U.S. patent application number 13/473353 was filed with the patent office on 2012-10-04 for antigens associated with inflammatory bowel disease including ulcerative colitis.
Invention is credited to Kathrin Schwager.
Application Number | 20120251439 13/473353 |
Document ID | / |
Family ID | 41786309 |
Filed Date | 2012-10-04 |
United States Patent
Application |
20120251439 |
Kind Code |
A1 |
Schwager; Kathrin |
October 4, 2012 |
Antigens Associated with Inflammatory Bowel Disease Including
Ulcerative Colitis
Abstract
Specific binding members that bind the ED-A isoform of
fibronectin for use in methods of diagnosis, detection, imaging
and/or treatment of inflammatory bowel disease such as ulcerative
colitis and/or for use in delivery to the neovasculature of
intestinal tissue of a molecule conjugated to the specific binding
member. Specific binding members that bind tenascin-C, especially
the A1, A2, A3, A4 and/or D domain tenascin-C large isoform, for
use in methods of diagnosis, detection, imaging and/or treatment of
ulcerative colitis and/or for use in delivery to the neovasculature
of inflammatory bowel disease tissue of a molecule conjugated to
the specific binding member.
Inventors: |
Schwager; Kathrin; (Aadorf,
CH) |
Family ID: |
41786309 |
Appl. No.: |
13/473353 |
Filed: |
May 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13140492 |
Jun 17, 2011 |
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PCT/EP09/09282 |
Dec 28, 2009 |
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13473353 |
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61142962 |
Jan 7, 2009 |
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Current U.S.
Class: |
424/1.11 ;
424/130.1; 424/135.1; 424/136.1; 424/85.1; 424/85.2; 424/85.5;
424/85.6; 424/85.7; 424/9.1; 514/1.1 |
Current CPC
Class: |
A61K 47/6843 20170801;
A61P 29/00 20180101; A61P 17/06 20180101; A61P 17/00 20180101; C07K
2317/56 20130101; A61K 51/1018 20130101; C07K 2317/626 20130101;
C07K 16/18 20130101; C07K 2317/622 20130101; C07K 2317/567
20130101; A61K 2039/505 20130101; C07K 2317/21 20130101; A61P 1/00
20180101; C07K 2317/565 20130101 |
Class at
Publication: |
424/1.11 ;
424/9.1; 424/85.1; 424/85.2; 424/85.5; 424/85.6; 424/85.7;
424/130.1; 424/135.1; 424/136.1; 514/1.1 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 38/19 20060101 A61K038/19; A61K 38/20 20060101
A61K038/20; A61K 38/00 20060101 A61K038/00; A61K 39/395 20060101
A61K039/395; A61P 1/00 20060101 A61P001/00; A61P 29/00 20060101
A61P029/00; A61K 49/00 20060101 A61K049/00; A61K 38/21 20060101
A61K038/21 |
Claims
1-86. (canceled)
87. A method of detecting or diagnosing inflammatory bowel disease
in a human or animal, wherein the method comprises the steps of (a)
administering a specific binding member which binds the ED-A domain
of fibronectin; and (b) determining the presence or absence of the
specific binding member in sites of bowel inflammation in the human
or animal body; wherein localization of the specific binding member
to sites of inflammation indicates the presence of inflammatory
bowel disease in said human or animal.
88. The method of claim 87, wherein the specific binding member is
conjugated to a detectable label or a radioisotope.
89. The method of claim 88 comprising imaging said site of
inflammation.
90. The method of claim 87, wherein the specific binding member
comprises a VH domain comprising a framework and a set of
complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein
HCDR1 has amino acid sequence SEQ ID NO: 42, HCDR2 has amino acid
sequence SEQ ID NO: 56, HCDR3 has amino acid sequence SEQ ID NO 60;
or wherein the VH domain comprises a set of complementarity
determining regions having ten or fewer amino acid substitutions
within the complementarity determining regions HCDR1, HCDR2 and
HCDR3.
91. The method of claim 90, wherein the VH domain framework is a
human germline framework, and wherein the VH domain optionally has
amino acid sequence SEQ ID NO: 14 or 16.
92. The method claim 87, wherein the specific binding member
further comprises a VL domain comprising a set of complementarity
determining regions LCDR1, LCDR2 and LCDR3 and a framework.
93. The method of claim 92, wherein LCDR1 has amino acid sequence
SEQ ID NO: 102, LCDR2 has amino acid sequence SEQ ID NO: 114, and
LCDR3 has amino acid sequence SEQ ID NO: 118; or wherein the VL
domain comprises a set of complementarity determining regions
having ten or fewer amino acid substitutions within the
complementarity determining regions LCDR1, LCDR2 and LCDR3.
94. The method of claim 92, wherein the VL domain framework is a
human germline framework, wherein the VL domain optionally has
amino acid sequence SEQ ID NO: 76 or 78.
95. The method of claim 92, wherein the specific binding member
comprises a single chain Fv.
96. The method of claim 95, wherein the binding member is a small
immunoprotein (SIP).
97. The method of claim 92, wherein the specific binding member is
a diabody.
98. The method of claim 87, wherein said inflammatory bowel disease
is ulcerative colitis and said binding member is anti-ED-A F8
antibody.
99. A method of treating inflammatory bowel disease in a patient,
the method comprising administering to a patient a therapeutically
effective amount of a medicament comprising a specific binding
member which binds the ED-A isoform of fibronectin.
100. The method of claim 99, wherein the specific binding member is
conjugated to a detectable label or a radioisotope.
101. The method of claim 99, wherein the specific binding member is
conjugated to a bioactive molecule, such as a cytokine, hormone, a
therapeutic radioisotope or a cytotoxic drug, and wherein the
bioactive molecule is optionally conjugated to the specific binding
member by a cleavable linker.
102. The method of claim 99, wherein said cytokine is selected from
the group consisting of a cytokine selected from the group
consisting of IL-10, TGF-beta, IL-2, IL-12, IL-15, IL-21, IL-24,
IL-33, tumour necrosis factor (TNF), interferon-alpha,
interferon-beta and interferon-gamma.
