] To verify whether the induced llama antibodies were EGFR
specific, antibody titers in serum was evaluated on mouse
fibroblasts expressing human EGFR (Her-14) and compared to the
parental mouse fibroblasts cell line NIH3T3 clone 2.2 (3T3),
similarly performed as described above (FIG. 6). Again, the serum
titer of antibodies binding to Her-14 was higher compared to the
titer for the parental 3T3 cells, indicating that circulating serum
antibodies were EGFR specific.
[0399] Finally, the serum response in immunized animals was
verified on solid-phase coated purified EGFR. Purified EGFR (Sigma)
and the irrelevant carcino embryonic antigen (CEA, Scripps), both
at 1 .mu.g/ml, were immobilized overnight at 4.degree. C. in a 96
well Maxisorp plate (Nunc). Wells were blocked with a casein
solution (1% in PBS). After addition of serum dilutions,
specifically bound immunoglobulins were detected using a rabbit
anti-llama antiserum followed by a goat anti-rabbit alkaline
phosphatase conjugate (Sigma), showing that for all animals a
significant antibody dependent immune response against EGFR was
induced (FIG. 7).
Example 27
Cloning of the Heavy-Chain Antibody Fragment (VHH) Repertoire
[0400] Since little is known on the immunoglobulin ontogeny of
camelids, B-cell containing tissues of distinct origin and of
different time points were collected for each animal (Table 9).
After tissue collection, total RNA was isolated according to the
procedure described by Chomczynski and Sacchi. (Chomczynski P and
Sacchi N. 1987. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem
162:156-159). The procedure to clone the VHH repertoire is based on
a method described in patent application WO 03/054016. cDNA was
prepared on total RNA with MMLV Reverse Transcriptase (Invitrogen)
using oligo d(T) oligonucleotides (de Heard H J, van Neer N, Reurs
A, Hufton S E, Roovers R C, Henderikx P, de Bruine A P, Arends J W,
Hoogenboom H R. 1999. A large non-immunized human Fab fragment
phage library that permits rapid isolation and kinetic analysis of
high affinity antibodies. J. Biol. Chem. 274:18218-30). The amounts
of RNA of the distinct tissues used for cDNA synthesis is listed in
Table 10. The cDNA was purified with a phenol/chloroform
extraction, followed by an ethanol precipitation and subsequently
used as template to amplify the VHH repertoire.
[0401] In a first PCR, the repertoire of both conventional (1.6 kb)
and heavy chain (1.3 kb) antibody gene segments were amplified
using a leader specific primer (ABL002) and ABL010, an oligo d(T)
primer (for a list of primers see Table 13). The resulting DNA
fragments were separated by agarose gel electrophoresis. The
amplified 1.3 kb fragment, encoding heavy chain antibody segments
was purified from the agarose gel and used as template in a nested
PCR using a mixture of FR1 primers (ABL037-ABL043) and ABL010. The
PCR products were digested with SfiI (introduced in the FR1 primer)
and BstEII (naturally occurring in FR4). Following gel
electrophoresis, the DNA fragment of approximately 400 basepairs
was purified from gel and 330 ng of amplified VHH repertoire was
ligated into the corresponding restriction sites of one microgram
of phagemid pAX004 to obtain a library after electroporation of
Escherichia coli TG1. pAX004 allows the production of phage
particles, expressing the individual VHHs as a fusion protein with
the geneIII product. The size of the libraries obtained from the
distinct tissues collected from the immunized llamas is described
in Table 10. As a quality control, a colony PCR using the M13
reverse and a geneIII primer was performed on 24 randomly picked
colonies of each library and the percentage of clones containing an
insert of the correct size was calculated (Table 10).
Example 28
Evaluation of the Cloned Repertoire
[0402] In a polyclonal phage ELISA, the specificity of the cloned
phage repertoire was evaluated on EGFR and on an irrelevant antigen
(TNF.alpha.). To generate recombinant virions expressing the VHH
repertoire as fusion proteins with the geneIII product, the library
was grown at 37.degree. C. in 10 ml 2.times.TY medium containing 2%
glucose, and 100 .mu.g/ml ampicillin, until the OD.sub.600nm
reached 0.5. M13KO7 phages (10.sup.12) were added and the mixture
was incubated at 37.degree. C. for 2.times.30 minutes, first
without shaking, then with shaking at 100 rpm. Cells were
centrifuged for 5 minutes at 4,500 rpm at room temperature. The
bacterial pellet was resuspended in 50 ml of 2.times.TY medium
containing 100 .mu.g/ml ampicillin and 25 .mu.g/ml kanamycin, and
incubated overnight at 37.degree. C. with vigorously shaking at 250
rpm. The overnight cultures were centrifuged for 15 minutes at
4,500 rpm at 4.degree. C. and supernatant was used to concentrate
the phages. Phages were PEG precipitated (20% poly-ethylene-glycol
and 1.5 M NaCl) for 30 minutes on ice and centrifuged for 20
minutes at 4,500 rpm. The pellet was resuspended in 1 ml PBS.
Phages were again PEG precipitated for 10 minutes on ice and
centrifuged for 10 minutes at 14,000 rpm and 4.degree. C. The
pellet was dissolved in 1 ml PBS. One .mu.g/ml of EGFR or
TNF.alpha. was immobilized in a 96 well Maxisorp plate (Nunc) and
incubated overnight at 4.degree. C. Plates were washed 5 times with
PBS/0.05% Tween-20 and wells were blocked with a casein solution
(1% in PBS) and phage dilutions were added for 2 hrs at room
temperature. Bound phages were detected using the anti-M13
gpVIII-HRP conjugated monoclonal antibody (Amersham Biosciences)
and ABTS/H.sub.2O.sub.2 as substrate. Plates were read at 405 nm
after 15 minutes incubation at room temperature. An example of a
phage response from a pool of phages rescued from PBL1 libraries of
animals 024 and 025 is depicted in FIG. 8.
Example 29
Multiple Selection Strategies to Identify EGFR Specific
Nanobodies
[0403] Libraries were rescued by growing the bacteria to
logarithmic phase (OD.sub.600=0.5), followed by infection with
helper phage to obtain recombinant phages expressing the repertoire
of cloned VHHs on tip of the phage as gpIII fusion protein (as
described in Example 18). When selecting for EGFR specific
antibodies, two distinct selection strategies have been
followed.
Selection by Epitope Specific Elution
[0404] A first selection strategy was based on the fact that EGFR
can be purified by affinity chromatography through ligand elution.
Four different elution conditions, applying an excess of molecules
that compete for the ligand binding site or overlapping epitope(s)
were carried out (Table 11). When selection was performed on A431
or Her-14 cells, unselected recombinant phages were mixed for 20
minutes at 4.degree. C. with 6.times.10.sup.6 blood cells (mainly
monocytes, T- and B-cells) or 2.times.10.sup.7 3T3s, respectively,
to deplete for recombinant phages that recognize common, non
EGFR-specific epitopes. Unbound phages were then incubated with
EGFR.sup.+ selection cells for 2 hours followed by 6 washes with
ice-cold PBS. Phages were subsequently eluted with an excess of EGF
ligand, mouse monoclonal 2e9 (Defize L H, Moolenaar W H, van der
Saag P T, de Laat S W 1986. Dissociation of cellular responses to
epidermal growth factor using anti-receptor monoclonal antibodies.
EMBO J. 5:1187-92) or EGFR antagonistic antibodies 225 and 528
(Sato J D, Kawamoto T, Le A D, Mendelsohn J, Polikoff J, Sato G H
1983. Biological effects in vitro of monoclonal antibodies to human
epidermal growth factor receptors. Mol. Biol. Med. 1:511-529). All
selection steps were performed at 4.degree. C. to avoid receptor
mediated phage internalization. Logarithmically grown E. coli TG1
was infected with the eluted phages and grown overnight at
37.degree. C. on selective medium 2.times.TY Ap100 and 2% glucose.
Cells were scraped and used in a next round of panning whenever
required. Two or three rounds of panning were performed to enrich
for EGFR specific recombinant phages (Table 11). Whenever purified
antigen was used for selection (Table 11), EGFR was immobilized at
1 .mu.g/ml on Maxisorp microtiter plates.
Selection for Internalizing VHH Fragments
[0405] A second selection strategy was based on the observation
that after binding of the ligand to the receptor, EGFR mediated
cell signaling can be downregulated by the mechanism of receptor
internalization. To identify recombinant phages that are able to
internalize through cell surface molecules, the protocol described
by Poul and colleagues (Poul M A, Becerril B, Nielsen U B, Morisson
P, Marks J D. 2000. Selection of tumor-specific internalizing human
antibodies from phage libraries. J. Mol. Biol. 301:1149-61.) was
followed. Unselected recombinant phages were added to approximately
2.times.10.sup.7 mouse fibroblast 3T3s for 30 minutes at 4.degree.
C. in ice cold binding medium (bicarbonate buffered DMEM; 10% FCS
(featal calf serum); 25 mM Hepes), supplemented with 2% skim milk
to deplete for non-specific VHHs. Unbound phages were subsequently
incubated with pre-cooled EGFR+selection cells (Her-14 or A431) in
binding medium for 1.5 hours at 4.degree. C., followed by six
washes with ice-cold PBS to remove non-bound phages. Cells were
covered with pre-warmed binding medium and immediately transferred
to 37.degree. C. for 20 minutes, to allow internalization.
Subsequently, cells were cooled down to 4.degree. C. and were
stripped with mild acid (500 mM NaCl; 100 mM glycine pH2.5)
incubations during 10 minutes to remove surface bound recombinant
phages. Cells were released from extracellular matrix by
trypsinization. Resuspended cells were then lyzed during 4 minutes
with 100 mM TEA at 4.degree. C. to release internalized phages.
Logarithmically grown E. coli TG1 was infected with the eluted
phages and grown overnight at 37.degree. C. on selective medium
(2.times.TY Ap100 with 2% glucose). The libraries used for a single
round of selection on A431 and in parallel on Her-14 are summarized
in Table 12.
Example 30
Characterization of EGFR Specific Nanobodies
[0406] To verify EGFR specificity of individual clones after the
epitope specific elution procedure of panning, a phage ELISA was
performed on individual clones. 47 randomly picked clones for each
selection procedure (1, 2, 3, 4, Ia and IIIa; Table 11) were grown
to logarithmic phase (OD.sub.600=0.5), followed by infection with
helper phage to obtain recombinant phages as described in Example
18. A phage ELISA was performed both on solid-phase coated EGFR
(comparing to non-coated well) as on gelatin coated Her-14 cells
(comparing to 3T3). The presence of EGFR specific VHH was verified
by using approximately 10.sup.9 recombinant phage particles of each
clone before detection with an anti-M13 gpVIII-HRP conjugated
monoclonal antibody. With clones that scored positive in phage
ELISA on cells and/or on solid-phase immobilized EGFR (Table 11), a
Hinfl fingerprint analysis was performed (data not shown). The
nucleotide sequence was determined for a representative clone of
each distinct fingerprint, resulting in 5, 8, 3, 4, 7, and 4
different sequences for conditions, 1, Ia, 2, IIIa, 3 and 4,
respectively. Amino acid sequence alignment of these 31 binders
(FIG. 9) indicated that 20 of them were unique (listed in Table 14
SEQ ID Nos 23 to 42). The EGFR specificity of the 20 unique clones
in phage ELISA (both on cells and on solid-phase coated EGFR) is
shown in FIG. 10.
[0407] For the selection according to the internalization protocol,
a phage ELISA on cells with a total of 84 individual clones was
performed, similarly as for the clones identified by the epitope
specific elution selection procedure. After Hinfl fingerprint
analysis, nucleotide sequence determination and amino acid sequence
alignment to the above described panel of 20 unique binders (data
not shown), 2 new anti-EGFR clones, EGFR-B11 and clone EGFR-F11,
were identified (Table 14 SEQ ID NOs: 43 to 44). The EGFR
specificity of both clones in phage ELISA on cells is shown in FIG.
10, panel A.
Example 31
EGF Receptor Mediated Internalization of Nanobodies
[0408] Her-14 and 3T3 cells were grown overnight on glass cover
slips, washed with binding medium (see Example 19) and cooled down
to 4.degree. C. for 20 minutes. Phages were prepared of nanobody
EGFR-IIIa42 as described in Example 18 and approximately 10.sup.12
recombinant virions, diluted in binding medium supplemented with 2%
skim milk, were added to the ice cold cells for 1 hour at 4.degree.
C. Cells were washed once with ice cold PBS to remove non bound
phages. Subsequently, the cells were shifted to 37.degree. C. for
20 minutes to allow phage internalization and again cooled down to
4.degree. C. Cells were washed twice with PBS. Following, cell
surface bound phages were removed by two acid washes with stripping
buffer (150 mM NaCl, 125 mM HAc) for seven minutes at room
temperature. After two washes with PBS, cells were fixed with 4%
paraformaldehyde in PBS for 30 minutes at room temperature, and
again washed twice with PBS. Fixed cells were then permeabilized in
0.2% Triton X-100 in PBS for 5 minutes at room temperature,
followed by two washes with PBS and remaining fixative was blocked
with 100 mM glycin in PBS for 10 minutes at room temperature. Cells
were washed with PBS-0.5% (w/v) gelatin and internalized phage was
visualized by staining with anti-M13 gpVIII-FITC (Amersham
Biosciences) followed by an anti-mouse FITC labeled monoclonal
antibody and subsequent visualization by fluorescence microscopy.
