U.S. patent application number 14/190788 was filed with the patent office on 2014-07-31 for methods and compositions for enhanced delivery of macromolecules.
This patent application is currently assigned to ESBATech, an Alcon Biomedical Research Unit LLC. The applicant listed for this patent is ESBATech, an Alcon Biomedical Research Unit LLC. Invention is credited to Esther Furrer, Valerie Hulmann-Cottier, David Urech.
Application Number | 20140212421 14/190788 |
Document ID | / |
Family ID | 41328751 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140212421 |
Kind Code |
A1 |
Hulmann-Cottier; Valerie ;
et al. |
July 31, 2014 |
METHODS AND COMPOSITIONS FOR ENHANCED DELIVERY OF
MACROMOLECULES
Abstract
The invention provides compositions and methods that enhance the
delivery of large macromolecules (i.e., greater than 10 kDa), such
as antigen-binding polypeptides, across tight junctions. Such
methods and compositions are particularly useful for delivering
therapeutic antigen-binding polypeptides to the CNS, via intranasal
administration, for the treatment of neurological disorders.
Inventors: |
Hulmann-Cottier; Valerie;
(Zurich, CH) ; Urech; David; (Jona, CH) ;
Furrer; Esther; (Forch, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ESBATech, an Alcon Biomedical Research Unit LLC |
Schlieren |
|
CH |
|
|
Assignee: |
ESBATech, an Alcon Biomedical
Research Unit LLC
Schlieren
CH
|
Family ID: |
41328751 |
Appl. No.: |
14/190788 |
Filed: |
February 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13000533 |
Dec 21, 2010 |
8697074 |
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PCT/CH2009/000248 |
Jul 10, 2009 |
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14190788 |
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61079586 |
Jul 10, 2008 |
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Current U.S.
Class: |
424/135.1 ;
424/130.1; 424/158.1; 424/172.1; 424/178.1 |
Current CPC
Class: |
C07K 16/241 20130101;
A61P 25/24 20180101; A61P 25/22 20180101; C07K 2317/622 20130101;
A61P 9/10 20180101; A61P 25/08 20180101; A61K 9/08 20130101; A61P
25/16 20180101; A61P 25/06 20180101; A61K 9/0043 20130101; A61P
9/00 20180101; A61P 25/20 20180101; A61K 47/42 20130101; A61K
2039/543 20130101; A61P 35/00 20180101; A61P 25/00 20180101; A61K
39/3955 20130101; A61K 2039/505 20130101; A61P 25/28 20180101; C07K
2317/24 20130101; Y02P 20/55 20151101; A61P 21/02 20180101; A61P
25/18 20180101; A61K 39/3955 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/135.1 ;
424/130.1; 424/172.1; 424/158.1; 424/178.1 |
International
Class: |
A61K 47/18 20060101
A61K047/18; A61K 39/395 20060101 A61K039/395; A61K 47/48 20060101
A61K047/48; A61K 9/00 20060101 A61K009/00 |
Claims
1. A method for selective intranasal delivery of an antigen-binding
polypeptide to the central nervous system of a patient, the method
comprising administering to a patient in need thereof a composition
comprising a penetration enhancer and an antigen-binding protein,
wherein the penetration enhancer is Pz-peptide.
2. The method of claim 1, wherein the antigen-binding polypeptide
is an scFv.
3. The method of claim 1, wherein the antigen-binding polypeptide
specifically binds to a target antigen selected from the group
consisting of TNF-alpha, amyloid beta, amyloid beta-derived
diffusible ligand receptor, monoamine oxidase-B,
L-3,4-dihydroxyphenylalanine decarboxylase, acetyl-coA carboxylase,
N-methyl-D-aspartate aeceptor (also known as GRIN1), GRINA, GRIN2D,
GRIN2C, GRIN3B, GRIN2A, GRIN2B, GRIN3A, histamine H1 Receptor,
muscarinic receptor (also known as CHRM1), CHRM2, CHRM3, CHRM4,
hypocretin receptor 1, hypocretin receptor 2, 5-hydroxytryptamine
(also known as HTR1A), dopamine receptor (also known as DRD1),
DRD2, DRD3, DRD4, DRD5, adrenergic beta 1 receptor, norepinephrin
transporter (NET), and dopamine D2 receptor.
4. The method of claim 2, wherein the scFv comprises an amino acid
sequence with at least 80% similarity to SEQ ID No: 20, SEQ ID No:
21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25, SEQ
ID No: 26, or SEQ ID No: 27.
5. The method of 2, wherein the scFv comprises a VH domain
comprising an amino acid sequence with at least 80% similarity to
SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No:
10, or SEQ ID No:35.
6. The method of claim 2, wherein the scFv comprises a VL domain
comprising an amino acid sequence with at least 80% similarity to
SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID
No: 15, or SEQ ID No:34.
7. The method of claim 2, wherein the scFv comprises an amino acid
sequence with at least 80% similarity to SEQ ID No: 1, SEQ ID No:
2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, or SEQ ID No:33.
8. The method of claim 2, wherein the scFv further comprises the
amino acid sequence with at least 80% similarity to SEQ ID NO: 16,
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 36 or SEQ
ID NO: 37.
9. The method of claim 1, wherein the antigen-binding polypeptide
is covalently linked to the penetration enhancer.
10. The method of claim 1, wherein the patient has a neurological
disorder.
11. The method of claim 10, wherein the disorder is selected from
the group consisting of migraine, depression, Alzheimer's disease,
Parkinson's disease, schizophrenia, epilepsy, stroke, meningitis,
amyotrophic lateral sclerosis, insomnia, meningitis, memory
impairment, multiple sclerosis, narcolepsy, stroke, traumatic brain
injury, and stress.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 13/000,533 (now allowed), filed Dec. 21, 2010; which is
the National Stage of International Application Serial No.
PCT/CH2009/000248, filed Jul. 10, 2009; which claims priority to
U.S. Provisional Application No. 61/079,586, filed Jul. 10, 2008,
the contents of which are hereby incorporated by reference.
FIELD OF INVENTION
[0002] The present disclosure relates to compositions and methods
that facilitate delivery of molecules across biological membranes,
particularly to delivery of antigen-binding polypeptides across the
blood-brain barrier into the central nervous system (CNS).
BACKGROUND OF THE INVENTION
[0003] According to a 2006 World Health Organization report, over 1
billion people worldwide are afflicted with a neurological
disorder, and such disorders result in nearly 6.8 million deaths
annually. Therapeutic antigen binding peptides, such as antibodies,
could be used for treatment of many, if not the majority, of these
neurological disorders. However, treatment of neurological
disorders using such therapeutic antigen-binding peptides is
frequently hampered by difficulties associated with delivering
drugs across the blood-brain barrier (BBB).
[0004] Although compounds that enhance the delivery of molecules
across epithelial cell layers have been discovered, they have
generally been shown to be only effective at enhancing the delivery
of small molecules. For example, the peptide 4-phenylazobenzyl
oxycarbonyl-Pro-Leu-Gly-Pro has been shown to enhance the transport
of small molecules across epithelial cell layers, whereas no
penetration enhancing effect was demonstrated for macromolecules of
10 kDa and larger (see U.S. Pat. No. 5,534,496; Yen et al. 1995, J
Control Release, 36:25-37). Despite being heavily investigated,
there is presently no convenient and efficient method for the
delivery of therapeutic antigen-binding polypeptides into the
CNS.
[0005] There is therefore a continuing need in the art for
compositions and methods that enhance the specific delivery of
therapeutic antigen-binding polypeptides across epithelial layers,
in particular to the CNS for the treatment of CNS disorders.
SUMMARY OF THE INVENTION
[0006] The present invention is based, at least in part, on the
surprising discovery that penetration enhancers (e.g., Pz-peptide
or FMOC-peptide) are capable of enhancing the specific delivery of
large macromolecules (i.e., greater than 10 kDa) such as
antigen-binding polypeptides (e.g. scFv) to the CNS, particularly
when administered to nasal mucosa. Accordingly, the invention
provides compositions and methods that enhance the delivery of
large macromolecules (i.e., greater than 10 kDa), such as
antigen-binding polypeptides (e.g., scFv), across epithelial
layers. Such methods and compositions are particularly advantageous
in that they enable the convenient, efficient, and selective
delivery of an antigen-binding polypeptide (e.g., scFv) to the CNS,
via intranasal administration, for the treatment of neurological
disorders.
[0007] In one aspect, the invention provides compositions
comprising one or more antigen-binding polypeptides, such as an
immunobinder (e.g., scFv), and one or more penetration enhancers
(e.g., Pz-peptide or FMOC-peptide). In a particular embodiment, an
antigen-binding polypeptide is covalently linked to a penetration
enhancer.
[0008] In certain embodiments, the antigen-binding polypeptide
specifically binds to a target antigen selected from the group
consisting of TNF-alpha, amyloid beta, amyloid beta-derived
diffusible ligand receptor, monoamine oxidase-B,
L-3,4-dihydroxyphenylalanine decarboxylase, acetyl-coA carboxylase,
N-methyl-D-aspartate receptor (also known as GRIN1), GRINA, GRIN2A,
GRIN2B, GRIN2C, GRIN2D, GRIN3A, GRIN3B, histamine H1 Receptor,
muscarinic receptor (also known as CHRM1), CHRM2, CHRM3, CHRM4,
hypocretin receptor 1, hypocretin receptor 2,5-hydroxytryptamine
(also known as HTR1A), dopamine receptor (also known as DRD1),
DRD2, DRD3, DRD4, DRD5, adrenergic beta 1 receptor, norepinephrin
transporter (NET), and dopamine D2 receptor, in particular to
TNFalpha.
[0009] In other embodiments, the antigen-binding polypeptide is a
scFv comprising an amino acid sequence with at least 80% preferably
85%, 90%, 95%, or 99% identity or similarity to one or more amino
acid sequences set forth in Tables 5, 6, and 7 herein.
[0010] In other embodiments, the penetration enhancer facilitates
the selective intranasal delivery of the antigen-binding
polypeptide to the central nervous system.
[0011] The compositions of the invention are particularly useful as
medicaments (or for the manufacture of medicaments), in particular,
for the treatment, prevention or delay of progression of a
neurological disorder, including, without limitation, migraine,
depression, Alzheimer's disease, Parkinson's disease,
schizophrenia, epilepsy, stroke, meningitis, amyotrophic lateral
sclerosis, insomnia, meningitis, memory impairment, multiple
sclerosis, narcolepsy, stroke, traumatic brain injury, and
stress.