103. The method of claim 99, wherein the specific binding member
comprises a VH domain comprising a framework and a set of
complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein
HCDR1 has amino acid sequence SEQ ID NO: 42, HCDR2 has amino acid
sequence SEQ ID NO: 56, HCDR3 has amino acid sequence SEQ ID NO 60;
or wherein the VH domain comprises a set of complementarity
determining regions having ten or fewer amino acid substitutions
within the complementarity determining regions HCDR1, HCDR2 and
HCDR3.
104. The method of claim 103, wherein the VH domain framework is a
human germline framework, and wherein the VH domain optionally has
amino acid sequence SEQ ID NO: 14 or 16.
105. The method claim 99, wherein the specific binding member
further comprises a VL domain comprising a set of complementarity
determining regions LCDR1, LCDR2 and LCDR3 and a framework.
106. The method of claim 105, wherein LCDR1 has amino acid sequence
SEQ ID NO: 102, LCDR2 has amino acid sequence SEQ ID NO: 114, and
LCDR3 has amino acid sequence SEQ ID NO: 118; or wherein the VL
domain comprises a set of complementarity determining regions
having ten or fewer amino acid substitutions within the
complementarity determining regions LCDR1, LCDR2 and LCDR3.
107. The method of claim 106, wherein the VL domain framework is a
human germline framework, wherein the VL domain optionally has
amino acid sequence SEQ ID NO: 76 or 78.
108. The method of claim 99, wherein the specific binding member
comprises a single chain Fv.
109. The method of claim 108, wherein the binding member is a small
immunoprotein (SIP).
110. The method of claim 99, wherein the specific binding member is
a diabody.
111. The method of claim 102, wherein said inflammatory bowel
disease is ulcerative colitis and said binding member is anti-ED-A
F8 antibody and said cytokine is IL-10.
112. The method of claim 109, wherein the binding member is a small
immunoprotein (SIP).
Description
[0001] The present invention relates to the detection and treatment
of endometriosis, psoriatic arthritis and psoriasis. The invention
involves use of a specific binding member that binds the ED-A
isoform of fibronectin, especially a specific binding member that
binds domain ED-A of fibronectin, or a specific binding member that
binds tenascin-C, especially the A1, A2, A3, A4, B and/or D domain
of tenascin C (the "long form" of tenascin-C).
[0002] Most conventional pharmaceuticals currently in use for the
treatment of angiogenesis-related diseases (such as cancer,
arthritis, etc.) do not selectively accumulate at the site of
disease [Bosslet et al., 58, 1195-1201 Cancer Res. (1998)]. For
example, intravenously administered drugs distribute evenly within
the different organs and tissues of the body, rather than
selectively accumulating at the site of disease.
[0003] One approach to circumvent the disadvantages of conventional
pharmacological therapies involves the preferential delivery of a
bioactive agent to the tumor site by means of a binding molecule
specific for a pathology-associated marker [Neri & Bicknell
(2005) Nature Rev. Cancer]. The selective targeting of the drug to
the diseased tissue will ultimately result in an increased local
concentration at its site of action, sparing normal organs from the
toxic effects of the bioactive agent used to confer a
pharmacological benefit (e.g., a cytotoxic drug, a cytokine, a
radionuclide, a photosensitizer). In most cases, this will lead to
an improved therapeutic index of the delivered pharmaceutical, i.e.
a higher efficacy with minimized side effects. Indeed, the
favourable toxicity profile of site-specific therapeutics may open
new avenues in the therapy of angiogenesis-related diseases,
allowing the systemic administration of highly potent and promising
agents, which are currently either given at suboptimal doses or
whose clinical application has to date been impeded by unacceptable
toxicities when applied in an unmodified form.
[0004] Ligand-based pharmacodelivery strategies fundamentally rely
on the identification of good-quality markers of pathology,
allowing a clear-cut discrimination between diseased tissues and
healthy organs. Monoclonal antibodies and their fragments represent
the preferred agents for pharmacodelivery applications [Rybak et
al. 2, 22-40 Chem. Med. Chem. (2007); Shrama et al., 5, 147-159
Nat. Rev. Drug Discovery (2006)], even though globular protein
mutants [Binz and Pluckthun, 23, 1257-1268 Nature Biotechnology
(2005)], peptides [Sergeeva et al., 58, 1622-1654, Adv. Drug.
Deliv. Rev. (2006)] and even small organic ligands [Low et al., 41,
120-129, Acc. Chem. Res. (2008)] are increasingly being used. Most
efforts in the field of disease targeting have been made using
specific markers expressed on the surface of diseased cells (e.g.,
on the surface of tumour cells in cancer). However, targeting
antigens on diseased cells themselves is a complex task for
blood-borne agents, facing a number of physical and kinetic
barriers, which may prevent efficient pharmacodelivery. These
barriers include interstitial pressure at site of disease,
relatively long diffusion distances within the interstitium,
heterogeneity of antigens at sites of disease, as well as the
so-called "antigen barrier" [Dennis et al., 67, 254-261, Cancer
Res. (2007)]. All these factors significantly impair deep tissue
penetration.
[0005] Proteins which are expressed around pathological blood
vessels at sites of disease, but which are either absent or present
at reduced levels in normal tissue, represent particularly
attractive targets for the development of selective and efficient
pharmacodelivery strategies [Rybak et al. (2007) ChemMedChem;
Trachsel et al., 9, R9, Arthritis Res. Ther. (2007)]. Vascular
targets are often more easily accessible from the bloodstream to
systemically administered agents, overcoming the problem of access
and allowing an efficient delivery of the compound to the site of
disease. Furthermore, the same vascular targeting agent could be
useful not only for the delivery of therapeutic agents but also for
molecular imaging applications.
[0006] As used herein, the term "vascular targeting" is not used to
indicate the inhibition of the target molecule in a signalling
pathway (e.g. the inhibition of VEGF signalling by bevacizumab or
the inhibition of the BCR/ABL kinase by imatinib). Instead, the
target molecule expressed on the vasculature at sites of
angiogenesis-related diseases is used as an easily accessible
binding site for specific ligands, exploiting the vasculature as a
scaffold to achieve a site-specific localization of effector
molecules at the tumour tissue. This fundamental conceptual
difference is clear from the fact that, while some vascular marker
molecules have been shown to be very suitable for ligand-directed
tumour targeting applications, their pathophysiological role is
still largely unknown (for example, the extra-domain B of
fibronectin; see below).