FIG. 11 shows that EGFRIIIa42 is able to internalize Her-14 (panel
A) but not 3T3 cells (panel B). Subsequently, FACS analysis
demonstrated that nanobody EGFR-IIIa42 is able to bind both A431
and Her-14, but not 3T3 (data not shown). To demonstrate the effect
of EGF receptor specific nanobodies on receptor signalling, cells
were seeded at 100,000 cells per well in 12-well tissue culture
plates in medium (DMEM) containing 10% (v/v) serum. After 8 hours,
cells were washed once with medium (DMEM) containing low (0.5% v/v)
serum and serum-starved overnight in the same medium. The day of
the assay, medium was refreshed with binding medium (DMEM/0.5%
FCS/25 mM Hepes and 2% skim milk) and when appropriate, ligand or
nanobody (mono- or bivalent) was added at 37.degree. C. After 15
minutes, cells were quickly cooled down on ice and washed twice
with ice-cold PBS (10 mM Na-phosphate; 150 mM NaCl, pH 7.4). Total
cell lysates were prepared by scraping the cells off the plate in
50 .mu.l protein sample buffer. Proteins were size-separated on 6%
(w/v) poly-acrylamide gels (20 .mu.l loaded per gel on two parallel
gels) and blotted to PVDF membrane (Roche). Blots were stained for
total amount of EGFR with a rabbit polyclonal antiserum to the
receptor (Santa Cruz) and for phosphorylated receptor using a
monoclonal anti phospho-tyrosine antibody (PY-20; Transduction
Labs), followed by an appropriate in donkey developped and
peroxidase conjugated secondary antibody (anti-rabbit or
anti-mouse). The detection was performed by enhanced
chemoluminiscence using Western Lightning.TM. substrate (Perkin
Elmer Life Sciences). Surprisingly, anti-EGFR-IIIa42 nanobody did
not activate EGFR.sup.+ cells deprived from EGF, indicated by the
lack of receptor Tyr kinase phosphorylation (FIG. 11, panel C). The
positive control, in which EGF was added in two concentrations to
the cells, clearly induced phosphorylation of the receptor and thus
induces activation of the cells.
PDK1
Example 32 (1)
Immunisation of Llamas
[0409] 2 llamas are immunised with a cocktail of recombinant EGF
receptor and with PDK1. The lamas are boosted with a cell line
overexpressing the EGF receptor. The immunization schemes are
summarised in Table 15.
Example 33
Repertoire Cloning
[0410] Different sources for RNA extraction are used: [0411] 150 ml
immune blood, between 4 and 10 days after the last antigen
injection [0412] lymph node biopsy 4 days after the last antigen
injection
[0413] Peripheral blood lymphocytes (PBLs) are isolated by
centrifugation on a density gradient (Ficoll-Paque Plus Amersham
Biosciences). PBLs and lymph node are used to extract total RNA
(Chomczynski and Sacchi 1987) followed by synthesis of cDNA using a
hexanucleotide random primer. The repertoire is amplified using two
hinge-specific primers:
AACAGTTAAGCTTCCGCTTGCGGCCGCGGAGCTGGGGTCTTCGCTGTGGTGCG (SEQ ID
NO:95) and AACAGTTAAGCTTCCGCTTGCGGCCGCTGGTTGTGGTTTTGGTGTCTTGGGTT
(SEQ ID NO:125) and a framework 1 specific primer:
GAGGTBCARCTGCAGGASTCYGG (SEQ ID NO:96). Fragments are digested with
PstI and NotI and cloned into a phagemid vector. The repertoire is
transformed in TG1 electrocompetent cells and plated on LB agar
plates containing 100 .mu.g/ml ampicillin and 2% glucose. Colonies
are screened for the presence of insert by PCR with vector specific
primers.
Example 34
Rescue of the Library, Phage Preparation
[0414] Libraries are grown at 37.degree. C. in 60 ml 2.times.TY
medium containing 2% glucose, and 100 .mu.g/ml ampicillin, until
the OD600 nm reached 0.5. M13KO7 phages (1012) are added and the
mixture is incubated at 37.degree. C. for 2.times.30 minutes, first
without shaking, then with shaking at 100 rpm. Cells are
centrifuged for 10 minutes at 4500 rpm at room temperature. The
bacterial pellet is resuspended in 300 ml of 2.times.TY medium
containing 100 .mu.g/ml ampicillin and 25 .mu.g/ml kanamycin, and
incubated overnight at 30.degree. C. with vigorously shaking at 250
rpm. The overnight cultures are centrifuged for 15 minutes at
10.000 rpm at 4.degree. C. Phages are PEG precipitated (20%
poly-ethylene-glycol and 1.5 M NaCl) and centrifuged for 30 minutes
at 10.000 rpm. The pellet is resuspended in 20 ml PBS. Phages are
again PEG precipitated and centrifuged for 30 minutes at 20,000 rpm
and 4.degree. C. The pellet is dissolved in 5 ml PBS. Phages are
titrated by infection of TG1 cells at OD600 nm=0.5 and plating on
LB agar plates containing 100 .mu.g/ml ampicillin and 2% glucose.
The number of transformants indicates the number of phages (pfu).
The phages are stored at -80.degree. C. with 15% glycerol.
Example 35
Selection
[0415] Immunotubes are coated with 2 .mu.g/ml EGFR, 2 .mu.g/ml PDK1
or with PBS containing 1% casein. After overnight incubation at
4.degree. C., the tubes are blocked with PBS containing 1% casein,
for 3 hours at RT. 200 .mu.l phages of the three libraries of llama
005 and of the three libraries of Ilama006 are pooled and added to
the immunotubes with a final volume of 2 ml in PBS for EGFR and in
50 mM Tris HCl (pH 7.4), 150 mM KCl, 1.0 mM DTT, 1 mM MgCl.sub.2
and 0.3 mg/ml BSA for PDK1.
[0416] After 2 hours incubation at RT, the immunotubes are washed
10.times. with PBS-Tween and 10.times. with PBS. Bound phages are
eluted with 2 ml 0.2 M glycin buffer pH=2.4. Eluted phages are
allowed to infect exponentially growing TG1 cells, and are then
plated on LB agar plates containing 100 .mu.g/ml ampicillin and 2%
glucose. Examples of results which might be obtained from the
panning are presented in Tables 16 and 17.
Example 36
Screening
[0417] A microtiter plate is coated with 2 .mu.g/ml EGFR or 2
.mu.g/ml PDK1, overnight at 4.degree. C. Plates are blocked for two
hours at room temperature with 300 .mu.l 1% casein in PBS. The
plates are washed three times with PBS-Tween. Periplasmic extracts
are prepared from single colonies and applied to the wells of the
microtiter plate. Plates are washed six times with PBS-Tween, after
which binding of VHH is detected by incubation with mouse
anti-Histidine mAB 1/1000 in PBS for 1 hour at RT followed by
anti-mouse-alkaline phosphatase conjugate 1/2000 in PBS, also for 1
hour at RT. Staining is performed with the substrate PNPP
(p-nitrophenyl-phosphate, 2 mg/ml in 1M diethanolamine, 1 mM
Mg.sub.2SO.sub.4, pH9.8) and the signals are measured after 30
minutes at 405 nm. An example of the expected number of positive
clones versus the number of clones tested in ELISA for each
selection is presented in Table 18.
Example 37
Screen for Internalised VHH
[0418] Individual clones specific for the EGFR are amplified by PCR
and cloned in a phage engineered to package the green fluorescent
protein reporter gene driven by the CMV promoter (Poul M A et al, J
Mol Biol, 1999, 288: 203-211). Phages are prepared and incubated
with tumor cells (A431) overexpressing EGFR. Phages that endergo
EGFR mediated endocytosis are be measured by GFP expression. 1 VHH
(EGFR-21) would be expected to show a very high expression of GFP
and would be used for further analysis. In another approach
internalised phage is stained with anti-phage antibodies (poly- or
monoclonal) after permeabilization of cells by treatment with cold
methanol as described by Larocca and colleagues (Larocco et al,
Molecular Therapy, 2001, 3: 476-484) and by Poul and colleagues
(Poul M A et al, J Mol Biol, 1999, 288: 203-211).
Example 38
Screen for VHH Inhibiting PDK1-Akt Interaction
[0419] PDK1 is coated in a microtiter plate as described above and
after blocking the plates, the wells are incubated with 100
.mu.g/ml Akt for one hour at RT. Then (without washing) 100 .mu.l
periplasmic extract is added to those wells and VHH binding is
measured as described above. VHH that are not able to bind to PDK1
would be scored as inhibitors for the interaction between PDK1 and
Akt. The expected number of inhibiting VHH versus the number of VHH
tested in inhibition ELISA is summarized in Table 19.
Example 39
Making a Bispecific Construct
[0420] A bispecific construct is prepared (Conrath et al, J Biol
Chem, 2001, 276: 7346-7350) of EGFR-21 and 5 different strong
inhibiting VHHs (PD-1, PD-7, PD-32, PD-33 and PD-72) for PDK1.
Protein is prepared and purified to homogeneity for the 5
bispecific constructs and shown to be stable by western blot
analysis.
Example 40
Endocytosis and Lysis of Tumor Cells
[0421] Bispecific constructs are incubated with tumor cells (A431)
overexpressing EGFR. All constructs that successfully endocytosed
would be shown by confocal microscopy. One of the constructs,
EGFR-21-PD-32, would be expected to able to inhibit cell growth and
finally lead to cell death.
Example 41
Calculation of Homologies Between Anti-Target-Single Domain
Antibodies of the Invention
[0422] 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
20 indicates the fraction homology between anti-TNF-alpha VHHs of
the invention. Table 21 indicates the percentage homology between
anti-IFN-gamma VHHs of the invention.
Example 42
Construction of a Bispecific Constructs Containing a VHH-CDR3
Fragment Fused to an Anti-Serum Albumin Vhh
[0423] A functional portion, the CDR3 region of MP2F6SR, was
amplified by using a sense primer located in the framework 4 region
(F6 CRD3 Forward:CTGGCCCCAGAAGTCATACC; SEQ ID NO:97) and an
anti-sense primer located in the framework 3 region (F6 CDR3
Reverse primer:TGTGCATGTGCAGCAAACC; SEQ ID NO:98).
[0424] In order to fuse the CDR-3 fragment with the anti-serum
albumin VHH MSA-21, a second round PCR amplification was performed
with following primers:
TABLE-US-00002 F6 CDR3 Reverse primer Sfi1: (SEQ ID NO: 99)
GTCCTCGCAACTGCGGCCCAGCCGGCCTGTGCATGTGCAGCAAACC F6 CDR3 Forward
primer Not1: (SEQ ID NO: 100)
GTCCTCGCAACTGCGCGGCCGCCTGGCCCCAGAAGTCATACC
[0425] 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).
[0426] After cleavage of the VHH gene of MSA clones with
restriction enzymes Pst1/BstEII the digested products were cloned
in pAX11 to obtain clones with a VHH at the C-terminus of the
multicloning site. The clones were examined by PCR using vector
based primers. From clones yielding a 650 bp product, DNA was
prepared and used as acceptor vector to clone the CDR3 of MP2F6SR,
after cleavage of the PCR product with restriction enzymes
Sfi1/Not1 to allow N-terminal expression of CDR3 in fusion with a
MSA VHH.