[0012] In another aspect, the invention provides a kit comprising
one or more antigen-binding polypeptides (e.g., scFv), one or more
penetration enhancers (e.g., Pz-peptide or FMOC-peptide), and
instructions for use.
DESCRIPTION OF THE DRAWINGS
[0013] The features and advantages of the present disclosure will
be better understood when reading the following detailed
description, taken together with the following drawings in
which:
[0014] FIG. 1 depicts time course experiments that track ESBA105
concentrations in the (A) olfactory bulb, (B) cerebrum, (C)
cerebellum, (D) brainstem, and (E) serum following intranasal
administration of 400 .mu.g scFv.
[0015] FIG. 2 compares ESBA105 concentrations in the (A) olfactory
bulb, (B) cerebrum, (C) cerebellum, (D) brainstem, and (E) serum
following either intranasal (400 .mu.g/mL) or intravenous (40
.mu.g/mL) administration of ESBA105, as well as in (F) serum
following either intranasal or intravenous administration of
ESBA105 at equal concentrations of 400 .mu.g/mL.
[0016] FIG. 3 shows (A) C.sub.max (mean values.+-.SEM, n=4) and (B)
exposure (AUC) brain tissue-to-blood concentration ratios of
ESBA105 in different brain regions following intranasal
administration with or without Pz peptide.
[0017] FIG. 4 depicts migration routes of ESBA105 from the nasal
cavity to the CNS following intranasal delivery. From the nasal
cavity, an administered compound may migrate into the blood and
pass the blood brain barrier to finally penetrate into the brain
tissue (lower route). Alternatively, the compound may migrate via
the N. olfactorius axonally (i.e. intracellular) or perineurnally
(i.e. extracellular) into the olfactory bulb and subsequently into
the cerebrum. The compound may also migrate via the N. trigeminus
(perineurally, i.e. extracellularly) into the brain stem and then
into the cerebellum.
DETAILED DESCRIPTION
Definitions
[0018] The term "penetration enhancer" encompasses any composition
that enhances the passage of a drug across a physical barrier such
as a tissue barrier (e.g. an epithelium). Suitable penetration
enhancers include, without limitation, the peptides
Pro-Leu-Gly-Pro-Arg [SEQ ID NO: 28], Pro-Leu-Gly-Pro-Lys [SEQ ID
NO: 29], Pro-Leu-Gly-Pro-Glu [SEQ ID NO: 30], Pro-Leu-Gly-Pro-Asp
[SEQ ID NO: 31], Pro-Leu-Gly-Pro [SEQ ID NO: 32], Pro-Leu-Gly and
Pro-Leu, N-terminally linked to a protective group such as
4-phenylazobenzyloxycarbonyl (Pz), N-methyl, t-butyloxcarbonyl
(t-Boc), fluoroenylmethyloxycarbonyl (FMOC), and carbobenzoxy (CBZ)
(see e.g. U.S. Pat. No. 5,534,496, which is hereby incorporated by
reference).
[0019] The term "Pz-peptide" refers to Pro-Leu-Gly-Pro-Arg [SEQ ID
NO: 28], N-terminally linked to a Pz group (see e.g. U.S. Pat. No.
5,534,496, which is hereby incorporated by reference).
[0020] The term "FMOC-peptide" refers to Pro-Leu-Gly-Pro-Arg [SEQ
ID NO: 28], N-terminally linked to a FMOC group (see e.g. U.S. Pat.
No. 5,534,496, which is hereby incorporated by reference).
[0021] The term "selective intranasal delivery" refers to the
intranasal application of a molecule (e.g., an antigen-binding
polypeptide) to a patient under conditions that result in higher
concentrations of the molecule in the CNS than in the serum of a
patient.
[0022] The term "antigen-binding polypeptide" refers to
polypeptides that are at least 10 kDa in size, and includes
immunobinders, monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), chimeric antibodies,
CDR-grafted antibodies, humanized antibodies, human antibodies,
single chain antibodies (scFvs), and antibody fragments, as well as
antigen-binding polypeptides based on alternative scaffolds known
in the art such as, but not limited to, CTLA-4, tendamistat,
fibronectin (FN3), neocarzinostatin, CBM4-2, lipocalins, T-cell
receptor, Protein A domain (protein Z), Im9, designed
ankyrin-repeat proteins (DARPins), designed TPR proteins, zinc
finger, pVIII, avian pancreatic polypeptide, GCN4, WW domain, Src
homology domain 3 (SH3), Src homology domain 2 (SH2), PDZ domains,
TEM-1 .beta.-lactamase, GFP, thioredoxin, staphylococcal nuclease,
PHD-finger, C.sub.1-2, BPT1 APPI, HPSTI, ecotin, LACI-D1, LDTI,
MTI-II, scorpion toxins, insect defensin A peptide, EETI-II,
Min-23, CBD, PBP, cytochrome b.sub.562, Ldl receptor domain A,
.gamma.-crystallin, ubiquitin, transferrin, and C-type lectin-like
domain (see e.g. Binz 2005, Curr Opin Biotechnol. Vol. 16 p.
459-69).
[0023] The term "immunobinder" refers to a molecule that contains
all or a part of the antigen binding site of an antibody, e.g., all
or part of the heavy and/or light chain variable domain, such that
the immunobinder specifically recognizes a target antigen.
Non-limiting examples of immunobinders include full-length
immunoglobulin molecules and scFvs, as well as antibody fragments,
including but not limited to (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fab' fragment, which is essentially a Fab
with part of the hinge region (see, Fundamental Immunology (Paul
ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the
V.sub.H and C.sub.H1 domains; (v) a Fv fragment consisting of the
V.sub.L and V.sub.H domains of a single arm of an antibody, (vi) a
single domain antibody such as a Dab fragment (Ward et al., (1989)
Nature 341:544-546)), which consists of a V.sub.H or V.sub.L
domain, a Camelid (see e.g. Hamers-Casterman, et al., Nature
363:446-448 (1993) and Dumoulin, et al., Protein Science 11:500-515
(2002)) or a Shark antibody (e.g., shark Ig-NARs Nanobodies.RTM.);
and (vii) a nanobody, a heavy chain variable region containing a
single variable domain and two constant domains.
[0024] The term "antibody" as used herein is a synonym for
"immunoglobulin." Antibodies according to the present invention may
be whole immunoglobulins or fragments thereof, comprising at least
one variable domain of an immunoglobulin, such as single variable
domains, Fv (Skerra A. and Pluckthun, A. (1988) Science
240:1038-41), scFv (Bird, R. E. et al. (1988) Science 242:423-26;
Huston, J. S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83),
Fab, (Fab').sub.2 or other fragments well known to a person skilled
in the art.
[0025] The term "single chain antibody" or "scFv" refers to a
molecule comprising an antibody heavy chain variable region
(V.sub.H) and an antibody light chain variable region (V.sub.L)
connected by a linker. Such scFv molecules may have the general
structures: NH.sub.2-V.sub.L-linker-V.sub.H-COOH or
NH.sub.2-V.sub.H-linker-V.sub.L-COOH.
[0026] The term "antibody framework" as used herein refers to the
part of the variable domain, either VL or VH, which serves as a
scaffold for the antigen binding loops of this variable domain
(Kabat, E. A. et al., (1991) Sequences of proteins of immunological
interest. NIH Publication 91-3242). Examples of suitable frameworks
are disclosed in PCT/CH2009/000219 and PCT/CH2009/000222, which are
hereby incorporated by reference herein.
[0027] The term "linker" refers to a linear amino acid sequence
linking two domains. Linkers of the invention may be genetically
and/or chemically fused to a domain. In certain embodiments,
linkers contain a loop formed via a disulfide bridge formed between
two cysteines present in the linker. The general structure of such
a linker is given in SEQ ID Nos. 18 and 19; SEQ ID Nos. 16 and 17
are exemplary embodiments of said linkers. A further suitable state
of the art linker consists of repeated GGGGS amino acid sequences
or variants thereof. In a preferred embodiment of the present
invention a (GGGGS).sub.4 linker (SEQ ID No: 36) or its derivative
(e.g. is used SEQ ID No: 37) is used, but variants of 1-3 repeats
are also possible (Holliger et al. (1993), Proc. Natl. Acad. Sci.
USA 90:6444-6448). Other linkers that may be used for the present
invention are described by Alfthan et al. (1995), Protein Eng.
8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et
al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J.
Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
Immunother. 50:51-59.
[0028] The term "modified" or "modifying," with respect to the
amino acid sequence of a polypeptide, refers to both the addition
of amino acids into the polypeptide sequence or the substitution of
existing amino acids in the polypeptide sequence. Amino acids
suitable for modifying a polypeptide include all known natural
amino acids, unnatural amino acids, and functionalized derivatives
thereof (see. e.g., U.S. Pat. Nos. 7,045,337 and 7,083,970, which
are hereby incorporate by reference in their entireties). In
certain embodiments, the term refers to the deletion of amino acids
from the polypeptide sequence.
[0029] A "target antigen" is a molecule (e.g., a soluble protein or
a membrane-bound protein, having one or more membrane-spanning
domains, a polypeptide, a peptide or a carbohydrate) containing an
antigenic determinant to which an antibody specifically binds.
[0030] The term "neurological disorder" includes diseases and
disorders that may affect the central nervous system (i.e. the
brain and spinal cord).
[0031] The term CNS disorder refers to a disorder that is
manifested in the CNS. By way of example, this may be a brain tumor
or a neurological disorder.
[0032] The term "effective amount" is defined as an amount of a
therapeutic (e.g. an antigen-binding polypeptide) sufficient to
partially, or completely prevent or arrest a disease or disorder
(e.g., a neurological disorder) in a patient. The effective amount
will depend upon the severity of the disease or disorder and a
variety of pharmacokinetic factors including the activity of the
particular compositions of the present invention employed, the
route of administration, the time of administration, the rate of
excretion of the particular compound being employed, the duration
of the treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0033] The term "patient" includes human and other mammalian
subjects that receive either prophylactic or therapeutic
treatment.
[0034] The terms "specific binding," "selective binding,"
"selectively binds," and "specifically binds," refer to antibody
binding to an epitope on a predetermined antigen. Typically, the
antibody binds with an affinity (K.sub.D) of approximately less
than about 10.sup.-7 M, such as approximately less than about
10.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M.
[0035] As used herein, "identity" refers to the sequence matching
between two polypeptides, molecules or between two nucleic acids.