[0007] The present inventors have previously performed extensive
work on the antibody-based targeting of markers of tumor
angiogenesis, such as the extra-domain B of fibronectin (ED-B)
(Schliemann and Neri, 1776, 175-192, Biochim Biophys Acta (2008).
ED-B is virtually undetectable in normal adult tissues, except for
the endometrium in the proliferative phase, but becomes
over-expressed in conditions involving tissue remodelling, with a
prominent peri-vascular pattern of staining. Three derivatives of
the human anti-EDB antibody L19 [Pini et al. (1998) J. Biol. Chem.]
are currently being investigated in Phase I and Phase II clinical
trials for cancer therapy (the radiolabelled product L19-.sup.131I
and the immunocytokines L19-IL2 and L19-TNF). The ability of L19 to
selectively localize at angiogenic sites in non-tumoral diseases
has been studied in animal models of ocular neo-vasculature
[Birchler et al. 17, 984-988, Nature Biotechnology (1999)],
rheumatoid arthritis [Trachsel et al., 9, R9, Arthritis Res. Ther.
(2007)], psoriasis [Trachsel et al., 127, 881-886, J. Inv.
Dermatol. (2007)] and atherosclerosis [Matter et al., 95, 1225-1233
Circulation Res. (2004)].
[0008] More recently, the present inventors have studied other
alternatively spliced domains of extracellular matrix components,
whose patterns of expression are less well characterized and which
have so far been used mainly as vascular targets for tumour
targeting applications: the ED-A domain of fibronectin [Rybak et
al., 67, 10948-10957 (2007) Cancer Res.] and the domain A1 of
tenascin-C [Brack et al., 12, 3200-3208, (2006) Clin. Cancer Res.].
ED-A is specifically recognized by the human monoclonal antibody F8
[Villa et al., 122, 2405-2413 (2008) Int. J. Cancer], while the
human monoclonal antibody F16 binds the A1 domain of tenascin-C
[Brack et al. (2006) Clin. Cancer Res.].
[0009] The pattern of expression of the extra-domains A1, A2, A3,
A4, B and D of tenascin-C are similar, being almost undetectable in
normal adult tissues, but strongly up-regulated in a multitude of
different tumours [Brack et al. (2006) Clin. Cancer Res.; Pedretti
et al. (2008) Lung Cancer, in press; Berndt et al., 132, 537-546, J
Cancer Res Clin Oncol (2006); Balza et al., 261, 175-178, FEBS
Lett. (1990)]. The term "tenascin-C large isoform" is often used to
indicate the form of tenascin-C containing the extra-domains A1,
A2, A3, A4, B and D [Borsi et al., 270, 6243-6245 (1995) J. Biol.
Chem.; Borsi et al., 66, 632-635 (1996) Int. J. Cancer; Carnemolla
et al., 154, 1345-1352 (1999) Am. J. Pathol.]. By contrast, the
expression of the extra-domain C of tenascin-C is more restricted,
being undetectable in normal adult tissues and being found only in
certain tumour types, mainly lung cancer and high-grade
astrocytomas [Carnemolla et al. (1999) Am. J. Pathol.].
[0010] Thus, antibody-based targeted delivery of bioactive agents
to sites of angiogenesis is an attractive therapeutic strategy for
cancer treatment, but is largely unexplored for chronic
inflammatory diseases. We have previously demonstrated that the
ED-B domain of fibronectin, a marker of angiogenesis, is expressed
in psoriatic lesions in patients and in a mouse model of psoriasis
as well as in arthritic paws in the collagen-induced mouse model of
rheumatoid arthritis. Using both radioactive and fluorescent
techniques, the human monoclonal antibody L19, specific to EDB, was
found to selectively localize at sites of inflammation in vivo,
following intravenous administration. These results suggest a
therapeutic potential for the L19-based selective delivery of
bioactive compounds to sites of inflammation (Trachsel, 2007;
PCT/EP2007/004044).
[0011] It has also previously been shown by in-situ-hybridisation
that the ED-A domain of fibronectin can be present in human
arthritic specimens (Berndt et al., 1998; Kriegsmann et al., 2004),
and the inventors have previously shown the expression of ED-A in
rheumatoid arthritis [PCT/EP2008/009070].
[0012] We show herein the patterns of expression of ED-A and of
tenascin-C large in other angiogenesis-related non-tumoural
diseases, such as multiple sclerosis, psoriatic arthritis,
psoriasis, inflammatory bowel diseases and endometriosis, using
identical concentrations of biotinylated versions of the F8 and F16
antibodies in SIP format [Borsi et al., 102, 75-85 (2002) Int. J.
Cancer; Villa et al., 122, 2405-2413 (2008) Int. J. Cancer; Brack
et al., 12, 3200-3208, (2006) Clin. Cancer Res.]. These diseases
are all associated with angiogenesis and are socially very
relevant. Ligand-based pharmacodelivery may open new diagnostic and
therapeutic opportunities for these diseases.
[0013] Multiple sclerosis is an autoimmune disease in which the
immune system attacks the nervous system, resulting in
demyelination of neurones (Compston and Coles, 359, 1221-1231,
Lancet, 2002). As well as demyelination, multiple sclerosis is also
characterised by inflammation. There is no known cure for multiple
sclerosis and many existing medications can have adverse side
effects or be poorly tolerated.
[0014] Psoriasis is a disease which affects the skin and joints,
usually by causing red, scaly psoriatic plaques to occur on the
skin. These psoriatic plaques are areas of inflammation. When
psoriasis causes inflammation of the joints, it is known as
psoriatic arthritis. More effective treatments of psoriasis and
psoriatic arthritis are required and one approach would be the
targeted delivery of anti-inflammatory cytokines, such as IL-10 or
TGF-.beta., photosensitisers or cytotoxic drugs with cleavable
linkers. Such an approach requires effective targeting of drugs to
areas of inflammation, and the identification of specific targets
expressed in these regions.
[0015] The main forms of inflammatory bowel disease are Crohn's
disease and ulcerative colitis. Crohn's disease can affect any part
of the gastrointestinal tract, whereas ulcerative colitis is
restricted to the colon and rectum (Summers et al., 2003).