Tables
TABLE-US-00003 [0427] TABLE 1 Immunization scheme as described in
Example 1 Day Llama 2 Llama 4 0 100 .mu.g 100 .mu.g 7 100 .mu.g 14
50 .mu.g 21 50 .mu.g 100 .mu.g 28 50 .mu.g 35 50 .mu.g 42 50 .mu.g
70 50 .mu.g
TABLE-US-00004 TABLE 2 Presence of insert by PCR with vector
specific primers as described in Example 1 #days after last
injection Source RNA Size of the library % insert Llama002 4 Lymph
1.3 .times. 10.sup.7 89 4 PBL 1.9 .times. 10.sup.7 95 10 PBL 1.1
.times. 10.sup.9 70 Llama004 4 PBL 1.7 .times. 10.sup.8 96 4 Lymph
4.9 .times. 10.sup.7 >95 10 PBL 2.2 .times. 10.sup.6 >95
TABLE-US-00005 TABLE 3 First selection as described in Example 1 5
.mu.g/ml 0.5 .mu.g/ml 0 .mu.g/ml (blanco) Llama 2 1.4 10.sup.6 2.7
10.sup.5 1.5 10.sup.4 (pool PBL day4, PBLday10, lymph node day4)
Enrichment compared to blanco 400 x 18 x Llama 4 3.3 10.sup.6 4.5
10.sup.5 7.2 10.sup.4 (pool PBL day4, PBLday10, lymph node day4)
Enrichment compared to blanco 140 x 6.25 x
TABLE-US-00006 TABLE 4 Second selection using the rescued phages
from the first selection as described in Example 1 1 .mu.g/ml 0
.mu.g/ml Elution 1 .mu.g/ml Elution 0 .mu.g/ml buffy Elution buffy
Elution coat cells Lysozyme coat cells Lysozyme Llama 2 1.2
10.sup.8 1.2 10.sup.8 6 10.sup.3 3 10.sup.3 (selection 5 .mu.g/ml
IgE: 400 x enrichment) Enrichment compared to No 2x lysozyme
elution enrich- ment Llama 4 1.3 10.sup.8 2 10.sup.7 3 10.sup.3 3
10.sup.3 (selection 5 .mu.g/ml IgE: 140 x enrichment) Enrichment
compared to 6.5 x No lysozyme elution enrich- ment
TABLE-US-00007 TABLE 5 Second round selection using neutravidine
coated tubes as described in Example 1 2 nM IgE 0 nM IgE Elution 2
nM IgE Elution 0 nM IgE buffy Elution buffy Elution coat cells
Lysozyme coat cells Lysozyme Llama 2 1.5 10.sup.8 1.5 10.sup.7 3
10.sup.5 3 10.sup.3 (selection 5 .mu.g/ml IgE: 400 x enrichment)
Enrichment compared to 10 x lysozyme elution Llama 4 3.3 10.sup.7
2.2 10.sup.7 3 10.sup.3 6 10.sup.3 (selection 5 .mu.g/ml IgE: 140 x
enrichment) Enrichment compared to 1.5 x lysozyme elution
TABLE-US-00008 TABLE 6 Number of clones that score positive for
binding to both human IgE and chimeric IgE versus the number of
clones tested in ELISA as described in Example 1 Selection with 5
.mu.g/ml Selection with 0.5 .mu.g/ml Llama 002 39/47 21/47 Llama
004 45/47 46/47
TABLE-US-00009 TABLE 7 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 rectal 100 .mu.l PBS rectal for
2 weeks 3 8 negative control 3 daily 100 .mu.l PBS intragastric
intragastric for 14 consecutive days 4 8 positive control 1 5 .mu.g
i.p. for 7 dexamethasone consecutive days 5 8 positive control 2
applied orally once IL10 expressing I. lactis per day for 14
consecutive days 6 8 bivalent VHH 3F daily 100 .mu.g bivalent
intra-gastric VHH 3F.sub.2 intragastric on 14 consecutive days 7 8
bivalent VHH 3F daily 100 .mu.g bivalent i.p. VHH 3F i.p. for 14
consecutive days 8 8 bivalent VHH 3F 100 .mu.g bivalent VHH
rectally 3F rectally in 100 .mu.l PBS every other day for two
weeks
TABLE-US-00010 TABLE 8 Overview of the libraries, their diversity
and % insert derived from different llama's and tissues as
described in Example 7 and 8 Animal Antigen Source Titer % Insert
Llama 5 Human MMP-12 PBL time 1 2.1 10.sup.8 94% Llama 5 Human
MMP-12 PBL time 2 7.5 10.sup.6 92% Llama 5 Human MMP-12 Lymph node
7.8 10.sup.8 100%
TABLE-US-00011 TABLE 9 Immunization schedule and tissue collections
Day Llama 024 Llama 025 Llama 026 Llama 027 0 intact cells intact
cells vesicles vesicles 7 intact cells intact cells vesicles
vesicles 14 intact cells intact cells vesicles vesicles 21 intact
cells intact cells vesicles vesicles 28 intact cells intact cells
vesicles vesicles 35 intact cells intact cells vesicles vesicles 42
intact cells intact cells vesicles vesicles 46 150 ml blood 150 ml
blood 150 ml blood 150 ml blood sample (PBL1) sample (PBL1) sample
(PBL1) sample (PBL1) lymph node lymph node 47 lymph node spleen
bone marrow 49 purified EGFR 150 ml blood 150 ml blood sample
(PBL2) sample (PBL2) 55 purified EGFR 59 150 ml blood sample (PBL2)
60 lymph node spleen bone marrow
TABLE-US-00012 TABLE 10 Overview of constructed libraries Animal
Tissue RNA (.mu.g) Size (.times.10.sup.8) % Insert Llama 024 PBL1
200 0.25 83 Llama 024 Lymph node ileum 40 2.3 78 Llama 024 Lymph
node bow 150 0.17 100 Llama 024 Bone marrow 97 1.5 83 Llama 024
Spleen 160 0.16 95 Llama 025 PBL1 200 0.06 95 Llama 025 Lymph node
200 0.8 96 (ileum + bow) Llama 025 Bone marrow 200 0.045 88 Llama
025 Spleen 200 2 86 Llama 025 PBL2 200 0.13 83 Llama 026 PBL1 +
lymph node 100 + 200 2.46 85 Llama 027 PBL1 + lymph node 100 + 200
1.08 92
TABLE-US-00013 TABLE 11 Overview of epitope specific elution
selection procedure Elution Elution Selection: antigen format
.PHI.ELISA .PHI.ELISA Binder condition molecule Round I Round II
Round III Her-14 EGFR families 1 EGF A431 Her-14 -- 1/47 24/47 6 Ia
EGFR 5/47 23/47 8 2 2e9 A431 Her-14 -- 2/47 32/47 5 IIIa EGFR 11/47
32/47 4 3 225 A431 A431 Her-14 8/47 28/47 5 EGFR 20/47 31/47 4 528
A431 A431 Her-14 16/47 10/47 5 EGFR 22/47 29/47
TABLE-US-00014 TABLE 12 Overview of `internalization` selection
procedure Library Selection cells Selected antibody fragment Pool
lymph node, bone Her-14 A2 marrow, spleen and PBL1 (024 + 025) Pool
bone marrow A431 A4, A9, B11 (024 + 025) Pool PBL1 (024 + 025) A431
F11
TABLE-US-00015 TABLE 13 Primer sequences SEQ ID Name NO Sequence 5'
- 3' ABL002 101 GGCTGAGCTCGGTGGTCCTGGCT ABL010 102
AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT ABL037 103
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAG TCTGG ABL038
104 CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCGATGTGCAGCTGGTGGAG TCTGG
ABL039 105 CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCGCGGTGCAGCTGGTGGAG
TCTGG ABL040 106
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCGCCGTGCAGCTGGTGGAT TCTGG ABL041
107 CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAG TCTGG
ABL042 108 CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAG
TCTGG ABL043 109
CATGCCATGACTCGCGGCCCAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAG TCTGG geneIII
110 CCACAGACAGCCCTCATAG M13 rev 111 GGATAACAATTTCACACAGG
TABLE-US-00016 TABLE 14 Sequence listing SEQ ID NO NAME SEQUENCE
Anti-IgE VHH 1 VHH#2C3
QVQLQDSGGGLVQPGGSLRLSCRASGRIFRINAMGWYRQAPGKQRELVATI
TSTGSTNFADSVKGRFTIYRDGAKRTVDLRLNSLKPEDTAVYFCNADVREY
DLGPWRQYWGQGTQVTVSS 2 VHH#4G12
QVQLQESGGGVVQPGGSLRLSCSVSGTSISNRVMAWFRQAPGKQRDFVAYI
TSAVNTDYADFVKGRFTISRDNAQNMVHLQMNSLKPEDTAVYYCNVLKDTW
FRTPYDYYWGQGTQVTVSS 3 VHH#2C1
QVQLQESGGGLVQPGDSLRLSCVVSGRTLSYSSLAWFRQAPGKERDFVAAL SLTTYY
ADSVKGRFTISRDNAKNTVYLQMNSLKPDDTADYFCATARTRTDYAPLLSA
ASTYDAWGQGTQVTVSL 4 VHH#2H3
QVQLQESGGGLVQAGGSLRLSCAASGRSSRYYAMGWFRQGPGKEREFVAAV
NWNGDSTYYADSVKGRFTISRGNAENTAYLQMNSLVPEDTAVYYCAMRMNA
GLGYSAASYQYWGQGTQVTVSL 5 VHH#2D12
QVQLQESGGGLVQAGDSLRLSCAASGLTFLEHVMAWFRQTPGKEREFVGAI
DWSGRRITYTDSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYYCAADRTY
SYSSTGYYYWGQGTQVTVSS 6 VHH#2G4
QVQLQDSGGGLVQAGDSLRLSCAASGLTFLEHVMAWFRQTPGKEREFVGAI
DWSGRRITYTDSVKGRFTISRDNAKNTVYLQMNTLKPEDTAVYYCAADRTY
SYSSTGYYYWGQGTQVTVSS 7 VHH#4C5
QVQLQESGGGLVQAGGSLRLSCAASGRTLSSYTMAWFRQAPGKEREFVASI
SSSGISTYYADSVKGRFTISRDIAKNTVYLQMNSLKPEDTAVYYCAAKYRY
YSTLYTKSGEYDYWGQGTQVTVSS 8 VHH#4A2
QVQLQDSGGGLVQAGGSLRLSCEASGRTISSYAMAWFRQAPGKEREFVASI
SSSGVSKHYADSVKGRFTISNDKVKNTVYLQMNSLKPEDTAVYFCAAKYRY
YSSYYTKSGDYDYWGQGTQVTVSS 9 VHH#2D4
QVQLQESGGGLVQAGGSLRLSCAASGLTFSTYAMGWFRQAPGKEREFVAAV
SYSGSYYADSVKGRFTISRDNAKNTVYLQMASLKPEDTAVYYCAARNRGYS
TYAGVYDYWGQGTQVTVSS 10 VHH#2B6
QVQLQDSGGGLVQAGGSLRLSCAASGVTFSSYAMGWFRQAPGKEREFVASI
TWIGGGTYYADSVKGRFTISRDHAGNTVYLQMNTLKPDDTAVYYCALDRRS
STYYLMKGEYDYRGRGTQVTVSS 11 VHH#2H11
QVQLQESGGGLVQAGGSLRLSCAASGVTFSSYAMGWFRQAPGKEREFVASI
TWTGTGTYYADSVKGRFTISRDHAGTTVYLQMNSLKPEDTAVYYCAVDRRS
STYYLMKGEYDYRGRGTQVTVSS Anti-TNF alpha VHH 12 VHH#3E-
QVQLQESGGGLVQPGGSLRLSCAASGRTFSDHSGYTYTIGWFRQAPKEREF His tag
VARIYWSSGNTYYADSVKGRFAISRDIAKNTVDLTMNNLEPEDTAVYYCAA
RDGIPTSRSVESYNYWGQGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH 13 VHH#3F
QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAV
SWSGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQ
KYNWASASYNVWGQGTQVTVSS 14 VHH#3F/
QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAV VHH#3F
SWSGGTTVYADSVLGRFEISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQ
KYNWASASYNVWGQGTQVTVSSEPKTPKPQPAAAQVQLQDSGGGLVQAGGS
LRLSCAASGGTFSSIIMAWFRQAPGKEREFVGAVSWSGGTTVYADSVLGRF
EISRDSARKSVYLQMNSLKPEDTAVYYCAARPYQKYNWASASYNVWGQGTQ VTVSS Human
MMP-12 specific VHH 15 MMP-12
QVQLQESGGGLVQPGGSLRLSCVASGFTFSDYPMAWVRQAPGKGLEWISVI P1-1
NSGGVNTSYAASVKGRFTISRDNAKNTLFLQMNSLKPEDTAVYYCAKYSLK
NEQYWRGQGTQVTVSS 16 MMP-12
QVQLQESGGGLVQPGGSLRLSCAASGSIFSIDGMGWYRQAPGKQRERKQRE P1-3
LVAAITSGGSTKYADSVKGRFTISRDNANDTVYLQMNTLKPEDTAVYYCNA
VLLRRGIVYDYWGQGKQVTVSS 17 MMP-12
QVQLQESGGGSVKAGGSLRLSCAASGSIFSIDGMGWYRQAPGKQRERKQRE P1-7
LVAAITSGGSTKYADSVKGRFTISRDNANDTVYLQMNTLKPEDTAVYYCNA
VLLRRGIVYDYWGQGKQVTVSS 18 MMP-12
QVQLQESGGGLVRAGGSLRLSCVASGRTLSKYRMGWFRQFPGKERELVAEI P1-26
EWKSSSTWYRDSVKGRFTISRDNAKNTVYLRMNSLKPEDTAVYYCAAATLG
EPLVKYTYWGQGTQVTVSS 19 MMP-12
QVQLQESGGGLVQPGGSLRLSCAASGSIFSIDGMGWYRQAPGKQRERKQRE P1-33
LVAAITSGGSTKYADSVKGRFTISRDNANDTVYLQMNTLKPEDTAVYYCNA
VLLRRGIVYDYWGQGKQVTVSS 20 MMP-12
QVQLQDSGGGLVRTGDSLRLSCVVFGGTISTYAMGWFRRAPGKEREFVAAI P1-41
DASGGFTEYADSVRGRFRIARDNPLSAVYLQMNSLKPEDTAFYYCAADKDR
DTVVRFTTTPNEYDYWGQGTQVTVSS 21 MMP-12
QVQLQESGGGLVQPGGSLRLSCAASGFTFNNHWLYWVRQAQGKGLEWVSAI P1-44
NPGGSTVYLDSVKGRFTISRGNTKNTLYLQMNSLKSEDTAVYYCTKAMAWA
TDWDEYDLWGQGTQVTVSS 22 MMP-12
QVQLQESGGGLVQAGGSLRLSCAASGRTFTVYTTGWFRQAPGKEREFVAAI P5-29
DWSGSSTYYTDSVKGRFTISRDNTKNTVYLQMNSLKPEDTAVYYCAARDAI
VGVTDTSGYRYWGQGTQVTVSS Anti-EGFR VHH 23 EGFR-1.4
EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYVMGWFRQAPGKERDFVVGI
GRSGGDNTYYADSVKGRFTISWDNAKNTMYLQMNSLKPEDTAVYYCAASTY
SRDTIFTKWANYNYWGQGTQVTVSS 24 EGFR-1.9
QVQLQESGGGLVKAGGSLRLSCAASGRTFSSYVMGWFRQAPGKEREFVGAI
HWSGGRTYYADSVKGRFTISSDNAKNTLYLQMNSLKPEDTAVYYCAASRII
YSYVNYVNPGEYDYWGQGTQVTVSS 25 EGFR-1.33
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSHYMSWFRQAPGKEREFVAAI
TSSSRTYYTESVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYCAADRTFY
GSTWSKYDYRGQGTQVTVSS 26 EGFR-1.34
QVQLQESGGGLVQAGGSLRLSCAASGRTFSKYAMGWFRQAPGKEREFVSAI
SWSDGSTYYADSVKGRFTISRDNAKNTVYLQVNSLKPEDTAVYYCAATYLV
DVWAVHVPIRPYEYDYWGQGTQVTVSS 27 EGFR-1.38
QVQLQDSGGGLVQAGDSLRLSCAASGRSFGGYAMGWFRQAPGKEREFVAAI
SWSGGSTYYADSLKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAAGLRP
SPNYNHERSYDYWGQGTQVTVSS 28 EGFR-Ia1
QVQLQESGGGLVQAGGSLLLSCAASGRTFSSYAMGWFRQAPGKEREFVAAI
NWSGGSTSYADSVKGRFTISRDNTKNTVYLQMNSLKPEDTAAFYCAATYNP
YSRDHYFPRMTTEYDYWGQGTQVTVSS 29 EGFR-Ia7
QVQLQESGGRLVQTGGSLRLSCAASGGTFGTYALGWFRQAPGKEREFVAAI
SRFGSTYYADSVKGRFTISRDNANNTVYLEMNSLKPEDTAVYYCAAREGVA
LGLRNDANYWGQGTQVTVSS 30 EGFR-Ia15
QVQLQDSGGGLVQAGGSLRLSCAASGGTFSSYAMGWFRQAPGKEREFVAAI
GLNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARTSGVVG
GTPKRYDYWGQGTQVTVSS 31 EGFR-
EVQLVESGGGSVQAGGSLKLSCAASGRSFSTYAMGWFRQAPGQDREFVATI IIIa42
SWTDSTDYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCAADRWAS
SRRNVDYDYWGQGTQVTVSS 32 EGFR-2.6
QVQLQESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVAAI
NWGGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASEWG
GSDYDHDYDYWGQGTQVTVSS 33 EGFR-2.20
EVQLVESGGGLVQAGGSLRLSCAASGRSFSSYAMAWFRQAPGKEREFVAAI
SWGGGSTYYAVSVKGRFTISRDNAKNTVYLQMNSLKPEDTARYYCAADETF
HSSAYGEYEYWGQGTQVTVSS 34 EGFR-
EVQLVESGGGLVQAGGSLRLSCTASGRTFSSYAMGWFRQTPGKEREFVAAI IIIa5
TSSGGSTYYADSVKGRFTISRDNAKSTMYLQMDSLMLDDTSVYYCAADSSR
PQYSDSALRRILSLSNSYPYWGQGTQVTVSS 35 EGFR-3.18
EVQLVESGGGLVQPGGSLRLSCVASGFTFADYAMSWVRQAPGKGLQWVSSI
SYNGDTTYYAESMKDRFTISRDNAKNTLYLQMNSLKSEDTAVYYCASSGSY
YPGHFESWGQGTQVTVSS 36 EGFR-3.32
QVQLQESGGGLVQAGGSLRLSCAASGRTFSGYAMGWFRQAPGEEREFVAAI
SWRGTSTYYGDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGSHS
DYAPDYDYWGQGTQVTVSS 37 EGFR-3.34
QVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAIGWFRQAPGKEREFVAAI
SWGGSNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGEVS
NSDYAYEYDYWGQGTQVTVSS 38 EGFR-3.39
QVQLQESGGGLVQTGGSLRLSCAASGRYIMGWFRQAPGKEREFVAGISRSG
ASTAYADSVKDRFTISRDSALNTVYLQMNSLKAEDTAVYFCAAALAIRLGI
PRGETEYEYWGQGTQVTVSS 39 EGFR-3.40
QVKLEESGGGLVQAGGSLRLSCSASGLTFSNYAMAWFRQAPGKEREFVATI
SQRGGMRHYLDSVKDRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAADLMY
GVDRRYDYWGRGTQVTVSS 40 EGFR-4.11
QVKLEESGGGLVQAGDSLRLSCAASGRSFSSITMGWFRQAPGKERQFVSAI
NSNGNRYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVQAYS
SSSDYYSQEGAYDYWGQGTQVTVSS 41 EGFR-4.21
EVQLVESGGGLVQAGGSLRLSCAVSGRTFSSMGWFRQAPGKEREFVATINL
SGDRTDYADSVKGRFTISRDNPKNTVYLQMDSLEPEDSAVYYCAGTSLYPS
NLRYYTLPGTYADWGQGTQVTVSS 42 EGFR-4.22
QVKLEESGGGLVQAGGSLRLSCAASGSIFSINAMGWYRQAPGKQRELVARI
TGTGTGITGAVSTNYADSVKGRFTISRDNARNTVYLQMNSLKPEDTAVYYC
AADRSRTIVVPDYWGQGTQVTVSS 43 EGFR-B11
QVQLQDSGGGLVQAGGSLRLSCAASRFSSAQYAIGWFRQAPGKEREGVSYI
TFSGGPTGYADSVKGRFTVSRDNAKNTVYLQMNSLKPEDTAVYYCAARPYT
RPGSMWVSSLYDNWGQGTQVTVSS 44 EGFR-F11
QVQLQESGGRLVQAGGSLRLSCAASEHTFRGYAIGWFRQAPGKEREFVSSI
TYDGTLTNYADSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYVCAAGYSY
RYTTLNQYDSWGQGTQVTVSS Anti - human IFN gamma VHH 45 MP3D2SRA
QVQLQDSGGGTVQAGGSLRLSCAASGRTFSDYAVGWFRQAPGKEREFVARI
LWTGASRSYANSVDGRFTVSTDNAKNTVYLQMNSLKPEDTAIYYCAALPSN
IITTDYLRVYYWGQGTQVTVSS 46 MP3A3SR
QVQLQDSGGGTVQAGGSLRLSCAASGRTFSNYAVGWFRQAPGKEREFVARI
KWSGGSRSYANSVDGRFTVSTDNAKNTVYLQMNSLKPEDTAIYYCAALPSN
IITTDYLRVYYWGQGTQVTVSS 47 MP3C5SR
QVQLQESGGGLVQAGGSLRLSCAAAGISGSVFSRTPMGWYRQAPGKQRELV
AGILTSGATSYAESVKGRFTISRDNAKNTVYLQMNSLSPEDTAEYYCNTYP
TWVLSWGQGTQVTVSS 48 MP3C1SR
QVQLQDSGGGLVQAGGSLRLSCAAAGISGSVFSRTPMGWYRQAPGKQRELV
AGILSSGATVYAESVKGRFTISRDNAKNTVYLQMNSLSPEDTAEYYCNTYP
TWVLSWGQGTQVTVSS 49 MP3G8SR
QVQLQESGGGLVQAGGSLRLSCAAAGISGSVFSRTPMGWYRQAPGKQRELV
AGILSSGATAYAESVKGRFTISRDNAKNTVYLQMNSLSPEDTAEYYCNTYP
TWVLSWGQGTQVTVSS 50 MP3D2BR
QVQLQESGGGLVQPGESLRLSCAASRGIFRFNAGGWYRQAPGKQRELVAFI
GVDNTTRYIDSVKGRFTISRDNAKTTVYLQMNSLQPEDTAVYYCNKVPYID WGQGTQVTVSS 51
MP3H6SRA QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYNMGWFRQAPGKEREFVAGI
SWNGGSIYYTSSVEGRFTISRDNAENTVYLQMNSLKPEDTGVYYCASKGRP
YGVPSPRQGDYDYWGQGTQVTVSS 52 MP3B4SRA
QVQLQESGGGLVQAGGSLRLSCAASGRTFSTYNMGWFRQAPGKEREFVAGI
SWNGGSIYYTSSVEGRFTISRDNAENTVYLQMNSLKPEDTGVYYCASKGRP
YGVPSPRQGDYDYWGQGTQVTVSS 53 MP4E4BR
QVQLQESGGGLVQAGGSLRLSCAASGRTFSIYNMGWFRQAPGKEREFVAAI
SWNGGSIYYTSSVEGRFTISRDNAINTVYLQMNSLKPEDTGVYYCASKGRP
YGVPSPRQGEYDYWGQGTQVTVSS 54 MP4H8SR
QVQLQESGGGLVQAGGSLRLSCAASGRTFNIYNMGWFRQAPGKERDFVAAI
SWNGGSIYYTSSVEGRFTISRDNAENTVYLQMNSLKPEDTGVYYCASKGRP
YGVPSPRQGDYDYWGQGTQVTVSS 55 MP2F6SR
QVKLEESGGGLVQAGGSLRLSCAASGRTENNYNMGWERQAPGKEREEVAAI
SWNGGSTYYDDSVKGRFTISRDNANNLVYLQMNSLNFEDTAVYYCACAANP
YGIPQYRENRYDFWGQGTQVTVSS 56 MP3D1BR
QVQLQESGGGLVQAGGSLRLSCAASGRTEDNYNMGWERQAPGKEREFVAAI
SWNGGSTYYDDSVKGRFTISRDNFQKLVYLQMNSLKLEDTAVYYCACAANP
YGIPQYRENRYDFWGQGTQVTVSS 57 MP2B5BR
QVQLVESGGRLVQAGGSLRLSCIASGRTISDYAAGWFRQAPGKEREFLASV
TWGFGSTSYADSVKGRFTISRDKAKDTVYLQMNTLEPDDTSVYYCASSPRY
CAGYRCYVTASEFDSWGQGTQVTVSS 58 MP2C1BR
QVKLEESGGRLVQAGGSLRLSCIASGRTISDYAAGWFRQAPGKEREFLASV
SWGFGSTYYADSVKGRFTISRDTAKDTVYLQMNTLEPDDTSVYYCASSPRY
CAGYRCYATASEFDSWGQGTQVTVSS 59 MP4A12SR
QVQLQESGGRLVQAGGSLRLSCIASGRTISDYAAGWFRQAPGKEREFLASV
TWGFGSTYYADSVKGRFTISRDKAKDTVYLQMNTLEPDDTSAYYCASSPRY
CAGYRCYVTASEFDSWGPGTQVTVSS
60 MP3F4SRA QVQLQDSGGGLVQAGDSLRLSCAASGRSFSSYGMGWFRQAPGKEHEFVAGI
WRSGVSLYYTDSVKGRFTISRDDAKMTVSLQMNSLKPEDTAVYYCAAEATF
PTWSRGRFADYDYRGQGTQVTVSS 61 MP3D3BR
QVQLQESGGGLVQAGDSLRLSCTASGRSFSSYGMGWFRQAPGKDHEFVAGI
WRSGVSLYYADSVKGRFTISRDDAKMTVSLQMNGLKPEDTAVYYCAAEATF
PTWNRGTFADYDYRGQGTQVTVSS 62 MP3E5BR
QVQLQESGGGLVQAGDSLRLSCAASGRSFSSYGMGWFRQAPGKEHEFVAGI
WRSGVSLYYADSVKGRFTISRDDAKMTVSLQMNGLKPEDTAVYYCAAEATF
PTWNRGSFADYDYRGQGTQVTVSS 63 MP3C7SRA
QVQLQESGGGLVQAGDSLRLSCAASGRSFSSYGMGWFRQAPGKEHEFVAGI
WRSGVSLYYADSVKGRFTISRDDAKMTVSLQMNSLKPEDTAVYYCAAEATF
PTWNRGRFADYDYSGQGTQVTVSS 64 MP2F1BR
AVQLVESGGGLVQTGDSLRLSCVASGGTESRYAMGWERQAPGKEREFVARI
GYSGRSISYATSVEGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCASLVSG
TLYQADYWGQGTQVTVSS 65 MP2C5BR
QVQLVESGGGLVQTGDSLRLSCVASGGTESRYAMGWERQPPGKERDEVARI
GYSGQSISYATSVEGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCASLVSG
TLYKPNYWGQGTQVTVSS 66 MP2C10BR
QVKLEESGGGLVQAGGSLRLSCAASGLTYTVGWERQAPGKEREEVAAISWS
GGSALYADSVKGRFTISRDNAKNTVYLQMGSLEPEDTAYYSCAAPGTRYYG
SNQVNYNYWGQGTQVTVSS 67 MP2G5SR
QVKLEESGGGLVQAGDSLRLSCAASGLTYTVGWFRQAPGKEREFVAAIDWS
GGSALYADSVKGRFTISRDNTKNTVYLQMGSLEPEDTAVYWCAAPGTRYHG
RNQVNYNYWGQGTQVTVSS 68 MP3B1SRA
QVQLQESGGGLVQPGGSLRLSCAASGFTSSNYAMSWVRQAPGKGLEWVSSI
NSRTGSITYADSVKGRFTITLDNAKNTLYLQMNSLKPEDTAVYYCASRVDD RVSRGQGTQVTVSS
69 MP2F10SR QVQLVESGGGLVQAGGSLRLSCAASGRTISSFRMGWFRRAPGEEREFVAFV
RSNGTSTYYADSVEGRFTITRDNAKNTVYLRMDSLKPEDTAVYYCAAATRD
YGGSFDYWGQGTQVTVSS 70 MP3A7SRA
QVQLQDSGGGLVQAGGSLRLSCAASGRTFSSFRMGWFRRAPGEEREFVAFV
RSNGTSTYYADSVEGRFTITRDNAKNTVYLRMDSLKPEDTAVYYCAAATRD
YGGSFDYWGQGTQVIVSS Anti-Mouse serum albumin VHH 71 MSA21
QVQLQESGGGLVQPGGSLRLSCEASGFTFSRFGMTWVRQAPGKGVEWVSGI
SSLGDSTLYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCTIGGSL NPGGQGTQVTVSS
72 MSAc16 AVQLVESGGGLVQAGDSLRLSCVVSGTTFSSAAMGWFRQAPGKEREFVGAI
KWSGTSTYYTDSVKGRFTISRDNVKNTVYLQMNNLKPEDTGVYTCAADRDR
YRDRMGPMTTTDFRFWGQGTQVTVSS 73 MSAc112
QVKLEESGGGLVQTGGSLRLSCAASGRTFSSFAMGWFRQAPGREREFVASI
GSSGITTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGLCYCAVNRYG
IPYRSGTQYQNWGQGTQVTVSS 74 MSAc110
EVQLEESGGGLVQPGGSLRLSCAASGLTFNDYAMGWYRQAPGKERDMVATI
SIGGRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCVAHRQTV
VRGPYLLWGQGTQVTVSS 75 MSAc114
QVQLVESGGKLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFVAGS
GRSNSYNYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAASTNL
WPRDRNLYAYWGQGTQVTVSS 76 MSAc116
EVQLVESGGGLVQAGDSLRLSCAASGRSLGIYRMGWFRQVPGKEREFVAAI
SWSGGTTRYLDSVKGRFTISRDSTKNAVYLQMNSLKPEDTAVYYCAVDSSG
RLYWTLSTSYDYWGQGTQVTVSS 77 MSAc119
QVQLVEFGGGLVQAGDSLRLSCAASGRSLGIYKMAWFRQVPGKEREFVAAI
SWSGGTTRYIDSVKGRFTLSRDNTKNMVYLQMNSLKPDDTAVYYCAVDSSG
RLYWTLSTSYDYWGQGTQVTVSS 78 MSAc15
EVQLVESGGGLVQAGGSLSLSCAASGRTFSPYTMGWFRQAPGKEREFLAGV
TWSGSSTFYGDSVKGRFTASRDSAKNTVTLEMNSLNPEDTAVYYCAAAYGG
GLYRDPRSYDYWGRGTQVTVSS 79 MSc111
AVQLVESGGGLVQAGGSLRLSCAASGFTLDAWPIAWFRQAPGKEREGVSCI
RDGTTYYADSVKGRFTISSDNANNTVYLQTNSLKPEDTAVYYCAAPSGPAT
GSSHTFGIYWNLRDDYDNWGQGTQVTVSS 80 MSAc115
EVQLVESGGGLVQAGGSLRLSCAASGFTFDHYTIGWFRQVPGKEREGVSCI
SSSDGSTYYADSVKGRFTISSDNAKNTVYLQMNTLEPDDTAVYYCAAGGLL
LRVEELQASDYDYWGQGIQVTVSS 81 MSAc18
AVQLVDSGGGLVQPGGSLRLSCTASGFTLDYYAIGWFRQAPGKEREGVACI
SNSDGSTYYGDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCATADRH
YSASHHPFADFAFNSWGQGTQVTVSS 82 MSAc17
EVQLVESGGGLVQAGGSLRLSCAAYGLTFWRAAMAWFRRAPGKERELVVAR
NWGDGSTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAVRTY
GSATYDIWGQGTQVTVSS 83 MSAc120
EVQLVESGGGLVQDGGSLRLSCIFSGRTFANYAMGWFRQAPGKEREFVAAI
NRNGGTTNYADALKGRFTISRDNTKNTAFLQMNSLKPDDTAVYYCAAREWP
FSTIPSGWRYWGQGTQVTVSS 84 MSAc14
DVQLVESGGGWVQPGGSLRLSCAASGPTASSHAIGWFRQAPGKEREFVVGI
NRGGVTRDYADSVKGRFAVSRDNVKNTVYLQMNRLKPEDSAIYICAARPEY
SFTAMSKGDMDYWGKGTLVTVSS
TABLE-US-00017 TABLE 15 Immunisation scheme according to Example 32
Day of Llama 005 Llama 006 Llama 005 immunization EGFr EGFr PDK1
Llama 006 PDK1 0 100 .mu.g 40 .mu.g 40 .mu.g 40 .mu.g 7 100 .mu.g
40 .mu.g 14 50 .mu.g 20 .mu.g 21 50 .mu.g 40 .mu.g 20 .mu.g 40
.mu.g 28 50 .mu.g 20 .mu.g 35 50 .mu.g 20 .mu.g 42 20 .mu.g 20
.mu.g 70 20 .mu.g 20 .mu.g
TABLE-US-00018 TABLE 16 Results of panning according to Example 35
Pfu Pfu Ilama Source RNA Elution conditions EGFr casein Enrichment
005 Pool of the 3 0.2M glycin, pH 1 .times. 10.sup.7 1 .times.
10.sup.4 1000 libraries 2.4 006 Pool of the 3 0.2M glycin, pH 5
.times. 10.sup.6 1 .times. 10.sup.4 500 libraries 2.4
TABLE-US-00019 TABLE 17 Results of panning according to Example 35
Pfu Pfu Ilama Source RNA Elution conditions PDK1 casein Enrichment
005 Pool of the 3 0.2M glycin, pH 1 .times. 10.sup.8 1 .times.
10.sup.4 10000 libraries 2.4 006 Pool of the 3 0.2M glycin, pH 9
.times. 10.sup.7 1 .times. 10.sup.4 9000 libraries 2.4
TABLE-US-00020 TABLE 18 Number of positive clones after screening
according to Example 36 target Llama005 Llama006 EGFr 26/95 38/95
PDK1 93/95 87/95
TABLE-US-00021 TABLE 19 Number of inhibiting VHH vs number of VHH
tested in inhibition ELISA according to Example 38. target Llama005
Llama006 PDK1 56/93 63/87
TABLE-US-00022 TABLE 20 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-00023 TABLE 21 Percentage homologies between
anti-IFN-gamma VHHs of the invention % Homology MP3D2SRA MP3A3SR
MP3C5SR MP3C1SR MP3G8SR P3D2BR MP3H6SRA MP3B4SRA MP4E4BR MP3D2SRA X
96 66 66 66 62 71 71 71 MP3A3SR -- X 66 66 66 62 72 72 72 MP3C5SR
-- -- X 97 98 73 65 65 64 MP3C1SR -- -- -- X 98 72 64 64 64 MP3G8SR
-- -- -- -- X 73 65 65 64 MP3D2BR -- -- -- -- -- X 63 63 63
MP3H6SRA -- -- -- -- -- -- X 100 97 MP3B4SRA -- -- -- -- -- -- -- X
97 MP4E4BR -- -- -- -- -- -- -- -- X MP4H8SR -- -- -- -- -- -- --
-- -- MP2F6SR -- -- -- -- -- -- -- -- -- MP3D1BR -- -- -- -- -- --
-- -- -- MP2B5BR -- -- -- -- -- -- -- -- -- MP2C1BR -- -- -- -- --
-- -- -- -- MP4A12SR -- -- -- -- -- -- -- -- -- MP3F4SRA -- -- --
-- -- -- -- -- -- MP3D3BR -- -- -- -- -- -- -- -- -- MP3E5BR -- --
-- -- -- -- -- -- -- MP3C7SRA -- -- -- -- -- -- -- -- -- MP2F1BR --
-- -- -- -- -- -- -- -- MP2C5BR -- -- -- -- -- -- -- -- -- MP2C10BR
-- -- -- -- -- -- -- -- -- MP2G5SR -- -- -- -- -- -- -- -- --
MP3B1SRA -- -- -- -- -- -- -- -- -- MP2F10SR -- -- -- -- -- -- --
-- -- MP3A7SRA -- -- -- -- -- -- -- -- -- MP4C10SR -- -- -- -- --
-- -- -- -- MP4D5BR -- -- -- -- -- -- -- -- -- MP3F1SRA -- -- -- --
-- -- -- -- -- MP6D6BR -- -- -- -- -- -- -- -- -- MP6B1BR -- -- --
-- -- -- -- -- -- MP6A8BR -- -- -- -- -- -- -- -- -- MP6B12BR -- --
-- -- -- -- -- -- -- MP6C11BR MP6B10BR % Homology MP4H8SR MP2F6SR
MP3D1BR MP2B5BR MP2C1BR MP4A12SR MP3F4SRA MP3D3BR MP3E5BR MP3D2SRA
70 68 69 65 63 64 68 66 67 MP3A3SR 71 70 71 65 63 64 68 66 67
MP3C5SR 63 63 63 60 58 59 64 64 65 MP3C1SR 62 62 62 58 57 58 65 64
64 MP3G8SR 63 63 63 59 58 59 64 64 65 MP3D2BR 62 63 64 59 58 58 62
61 62 MP3H6SRA 97 80 81 67 68 67 75 71 73 MP3B4SRA 97 80 81 67 68
67 75 71 73 MP4E4BR 97 81 82 68 69 68 73 70 71 MP4H8SR X 81 81 66
66 66 72 69 71 MP2F6SR -- X 94 65 68 64 70 67 69 MP3D1BR -- -- X 65
66 65 71 69 71 MP2B5BR -- -- -- X 95 97 63 64 64 MP2C1BR -- -- --
-- X 95 63 64 64 MP4A12SR -- -- -- -- -- X 63 64 64 MP3F4SRA -- --
-- -- -- -- X 94 96 MP3D3BR -- -- -- -- -- -- -- X 98 MP3E5BR -- --
-- -- -- -- -- -- X MP3C7SRA -- -- -- -- -- -- -- -- -- MP2F1BR --
-- -- -- -- -- -- -- -- MP2C5BR -- -- -- -- -- -- -- -- -- MP2C10BR
-- -- -- -- -- -- -- -- -- MP2G5SR -- -- -- -- -- -- -- -- --
MP3B1SRA -- -- -- -- -- -- -- -- -- MP2F10SR -- -- -- -- -- -- --
-- -- MP3A7SRA -- -- -- -- -- -- -- -- -- MP4C10SR -- -- -- -- --
-- -- -- -- MP4D5BR -- -- -- -- -- -- -- -- -- MP3F1SRA -- -- -- --
-- -- -- -- -- MP6D6BR -- -- -- -- -- -- -- -- -- MP6B1BR -- -- --
-- -- -- -- -- -- MP6A8BR -- -- -- -- -- -- -- -- -- MP6B12BR -- --
-- -- -- -- -- -- -- MP6C11BR MP6B10BR % Homology MP3C7SRA MP2F1BR
MP2C5BR MP2C10BR MP2G5SR MP3B1SRA MP2F10SR MP3A7SRA MP4C10SR
MP3D2SRA 68 71 70 68 67 63 67 68 60 MP3A3SR 68 72 72 69 67 64 66 67
60 MP3C5SR 66 65 65 65 63 63 64 64 61 MP3C1SR 65 64 63 64 62 63 64
65 60 MP3G8SR 66 65 64 65 63 63 65 65 61 MP3D2BR 63 64 63 63 63 64
63 63 63 MP3H6SRA 75 73 71 73 71 66 75 75 63 MP3B4SRA 75 73 71 73
71 66 75 75 63 MP4E4BR 73 73 71 73 71 66 75 75 63 MP4H8SR 72 71 71
72 71 64 73 73 62 MP2F6SR 71 67 65 73 71 63 71 70 62 MP3D1BR 72 67
65 70 69 63 71 71 62 MP2B5BR 64 65 63 64 63 60 66 63 57 MP2C1BR 64
63 61 66 65 59 66 63 56 MP4A12SR 64 62 60 63 62 59 65 63 56
MP3F4SRA 97 69 67 68 68 62 67 69 60 MP3D3BR 96 70 68 67 67 62 67 67
60 MP3E5BR 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
REFERENCES
[0428] Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y,
Nakaya R. A novel method in the induction of reliable experimental
acute and chronic ulcerative colitis in mice. Gastroenterology
1990; 98:694-702. [0429] Kojouharoff G, Hans W, Obermeier F, Mannel
D N, Andus T, Scholmerich J, Gross V, Falk W. Neutralization of
tumour necrosis factor (TNF) but not of IL-1 reduces inflammation
in chronic dextran sulphate sodium-induced colitis in mice. Clin
Exp Immunol 1997; 107:353-8.