When a position in both of the two compared sequences is occupied
by the same base or amino acid monomer subunit (for instance, if a
position in each of the two DNA molecules is occupied by adenine,
or a position in each of two polypeptides is occupied by a lysine),
then the respective molecules are identical at that position. The
"percentage identity" between two sequences is a function of the
number of matching positions shared by the two sequences divided by
the number of positions compared .times.100. For instance, if 6 of
10 of the positions in two sequences are matched, then the two
sequences have 60% identity. By way of example, the DNA sequences
CTGACT and CAGGTT share 50% identity (3 of the 6 total positions
are matched). Generally, a comparison is made when two sequences
are aligned to give maximum identity. Such alignment may be
provided using, for instance, the method of Needleman et al. (1970)
J. Mol. Biol. 48: 443-453, implemented conveniently by computer
programs such as the Align program (DNAstar, Inc.). The percent
identity between two amino acid sequences may also be determined
using the algorithm of E. Meyers and W. Miller (Comput. Appi.
Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN
program (version 2.0), using a PAM120 weight residue table, a gap
length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences may be determined
using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970))
algorithm which has been incorporated into the GAP program in the
GCG software package (available at www.gcg.com), using either a
Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14,
12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0036] "Similar" sequences are those which, when aligned, share
identical and similar amino acid residues, where similar residues
are conservative substitutions for corresponding amino acid
residues in an aligned reference sequence. In this regard, a
"conservative substitution" of a residue in a reference sequence is
a substitution by a residue that is physically or functionally
similar to the corresponding reference residue, e.g., that has a
similar size, shape, electric charge, chemical properties,
including the ability to form covalent or hydrogen bonds, or the
like. Thus, a "conservative substitution modified" sequence is one
that differs from a reference sequence or a wild-type sequence in
that one or more conservative substitutions are present. The
"percentage similarity" between two sequences is a function of the
number of positions that contain matching residues or conservative
substitutions shared by the two sequences divided by the number of
positions compared .times.100. For instance, if 6 of 10 of the
positions in two sequences are matched and 2 of 10 positions
contain conservative substitutions, then the two sequences have 80%
positive similarity.
[0037] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not negatively affect or alter the binding characteristics
of the antibody containing the amino acid sequence. Such
conservative sequence modifications include nucleotide and amino
acid substitutions, additions and deletions. For example,
modifications may be introduced by standard techniques known in the
art, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Conservative amino acid substitutions include ones in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a human anti-VEGF
antibody is preferably replaced with another amino acid residue
from the same side chain family. Methods of identifying nucleotide
and amino acid conservative substitutions which do not eliminate
antigen binding are well-known in the art (see, e.g., Brummell et
al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng.
12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA
94:412-417 (1997))
[0038] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein may be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the present specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0039] Various aspects of the invention are described in further
detail in the following subsections. It is understood that the
various embodiments may be combined at will.
Improved Antigen-Binding Polypeptide Compositions
[0040] In one aspect, the invention provides compositions for
enhancing the delivery of therapeutic polypeptides, such as
antigen-binding polypeptides (e.g., scFv) across tissue barriers,
more particularly across the nasal mucosa into the CNS. Such
compositions generally comprise an antigen-binding polypeptide and
a penetration enhancer. These compositions are particularly
advantageous in that they are capable of selective intranasal
delivery of the antigen-binding polypeptide to the central nervous
system. Today, biologics are typically systemically administered
requiring thus a higher dose of the drug and/or subjecting the
organism in need thereof to the drug; alternatively, the biologic
may be administered via a cranial cannula. Therefore, the present
invention significantly improves life quality of a subject in need
of the antigen-binding polypeptide.
[0041] Any antigen-binding polypeptide is suitable for use in the
methods of the invention. In certain embodiments, the
antigen-binding polypeptide is an immunobinder, such as an scFv.
Such scFv preferably comprise highly stable and soluble framework
regions such as those set forth in WO09/000,098, the contents of
which are incorporated herein by reference. In a particularly
preferred embodiment, the scFv comprises an amino acid sequence
with at least 80% similarity (e.g., 85%, 90%, 95%, or 99%) to one
or more amino acid sequences set forth in Tables 5, 6, and 7. Most
preferably, the scFv comprises an amino acid sequence with at least
80% identity, preferably 85%, 90%, 95%, or 99% identity, to one or
more amino acid sequences set forth in Tables 5, 6, and 7.
[0042] In a preferred embodiment, said scFv comprises a framework
sequence having at least at least 80% similarity (e.g., 85%, 90%,
95%, or 99%), more preferably at least 80% identity, even more
preferably 85%, 90%, 95%, or 99% identity, to SEQ ID No: 20, SEQ ID
No: 21, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 24, SEQ ID No: 25,
SEQ ID No: 26, or SEQ ID No: 27.
[0043] In another embodiment, said scFv comprises a VH domain
comprising an amino acid sequence with at least 80% similarity
(e.g., 85%, 90%, 95%, or 99%), more preferably at least 80%
identity, even more preferably 85%, 90%, 95%, or 99% identity, to
SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No:
10, or SEQ ID No:35. Additionally or alternatively, said scFv
comprises a VL domain comprising an amino acid sequence with at
least 80% similarity (e.g., 85%, 90%, 95%, or 99%), more preferably
at least 80% identity, even more preferably 85%, 90%, 95%, or 99%
identity, to SEQ ID No: 11, SEQ ID No: 12, SEQ ID No: 13, SEQ ID
No: 14, SEQ ID No: 15, or SEQ ID No:34. In one embodiment, said VH
and/or VL are linked by a linker to yield a molecule having the
general structure NH2-VH-linker-VL-COOH or NH2-VL-linker-VH-COOH.
Said linker molecule may e.g. be selected by the group consisting
of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ
ID NO:36 and SEQ ID NO: 37 or is a sequence having at least 80%
similarity thereto.
[0044] In a preferred embodiment, the scFv comprises an amino acid
sequence with at least 80% similarity (e.g., 85%, 90%, 95%, or
99%), more preferably at least 80% identity, even more preferably
85%, 90%, 95%, or 99% identity, to SEQ ID No: 1, SEQ ID No: 2, SEQ
ID No: 3, SEQ ID No: 4, SEQ ID No: 5, or SEQ ID No:33.
[0045] Any penetration enhancer may be used in the compositions of
the invention. In certain embodiments, the penetration enhancer is
a peptide or peptidomimetic linked to a protective group. Peptides
suitable for use in the invention may contain any known amino
acids, including natural amino acids, non-natural amino acids,
D-amino acids, and amino-acid derivatives. In a particular
embodiment, the penetration enhancer is a peptide selected from the
group consisting of Pro-Leu-Gly-Pro-Arg [SEQ ID NO: 28],
Pro-Leu-Gly-Pro-Lys [SEQ ID NO: 29], Pro-Leu-Gly-Pro-Glu [SEQ ID
NO: 30], Pro-Leu-Gly-Pro-Asp [SEQ ID NO: 31], Pro-Leu-Gly-Pro [SEQ
ID NO: 32], Pro-Leu-Gly and Pro-Leu, N-terminally linked to a
protective group such as 4-phenylazobenzyloxycarbonyl (Pz),
N-methyl, t-butyloxcarbonyl (t-Boc), fluoroenylmethyloxycarbonyl
(FMOC), and carbobenzoxy (CBZ) (see e.g. U.S. Pat. No. 5,534,496,
which is hereby incorporated by reference). In a preferred
embodiment, the penetration enhancer is Pro-Leu-Gly-Pro-Arg [SEQ ID
NO: 28] N-terminally linked to a Pz or FMOC group (see e.g. U.S.
Pat. No. 5,534,496, which is hereby incorporated by reference).
[0046] It is contemplated within the scope of the invention that
the penetration enhancers and antigen-binding proteins may
co-delivered to a target tissue in a single pharmaceutical
composition, or their delivery may be temporally separated by
administration in distinct compositions.
[0047] It is further contemplated within the scope of the invention
that a penetration enhancer may be conjugated to an antigen-binding
protein. All modes of physical or chemical conjugation known in the
art are contemplated. For conjugating a chemical group to an amino
acid, amino acid derivative, or amino acid mimetic, any suitable
chemistry known in the art may be employed. Conjugation may be to
any amino acid residue of an antigen-binding protein, including
Lysine, Cysteine and Histidine residues.
[0048] In certain embodiments, the compositions of the invention
may comprise additional compounds suitable for co-delivery with the
above-mentioned antigen-binding proteins. Such drugs include, but
are not limited to, small molecules, nootropics, polypeptides, and
oligonucleotides.
[0049] The compositions of the invention may be used to deliver
antigen-binding proteins across the tight junctions of any
biological membrane including, but are not limited to, mucosal
epithelium (e.g., nasal epithelium), and corneal tissue. A
particularly preferred target membrane is the nasal epithelium
because administration of a composition of the invention to the
nasal epithelium results in the direct and specific delivery of an
antigen-binding protein into the CNS, preferably without first
entering the blood stream.
Treatment of CNS Disorders
[0050] The compositions of the invention are particularly suited to
treating, preventing and or delaying the progression of CNS
disorders because such compositions allow for the direct and
selective delivery of antigen-binding polypeptides into the CNS via
the nasal mucosa. Suitable disorders for treatment with using the
compositions of the invention include, but are not limited to,
behavioral/cognitive syndromes, headache disorders (e.g. migraine,
cluster headache and tension headache), epilepsy, traumatic brain
injury, neurodegenerative disorders (e.g., Adrenoleukodystrophy,
Alcoholism, Alexander's disease, Alper's disease, Alzheimer's
disease, Amyotrophic lateral sclerosis (also known as Lou Gehrig's
Disease), Ataxia telangiectasia, Batten disease (also known as
Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform
encephalopathy, Canavan disease, Cerebral palsy, Cockayne syndrome,
Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial
Fatal Insomnia, Frontotemporal lobar degeneration, Huntington's
disease, HIV-associated dementia, Kennedy's disease, Krabbe's
disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph
disease (Spinocerebellar ataxia type 3), Multiple System Atrophy,
Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's
disease, Pelizaeus-Merzbacher Disease, Pick's disease, Primary
lateral sclerosis, Prion diseases, Progressive Supranuclear Palsy,
Refsum's disease, Sandhoff disease, Schilder's disease, Sub-acute
combined degeneration of spinal cord secondary to Pernicious
Anaemia, Spinocerebellar ataxia, Spinal muscular atrophy,
Steele-Richardson-Olszewski disease, Tabes dorsalis, and Toxic
encephalopathy, cerebrovascular disease (e.g. transient ischemic
attack and stroke), sleep disorders, cerebral palsy, infections
(e.g. encephalitis, meningitis, and myelitis), neoplasms (e.g.
brain and spinal cord tumors), movement disorders (e.g.
hemiballismus, tic disorder, and Gilles de la Tourette syndrome),
demyelinating diseases of the CNS (e.g. multiple sclerosis
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy), disorders of peripheral nerves (e.g. myopathy and
neuromuscular junctions), altered mental status (e.g.
encephalopathy, stupor, and coma), speech and language disorders,
paraneoplastic neurological syndromes, and syndromes having
functional neurological symptoms with no apparent physiological
cause.