Depending on its severity, treatment of ulcerative colitis may
require immunosuppression to control its symptoms and treatment
usually involves the administration of anti-inflammatory
molecules.
[0016] Endometriosis is a common medical condition in women, and is
characterized by growth beyond or outside the uterus of tissue
resembling endometrium, which normally lines the uterus (Rock and
Markham, 340, 1264-1267 (1992) Lancet).
[0017] Endometriosis is typically seen during the reproductive
years, and it has been estimated that it occurs in approximately 5%
to 10% of women. Its main, but not universal, symptom is pelvic
pain in various manifestations. Further, endometriosis is common in
women with infertility (Buyalos and Agarwal, 12, 377-381, (2000)
Curr Opin Obstet Gynecol).
[0018] A major symptom of endometriosis is severe recurring pelvic
pain. The amount of pain a woman feels is not necessarily related
to the extent or stage (1 through to 4) of endometriosis. Some
women will have little or no pain despite having extensive
endometriosis affecting large areas or having endometriosis with
scarring. On the other hand, women may have severe pain even though
they have only a few small areas of endometriosis (Muse K, 31,
813-822 (1988) Clin Obstet Gynecol).
[0019] Typical endometriotic lesions show histological features
similar to endometrium, namely stroma and endometrial epithelium
and glands that respond to hormonal stimuli. Older lesions may
display no glands, but residual hemosiderin deposits. To the eye,
lesions appear dark blue or powder-burn black and vary in size;
some other lesions are red, white, or non-pigmented.
[0020] Additionally other lesions may be present, notably
endometriomas of the ovary, scar formation, and peritoneal defects
or pockets. As normal appearing peritoneum of infertile women
reveals endometriosis on biopsy in 6-13% of cases, some lesions may
not be visible to the eye.
[0021] A health history and a physical examination can in many
patients lead the physician to suspect endometriosis.
[0022] Use of imaging tests may identify larger endometriotic
areas, such as nodules or endometriotic cysts. The two most common
imaging tests are ultrasound and magnetic resonance imaging (MRI).
However, normal results on these tests do not eliminate the
possibility of endometriosis, as areas of endometriosis are often
too small to be seen by these tests.
[0023] The only way to confirm and diagnose endometriosis is by
laparoscopy, or other types of surgery. The diagnosis is based on
the characteristic appearance of the disease, and is corroborated
by a biopsy, if necessary. Laparoscopy also allows for surgical
treatment of endometriosis (Brosens I., 15, 229-233 (1997) Semin
Reprod Endocrinol).
[0024] For this reason, the discovery of good-quality vascular
markers of endometriosis provides new opportunities for imaging
endometriosis (e.g. through the ligand-mediated delivery of
radionuclides and radioisotopes), and for the pharmacodelivery to
endometriotic tissue of bioactive molecules (such as cytokines,
hormones, therapeutic radionuclides, or drugs with cleavable
linkers).
[0025] We show herein that an anti-tenascin-C antibody, such as the
F16 antibody disclosed herein, is able to give a stronger staining
pattern on human endometriotic tissue than the anti-ED-B antibody
L19. Similarly, an anti-ED-A antibody, such as the F8 antibody
disclosed herein, is also able to give a stronger staining pattern
on human endometriotic tissue than the anti-ED-B antibody L19,
although the staining seen with the F8 antibody is not as intense
as that seen with the F16 antibody.
[0026] We also shown herein that an anti-tenascin-C antibody, such
as the F16 antibody disclosed herein, is able to give a stronger
staining pattern on human psoriatic arthritic tissue than the
anti-ED-B antibody L19, or the anti-ED-A antibody F8.
[0027] However, we show herein that staining of samples from
patients with ulcerative colitis is virtually negative for the
anti-tenascin-C antibody F16, the anti-ED-A antibody F8 and the
anti-ED-B antibody L19, with only a weak positivity being observed
with F8 in some specimens.
[0028] Analysis of tissue sections from pathological specimens of
patients with multiple sclerosis also revealed only very weak
positivity with the anti-tenascin-C antibody F16, the anti-ED-A
antibody F8 and the anti-ED-B antibody L19.
[0029] In the case of components of the modified subendothelial
matrix, in vivo targeting performance of antibody derivatives
correlates with abundant antigen expression [Borsi et al., 102,
75-85 (2002) Int. J. Cancer; Demartis et al., 28, 534-539 (2001)
Eur. J. Nucl. Med.; Tarli et al., 94, 192-198 (1999) Blood; Viti et
al., 59, 347-352, (1999) Cancer Res.]. Based on the findings
described above, we found that the expression of ED-A and of the
tenascin-C "large" isoform is not found in all angiogenesis-related
diseases, and that endometriosis appears to be particularly suited
for pharmacodelivery using anti-ED-A antibody molecules, while
endometriosis, psoriatic arthritis and psoriasis appear to be
particularly suited for pharmacodelivery using anti-tenascin-C
antibody molecules.
[0030] Accordingly, ED-A of fibronectin is indicated as a vascular
marker of endometriosis, while tenascin-C (in particular, the
"large" isoform of tenascin-C) is indicated as a vascular marker of
endometriosis, psoriatic arthritis and psoriasis.
[0031] Specific binding members, such as antibody molecules that
bind the ED-A of fibronectin, represent novel agents which may be
used for the treatment of endometriosis, while specific binding
members, such as antibody molecules that bind the "large" isoform
of tenascin-C, represent novel agents which may be used for the
treatment of endometriosis, psoriatic arthritis, or psoriasis.
[0032] In a first aspect, the invention provides a specific binding
member, e.g. an antibody molecule, that binds the Extra Domain-A
(ED-A) isoform of fibronectin (A-FN) for use in a method of
treatment of endometriosis. The invention also provides the use of
a specific binding member, e.g. an antibody molecule, that binds
the Extra Domain-A (ED-A) isoform of fibronectin for the
manufacture of a medicament for treating endometriosis. The
invention also provides a method of treating endometriosis in a
patient, the method comprising administering to a patient a
therapeutically effective amount of a medicament comprising a
specific binding member which binds the ED-A isoform of
fibronectin. Preferably, the specific binding member binds the ED-A
isoform of human fibronectin.
[0033] The specific binding member, e.g. an antibody molecule, for
use in this first aspect of the invention, may bind the ED-A of
fibronectin.