MMP12
[0429] [0430] Salmela M T, Pender S L, Reunala T, MacDonald T,
Saarialho-Kere U. Gut, 2001; 48(4):496-502 Parallel expression of
macrophage metalloelastase (MMP-12) in duodenal and skin lesions of
patients with dermatitis herpetiformis. [0431] Chavey C, Mari B,
Monthouel M N, Bonnafous S, Anglard P, Van Obberghen E,
Tartare-Deckert S. J. Biol. Chem., 2003; 278: 11888-11896. Matrix
metalloproteinases are differentially expressed in adipose tissue
during obesity and modulate adipocyte differentiation. [0432] Churg
A, Wang R.sup.D, Tai H, Wang X, Xie C, Dai J, Shapiro S D, Wright J
L. Am. J. Respir. Crit. Care Med., 2003; 167: 1083-1089. Macrophage
Metalloelastase Mediates Acute Cigarette Smoke-Induced Inflammation
Via TNF-alpha Release. [0433] R Lang, A Kocourek, M Braun, H
Tschesche, R Huber, W Bode, K Maskos J Mol Biol, September 2001;
312(4): 731-42. Substrate specificity determinants of human
macrophage elastase (MMP-12) based on the 1.1 A crystal structure.
[0434] Yoshikatsu Kaneko, Minoru Sakatsume, Yuansheng Xie, Takeshi
Kuroda, Michiko Igashima, Ichiei Narita and Fumitake Gejyo The
Journal of Immunology, 2003, 170: 3377-3385. Macrophage
Metalloelastase as a Major Factor for Glomerular Injury in
Anti-Glomerular Basement Membrane Nephritis [0435] Ding Y, Shimada
Y, Gorrin-Rivas MJ, Itami A, Li Z, Hong T, Maeda M, Komoto I,
Kawabe A, Kaganoi J, Imamura M. Oncology 2002; 63(4):378-84.
Clinicopathological significance of human macrophage
metalloelastase expression in esophageal squamous cell carcinoma.
[0436] Kerkela E, Ala-Aho R, Jeskanen L, Rechardt O, Grenman R,
Shapiro S D, Kahari V M, Saarialho-Kere U. J Invest Dermatol 2000
June; 114(6):1113-9 Expression of human macrophage metalloelastase
(MMP-12) by tumor cells in skin cancer.
Sequence CWU 1
1
1431121PRTLama glama 1Gln Val Gln Leu Gln Asp Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly
Arg Ile Phe Arg Ile Asn 20 25 30Ala Met Gly Trp Tyr Arg Gln Ala Pro
Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Thr Ile Thr Ser Thr Gly Ser
Thr Asn Phe Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Tyr Arg
Asp Gly Ala Lys Arg Thr Val Asp Leu65 70 75 80Arg Leu Asn Ser Leu
Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Asp Val Arg
Glu Tyr Asp Leu Gly Pro Trp Arg Gln Tyr Trp Gly 100 105 110Gln Gly
Thr Gln Val Thr Val Ser Ser 115 1202121PRTLama glama 2Gln Val Gln
Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ser Val Ser Gly Thr Ser Ile Ser Asn Arg 20 25 30Val
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Asp Phe Val 35 40
45Ala Tyr Ile Thr Ser Ala Val Asn Thr Asp Tyr Ala Asp Phe Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Met Val His
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Asn 85 90 95Val Leu Lys Asp Thr Trp Phe Arg Thr Pro Tyr Asp
Tyr Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115
1203125PRTLama glama 3Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys Val Val Ser Gly
Arg Thr Leu Ser Tyr Ser 20 25 30Ser Leu Ala Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Asp Phe Val 35 40 45Ala Ala Leu Ser Leu Thr Thr Tyr
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 80Asn Ser Leu Lys Pro
Asp Asp Thr Ala Asp Tyr Phe Cys Ala Thr Ala 85 90 95Arg Thr Arg Thr
Asp Tyr Ala Pro Leu Leu Ser Ala Ala Ser Thr Tyr 100 105 110Asp Ala
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Leu 115 120 1254124PRTLama
glama 4Gln 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 Ser Ser Arg
Tyr Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Ala Ala Val Asn Trp Asn Gly Asp Ser Thr Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Gly Asn Ala
Glu Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Val Pro Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Met Arg Met Asn Ala Gly Leu
Gly Tyr Ser Ala Ala Ser Tyr Gln 100 105 110Tyr Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Leu 115 1205122PRTLama glama 5Gln 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 Leu Thr Phe Leu Glu His 20 25 30Val Met
Ala Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly
Ala Ile Asp Trp Ser Gly Arg Arg Ile Thr Tyr Thr 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 Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Asp Arg Thr Tyr Ser Tyr Ser Ser Thr Gly Tyr Tyr
Tyr Trp 100 105 110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
1206122PRTLama glama 6Gln 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
Leu Thr Phe Leu Glu His 20 25 30Val Met Ala Trp Phe Arg Gln Thr Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala Ile Asp Trp Ser Gly Arg
Arg Ile Thr Tyr Thr 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 Thr
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Asp Arg
Thr Tyr Ser Tyr Ser Ser Thr Gly Tyr Tyr Tyr Trp 100 105 110Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 1207126PRTLama glama 7Gln 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 Leu Ser Ser Tyr 20 25
30Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45Ala Ser Ile Ser Ser Ser Gly Ile Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile 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 Lys Tyr Arg Tyr Tyr Ser Thr Leu Tyr
Thr Lys Ser Gly Glu 100 105 110Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 120 1258126PRTLama 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 Glu Ala Ser Gly Arg Thr Ile Ser Ser Tyr 20 25 30Ala Met
Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Ser Ile Ser Ser Ser Gly Val Ser Lys His Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Asn Asp Lys Val Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys 85 90 95Ala Ala Lys Tyr Arg Tyr Tyr Ser Ser Tyr Tyr Thr Lys Ser
Gly Asp 100 105 110Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 1259121PRTLama glama 9Gln 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 Leu Thr Phe Ser Thr Tyr 20 25 30Ala Met Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Val Ser
Tyr Ser Gly Ser Tyr Tyr Ala Asp Ser Val Lys Gly 50 55 60Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln65 70 75 80Met
Ala Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 85 90
95Arg Asn Arg Gly Tyr Ser Thr Tyr Ala Gly Val Tyr Asp Tyr Trp Gly
100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12010125PRTLama
glama 10Gln 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 Val Thr Phe Ser
Ser Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Ala Ser Ile Thr Trp Ile Gly Gly Gly Thr Tyr Tyr
Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp His Ala
Gly Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Thr Leu Lys Pro Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Leu Asp Arg Arg Ser Ser Thr
Tyr Tyr Leu Met Lys Gly Glu Tyr 100 105 110Asp Tyr Arg Gly Arg Gly
Thr Gln Val Thr Val Ser Ser 115 120 12511125PRTLama glama 11Gln 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 Val Thr Phe Ser Ser Tyr 20 25
30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45Ala Ser Ile Thr Trp Thr Gly Thr Gly Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp His Ala Gly 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 Val Asp Arg Arg Ser Ser Thr Tyr Tyr Leu
Met Lys Gly Glu Tyr 100 105 110Asp Tyr Arg Gly Arg Gly Thr Gln Val
Thr Val Ser Ser 115 120 12512151PRTLama glama 12Gln 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 Lys Glu 35 40 45Arg Glu
Phe Val Ala Arg Ile Tyr Trp Ser Ser Gly Asn Thr Tyr Tyr 50 55 60Ala
Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp Ile Ala Lys65 70 75
80Asn Thr Val Asp Leu Thr Met Asn Asn Leu Glu Pro Glu Asp Thr Ala
85 90 95Val Tyr Tyr Cys Ala Ala Arg Asp Gly Ile Pro Thr Ser Arg Ser
Val 100 105 110Glu Ser Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 125Ala Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu
Asp Leu Asn Gly Ala 130 135 140Ala His His His His His His145
15013124PRTLama glama 13Gln 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 12014260PRTLama glama
14Gln 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 245 250 255 Thr Val Ser
Ser 26015118PRTLama glama 15Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser
Gly Phe Thr Phe Ser Asp Tyr 20 25 30Pro Met Ala Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Ser Val Ile Asn Ser Gly Gly
Val Asn Thr Ser Tyr Ala Ala Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Tyr
Ser Leu Lys Asn Glu Gln Tyr Trp Arg Gly Gln Gly Thr 100 105 110Gln
Val Thr Val Ser Ser 11516124PRTLama glama 16Gln 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 Ser Ile Asp 20 25 30Gly Met Gly Trp
Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Arg Lys 35 40 45Gln Arg Glu
Leu Val Ala Ala Ile Thr Ser Gly Gly Ser Thr Lys Tyr 50 55 60Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn65 70 75
80Asp Thr Val Tyr Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala
85 90 95Val Tyr Tyr Cys Asn Ala Val Leu Leu Arg Arg Gly Ile Val Tyr
Asp 100 105 110Tyr Trp Gly Gln Gly Lys Gln Val Thr Val Ser Ser 115
12017124PRTLama glama 17Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser
Val Lys Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Ser Ile Phe Ser Ile Asp 20 25 30Gly Met Gly Trp Tyr Arg Gln Ala Pro
Gly Lys Gln Arg Glu Arg Lys 35 40 45Gln Arg Glu Leu Val Ala Ala Ile
Thr Ser Gly Gly Ser Thr Lys Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Asn65 70 75 80Asp Thr Val Tyr Leu
Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala 85 90 95Val Tyr Tyr Cys
Asn Ala Val Leu Leu Arg Arg Gly Ile Val Tyr Asp 100 105 110Tyr Trp
Gly Gln Gly Lys Gln Val Thr Val Ser Ser 115 12018121PRTLama glama
18Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Arg Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Leu Ser Lys
Tyr 20 25 30Arg Met Gly Trp Phe Arg Gln Phe Pro Gly Lys Glu Arg Glu
Leu Val 35 40 45Ala Glu Ile Glu Trp Lys Ser Ser Ser Thr Trp Tyr Arg
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr65 70 75 80Leu Arg Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ala Thr Leu Gly Glu Pro Leu
Val Lys Tyr Thr Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val
Ser Ser 115 12019124PRTLama glama 19Gln 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 Ser Ile Asp 20 25 30Gly Met Gly Trp Tyr Arg
Gln Ala Pro Gly Lys Gln Arg Glu Arg Lys 35 40 45Gln Arg Glu Leu Val
Ala Ala Ile Thr Ser Gly Gly Ser Thr Lys
Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Asn65 70 75 80Asp Thr Val Tyr Leu Gln Met Asn Thr Leu Lys Pro
Glu Asp Thr Ala 85 90 95Val Tyr Tyr Cys Asn Ala Val Leu Leu Arg Arg
Gly Ile Val Tyr Asp 100 105 110Tyr Trp Gly Gln Gly Lys Gln Val Thr
Val Ser Ser 115 12020128PRTLama glama 20Gln Val Gln Leu Gln Asp Ser
Gly Gly Gly Leu Val Arg Thr Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys
Val Val Phe Gly Gly Thr Ile Ser Thr Tyr 20 25 30Ala Met Gly Trp Phe
Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Asp
Ala Ser Gly Gly Phe Thr Glu Tyr Ala Asp Ser Val 50 55 60Arg Gly Arg
Phe Arg Ile Ala Arg Asp Asn Pro Leu Ser Ala Val Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90
95Ala Ala Asp Lys Asp Arg Asp Thr Val Val Arg Phe Thr Thr Thr Pro
100 105 110Asn Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 12521121PRTLama 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 Phe Thr Phe Asn Asn His 20 25 30Trp Leu Tyr Trp Val
Arg Gln Ala Gln Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Asn
Pro Gly Gly Ser Thr Val Tyr Leu Asp Ser Val Lys 50 55 60Gly Arg Phe
Thr Ile Ser Arg Gly Asn Thr Lys Asn Thr Leu Tyr Leu65 70 75 80Gln
Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr 85 90
95Lys Ala Met Ala Trp Ala Thr Asp Trp Asp Glu Tyr Asp Leu Trp Gly
100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser 115 12022124PRTLama
glama 22Gln 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 Thr
Val Tyr 20 25 30Thr Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val 35 40 45Ala Ala Ile Asp Trp Ser Gly Ser Ser Thr Tyr Tyr
Thr Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr
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 Arg Asp Ala Ile Val Gly
Val Thr Asp Thr Ser Gly Tyr Arg 100 105 110Tyr Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 12023127PRTLama glama 23Glu 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 Phe Ser Asn Tyr 20 25 30Val Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val 35 40 45Val
Gly Ile Gly Arg Ser Gly Gly Asp Asn Thr Tyr Tyr Ala Asp Ser 50 55
60Val Lys Gly Arg Phe Thr Ile Ser Trp Asp Asn Ala Lys Asn Thr Met65
70 75 80Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr 85 90 95Cys Ala Ala Ser Thr Tyr Ser Arg Asp Thr Ile Phe Thr Lys
Trp Ala 100 105 110Asn Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 12524127PRTLama glama 24Gln Val Gln Leu Gln Glu
Ser Gly Gly Gly Leu Val Lys Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Val Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala Ile
His Trp Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Ser 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 Ala Ser Arg Ile Ile Tyr Ser Tyr Val Asn Tyr Val Asn Pro
Gly 100 105 110Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120 12525122PRTLama glama 25Glu 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 Ser His 20 25 30Tyr Met Ser Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Thr
Ser Ser Ser Arg Thr Tyr Tyr Thr Glu 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 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Ala Asp Arg Thr Phe Tyr Gly Ser Thr Trp Ser Lys Tyr Asp Tyr Arg
100 105 110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
12026129PRTLama glama 26Gln 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 Lys Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ser Ala Ile Ser Trp Ser Asp Gly