[0051] Accordingly, in another aspect the invention provides a
method of treating or preventing a disease or disorder of the
central nervous system, the method comprising administering to the
nasal mucosa of a subject in need of treatment thereof, an
effective amount of a composition comprising an antigen-binding
polypeptide (e.g., an scFv) and a penetration enhancer (e.g.,
Pz-peptide) such that the disease or disorder is treated or
prevented.
[0052] In yet another aspect the invention provides a method of
selectively delivering an antigen-binding polypeptide to the
central nervous system of a subject, the method comprising
contacting a composition comprising an antigen-binding polypeptide
(e.g., an scFv) and a penetration enhancer (e.g., Pz-peptide) with
the nasal mucosa of a subject, whereby the antigen-binding
polypeptide is directly and selectively delivered to the central
nervous system.
Target Antigens
[0053] The antigen-binding polypeptides used in the methods of the
invention may bind to one or more specific target antigens.
Suitable target antigens include, but are not limited to, TNF-alpha
(e.g. Genbank Accession Numbers: NP.sub.--000585.2), amyloid beta
(e.g. Genbank Accession Number: NP.sub.--000475.1), amyloid
beta-derived diffusible ligand receptor (see e.g., WO/2004/031400),
monoamine oxidase-B (e.g. Genbank Accession Number:
NP.sub.--000889.3), L-3,4-dihydroxyphenylalanine decarboxylase
(e.g. Genbank Accession Number: NP.sub.--000781.1), acetyl-coA
carboxylase (e.g. Genbank Accession Number: NP.sub.--942131.1),
N-methyl-D-aspartate aeceptor (also known as GRIN1) (e.g. Genbank
Accession Number: NP.sub.--000823.4)), GRINA (e.g. Genbank
Accession Number: NP.sub.--000828.1), GRIN2D (e.g. Genbank
Accession Number: NP.sub.--000827.2), GRIN2C (e.g. Genbank
Accession Number: NP.sub.--000826.2), GRIN3B (e.g. Genbank
Accession Number: NP.sub.--619635.1), GRIN2A (e.g. Genbank
Accession Number: NP.sub.--000824.1), GRIN2B (e.g. Genbank
Accession Number: NP.sub.--000825.2), GRIN3A (e.g. Genbank
Accession Number: NP.sub.--597702.2), histamine H1 Receptor (e.g.
Genbank Accession Number: NP.sub.--000852.1), muscarinic receptor
(also known as CHRM1) (e.g. Genbank Accession Number:
NP.sub.--000729.2), CHRM2 (NP.sub.--000730.1), CHRM3
(NP.sub.--000731.1), CHRM4 (NP.sub.--000732.2), hypocretin receptor
1 (e.g. Genbank Accession Number: NP.sub.--001516.2), hypocretin
receptor 2 (e.g. Genbank Accession Number: NP.sub.--001517.2),
5-hydroxytryptamine (also known as HTR1A) (e.g. Genbank Accession
Number: NP.sub.--000515.2), dopamine receptor (also known as DRD1)
(e.g. Genbank Accession Number: NP.sub.--000785.1), DRD2 (e.g.
Genbank Accession Number: NP.sub.--000786.1), DRD3 (e.g. Genbank
Accession Number: NP.sub.--000787.2), DRD4 (e.g. Genbank Accession
Number: NP.sub.--000788.2), DRD5 (e.g. Genbank Accession Number:
NP.sub.--000789.1), norepinephrine transporter (NET) (e.g. Genbank
Accession Number: NP.sub.--001034.1), adrenergic beta 1 receptor
(e.g. Genbank Accession Number: NP.sub.--000675.1), and dopamine D2
receptor (e.g. Genbank Accession Number: NP.sub.--000786.1).
Formulations
[0054] Another aspect of the invention pertains to pharmaceutical
formulations of the antigen-binding polypeptide/penetration
enhancer compositions of the invention. Such formulations typically
comprise one or more antigen-binding polypeptide, one or more
penetration enhancer, and a pharmaceutically acceptable carrier. As
used herein, "pharmaceutically acceptable carrier" includes any and
all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Preferably, the carrier
is suitable for, for example, intravenous, intramuscular,
subcutaneous, topical (e.g., to eye, skin, or epidermal layer),
inhalation, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the antigen-binding polypeptide/penetration
enhancer composition may be coated in a material to protect the
compounds from the action of acids and other natural conditions
that may inactivate the compound.
[0055] The pharmaceutical compositions of the invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al.
(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0056] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0057] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity may be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0058] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents that delay absorption such
as aluminum monostearate and gelatin.
[0059] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds may also be incorporated into the
compositions.
[0060] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition may be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier may be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity may be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
may be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0061] Sterile injectable solutions may be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0062] The amount of active ingredient that may be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient that may be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0063] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0064] Another aspect of the invention is a method of administering
the pharmaceutical compositions of the invention. It is
contemplated within the scope of the invention that representative
delivery regimens may include oral parenteral (including
subcutaneous, intramuscular, and intravenous), rectal, buccal,
sublingual, pulmonary, transdermal, intranasal, and oral. The
preferred delivery regimen is nasal.
[0065] For nasal administration, either a solid or a liquid carrier
may be used. The solid carrier includes a coarse powder having
particle size in the range of, for example, from about 20 to about
500 microns and such formulation is administered by rapid
inhalation through the nasal passages. Where the liquid carrier is
used, the formulation may be administered as a nasal spray or drops
and may include oil or aqueous solutions of the active
ingredients.
[0066] Formulations suitable for nasal administration are presented
such that particles containing an active compound and desirably
having a diameter in the range of 0.5 to 7 microns are delivered in
the bronchial tree of the recipient. As one possibility such
formulations are in the form of finely comminuted powders which may
conveniently be presented either in a pierceable capsule, suitably
of, for example, gelatin, for use in an inhalation device, or
alternatively as a self-propelling formulation comprising an active
compound, a suitable liquid or gaseous propellant and optionally
other ingredients such as a surfactant and/or a solid diluent.
Suitable liquid propellants include propane and the
chlorofluorocarbons, and suitable gaseous propellants include
carbon dioxide. Self-propelling formulations may also be employed
wherein an active compound is dispensed in the form of droplets of
solution or suspension. Such self-propelling formulations are
analogous to those known in the art and may be prepared by
established procedures. Suitably they are presented in a container
provided with either a manually-operable or automatically
functioning valve having the desired spray characteristics;
advantageously the valve is of a metered type delivering a fixed
volume, for example, 25 to 100 .mu.ls, upon each operation thereof.
As a further possibility an active compound may be in the form of a
solution or suspension for use in an atomizer or nebuliser whereby
an accelerated airstream or ultrasonic agitation is employed to
produce a fine droplet mist for inhalation. When dispensed such
formulations should desirably have a particle diameter in the range
10 to 200 microns to enable retention in the nasal cavity; this may
be achieved by, as appropriate, use of a powder of a suitable
particle size or choice of an appropriate valve. Other suitable
formulations include coarse powders having a particle diameter in
the range 20 to 500 microns, for administration by rapid inhalation
through the nasal passage from a container held close up to the
nose, and nasal drops comprising 0.2 to 5% w/v of an active
compound in aqueous or oily solution or suspension.
Use of the Compositions
[0067] The compositions of the present invention may be used as a
medicament, for example for the treatment, prevention and/or delay
of progression of a neurological disorder. Accordingly, the
composition disclosed herein may be used for the manufacture of a
medicament useful for the treatment or prevention of a neurological
disorder.
[0068] In a preferred embodiment, such a disorder is selected from
the group consisting of migraine, depression, Alzheimer's disease,
Parkinson's disease, schizophrenia, epilepsy, stroke, meningitis,
amyotrophic lateral sclerosis, insomnia, meningitis, memory
impairment, multiple sclerosis, narcolepsy, stroke, traumatic brain
injury, and stress.
[0069] Preferably, the composition is formulated for intranasal
delivery.
EXAMPLES
[0070] The present disclosure is further illustrated by the
following examples, which should not be construed as further
limiting.
[0071] In general, the practice of the present invention employs,
unless otherwise indicated, conventional techniques of chemistry,
molecular biology, recombinant DNA technology, and immunology
(especially, e.g., immunoglobulin technology). See, e.g., Sambrook,
Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor
Laboratory Press (1989); Antibody Engineering Protocols (Methods in
Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody
Engineering: A Practical Approach (Practical Approach Series, 169),
McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual,
Harlow et al., C.S.H.L. Press, Pub. (1999); Current Protocols in
Molecular Biology, eds. Ausubel et al., John Wiley & Sons
(1992). See also, e.g., Polytherics US6803438; EP1701741A2;
EP1648518A2; WO05065712A2; WO05007197A2; EP1496941A1; EP1222217B1;
EP1210093A4; EP1461369A2; WO03089010A1; WO03059973A2; and
EP1210093A1); Genentech US20070092940A1 and EP1240337B1; and
ESBATech U.S. Ser. No. 60/899,907, PCT/CH2009/000225,
PCT/CH2009/000222, PCT/CH2009/000222, WO 06/131013 and
WO03097697A2.
Purification of ESBA105
[0072] ESBA105, an anti-TNF-alpha single chain antibody fragment
with a molecular weight of 26.3 kDa, was purified from Escherichia
coli host cells as previously described (Furrer et al. (2009)
Invest Opthalmol V is Sci 50, 771-778; Ottiger et al. (2009) Invest
Opthalmol V is Sci 50, 779-786). Briefly, ESBA105 was produced by
recombinant expression in E. coli BL21(DE3), refolding from
inclusion bodies, and subsequent size-exclusion chromatography. For
animal studies ESBA105 was formulated at 10 mg/ml (for intranasal
administration) or 0.5 mg/ml (for intravenous injection) in 50 mM
sodium phosphate, 150 mM NaCl, pH 6.5. The endotoxin content as
determined in the LAL clotting assay was below 0.1 EU in all
formulations used for in vivo experiments.