[0034] The specific binding member e.g. an antibody molecule, for
use in this first aspect of the invention, may be conjugated to a
detectable label, a radioisotope, or a bioactive molecule, such as
a cytokine, a hormone, a therapeutic radioisotope or a cytotoxic
drug. The specific binding member may be conjugated to the
bioactive molecule by a cleavable linker.
[0035] In a second aspect, the invention provides a specific
binding member, e.g. an antibody molecule, that binds the ED-A
isoform of fibronectin for use in the delivery to the
neovasculature of endometriotic tissue of a molecule conjugated to
the specific binding member. The invention also provides the use of
a specific binding member, e.g. an antibody molecule, that binds
the ED-A isoform of fibronectin for the manufacture of a medicament
for delivery to the neovasculature of endometriotic tissue of a
molecule conjugated to the specific binding member. The invention
also provides a method of delivering a molecule to the
neovasculature of endometriotic tissue in a human or animal,
wherein the molecule is conjugated to a specific binding member
which binds the ED-A isoform of fibronectin to form a conjugate and
the method comprises administering the conjugate to the human or
animal. Preferably, the specific binding member binds the ED-A
isoform of human fibronectin.
[0036] The specific binding member, e.g. an antibody molecule, for
use in this second aspect of the invention, may bind the ED-A of
fibronectin.
[0037] The specific binding member e.g. an antibody molecule, for
use in this second aspect of the invention, may be conjugated to a
detectable label, a radioisotope, or a bioactive molecule, such as
a cytokine, a hormone, a therapeutic radioisotope or a cytotoxic
drug. The specific binding member may be conjugated to the
bioactive molecule by a cleavable linker.
[0038] In a third aspect, the invention provides a specific binding
member, e.g. an antibody molecule, that binds the ED-A isoform of
fibronectin for use in a method of diagnosis of endometriosis. The
invention also provides use of a specific binding member that binds
the ED-A isoform of fibronectin for the manufacture of a diagnostic
product for diagnosing endometriosis. The invention also provides a
method of detecting or diagnosing endometriosis in a human or
animal, wherein the method comprises the steps of: [0039] (a)
administering to the human or animal a specific binding member
which binds the ED-A domain of fibronectin, and [0040] (b)
determining the presence or absence of the specific binding member
in sites of endometriosis of the human or animal body, [0041]
wherein localisation of the specific binding member to site of
endometriosis indicates the presence of endometriosis.
[0042] Preferably, the specific binding member binds the ED-A
isoform of human fibronectin.
[0043] The specific binding member, e.g. an antibody molecule, for
use in this third aspect of the invention, may bind the ED-A of
fibronectin.
[0044] The specific binding member e.g. an antibody molecule, for
use in this third aspect of the invention, may be conjugated to a
detectable label, or a radioisotope.
[0045] In a fourth aspect, the invention provides a specific
binding member that binds the ED-A isoform of fibronectin for use
in a method of imaging endometriotic tissue. The invention also
provides use of a specific binding member that binds the ED-A
isoform of fibronectin for the manufacture of an imaging agent for
imaging endometriotic tissue. The invention also provides a method
of detecting or imaging endometriotic tissue in a human or animal,
wherein the method comprises the steps of: [0046] (a) administering
to the human or animal a specific binding member which binds the
ED-A domain of fibronectin, and [0047] (b) detecting the binding of
the specific binding member to endometriotic tissue in the human or
animal body.
[0048] Preferably, the specific binding member binds the ED-A
isoform of human fibronectin.
[0049] The specific binding member, e.g. an antibody molecule, for
use in this fourth aspect of the invention, may bind the ED-A of
fibronectin.
[0050] The specific binding member e.g. an antibody molecule, for
use in this fourth aspect of the invention, may be conjugated to a
detectable label, or a radioisotope.
[0051] A specific binding member for use in the invention may be an
antibody which binds the ED-A isoform of fibronectin and/or the
ED-A of fibronectin, wherein the antibody comprises one or more
complementarity determining regions (CDRs) of antibody H1, B2, C5,
D5, E5, C8, F8, F1, B7, E8 or G9, or variants thereof. F8, D5 and
B7 are disclosed in Villa et al., 122, 2405-2413 (2008) Int. J.
Cancer, while H1, B2, C5, E5, C8, F1, E8 and G9 (and also F8, D5
and B7) are disclosed in WO 2008/120101. Preferably, a specific
binding member for use in the invention is an antibody which binds
the ED-A isoform of fibronectin and/or the ED-A of fibronectin,
comprising one or more complementarity determining regions (CDRs)
of antibody B2, C5, D5, C8, F8, B7 or G9, or variants thereof.
Preferably, the specific binding member binds the ED-A isoform of
human fibronectin. Most preferably, a specific binding member for
use in the invention is an antibody which binds the ED-A isoform of
fibronectin and/or the ED-A of fibronectin, comprising one or more
complementarity determining regions (CDRs) of antibody F8, or
variants thereof.
[0052] Preferably, a suitable variant for use as described herein
comprises an antibody antigen binding site comprising a VH domain
and a VL domain of any one of antibodies F8, H1, 82, C5, D5, E5,
C8, F1, B7, E8 and G9, wherein the valine (V) residue at position
of the VH domain is substituted with leucine (L), and/or the lysine
(K) residue at position 18 of the VL domain is substituted with
arginine (R). Most preferably, a suitable variant for use as
described herein comprises an antibody antigen binding site
comprising the F8 VH V5L domain of SEQ ID NO: 16 and the F8 VL K18R
domain of SEQ ID NO: 78, wherein the valine (V) residue at position
5 of the VH domain is substituted by leucine (L), and/or the lysine
(K) residue at position 18 of the VL domain is substituted by
arginine (R).
[0053] A specific binding member for use in the invention may
comprise a set of H and/or L CDRs of antibody H1, B2, C5, D5, E5,
C8, F8, F1, B7, E8 or G9, or a set of H and/or L CDRs of antibody
H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9 with ten or fewer,
e.g. one, two, three, four, or five, amino acid substitutions
within the disclosed set of H and/or L CDRs. Preferably, a specific
binding member for use in the invention comprises a set of H and/or
L CDRs of antibody B2, C5, D5, C8, F8, B7 or G9 with ten or fewer,
e.g. one, two, three, four, or five, amino acid substitutions
within the disclosed set of H and/or L CDRs. Preferably, a specific
binding member for use in the invention comprises a set of H and/or
L CDRs of antibody F8 with ten or fewer, e.g. one, two, three,
four, or five, amino acid substitutions within the disclosed set of
H and/or L CDRs.