Ser Thr Tyr 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 Val Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Thr Tyr
Leu Val Asp Val Trp Ala Val His Val Pro Ile Arg 100 105 110Pro Tyr
Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120
125Ser27125PRTLama glama 27Gln 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 Gly Gly Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Leu 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 Leu Tyr Tyr Cys 85 90 95Ala Ala Gly
Leu Arg Pro Ser Pro Asn Tyr Asn His Glu Arg Ser Tyr 100 105 110Asp
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
12528129PRTLama glama 28Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Leu Leu Ser Cys Ala Ala Ser Gly
Arg Thr Phe Ser Ser 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 Trp Ser Gly Gly
Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Thr Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Ala Phe Tyr Cys 85 90 95Ala Ala Thr Tyr
Asn Pro Tyr Ser Arg Asp His Tyr Phe Pro Arg Met 100 105 110Thr Thr
Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120
125Ser29122PRTLama glama 29Gln Val Gln Leu Gln Glu Ser Gly Gly Arg
Leu Val Gln Thr Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Gly Thr Phe Gly Thr Tyr 20 25 30Ala Leu Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Arg Phe Gly
Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Asn Asn Thr Val Tyr Leu65 70 75 80Glu Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Arg Glu
Gly Val Ala Leu Gly Leu Arg Asn Asp Ala Asn Tyr Trp 100 105 110Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 12030121PRTLama glama 30Gln
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 Tyr
20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Ala Ile Gly Leu Asn Thr Tyr 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 80Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Ala Arg 85 90 95Thr Ser Gly Val Val Gly Gly Thr Pro Lys
Arg Tyr Asp Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser
Ser 115 12031122PRTLama glama 31Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Arg Ser Phe Ser Thr Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln
Ala Pro Gly Gln Asp Arg Glu Phe Val 35 40 45Ala Thr Ile Ser Trp Thr
Asp Ser Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Asp
Arg Trp Ala Ser Ser Arg Arg Asn Val Asp Tyr Asp Tyr Trp 100 105
110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12032123PRTLama
glama 32Gln 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
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 Trp Gly Gly Gly Asn Thr Tyr 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 Ser Glu Trp Gly Gly Ser
Asp Tyr Asp His Asp Tyr Asp Tyr 100 105 110Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 115 12033123PRTLama glama 33Glu 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 Ser Phe Ser Ser Tyr 20 25 30Ala Met Ala
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala
Ile Ser Trp Gly Gly Gly Ser Thr Tyr Tyr Ala Val 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 Arg Tyr Tyr Cys
85 90 95Ala Ala Asp Glu Thr Phe His Ser Ser Ala Tyr Gly Glu Tyr Glu
Tyr 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
12034133PRTLama glama 34Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly
Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Thr Pro
Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Thr Ser Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Ser Thr Met Tyr65 70 75 80Leu Gln Met Asp Ser
Leu Met Leu Asp Asp Thr Ser Val Tyr Tyr Cys 85 90 95Ala Ala Asp Ser
Ser Arg Pro Gln Tyr Ser Asp Ser Ala Leu Arg Arg 100 105 110Ile Leu
Ser Leu Ser Asn Ser Tyr Pro Tyr Trp Gly Gln Gly Thr Gln 115 120
125Val Thr Val Ser Ser 13035120PRTLama glama 35Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Val Ala Ser Gly Phe Thr Phe Ala Asp Tyr 20 25 30Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Gln Trp Val 35 40 45Ser Ser
Ile Ser Tyr Asn Gly Asp Thr Thr Tyr Tyr Ala Glu Ser Met 50 55 60Lys
Asp 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 Ser Ser Gly Ser Tyr Tyr Pro Gly His Phe Glu Ser Trp Gly
Gln 100 105 110Gly Thr Gln Val Thr Val Ser Ser 115 12036121PRTLama
glama 36Gln 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
Gly Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg
Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Arg Gly Thr Ser Thr Tyr Tyr
Gly Asp Ser Ala 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 Gly Ser His Ser Asp Tyr
Ala Pro Asp Tyr Asp Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr
Val Ser Ser 115 12037123PRTLama glama 37Gln 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 Ser Tyr 20 25 30Ala Ile Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser
Trp Gly Gly Ser Asn Thr Tyr 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 Gly Glu Val Ser Asn Ser Asp Tyr Ala Tyr Glu Tyr Asp Tyr
100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
12038122PRTLama glama 38Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu
Val Gln Thr Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Arg Tyr Ile Met Gly Trp 20 25 30Phe Arg Gln Ala Pro Gly Lys Glu Arg
Glu Phe Val Ala Gly Ile Ser 35 40 45Arg Ser Gly Ala Ser Thr Ala Tyr
Ala Asp Ser Val Lys Asp Arg Phe 50 55 60Thr Ile Ser Arg Asp Ser Ala
Leu Asn Thr Val Tyr Leu Gln Met Asn65 70 75 80Ser Leu Lys Ala Glu
Asp Thr Ala Val Tyr Phe Cys Ala Ala Ala Leu 85 90 95Ala Ile Arg Leu
Gly Ile Pro Arg Gly Glu Thr Glu Tyr Glu Tyr Trp 100 105 110Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 12039121PRTLama glama 39Gln Val
Lys Leu Glu Glu Ser Gly
Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ser
Ala Ser Gly Leu Thr Phe Ser Asn Tyr 20 25 30Ala Met Ala Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Thr Ile Ser Gln
Arg Gly Gly Met Arg His Tyr Leu Asp Ser Val 50 55 60Lys Asp Arg Phe
Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln
Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Ala Asp Leu Met Tyr Gly Val Asp Arg Arg Tyr Asp Tyr Trp Gly 100 105
110Arg Gly Thr Gln Val Thr Val Ser Ser 115 12040127PRTLama glama
40Gln 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 Arg Ser Phe Ser Ser
Ile 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln
Phe Val 35 40 45Ser Ala Ile Asn Ser Asn Gly Asn Arg 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 Val Tyr Tyr Cys Ala 85 90 95Ala Val Gln Ala Tyr Ser Ser Ser Ser
Asp Tyr Tyr Ser Gln Glu Gly 100 105 110Ala Tyr Asp Tyr Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 12541126PRTLama glama 41Glu
Val Gln Leu Val Glu 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 Ser Met
20 25 30Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala
Thr 35 40 45Ile Asn Leu Ser Gly Asp Arg Thr Asp Tyr Ala Asp Ser Val
Lys Gly 50 55 60Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Val
Tyr Leu Gln65 70 75 80Met Asp Ser Leu Glu Pro Glu Asp Ser Ala Val
Tyr Tyr Cys Ala Gly 85 90 95Thr Ser Leu Tyr Pro Ser Asn Leu Arg Tyr
Tyr Thr Leu Pro Gly Thr 100 105 110Tyr Ala Asp Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 120 12542126PRTLama glama 42Gln 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 Ser Ile Phe Ser Ile Asn 20 25 30Ala
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45Ala Arg Ile Thr Gly Thr Gly Thr Gly Ile Thr Gly Ala Val Ser Thr
50 55 60Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn65 70 75 80Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu Asp 85 90 95Thr Ala Val Tyr Tyr Cys Ala Ala Asp Arg Ser Arg
Thr Ile Val Val 100 105 110Pro Asp Tyr Trp Gly Gln Gly Thr Gln Val
Thr Val Ser Ser 115 120 12543126PRTLama glama 43Gln 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 Arg Phe Ser Ser Ala Gln Tyr 20 25 30Ala Ile Gly
Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ser Tyr
Ile Thr Phe Ser Gly Gly Pro Thr Gly Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Val 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 Arg Pro Tyr Thr Arg Pro Gly Ser Met Trp Val Ser Ser
Leu 100 105 110Tyr Asp Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120 12544123PRTLama glama 44Gln Val Gln Leu Gln Glu Ser Gly
Gly Arg Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Glu His Thr Phe Arg Gly Tyr 20 25 30Ala Ile Gly Trp Phe Arg
Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ser Ser Ile Thr Tyr
Asp Gly Thr Leu Thr Asn Tyr Ala Asp Ser Val 50 55 60Thr 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 Val Cys 85 90 95Ala
Ala Gly Tyr Ser Tyr Arg Tyr Thr Thr Leu Asn Gln Tyr Asp Ser 100 105
110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 12045124PRTLama
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
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 12046123PRTLama glama 46Gln 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
12047118PRTLama glama 47Gln 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 11548118PRTLama glama 48Gln 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 11549118PRTLama glama 49Gln 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
11550113PRTLama glama 50Gln 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 110Ser
51126PRTLama 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 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
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 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
12554126PRTLama glama 54Gln 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
12555126PRTLama glama 55Gln 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
12556126PRTLama glama 56Gln 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
12557128PRTLama glama 57Gln 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
12558128PRTLama glama 58Gln 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 12559128PRTLama glama 59Gln 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 12560126PRTLama glama 60Gln 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 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 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
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 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
12563126PRTLama glama 63Gln 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
12564120PRTLama glama 64Ala 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 12065120PRTLama glama 65Gln 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
12066121PRTLama glama 66Gln 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 12067121PRTLama glama 67Gln 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
12068116PRTLama glama 68Gln 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 11569120PRTLama glama 69Gln 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 12070120PRTLama glama 70Gln
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 12071115PRTLama glama 71Gln 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 11572128PRTLama glama 72Ala 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 12573124PRTLama glama 73Gln 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
12074120PRTLama glama 74Glu 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 12075123PRTLama glama 75Gln 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
12076125PRTLama glama 76Glu 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 12577125PRTLama
glama 77Gln 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 12578124PRTLama glama 78Glu 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 12079131PRTLama glama 79Ala 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 13080126PRTLama glama 80Glu 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 12581128PRTLama glama 81Ala 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 12582120PRTLama glama 82Glu 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 12083123PRTLama
glama 83Glu 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 12084125PRTLama glama 84Asp 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 1258523DNALama glama 85ggctgagctc ggtggtcctg gct
238645DNALama glama 86aactggaaga attcgcggcc gcaggaattt tttttttttt
ttttt 458723DNALama glama 87ggctgagctc ggtggtcctg gct 238845DNALama
glama 88aactggaaga attcgcggcc gcaggaattt tttttttttt ttttt
458923DNALama glama 89ggctgagctc ggtggtcctg gct 239045DNALama glama
90aactggaaga attcgcggcc gcaggaattt tttttttttt ttttt 459123DNALama
glama 91cccctggccc cagtagttat acg 239217DNALama glama 92tgtgcagcaa
gagacgg 179344DNALama glama 93gtcctcgcaa ctgcggccca gccggcctgt
gcagcaagag acgg 449445DNALama glama 94gtcctcgcaa ctgcgcggcc
gccccctggc cccagtagtt atacg 459553DNALama glama 95aacagttaag
cttccgcttg cggccgcgga gctggggtct tcgctgtggt gcg 539623DNALama glama
96gaggtbcarc tgcaggastc ygg 239720DNALama glama 97ctggccccag
aagtcatacc 209819DNALama glama 98tgtgcatgtg cagcaaacc 199946DNALama
glama 99gtcctcgcaa ctgcggccca gccggcctgt gcatgtgcag caaacc
4610042DNALama glama 100gtcctcgcaa ctgcgcggcc gcctggcccc agaagtcata
cc 4210123DNALama glama 101ggctgagctc ggtggtcctg gct 2310245DNALama
glama 102aactggaaga attcgcggcc gcaggaattt tttttttttt ttttt
4510357DNALama glama 103catgccatga ctcgcggccc agccggccat ggccgaggtg
cagctggtgg agtctgg 5710457DNALama glama 104catgccatga ctcgcggccc
agccggccat ggccgatgtg cagctggtgg agtctgg 5710557DNALama glama
105catgccatga ctcgcggccc agccggccat ggccgcggtg cagctggtgg agtctgg
5710657DNALama glama 106catgccatga ctcgcggccc agccggccat ggccgccgtg
cagctggtgg attctgg 5710757DNALama glama 107catgccatga ctcgcggccc
agccggccat ggcccaggtg cagctggtgg agtctgg 5710857DNALama glama
108catgccatga ctcgcggccc agccggccat ggcccaggta cagctggtgg agtctgg