Intranasal Administration of Evans Blue
[0073] Optimal conditions for targeting proteins to the CNS were
initially determined by administering 0.3% Evans blue in 0.9% NaCl
via an intranasal route to Balb/c mice. Animals were then
sacrificed by CO.sub.2 inhalation, at predefined time points, and
their lungs and stomachs were harvested and visually inspected for
the presence of Evans blue. Optimal conditions were obtained by
keeping the animals under isoflurane (Provet, Lyssach, Switzerland)
anaesthesia in a supine position and treating each nare with 2
.mu.l Evans blue at five minute intervals until a total of 40 .mu.l
was reached (45 min) (Table 1). Consequently, this protocol was
used for intranasal administration of ESBA105 in all embodiments
described herein.
Intranasal and Intravenous Administration of ESBA105
[0074] Prebleeds of all animals were collected ten days before the
intranasal or intravenous dosing with ESBA105. Intranasal
administration of ESBA105 was carried out under isoflurane (Provet,
Lyssach, Switzerland) anaesthesia. Mice were placed in a supine
position and a total of 40 .mu.l (400 .mu.g) ESBA105 was
administered by pipette in 2 .mu.l drops, treating each nare every
five minutes over a total of 45 minutes. For the intranasal PK
study, four animals were sacrificed at 1, 2, 4, 6, 8, 10, 12, and
24 hours after the first intranasal instillation. In some
experiments 3 mM Pz-peptide
(4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-D-Arg; Bachem,
Bubendorf, Switzerland), a penetration enhancer that facilitates
the transport of paracellular markers by triggering opening of
tight junctions in a transient, reversible manner (Yen and Lee
(1994) Journal of Controlled Release 28, 97-109), was added to the
ESBA105 formulation. Four animals were sacrificed at 1, 2, and 4
hours after the first administration. For intravenous injection,
mice were placed in a restrainer and 40 .mu.g (80 .mu.l) ESBA105
were injected into the tail vein. The intravenous dose was chosen
to best approximate the systemic exposure according to the area
under the blood concentration-time curve (AUC) observed over a 4
hour period with intranasal administration of 400 .mu.g ESBA105.
Two animals were sacrificed at each time point (1, 2, and 4 hours).
At time of sacrifice mice were deeply anaesthetized with a mixture
of ketamine (Ketasol100, 65 mg/kg; Pharmacy, Schlieren,
Switzerland), xylazine (Rompun, 13 mg/kg; Provet, Lyssach,
Switzerland) and acepromazine (Prequillan, 2 mg/kg; Arovet,
Zollikon, Switzerland). A blood sample was collected by heart
puncture before perfusing the mice with 20 ml PBS. The brains were
carefully harvested and dissected into olfactory bulb, cerebrum
including thalamus and hypothalamus, cerebellum and brainstem. The
tissues were weighed, frozen on dry ice and stored at -80.degree.
C. until analysis.
Tissue Preparation
[0075] Tissues were prepared for analysis as follows. 100 .mu.l
lysis buffer (10 mM Tris, pH 7.4, 0.1% SDS, with proteinase
inhibitor cocktail (Roche Diagnostics, Rotkreuz, Switzerland)) was
added to 15 mg of brain tissue. Tissues were sonicated for 5
seconds (8 cycles, 100% intensity) (Sonoplus, Bandelin, Berlin,
Germany), centrifuged, and the supernatants were subjected to ELISA
based determination of ESBA105 concentrations.
Quantification of ESBA105 in Serum and Brain Tissue
[0076] ESBA105 concentrations were determined by triplicate
measurements of each sample in a direct ELISA. 96-well plates (NUNC
MaxiSorp; Omnilab, Mettmenstetten, Switzerland) were coated with
0.5 .mu.g/ml human TNF-alpha (Peprotech, London, UK) in PBS
overnight at 4.degree. C. Between each of the following steps
plates were washed three times with TBS-T (0.005% Tween20; Axon
Lab, Baden-Dattwyl, Switzerland) using a micro plate washer (ASYS
Atlantis, Salzburg, Austria). Unspecific binding sites were
saturated by 1.5 hour incubation in PBS/1% BSA/0.2% Tween20.
Predilutions of each sample were prepared in dilution buffer (PBS,
0.1% BSA, 0.2% Tween20) containing 10% of the respective matrix
(olfactory bulb, cerebrum, cerebellum, brainstem or serum).
Standard reference dilution series (50--0.5 ng/ml) of ESBA105 were
prepared in dilution buffer/10% respective matrix. Prediluted
samples and standard reference dilutions were then added to the
wells and plates were incubated for 1.5 hours at room temperature.
Bound ESBA105 was detected with a biotinylated affinity purified
polyclonal rabbit anti-ESBA105 antibody (AK3A, ESBATech, Schlieren,
Switzerland) that was diluted 1:20'000 in dilution buffer (1.5 h,
room temperature). AK3A, in turn, was detected with
poly-horseradish peroxidase streptavidin (Stereospecific Detection
Technologies, Baesweiler, Germany) at a concentration of 0.2 ng/ml
dilution buffer. POD (Roche Diagnostics, Rotkreuz, Switzerland) was
used as peroxidase substrate and the color reaction was stopped
after 2 to 20 minutes (depending on color intensity) by addition of
1 M HCl. Absorbance was measured at 450 nm in a plate reader
(Sunrise; Tecan, Maennedorf, Switzerland) and ESBA105
concentrations in samples were calculated by polynomial regression
from a standard curve (GraphPad Prism 4,03; GraphPad Software,
Inc., San Diego, Calif.). The minimum quantifiable concentration
(LOQ) of ESBA105 was 5 ng/ml in serum and 33 ng/ml in brain tissue,
respectively. Undiluted samples that resulted in signals below the
lower limit of quantitation were set to LOQ for mathematical
evaluation and graphical display.
Example 1
Mode of Intranasal Administration
[0077] This example demonstrates that low volume nasal
administration results in specific delivery to the CNS. For
efficient and specific drug delivery into the CNS, an applied
substance should remain in the nasal cavity; however, several
studies have shown that an intranasally applied substance may
migrate to the respiratory system and the gastrointestinal tract
due to breathing and ingestion (Eyles et al. (1999) Int J Pharm
189, 75-79; Klavinskis et al. (1999) J Immunol 162, 254-262;
Lundholm et al. (1999) Vaccine 17, 2036-2042; Trolle et al. (2000)
Vaccine 18, 2991-2998). One of skill in the art will appreciate
that a number of aspects (e.g. anesthesia, animal position, and
volume and frequency of administration) may influence the residence
time of the administered compound in the nasal cavity. Using the
intranasal administration protocol described above, Evans Blue was
used as a tracer to assess the post-administration distribution of
the dye after intranasal application. 40-50 .mu.l Evans blue was
administered intranasally via several different methods. First, a
single dose was given to either anaesthetized or alert mice held in
a supine position, which resulted in dye migrating to both the
lungs and stomach in both cases. Second, a single dose was given to
anaesthetized animals for 30-50 minutes in the supine position
instead of only 3 minutes. Third, the volume of die was
administered in two separate 10 .mu.l doses that were administered
at 5 minutes intervals. Neither of these methods reduced Evans blue
migration to the lungs and stomach. Finally, volumes as low as 2
.mu.l were applied to anaesthetized animals in the supine position,
which resulted in only minimal traces of Evans blue in the lungs
and blue staining was totally absent in the stomach (Table 1).
Example 2
Delivery of ESBA105 to the CNS
[0078] This example demonstrates that intranasal administration of
a scFv results in delivery of the scFv to the CNS. ESBA105 (SEQ ID
NO: 1) is a single chain antibody that specifically binds and
inhibits TNFalpha (see e.g. WO 06/131013, which is hereby
incorporated by reference). Following intranasal administration by
the above described protocol, ESBA105 reached significant
concentrations in all regions of the brain analyzed, and displayed
a bimodal distribution over time. Maximum ESBA105 concentrations
(C.sub.max) in cerebellum and brainstem were reached one hour after
the first instillation, with concentrations in the olfactory bulb
and cerebrum peaking one hour later. ESBA105 levels then declined
in all brain regions, but rose again after 6-12 hours to produce a
clear second, lower level, concentration peak in the olfactory
bulb, cerebellum, and brainstem (FIG. 1), which demonstrates that
two different migration routes are likely to exist. Highest
concentrations were measured in the olfactory bulb and the
brainstem. In the olfactory bulb, which is connected to the nasal
cavity through the olfactory system (N olfactorius), concentrations
culminated at 9455 ng/ml. Concentrations were even higher in the
brainstem (11067 ng/ml), which is connected to the nasal passages
through the peripheral trigeminal system (N. trigeminus) (Table 2).
C.sub.max in the cerebrum (975 ng/ml) was slightly delayed (2
hours), and about seven to ten times lower than in the cerebellum
or the olfactory bulb, respectively. These results demonstrate that
ESBA 105 first reaches the olfactory bulb and the brainstem and
from there distributes to cerebrum and cerebellum. Similar to the
brainstem and cerebellum, C.sub.max in serum was reached at one
hour after the first administration of ESBA105 and peaked a second
time between five and 10 hours. Interestingly, ESBA105 levels
remained almost constant during the last 12 hours (FIG. 1).
Example 3
ESBA105 Delivery to the CNS is Direct
[0079] This example demonstrates that intranasal administration of
ESBA105 results in direct delivery to the CNS, rather than via the
bloodstream. To determine whether ESBA105 migrates directly through
the BBB to the CNS from the nasal cavity, or indirectly via
systemic absorption and subsequent trans-BBB delivery to the brain,
intranasal administration was compared side by side with
intravenous injection as described above. Following intravenous
injection, considerable concentrations of ESBA105 were reached in
all analyzed regions, except the cerebrum where concentrations were
below the lower limit of quantitation. However, considerably higher
drug concentrations were measured in all brain regions following
intranasal administration (FIG. 2). Maximum ESBA105 levels in
cerebellum and brainstem upon intranasal dosing were about 10-18
fold higher than those following intravenous injection. Moreover,
C.sub.max in the olfactory bulb was more than 60-fold higher for
intranasal versus intravenous administration (Table 3).