[0054] Substitutions may potentially be made at any residue within
the set of CDRs, and may be within CDR1, CDR2 and/or CDR3.
[0055] For example, a specific binding member for use in the
invention may comprise one or more CDRs as described herein, e.g. a
CDR3, and optionally also a CDR1 and CDR2 to form a set of
CDRs.
[0056] A specific binding member for use in the invention may also
comprise an antibody molecule, e.g. a human antibody molecule. The
specific binding member normally comprises an antibody VH and/or VL
domain. VH domains of specific binding members are also provided
for use in the invention. Within each of the VH and VL domains are
complementarity determining regions, ("CDRs"), and framework
regions, ("FRs"). A VH domain comprises a set of HCDRs, and a VL
domain comprises a set of LCDRs. An antibody molecule may comprise
an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a
framework. It may alternatively or also comprise an antibody VL
domain comprising a VL CDR1, CDR2 and CDR3 and a framework. The VH
and VL domains and CDRs of antibodies H1, B2, C5, D5, E5, C8, F8
(and its variant comprising V5L VH and K18R VL), F1, B7, E8 and G9
are described herein. All VH and VL sequences, CDR sequences, sets
of CDRs and sets of HCDRs and sets of LCDRs disclosed herein
represent embodiments of a specific binding member for use in the
invention. As described herein, a "set of CDRs" comprises CDR1,
CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and
HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless
otherwise stated, a "set of CDRs" includes HCDRs and LCDRs.
[0057] A specific binding member for use in the invention may
comprise an antibody VH domain comprising complementarity
determining regions HCDR1, HCDR2 and HCDR3 and a framework, wherein
HCDR1 is SEQ ID NO: 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50,
and wherein optionally HCDR2 is SEQ ID NO: 56, and/or HCDR3 is SEQ
ID NO: 60. Preferably, the HCDR1 is SEQ ID NO: 42.
[0058] Typically, a VB domain is paired with a VL domain to provide
an antibody antigen-binding site, although as discussed further
below, a VH or VL domain alone may be used to bind antigen. Thus, a
specific binding member for use in the invention may further
comprise an antibody VL domain comprising complementarity
determining regions LCDR1, LCDR2 and LCDR3 and a framework, wherein
LCDR1 is SEQ ID NO: 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or
110, and wherein optionally LCDR2 is SEQ ID NO: 114 and/or LCDR3 is
SEQ ID NO: 118. Preferably, the LCDR1 is SEQ ID NO: 102.
[0059] A specific binding member for use in the invention may be an
isolated antibody molecule for the ED-A of fibronectin, comprising
a VH domain and a VL domain, wherein the VH domain comprises a
framework and a set of complementarity determining regions HCDR1,
HCDR2 and HCDR3 and wherein the VL domain comprises complementarity
determining regions LCDR1, LCDR2 and LCDR3 and a framework, and
wherein
[0060] HCDR1 has amino acid sequence SEQ ID NO: 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, or 50;
[0061] HCDR2 has amino acid sequence SEQ ID NO: 56;
[0062] HCDR3 has amino acid sequence SEQ ID NO: 60;
[0063] LCDR1 has amino acid sequence SEQ ID NO: 90, 92, 94, 96, 98,
100, 102, 104, 106, 108, or 110;
[0064] LCDR2 has amino acid sequence SEQ ID NO: 114; and
[0065] LCDR3 has amino acid sequence SEQ ID NO: 118.
[0066] Preferably, the HCDR1 is SEQ ID NO: 42, and the LCDR1 is SEQ
ID NO: 102.
[0067] One or more CDRs or a set of CDRs of an antibody may be
grafted into a framework (e.g. human framework) to provide an
antibody molecule for use in the invention. Framework regions may
comprise human germline gene segment sequences. Thus, the framework
may be germlined, whereby one or more residues within the framework
are changed to match the residues at the equivalent position in the
most similar human germline framework. A specific binding member
for use in the invention may be an isolated antibody molecule
having a VH domain comprising a set of HCDRs in a human germline
framework, e.g. DP47. Normally the specific binding member also has
a VL domain comprising a set of LCDRs, e.g. in a human germline
framework. The human germline framework of the VL domain may be
DPK22.
[0068] A VH domain for use in the invention may have amino acid
sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or
24.
[0069] Preferably, a VH domain for use in the invention has amino
acid sequence SEQ ID NO: 14 or 16. A VL domain for use in the
invention may have the amino acid SEQ ID NO: 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, or 86. Preferably, a VL domain for use in
the invention has amino acid SEQ ID NO: 76 or 78.
[0070] A specific binding member for use in the invention may be or
comprise a single chain Fv (scFv), comprising a VH domain and a VL
domain joined via a peptide linker. The skilled person may select
an appropriate length and sequence of linker, e.g. at least 5 or at
least 10 amino acids in length, up to about 15, up to about 20 or
up to about 25 amino acids in length. The linker may have the amino
acid sequence GSSGG (SEQ ID NO: 122).
[0071] The specific binding member may be a diabody, which is a
multivalent or multispecific fragment constructed by gene fusion
(WO94/13804; Holliger 1993a).
[0072] A single chain Fv (scFv) may be comprised within a
mini-immunoglobulin or small immunoprotein (SIP), e.g. as described
in (Li et al., 1997). An SIP may comprise an scFv molecule fused to
the CH4 domain of the human IgE secretory isoform IgE-S2
(.epsilon..sub.s2-CH4; Batista et al., 1996) forming an
homo-dimeric mini-immunoglobulin antibody molecule.
[0073] Alternatively, a specific binding member for use in the
invention may comprise an antigen-binding site within anon-antibody
molecule, normally provided by one or more CDRs e.g. a set of CDRs
in a non-antibody protein scaffold. Specific binding members,
including non-antibody and antibody molecules, are described in
more detail elsewhere herein.