5710957DNALama glama 109catgccatga ctcgcggccc agccggccat ggcccaggta
aagctggagg agtctgg 5711019DNALama glama 110ccacagacag ccctcatag
1911120DNALama glama 111ggataacaat ttcacacagg 20112122PRTLama glama
112Ala 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 Ser
His 20 25 30Tyr Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Ala Ile Thr Ser Ser Ser Arg Thr Tyr Tyr Thr Glu
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 Ser Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Ala Asp Arg Thr Phe Tyr Gly Ser Thr
Trp Ser Lys Tyr Asp Tyr Arg 100 105 110Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120113122PRTLama glama 113Gln Val Lys Leu Glu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Asp1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His 20 25 30Tyr Met Ser Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile
Thr Ser Ser Ser Arg Thr Tyr Tyr Thr Glu 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 Asp Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Ala Asp Arg Thr Phe Tyr Gly Ser Thr Trp Ser Lys Tyr Asp Tyr
Arg 100 105 110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120114127PRTLama glama 114Gln 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 Phe Ser Asn Tyr 20 25 30Val Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Asp Phe Val 35 40 45Val Gly Ile Gly Arg Ser Gly
Gly Asp Asn Thr Tyr Tyr Ala Asp Ser 50 55 60Val Lys Gly Arg Phe Thr
Ile Ser Trp Asp Asn Ala Lys Asn Thr Met65 70 75 80Tyr Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Ala
Ser Thr Tyr Ser Arg Asp Thr Ile Phe Thr Lys Trp Ala 100 105 110Asn
Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120
125115122PRTLama glama 115Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Arg Gly Phe Ser Arg Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala
Pro Gly Gln Asp Arg Glu Phe Val 35 40 45Ala Thr Ile Ser Trp Thr Asn
Ser Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Ala Ile Ser
Arg Asp Asn Ala Lys Asn Thr Ala Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Asp Lys
Trp Ala Ser Ser Thr Arg Ser Ile Asp Tyr Asp Tyr Trp 100 105 110Gly
Gln Gly Ile Gln Val Thr Val Ser Ser 115 120116122PRTLama glama
116Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Arg Arg Phe Ser Thr
Tyr 20 25 30Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Gln Asp Arg Glu
Phe Val 35 40 45Ala Thr Ile Ser Trp Thr Asn Ser Thr Asp Tyr Ala Asp
Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Gly Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ser Val Tyr Val Cys Ala 85 90 95Ala Asp Lys Trp Ser Ser Ser Arg Arg
Ser Val Asp Tyr Asp Tyr Trp 100 105 110Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120117122PRTLama glama 117Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu Lys Leu Ser
Cys Thr Ala Ser Gly Arg Arg Phe Ser Thr Tyr 20 25 30Ala Val Gly Trp
Phe Arg Gln Ala Pro Gly Gln Asp Arg Glu Phe Val 35 40 45Ala Thr Ile
Ser Trp Thr Asn Ser Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr Leu65 70 75
80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ser Val Tyr Val Cys Ala
85 90 95Ala Asp Lys Trp Ser Ser Ser Arg Arg Ser Val Asp Tyr Asp Tyr
Trp 100 105 110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120118129PRTLama glama 118Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Leu Leu Ser Cys Ala Ala Ser
Gly Arg Thr Phe Ser Ser 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 Trp Ser Gly
Gly Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Lys Asp Asn Thr Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Ala Phe Tyr Cys 85 90 95Ala Ala Thr
Tyr Asn Pro Tyr Ser Arg Asp His Tyr Phe Pro Arg Met 100 105 110Thr
Thr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120
125Ser119129PRTLama glama 119Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Leu Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser 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 Trp Ser
Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Ala Phe Tyr Cys 85 90 95Ala Ala
Thr Tyr Asn Pro Tyr Ser Arg Asp His Tyr Phe Pro Arg Met 100 105
110Thr Thr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125Ser12072PRTLama glama 120Gln 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 Ser Ile Phe Ser Ile Asn 20 25 30Ala Met Gly Trp Tyr Arg
Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Arg Ile Thr Gly
Thr Gly Thr Gly Ile Thr Gly Ala Val Ser Thr 50 55 60Asn Tyr Ala Asp
Ser Val Lys Gly65 7012165PRTLama glama 121 Gln 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 Arg Ser Phe Ser Ser Ile 20 25 30Thr Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val 35 40 45Ser Ala Ile
Asn Ser Asn Gly Asn Arg Tyr Tyr Ala Asp Ser Val Lys 50 55
60Gly6512264PRTLama glama 122Glu Val Gln Leu Val Glu 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 Ser Met 20 25 30Gly Trp Phe Arg Gln Ala Pro
Gly Lys Glu Arg Glu Phe Val Ala Thr 35 40 45Ile Asn Leu Ser Gly Asp
Arg Thr Asp Tyr Ala Asp Ser Val Lys Gly 50 55 6012366PRTLama glama
123Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser
Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Ala Ile Thr Ser Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Val 50 55 60Lys Gly6512466PRTLama glama 124Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ala Asp Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Gln Trp Val 35 40 45Ser Ser Ile Ser Tyr Asn Gly Asp
Thr Thr Tyr Tyr Ala Glu Ser Met 50 55 60Lys Asp6512553DNALama glama
125aacagttaag cttccgcttg cggccgctgg ttgtggtttt ggtgtcttgg gtt
53126122PRTLama glama 126Glu 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 Ser His 20 25 30 Tyr Met Ser Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Thr Ser Ser
Ser Arg Thr Tyr Tyr Thr Glu 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 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Asp
Arg Thr Phe Tyr Gly Ser Thr Trp Ser Lys Tyr Asp Tyr Arg 100 105
110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120127127PRTLama
glama 127Glu 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 Phe
Ser Asn Tyr 20 25 30Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Asp Phe Val 35 40 45Val Gly Ile Gly Arg Ser Gly Gly Asp Asn Thr
Tyr Tyr Ala Asp Ser 50 55 60Val Lys Gly Arg Phe Thr Ile Ser Trp Asp
Asn Ala Lys Asn Thr Met65 70 75 80Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Ala Ser Thr Tyr Ser
Arg Asp Thr Ile Phe Thr Lys Trp Ala 100 105 110Asn Tyr Asn Tyr Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125128123PRTLama
glama 128Glu 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 Ser Phe
Ser Ser Tyr 20 25 30Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Gly Gly Gly Ser Thr Tyr
Tyr Ala Val 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 Arg Tyr Tyr Cys 85 90 95Ala Ala Asp Glu Thr Phe His
Ser Ser Ala Tyr Gly Glu Tyr Glu Tyr 100 105 110Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 120129123PRTLama 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 Arg Ser Phe Ser Ser Tyr 20 25 30Ala
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Ala Ile Ser Trp Gly Gly Gly Ser Thr Tyr Tyr Ala Val 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 Arg
Tyr Tyr Cys 85 90 95Ala Ala Asp Glu Thr Phe His Ser Ser Ala Tyr Gly
Glu Tyr Glu Tyr 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser
Ser 115 120130122PRTLama glama 130Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala
Ala Ser Gly Arg Ser Phe Ser Thr Tyr 20 25 30Ala Met Gly Trp Phe Arg
Gln Ala Pro Gly Gln Asp Arg Glu Phe Val 35 40 45Ala Thr Ile Ser Trp
Thr Asp Ser Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr Leu65 70 75 80Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala
Asp Arg Trp Ala Ser Ser Arg Arg Asn Val Asp Tyr Asp Tyr Trp 100 105
110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120131129PRTLama
glama 131Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala
Gly Gly1 5 10 15Ser Leu Leu Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe
Ser Ser 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 Trp Ser Gly Gly Ser Thr Ser
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Thr Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Ala Phe Tyr Cys 85 90 95Ala Ala Thr Tyr Asn Pro Tyr
Ser Arg Asp His Tyr Phe Pro Arg Met 100 105 110Thr Thr Glu Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120
125Ser132127PRTLama glama 132Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly Leu Val Lys Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Val Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Gly Ala Ile His Trp Ser
Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ser 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 Ala
Ser Arg Ile Ile Tyr Ser Tyr Val Asn Tyr Val Asn Pro Gly 100 105
110Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120 125133129PRTLama glama 133Gln 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 Lys Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ser Ala Ile Ser Trp Ser
Asp Gly Ser Thr Tyr 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 Val
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Thr Tyr Leu Val Asp Val Trp Ala Val His Val Pro Ile Arg 100 105
110Pro Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125Ser134129PRTLama glama 134Gln 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 Lys Tyr 20 25 30Ala Met Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ser Ala Ile Ser
Trp Ser Asp Gly Ser Thr Tyr 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 Val Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Ala Thr Tyr Leu Val Asp Val Trp Ala Val His Val Pro Ile Arg
100 105 110Pro Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser 115 120 125Ser135123PRTLama glama 135Gln 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 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 Trp Gly Gly Gly Asn Thr Tyr 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 Ser Glu Trp Gly Gly Ser Asp Tyr Asp His Asp Tyr Asp
Tyr 100 105 110Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120136123PRTLama glama 136Gln 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 Ser Tyr 20 25 30Ala Ile Gly Trp Phe Arg Gln Ala
Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Gly Gly
Ser Asn Thr Tyr 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 Gly
Glu Val Ser Asn Ser Asp Tyr Ala Tyr Glu Tyr Asp Tyr 100 105 110Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120137125PRTLama glama
137Gln 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 Gly Gly
Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala
Asp Ser Leu 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 Leu Tyr Tyr Cys 85 90 95Ala Ala Gly Leu Arg Pro Ser Pro Asn
Tyr Asn His Glu Arg Ser Tyr 100 105 110Asp Tyr Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 120 125138121PRTLama glama 138Gln 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 Gly Tyr 20 25
30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Glu Arg Glu Phe Val
35 40 45Ala Ala Ile Ser Trp Arg Gly Thr Ser Thr Tyr Tyr Gly Asp Ser
Ala 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 Gly Ser His Ser Asp Tyr Ala Pro Asp
Tyr Asp Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val Ser Ser
115 120139121PRTLama glama 139Gln 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 Gly Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln
Ala Pro Gly Glu Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Arg
Gly Thr Ser Thr Tyr Tyr Gly Asp Ser Ala 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
Gly Ser His Ser Asp Tyr Ala Pro Asp Tyr Asp Tyr Trp Gly 100 105
110Gln Gly Thr Gln Val Thr Val Ser Ser 115 120140121PRTLama glama
140Gln 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
Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Ala Ile Gly Leu Asn Thr Tyr 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 80Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Ala Arg 85 90 95Thr Ser Gly Val Val Gly Gly Thr Pro
Lys Arg Tyr Asp Tyr Trp Gly 100 105 110Gln Gly Thr Gln Val Thr Val
Ser Ser 115 120141122PRTLama glama 141Gln Val Gln Leu Gln Glu Ser
Gly Gly Arg Leu Val Gln Thr Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Gly Thr Phe Gly Thr Tyr 20 25 30Ala Leu Gly Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser
Arg Phe Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Val Tyr Leu65 70 75 80Glu
Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95Ala Arg Glu Gly Val Ala Leu Gly Leu Arg Asn Asp Ala Asn Tyr Trp
100 105 110Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115
120142122PRTLama glama 142Gln Val Gln Leu Gln Glu Ser Gly Gly Gly
Leu Val Gln Thr Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Arg Tyr Ile Met Gly Trp 20 25 30Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val Ala Gly Ile Ser 35 40 45Arg Ser Gly Ala Ser Thr Ala
Tyr Ala Asp Ser Val Lys Asp Arg Phe 50 55 60Thr Ile Ser Arg Asp Ser
Ala Leu Asn Thr Val Tyr Leu Gln Met Asn65 70 75 80Ser Leu Lys Ala
Glu Asp Thr Ala Val Tyr Phe Cys Ala Ala Ala Leu 85 90 95Ala Ile Arg
Leu Gly Ile Pro Arg Gly Glu Thr Glu Tyr Glu Tyr Trp 100 105 110Gly
Gln Gly Thr Gln Val Thr Val Ser Ser 115 120143121PRTLama glama
143Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Leu Thr Phe Ser Asn
Tyr 20 25 30Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Thr Ile Ser Gln Arg Gly Gly Met Arg His Tyr Leu
Asp Ser Val 50 55 60Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Asp Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Asp Leu Met Tyr Gly Val Asp
Arg Arg Tyr Asp Tyr Trp Gly 100 105 110Arg Gly Thr Gln Val Thr Val
Ser Ser 115 120
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