Surprisingly, although dosing was set to produce similar systemic
exposures for both routes, serum concentrations were clearly lower
after intranasal administration (FIG. 2), reaching 6006 ng/ml while
C.sub.max following intravenous injection was more than 10-fold
higher (63709 ng/ml) (Table 3). Following intravenous injection,
maximal concentrations (C.sub.max) and exposures (AUC) in olfactory
bulb, cerebellum and brainstem reached similar values with 202,
257, and 174 ng/ml for C.sub.max and 448, 567, and 416 ng-h/ml for
AUC, respectively. C.sub.max in brain tissues following intravenous
injection was at 2 hours and no ESBA105 could be detected at 4
hours. In contrast, following intranasal administration clearly
higher concentrations were measured in all brain regions. Highest
values were obtained for the olfactory bulb (C.sub.max: 12586
ng/ml; AUC: 23130 ng-h/ml) followed by brainstem (C.: 3169 ng/ml;
AUC: 7942 ng-h/ml), cerebellum (C.sub.max: 2819 ng/ml; AUC: 5908
ng-h/ml) and cerebrum (C.sub.max: 1831 ng/ml; AUC: 2951 ng-h/ml).
Moreover, in contrast to intravenous injection, there were still
detectable concentrations of ESBA105 in all brain regions four
hours after intranasal administration. These results demonstrate
that ESBA105 is able to penetrate from the blood across the BBB
into the CNS, and that the most efficient route of delivery is via
intranasal administration (Table 3).
Example 4
Pz-Peptide Improves ESBA105 Delivery Across the BBB
[0080] This example demonstrates that the Pz-peptide significantly
enhances the intranasal delivery of a scFv to the CNS.
Specifically, the ability of the Pz-peptide to function as a
penetration enhancer for transport of drugs through the BBB was
examined by adding 3 mM Pz-peptide to ESBA105 and assessing
transport to the brain. In presence of Pz-peptide, C.sub.max in
olfactory bulb, cerebrum and cerebellum was reached earlier than
ESBA105 alone (one instead of two hours after first dosing) (Table
4). Furthermore, addition of Pz-peptide resulted in a 2- to 3-fold
increase in C.sub.max in olfactory bulb and cerebrum (7309 to 15786
ng/ml and 1133 to 3417 ng/ml, respectively), while C.sub.max in the
brainstem remained unchanged. Tissue-to-blood ratios for C.sub.max
were clearly higher in olfactory bulb and cerebrum with
co-administration of ESBA105 and Pz-peptide than with ESBA105 alone
(FIG. 3A). The effect on delivery to cerebellum, brainstem and
serum was, however, less pronounced. In summary, Pz-peptide can
enhance delivery of large molecular weight proteins to the
olfactory bulb and the cerebrum without increasing systemic
exposure (FIG. 3). Therefore, for therapeutic applications,
Pz-peptide may enhance drug delivery without increasing the risk of
systemic side effects (Table 4).
TABLE-US-00001 TABLE 1 Administration scheme and presence of dye
following intranasal delivery of 0.3% Evans blue dye. sacrificed
(after first application anaesthesia admin- Evans blue volume
interval nare isoflurane duration istration) lungs stomach 1
.times. 40 .mu.l -- both no -- 50 min + ++ 1 .times. 40 .mu.l --
both yes 3 min 50 min + (+) 1 .times. 50 .mu.l -- both yes 3 min 3
min + + 1 .times. 50 .mu.l -- both yes 3 min 30 min ++ ++ 1 .times.
50 .mu.l -- both yes 3 min 50 min ++ ++ 10 .times. 10 .mu.l 5 min
alternating yes 45 min 55 min +++ +++ 10 .times. (2 + 2 .mu.l) 5
min 2 .mu.l per nare yes 45 min 50 min - -
TABLE-US-00002 TABLE 2 Pharmacokinetic parameters after intranasal
administration of 400 .mu.g ESBA105. C.sub.max ng/mg AUC/ of total
T.sub.max AUC mg ng/ml protein hours ng-h/ml ng-h/ml Olfactory 9455
.+-. 6465 10459 .+-. 7152 2 51913 129'782 bulb Cerebrum 975 .+-.
1806 1079 .+-. 1998 2 2576 6'440 Cerebellum 7354 .+-. 8240 8134
.+-. 9115 1 13249 33'123 Brainstem 11067 .+-. 18530 12242 .+-.
20498 1 21770 54'425 Serum 7191 .+-. 975 nd 1 26257 65'643
TABLE-US-00003 TABLE 3 Pharmacokinetic parameters after intranasal
or intravenous administration of ESBA105. Intranasal.sup.a
Intravenous.sup.a AUC/ AUC/ AUC.sub.(in)/ AUC mg.sup.b AUC mg.sup.b
AUC.sub.(iv) C.sub.max T.sub.max (ng- (ng- C.sub.max T.sub.max (ng-
(ng- (per (ng/ml) (h) h/ml) h/ml) (ng/ml) (h) h/ml) h/ml) mg) Olf.
bulb 12586 .+-. 24693 2 23130 57825 202 .+-. 238 2 448 11200 5.16
Cerebrum 1831 .+-. 3476 2 2951 7378 0 nd 0 nd nd Cerebellum 2819
.+-. 3830 2 5908 14770 257 .+-. 316 2 567 14175 1.04 Brainstem 3169
.+-. 2211 1 7942 19855 174 .+-. 28 2 416 10400 1.91 Serum 6006 .+-.
1896 2 15709 39273 nd nd 54254 1356350 0.03 Cerebrum.sup.c 1831
.+-. 3476 2 2951 7378 142 .+-. 126 2 375 938 7.87 .sup.aThe doses
of 400 .mu.g (intranasal) and 40 .mu.g (intravenous) were chosen to
reach a similar systemic exposure. .sup.bAUC normalized to 1 mg
assuming that the AUC increases linearly with different doses.
.sup.cIntranasal or intravenous administration of an equal dose of
EBSA105 (400 .mu.g).
TABLE-US-00004 TABLE 4 Pharmacokinetic parameters after intranasal
delivery of 400 .mu.g ESBA105 with or without 3 mM Pz-peptide.
ESBA105 ESBA105/Pz-peptide AUC AUC C.sub.max T.sub.max (ng-
C.sub.max T.sub.max (ng- C.sub.maxE105/ AUC.sub.E105/ (ng/ml) (h)
h/ml) (ng/ml) (h) h/ml) C.sub.maxE105/Pz AUC.sub.E105/Pz Olf. bulb
7309 .+-. 17233 2 16854 15786 .+-. 29556 1 22210 0.46 0.76 Cerebrum
1133 .+-. 2403 2 2792 3417 .+-. 7832 1 4303 0.33 0.65 Cerebellum
1714 .+-. 2796 2 3922 2119 .+-. 2355 1 4367 0.81 0.90 Brainstem
1850 .+-. 2053 1 4578 3454 .+-. 2818 1 4157 0.54 1.10 Serum 4645
.+-. 2422 2 12656 4109 .+-. 2943 2 12010 1.13 1.05
TABLE-US-00005 TABLE 5 scFv, VH, and VL sequences SEQ ID NO: Name:
Sequence: 1 ESBA105 MADIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQ scFv
QRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTIS (.alpha.-
SLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKRGGGGSGG TNF-
GGSGGGGSSGGGSQVQLVQSGAEVKKPGASVKVSCTASG alpha)/
YTFTHYGMNWVRQAPGKGLEWMGWINTYTGEPTYADKFK WO
DRFTFSLETSASTVYMELTSLTSDDTAVYYCARERGDAM 2006/ DYWGQGTLVTVSS 131013
2 EP34max EIVMTQSPSTLSASLGDRVIITCQSSQSVYGNIWMAWYQ scFv
QKSGKAPKLLIYQASKLASGVPSRFSGSGSGAEFSLTIS (.alpha.-
SLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVLGGGGGS TNF-
GGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTA alpha)
SGFTISRSYWICWVRQAPGKGLEWVACIYGDNDITPLYA
NWAKGRFPVSTDTSKNTVYLQMNSLRAEDTAVYYCARLG YADYAYDLWGQGTLVTVSS 3
EP43max EIVMTQSPSTLSASVGDRVIIKCQASQSISDWLAWYQQK scFv
PGKAPKLLIYGASRLASGFPSRFSGSGSGAEFTLTISGL (.alpha.-
EPADFATYYCQQGWSDSYVDNLFGQGTKLTVLGGGGGSG TNF-
GGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVS alpha)
GFSLSSGAMSWVRQAPGKGLEWVGVIISSGATYYASWAK
GRFTISKDTSKNTVYLQMNSLRAEDTAVYYCARGGPDDS NSMGTFDPWGQGTLVTVSS 4
ESBA212 ADIVLTQSPSSLSASVGDRVTLTCRASSSVNYMHWYQQR scFv
PGKPPKALIYATSNLASGVPSRFSGSGSGTEFTLTISSL VL (.alpha.-
QPEDVAVYYCQQWRTNPPTFGQGTKLEVKRGGGGSGGGG Abeta)/
SGGGGSGGGGSQVQLVQSGPEVKKPGASVKVSCTASGYT WO
FTEYTMHWVRQAPGQGLEWMGGVNPYNDNTSYIRKLQGR 2009/
VTLTVDRSSSTAYMELTSLTSDDTAVYYCARYGGLRPYY 033309 FPMDFWGQGTLVTVSS 5
ESBA521 QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQ scFv
LPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITG (.alpha.-
LQTGDEADYYCGTWDSSLSGVVFGGGTKLTVLGGGGGSG ALK1)/
GGGSGGGGSSGGGSEVQLVESGGGLVQPGGSLRLSCAAS WO
GFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSV 2007/
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGIA 124610 VAGTGFDYWGQGTLVTVSS
33 ESBA903 EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQK scFv
PGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSL (.alpha.-
QPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGGS VEGF)
GGGGSGGGGSSGGGSEVQLVESGGGLVQPGGSLRLSCTA
SGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATW
AKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAGGDHN SGWGLDIWGQGTLVTVSS 6
ESBA105 QVQLVQSGAEVKKPGASVKVSCTASGYTFTHYGMNWVRQ VH
APGKGLEWMGWINTYTGEPTYADKFKDRFTFSLETSAST
VYMELTSLTSDDTAVYYCARERGDAMDYWGQGTLVTVSS 7 EP34max
EVQLVESGGGLVQPGGSLRLSCTASGFTISRSYWICWVR VH
QAPGKGLEWVACIYGDNDITPLYANWAKGRFPVSTDTSK
NTVYLQMNSLRAEDTAVYYCARLGYADYAYDLWGQGTLV TVSS 8 EP43max
EVQLVESGGGLVQPGGSLRLSCTVSGFSLSSGAMSWVRQ VH
APGKGLEWVGVIISSGATYYASWAKGRFTISKDTSKNTV
YLQMNSLRAEDTAVYYCARGGPDDSNSMGTFDPWGQGTL VTVSS 9 ESBA212
QVQLVQSGPEVKKPGASVKVSCTASGYTFTEYTMHWVRQ VH
APGQGLEWMGGVNPYNDNTSYIRKLQGRVTLTVDRSSST
AYMELTSLTSDDTAVYYCARYGGLRPYYFPMDFWGQGTL VTVSS 10 ESBA521
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQ VH
APGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCARDAGIAVAGTGFDYWGQGTL VTVSS 35 ESBA903
EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVR VH
QAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDTSKNT
VYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVT VSS 11 ESBA105
MADIVMTQSPSSLSASVGDRVTLTCTASQSVSNDVVWYQ VL
QRPGKAPKLLIYSAFNRYTGVPSRFSGRGYGTDFTLTIS
SLQPEDVAVYYCQQDYNSPRTFGQGTKLEVKR 12 EP34max
EIVMTQSPSTLSASLGDRVIITCQSSQSVYGNIWMAWYQ VL
QKSGKAPKLLIYQASKLASGVPSRFSGSGSGAEFSLTIS
SLQPDDFATYYCQGNFNTGDRYAFGQGTKLTVL 13 EP43max
EIVMTQSPSTLSASVGDRVIIKCQASQSISDWLAWYQQK VL
PGKAPKLLIYGASRLASGFPSRFSGSGSGAEFTLTISGL
EPADFATYYCQQGWSDSYVDNLFGQGTKLTVLG 14 ESBA212
ADIVLTQSPSSLSASVGDRVTLTCRASSSVNYMHWYQQR
PGKPPKALIYATSNLASGVPSRFSGSGSGTEFTLTISSL
QPEDVAVYYCQQWRTNPPTFGQGTKLEVKR 15 ESBA521
QSVLTQPPSVSAAPGQKVTISCSGSTSNIGDNYVSWYQQ VL
LPGTAPQLLIYDNTKRPSGIPDRFSGSKSGTSATLGITG
LQTGDEADYYCGTWDSSLSGVVFGGGTKLTVLG 34 ESBA903
EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQK VL (.alpha.-
PGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSL VEGF)
QPDDFATYYCQNVYLASTNGANFGQGTKLTVLG
TABLE-US-00006 TABLE 6 Linker Sequences Modified (MLS) and SEQ
Standard ID (SLS) Linker NO: Sequence Name: Sequence: 16 MLS 1
GGGGSGGGGSCGGGSGGGCGGGGSGGGGS 17 MLS 2 (Pep1)
GGGGSGGGGSCGAHWQFNALTVRCGGGGSG GGGS 18 MLS 3
GGGGSGGGGSC(X).sub.3-50CGGGGSGGGGS 19 MLS 4
(X).sub.3-15C(X).sub.3-50CG(X).sub.3-15 36 SLS 1
GGGGSGGGGSGGGGSGGGGS 37 SLS 2 GGGGSGGGGSGGGGSSGGGS
TABLE-US-00007 TABLE 7 Framework Sequences SEQ Frame- work ID
Sequence NO: Name: Sequence: 20 FW1.4
EVQLVESGGGLVQPGGSLRLSCAAS(X).sub.n=1- VH
.sub.50WVRQAPGKGLEWVS(X).sub.n=1- (a43)
.sub.50RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK(X).sub.n=1-
.sub.50WGQGTLVTVSS 21 FW1.4 EIVMTQSPSTLSASVGDRVIITC(X).sub.n=1- VL
.sub.50WYQQKPGKAPKLLIY(X).sub.n=1-50 (K127)
GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X).sub.n=1- .sub.50FGQGTKLTVLG 22
FW1.4 EIVMTQSPSTLSASVGDRVIITC(X).sub.n=1- scFv
.sub.50WYQQKPGKAPKLLIY(X).sub.n=1-50
GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X).sub.n=1- .sub.50FGQGTKLTVLG
GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR
LSCAAS(X).sub.n=1-50WVRQAPGKGLEWVS(X).sub.n=1-
.sub.50RFTISRDNSKNTLYLQMNSLRAEDTA VYYCAK(X).sub.n=1-5WGQGTLVTVSS 23
rFW1.4 EVQLVESGGGLVQPGGSLRLSCTAS(X).sub.n=1- VH
.sub.50WVRQAPGKGLEWVG(X).sub.n=1-50
RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X).sub.n=1- .sub.50WGQGTLVTVSS 24
rFW1.4 EIVMTQSPSTLSASVGDRVIITC(X).sub.n=1- VL =
.sub.50WYQQKPGKAPKLLIY(X).sub.n=1-50 rFW1.4v2
GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X).sub.n=1- VL .sub.50FGQGTKLTVLG
25 rFW1.4 EIVMTQSPSTLSASVGDRVIITC(X).sub.n=1- scFv
.sub.50WYQQKPGKAPKLLIY(X).sub.n=1-50
GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X).sub.n=1- .sub.50FGQGTKLTVLG
GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLR
LSCTAS(X).sub.n=1-50WVRQAPGKGLEWVG(X).sub.n=1-
.sub.50RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR(X).sub.n=1-
.sub.50WGQGTLVTVSS 26 rFW1.4 EVQLVESGGGLVQPGGSLRLSCTVS(X).sub.n=1-
(V2) .sub.50WVRQAPGKGLEWVG(X).sub.n=1-50 VH
RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR(X).sub.n=1- .sub.50WGQGTLVTVSS 27
rFW1.4 EIVMTQSPSTLSASVGDRVIITC(X).sub.n=1- (V2)
.sub.50WYQQKPGKAPKLLIY(X).sub.n=1-50 scFv
GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(X).sub.n=1-
.sub.50FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGS
EVQLVESGGGLVQPGGSLRLSCTVS(X).sub.n=1-50 WVRQAPGKGLEWVG(X).sub.n=1-
.sub.50RFTISKDTSKNIVYLQMNSLRAEDTAVYYCAR(X).sub.n=1-
.sub.50WGQGTLVTVSS
TABLE-US-00008 TABLE 8 Penetration Enhancer Sequences SEQ ID NO:
Sequence: 28 PLGPR 29 PLGPK 30 PLGPE 31 PLGPD 32 PLGP
EQUIVALENTS
[0081] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
371247PRTArtificial SequenceESBA105 scFv 1Met Ala Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg
Val Thr Leu Thr Cys Thr Ala Ser Gln Ser Val Ser 20 25 30 Asn Asp
Val Val Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu 35 40 45
Leu Ile Tyr Ser Ala Phe Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe 50
55 60 Ser Gly Arg Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu 65 70 75 80 Gln Pro Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp
Tyr Asn Ser 85 90 95 Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu
Val Lys Arg Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ser Gly Gly 115 120 125 Gly Ser Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro 130 135 140 Gly Ala Ser Val Lys
Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe Thr 145 150 155 160 His Tyr
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175
Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp 180
185 190 Lys Phe Lys Asp Arg Phe Thr Phe Ser Leu Glu Thr Ser Ala Ser
Thr 195 200 205 Val Tyr Met Glu Leu Thr Ser Leu Thr Ser Asp Asp Thr
Ala Val Tyr 210 215 220 Tyr Cys Ala Arg Glu Arg Gly Asp Ala Met Asp
Tyr Trp Gly Gln Gly 225 230 235 240 Thr Leu Val Thr Val Ser Ser 245
2253PRTArtificial SequenceEP34max scFv 2Glu Ile Val Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Ile Ile
Thr Cys Gln Ser Ser Gln Ser Val Tyr Gly Asn 20 25 30 Ile Trp Met
Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu 35 40 45 Leu
Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55
60 Ser Gly Ser Gly Ser Gly Ala Glu Phe Ser Leu Thr Ile Ser Ser Leu
65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Asn Phe
Asn Thr 85 90 95 Gly Asp Arg Tyr Ala Phe Gly Gln Gly Thr Lys Leu
Thr Val Leu Gly 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu
Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr 145 150 155 160 Ile Ser Arg
Ser Tyr Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys 165 170 175 Gly
Leu Glu Trp Val Ala Cys Ile Tyr Gly Asp Asn Asp Ile Thr Pro 180 185
190 Leu Tyr Ala Asn Trp Ala Lys Gly Arg Phe Pro Val Ser Thr Asp Thr
195 200 205 Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Tyr Ala
Asp Tyr Ala Tyr 225 230 235 240 Asp Leu Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 245 250 3253PRTArtificial SequenceEP43max scFv 3Glu
Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Ile Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Asp Trp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Phe Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr
Ile Ser Gly Leu Glu Pro 65 70 75 80 Ala Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe Gly
Gln Gly Thr Lys Leu Thr Val Leu Gly Gly 100 105 110 Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 130 135 140
Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Phe Ser Leu 145
150 155 160 Ser Ser Gly Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 165 170 175 Glu Trp Val Gly Val Ile Ile Ser Ser Gly Ala Thr
Tyr Tyr Ala Ser 180 185 190 Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys
Asp Thr Ser Lys Asn Thr 195 200 205 Val Tyr Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr 210 215 220 Tyr Cys Ala Arg Gly Gly
Pro Asp Asp Ser Asn Ser Met Gly Thr Phe 225 230 235 240 Asp Pro Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 4250PRTArtificial
SequenceESBA212 scFv 4Ala Asp Ile Val Leu Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Leu Thr Cys Arg
Ala Ser Ser Ser Val Asn Tyr 20 25 30 Met His Trp Tyr Gln Gln Arg
Pro Gly Lys Pro Pro Lys Ala Leu Ile 35 40 45 Tyr Ala Thr Ser Asn
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Val Ala Val Tyr Tyr Cys Gln Gln Trp Arg Thr Asn Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Val Lys Arg Gly Gly Gly Gly
100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 115 120 125 Gln Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys
Lys Pro Gly Ala 130 135 140 Ser Val Lys Val Ser Cys Thr Ala Ser Gly
Tyr Thr Phe Thr Glu Tyr 145 150 155 160 Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 165 170 175 Gly Gly Val Asn Pro
Tyr Asn Asp Asn Thr Ser Tyr Ile Arg Lys Leu 180 185 190 Gln Gly Arg
Val Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 195 200 205 Met
Glu Leu Thr Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys 210 215
220 Ala Arg Tyr Gly Gly Leu Arg Pro Tyr Tyr Phe Pro Met Asp Phe Trp
225 230 235 240 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250
5253PRTArtificial SequenceESBA521 scFv 5Gln Ser Val Leu Thr Gln Pro
Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser
Cys Ser Gly Ser Thr Ser Asn Ile Gly Asp Asn 20 25 30 Tyr Val Ser
Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Gln Leu Leu 35 40 45 Ile
Tyr Asp Asn Thr Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln
65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser
Ser Leu 85 90 95 Ser Gly Val Val Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ser Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln 130 135 140 Pro Gly Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 145 150 155 160 Ser Ser Tyr
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 165 170 175 Glu
Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala 180 185
190 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
195 200 205 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Asp Ala Gly Ile Ala Val Ala
Gly Thr Gly Phe 225 230 235 240 Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 245 250 6117PRTArtificial SequenceESBA105 VH 6Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe Thr His Tyr
20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr
Ala Asp Lys Phe 50 55 60 Lys Asp Arg Phe Thr Phe Ser Leu Glu Thr
Ser Ala Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Thr Ser Leu Thr Ser
Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Gly Asp
Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser
Ser 115 7121PRTArtificial SequenceESBA34max VH 7Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Thr Ala Ser Gly Phe Thr Ile Ser Arg Ser 20 25 30 Tyr
Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40
45 Val Ala Cys Ile Tyr Gly Asp Asn Asp Ile Thr Pro Leu Tyr Ala Asn
50 55 60 Trp Ala Lys Gly Arg Phe Pro Val Ser Thr Asp Thr Ser Lys
Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Leu Gly Tyr Ala Asp Tyr
Ala Tyr Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 8122PRTArtificial SequenceESBA43max VH 8Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Gly 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Gly Val Ile Ile Ser Ser Gly Ala Thr Tyr Tyr Ala Ser Trp Ala
Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr
Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Pro Asp Asp Ser Asn Ser
Met Gly Thr Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 9122PRTArtificial SequenceESBA212 VH 9Gln Val
Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Val Ser Cys Thr Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20
25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Gly Val Asn Pro Tyr Asn Asp Asn Thr Ser Tyr Ile
Arg Lys Leu 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Arg Ser
Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Thr Ser Leu Thr Ser Asp
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Gly Gly Leu Arg
Pro Tyr Tyr Phe Pro Met Asp Phe Trp 100 105 110 Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 10122PRTArtificial SequenceESBA521 VH
10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Gly
Ile Ala Val Ala Gly Thr Gly Phe Asp Tyr Trp 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 11110PRTArtificial
SequenceESBA105 VL 11Met Ala Asp Ile Val Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Leu Thr Cys
Thr Ala Ser Gln Ser Val Ser 20 25 30 Asn Asp Val Val Trp Tyr Gln
Gln Arg Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Ala
Phe Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Arg
Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln
Pro Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Ser 85 90
95 Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Val Lys Arg 100 105
110 12111PRTArtificial SequenceEP34max VL 12Glu Ile Val Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Ile
Ile Thr Cys Gln Ser Ser Gln Ser Val Tyr Gly Asn 20 25 30 Ile Trp
Met Ala Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys Leu 35 40 45
Leu Ile Tyr Gln Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50
55 60 Ser Gly Ser Gly Ser Gly Ala Glu Phe Ser Leu Thr Ile Ser Ser
Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Asn
Phe Asn Thr 85 90 95 Gly Asp Arg Tyr Ala Phe Gly Gln Gly Thr Lys
Leu Thr Val Leu 100 105 110 13111PRTArtificial SequenceEP43max VL
13Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Ile Ile Lys Cys Gln Ala Ser Gln Ser Ile Ser Asp
Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Arg Leu Ala Ser Gly Phe Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu
Thr Ile Ser Gly Leu Glu Pro 65 70 75 80 Ala Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Gly Trp Ser Asp Ser Tyr 85 90 95 Val Asp Asn Leu Phe
Gly Gln Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
14108PRTArtificial SequenceESBA212 VL 14Ala Asp Ile Val Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr
Leu Thr Cys Arg Ala Ser Ser Ser Val Asn Tyr 20 25 30 Met His Trp
Tyr Gln Gln Arg Pro Gly Lys Pro Pro Lys Ala Leu
Ile 35 40 45 Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln
Gln Trp Arg Thr Asn Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Leu Glu Val Lys Arg 100 105 15111PRTArtificial SequenceESBA521 VL
15Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1
5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Thr Ser Asn Ile Gly Asp
Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Gln Leu Leu 35 40 45 Ile Tyr Asp Asn Thr Lys Arg Pro Ser Gly Ile
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr
Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr
Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Gly Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
1629PRTArtificial Sequencelinker 16Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Cys Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Cys Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser 20 25 1734PRTArtificial Sequencelinker
17Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Gly Ala His Trp Gln 1
5 10 15 Phe Asn Ala Leu Thr Val Arg Cys Gly Gly Gly Gly Ser Gly Gly
Gly 20 25 30 Gly Ser 1872PRTArtificialSynthetic peptide 18Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Cys Gly Gly 50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser 65 70
1983PRTArtificialSynthetic peptide 19Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55
60 Xaa Xaa Cys Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80 Xaa Xaa Xaa 20232PRTArtificialSynthetic peptide 20Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa
20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Trp Val Arg Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Val
Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser 130 135 140
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 145
150 155 160 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Xaa Xaa Xaa
Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Trp Gly Gln 210 215 220 Gly Thr Leu Val Thr Val
Ser Ser 225 230 21231PRTArtificialSynthetic peptide 21Glu Ile Val
Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Ile Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln
Gln Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly
Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155
160 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa
165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly 225
230 22483PRTArtificialSynthetic peptide 22Glu Ile Val Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile
Ile Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50
55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro
Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser
Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180
185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe
Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly
Ser Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 260 265 270 Cys Ala Ala Ser
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305
310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 325 330 335 Glu Trp Val Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 385 390 395 400 Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 405 410 415 Val
Tyr Tyr Cys Ala Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425
430 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
435 440 445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 450 455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly
Thr Leu Val Thr 465 470 475 480 Val Ser Ser
23232PRTArtificialSynthetic peptide 23Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Thr Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55
60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala
65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Val Gly Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Arg Phe Thr Ile Ser 130 135 140 Arg Asp Thr Ser Lys Asn
Thr Val Tyr Leu Gln Met Asn Ser Leu Arg 145 150 155 160 Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185
190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp
Gly Gln 210 215 220 Gly Thr Leu Val Thr Val Ser Ser 225 230
24231PRTArtificialSynthetic peptide 24Glu Ile Val Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile
Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55
60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly
65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp
Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185
190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly
Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly 225 230
25483PRTArtificialSynthetic peptide 25Glu Ile Val Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile
Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55
60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly
65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp
Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185
190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly
Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser
Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser 260 265 270 Cys Thr Ala Ser Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310
315 320 Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 325 330 335 Glu Trp Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Arg
Phe Thr Ile Ser Arg Asp Thr Ser Lys 385 390 395 400 Asn Thr Val Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 405 410 415 Val Tyr
Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435 440 445 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 450 455 460 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Trp Gly Gln Gly Thr Leu Val Thr 465 470 475 480 Val
Ser Ser 26232PRTArtificialSynthetic peptide 26Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Thr Val Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40
45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg
Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Val Gly Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Arg Phe Thr Ile Ser 130 135 140 Lys Asp Thr Ser
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg 145 150 155 160 Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa 165 170
175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Trp Gly Gln 210 215 220 Gly Thr Leu Val Thr Val Ser Ser 225 230
27483PRTArtificialSynthetic peptide 27Glu Ile Val Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Ile Ile
Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55
60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln Lys Pro Gly
65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160 Gln Pro Asp
Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 180 185
190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly
Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly Gly Gly Gly Gly Ser
Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser 260 265 270 Cys Thr Val Ser Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310
315 320 Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 325 330 335 Glu Trp Val Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Arg
Phe Thr Ile Ser Lys Asp Thr Ser Lys 385 390 395 400 Asn Thr Val Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 405 410 415 Val Tyr
Tyr Cys Ala Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 420 425 430
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 435
440 445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 450 455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly Gln Gly Thr
Leu Val Thr 465 470 475 480 Val Ser Ser 285PRTArtificial
Sequencepenetration enhancer 28Pro Leu Gly Pro Arg 1 5
295PRTArtificial Sequencepenetration enhancer 29Pro Leu Gly Pro Lys
1 5 305PRTArtificial Sequencepenetration enhancer 30Pro Leu Gly Pro
Glu 1 5 315PRTArtificial Sequencepenetration enhancer 31Pro Leu Gly
Pro Asp 1 5 324PRTArtificial Sequencepenetratin enhancer 32Pro Leu
Gly Pro 1 33252PRTArtificial SequenceESBA903 scFv 33Glu Ile Val Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Ile Ile Thr Cys Gln Ala Ser Glu Ile Ile His Ser Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val
Tyr Leu Ala Ser Thr 85 90 95 Asn Gly Ala Asn Phe Gly Gln Gly Thr
Lys Leu Thr Val Leu Gly Gly 100 105 110 Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ser 115 120 125 Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser 145 150 155 160
Leu Thr Asp Tyr Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys 165
170 175 Gly Leu Glu Trp Val Gly Phe Ile Asp Pro Asp Asp Asp Pro Tyr
Tyr 180 185 190 Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp
Thr Ser Lys 195 200 205 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala 210 215 220 Val Tyr Tyr Cys Ala Gly Gly Asp His
Asn Ser Gly Trp Gly Leu Asp 225 230 235 240 Ile Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 245 250 34111PRTArtificial SequenceESBA903
VL 34Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
Gly 1 5 10 15 Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Glu Ile Ile
His Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45 Tyr Leu Ala Ser Thr Leu Ala Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ala Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr
Tyr Tyr Cys Gln Asn Val Tyr Leu Ala Ser Thr 85 90 95 Asn Gly Ala
Asn Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly 100 105 110
35120PRTArtificial SequenceESBA903 VH 35Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Thr Ala Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30 Tyr Tyr Met
Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val
Gly Phe Ile Asp Pro Asp Asp Asp Pro Tyr Tyr Ala Thr Trp Ala 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Gly Gly Asp His Asn Ser Gly Trp Gly Leu Asp
Ile Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
3620PRTArtificial Sequencelinker 36Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20
3720PRTArtificial Sequencelinker 37Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ser 1 5 10 15 Gly Gly Gly Ser 20
* * * * *
References