[0074] According to a fifth aspect, the invention provides a
specific binding member that binds tenascin-C for use in a method
of treatment of endometriosis, psoriatic arthritis or psoriasis.
The invention also provides use of a specific binding member that
binds tenascin-C for the manufacture of a medicament for treating
endometriosis, psoriatic arthritis or psoriasis. The invention also
provides a method of treating endometriosis, psoriatic arthritis or
psoriasis in a patient, the method comprising administering to a
patient a therapeutically effective amount of a medicament
comprising a specific binding member which binds tenascin-C.
Preferably, the specific binding member binds human tenascin-C.
[0075] The specific binding member, e.g. an antibody molecule, for
use in this fifth aspect of the invention may bind specifically to
tenascin-C large isoform. For example, the specific binding member
may bind preferentially to tenascin-C large isoform relative to
tenascin-C small isoform. Preferably, the specific binding member
binds to the A1, A2, A3, A4, B and/or D domain of tenascin-C large
isoform. Most preferably, the specific binding member binds to the
A1 domain of tenascin-C.
[0076] The specific binding member, e.g. an antibody molecule, for
use in this fifth aspect of the invention, may be conjugated to a
detectable label, a radioisotope, or a bioactive molecule, such as
a cytokine, hormone, a therapeutic isotope, or a cytotoxic drug.
For example, the specific binding member may be conjugated to a
cytokine, such as IL-10, TGF-.beta., IL-2, IL-12, IL-15, IL-21,
IL-24, IL-33, tumour necrosis factor (TNF), or interferon-.alpha.,
-.beta. or -.gamma.. The specific binding member may be conjugated
to the bioactive molecule by a cleavable linker.
[0077] According to a sixth aspect, the invention provides a
specific binding member that binds tenascin-C, for use in the
delivery to the neovasculature of endometriotic, psoriatic
arthritic or psoriatic tissue of a molecule conjugated to the
specific binding member. The invention also provides use of a
specific binding member that binds tenascin-C for the manufacture
of a medicament for delivery of a molecule conjugated to the
specific binding member to the neovasculature of endometriotic,
psoriatic arthritic or psoriatic tissue. The invention also
provides a method of delivering a molecule to the neovasculature of
endometriotic, psoriatic arthritic or psoriatic tissue in a human
or animal, wherein the molecule is conjugated to a specific binding
member which binds tenascin-C to form a conjugate and the method
comprises administering the conjugate to the human or animal.
Preferably, the specific binding member binds human tenascin-C.
[0078] The specific binding member, e.g. an antibody molecule, for
use in this sixth aspect of the invention may bind specifically to
tenascin-C large isoform. For example, the specific binding member
may bind preferentially to tenascin-C large isoform relative to
tenascin-C small isoform. Preferably, the specific binding member
binds to the A1, A2, A3, A4, B and/or D domain of tenascin-C large
isoform. Most preferably, the specific binding member binds to the
A1 domain of tenascin-C.
[0079] The specific binding member, e.g. an antibody molecule, for
use in this sixth aspect of the invention, may be conjugated to a
detectable label, a radioisotope, or a bioactive molecule, such as
a cytokine, hormone, a therapeutic isotope, or a cytotoxic drug.
For example, the specific binding member may be conjugated to a
cytokine, such as IL-10, TGF-.beta., IL-2, IL-12, IL-15, IL-21,
IL-24, IL-33, tumour necrosis factor (TNF), or interferon-.alpha.,
-.beta. or -.gamma.. The specific binding member may be conjugated
to the bioactive molecule by a cleavable linker.
[0080] According to a seventh aspect, the invention provides a
specific binding member that binds tenascin-C for use in a method
of diagnosis of endometriosis, psoriatic arthritis or psoriasis.
The invention also provides use of a specific binding member that
binds tenascin-C for the manufacture of a diagnostic product for
diagnosing endometriosis, psoriatic arthritis or psoriasis. The
invention also provides a method of detecting or diagnosing
endometriosis, psoriatic arthritis or psoriasis in a human or
animal, wherein the method comprises the steps of: [0081] (a)
administering to the human or animal a specific binding member
which binds tenascin-C, and [0082] (b) determining the presence or
absence of the specific binding member in sites of endometriosis,
psoriatic arthritis or psoriasis of the human or animal body;
wherein localisation of the specific binding member to sites of
endometriosis, psoriatic arthritis or psoriasis indicates the
presence of endometriosis, psoriatic arthritis or psoriasis.
[0083] Preferably, the specific binding member binds human
tenascin-C.
[0084] The specific binding member, e.g. an antibody molecule, for
use in this seventh aspect of the invention may bind specifically
to tenascin-C large isoform. For example, the specific binding
member may bind preferentially to tenascin-C large isoform relative
to tenascin-C small isoform. Preferably, the specific binding
member binds to the A1, A2, A3, A4, B and/or D domain of tenascin-C
large isoform. Most preferably, the specific binding member binds
to the A1 domain of tenascin-C.
[0085] The specific binding member, e.g. an antibody molecule, for
use in this seventh aspect of the invention, may be conjugated to a
detectable label, or a radioisotope.
[0086] In an eighth aspect, the invention provides a specific
binding member that binds tenascin-C for use in a method of imaging
endometriotic, psoriatic arthritic or psoriatic tissue. The
invention also provides use of a specific binding member that binds
tenascin-C for the manufacture of an imaging agent for imaging
endometriotic, psoriatic arthritic or psoriatic tissue. The
invention also provides a method of detecting or imaging
endometriotic, psoriatic arthritic or psoriatic tissue in a human
or animal, wherein the method comprises the steps of: [0087] (a)
administering to the human or animal a specific binding member
which binds tenascin-C, and [0088] (b) detecting the binding of the
specific binding member to endometriotic, psoriatic arthritic or
psoriatic tissue in the human or animal body.
[0089] Preferably, the specific binding member binds human
tenascin-C. The specific binding member, e.g. an antibody molecule,
for use in this eighth aspect of the invention may bind
specifically to tenascin-C large isoform. For example, the specific
binding member may bind preferentially to tenascin-C large isoform
relative to tenascin-C small isoform. Preferably, the specific
binding member binds to the A1, A2, A3, A4, B and/or D domain of
tenascin-C large isoform. Most preferably, the specific binding
member binds to the A1 domain of tenascin-C.
[0090] The specific binding member, e.g. an antibody molecule, for
use in this eighth aspect of the invention, may be conjugated to a
detectable label, or a radioisotope.
[0091] A specific binding member for use in the invention may be an
antibody which binds tenascin-C, and/or the A1, A2, A3, A4, B
and/or D domain of the tenascin-C large isoform, wherein the
antibody comprises one or more complementarity determining regions
(CDRs) of antibody F16 or 4A1-F16 (Brack et al., 12, 3200-3208,
(2006) Clin. Cancer Res.), or variants thereof.
[0092] A specific binding member for use in the invention may
comprise a set of H and/or L CDRs of antibody F16 or 4A1-F16, or a
set of H and/or L CDRs of antibody F16 or 4A1-F16 with ten or
fewer, e.g. one, two, three, four, or five, amino acid
substitutions within the disclosed set of H and/or L CDRs.
Preferably, a specific binding member for use in the invention
comprises a set of H and/or L CDRs of antibody F16 or 4A1-F16 with
ten or fewer, e.g. one, two, three, four, or five, amino acid
substitutions within the disclosed set of H and/or L CDRs.
Preferably, a specific binding member for use in the invention
comprises a set of H and/or L CDRs of antibody F16 or 4A1-F16 with
ten or fewer, e.g. one, two, three, four, or five, amino acid
substitutions within the disclosed set of H and/or L CDRs.
[0093] Substitutions may potentially be made at any residue within
the set of CDRs, and may be within CDR1, CDR2 and/or CDR3.
[0094] For example, a specific binding member for use in the
invention may comprise one or more CDRs as described herein, e.g. a
CDR3, and optionally also a CDR1 and CDR2 to form a set of
CDRs.
[0095] A specific binding member for use in the invention may also
comprise an antibody molecule, e.g. a human antibody molecule. The
specific binding member normally comprises an antibody VH and/or VL
domain. VH domains of specific binding members are also provided
for use in the invention. Within each of the VH and VL domains are
complementarity determining regions, ("CDRs"), and framework
regions, ("FRs"). A VH domain comprises a set of HCDRs, and a VL
domain comprises a set of LCDRs. An antibody molecule may comprise
an antibody VH domain comprising a VH CDR1, CDR2 and CDR3 and a
framework. It may alternatively or also comprise an antibody VL
domain comprising a VL CDR1, CDR2 and CDR3 and a framework. The VH
and VL domains and CDRs of antibody F16 and 4A1-F16 are described
herein. All VH and VL sequences, CDR sequences, sets of CDRs and
sets of HCDRs and sets of LCDRs disclosed herein represent
embodiments of a specific binding member for use in the invention.
As described herein, a "set of CDRs" comprises CDR1, CDR2 and CDR3.
Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of
LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a
"set of CDRs" includes HCDRs and LCDRs.
[0096] A specific binding member for use in the invention may
comprise an antibody VH domain comprising complementarity
determining regions HCDR1, HCDR2 and HCDR3 and a framework, wherein
HCDR1 is SEQ ID NO: 52 or 54, and wherein optionally HCDR2 is SEQ
ID NO: 58, and/or HCDR3 is SEQ ID NO: 62. Preferably, the HCDR1 is
SEQ ID NO: 52.
[0097] Typically, a VH domain is paired with a VL domain to provide
an antibody antigen-binding site, although as discussed further
below, a VH or VL domain alone may be used to bind antigen. Thus, a
specific binding member for use in the invention may further
comprise an antibody VL domain comprising complementarity
determining regions LCDR1, LCDR2 and LCDR3 and a framework, wherein
LCDR1 is SEQ ID NO: 112, and wherein optionally LCDR2 is SEQ ID NO:
116, and/or LCDR3 is SEQ ID NO: 120.
[0098] A specific binding member for use in the invention may be an
isolated antibody molecule for the A1 domain of tenascin-C,
comprising a VH domain and a VL domain, wherein the VH domain
comprises a framework and a set of complementarity determining
regions HCDR1, HCDR2 and HCDR3 and wherein the VL domain comprises
complementarity determining regions LCDR1, LCDR2 and LCDR3 and a
framework, and wherein
[0099] HCDR1 has amino acid sequence SEQ ID NO: 52 or 54;
[0100] HCDR2 has amino acid sequence SEQ ID NO: 58;
[0101] HCDR3 has amino acid sequence SEQ ID NO: 62;
[0102] LCDR1 has amino acid sequence SEQ ID NO: 112;
[0103] LCDR2 has amino acid sequence SEQ ID NO: 116; and
[0104] LCDR3 has amino acid sequence SEQ ID NO: 120.
[0105] One or more CDRs or a set of CDRs of an antibody may be
grafted into a framework (e.g. human framework) to provide an
antibody molecule for use in the invention. Framework regions may
comprise human germline gene segment sequences. Thus, the framework
may be germlined, whereby one or more residues within the framework
are changed to match the residues at the equivalent position in the
most similar human germline framework. A specific binding member
for use in the invention may be an isolated antibody molecule
having a VH domain comprising a set of HCDRs in a human germline
framework, e.g. DP47. Normally the specific binding member also has
a VL domain comprising a set of LCDRs, e.g. in a human germline
framework. The human germline framework of the VL domain may be
DPK22.
[0106] A VH domain for use in the invention may have amino acid
sequence SEQ ID NO: 26 or 28. Preferably, the VH domain has the
amino acid sequence SEQ ID NO: 26. A VL domain for use in the
invention may have the amino acid SEQ ID NO: 88.
[0107] The VL domain of F16 and 4A1-F16 (SEQ ID NO: 88) may
optionally include an extra glycine residue at its C-terminal
end.
[0108] A specific binding member for use in the invention may be or
comprise a single chain Fv (scFv), comprising a VH domain and a VL
domain joined via a peptide linker. The skilled person may select
an appropriate length and sequence of linker, e.g. at least 5 or at
least 10 amino acids in length, up to about 15, up to about 20 or
up to about 25 amino acids in length. The linker may have the amino
acid sequence GSSGG (SEQ ID NO: 122).
[0109] The specific binding member may be a diabody, which is a
multivalent or multispecific fragment constructed by gene fusion
(WO94/13804; Holliger 1993a).
[0110] A single chain Fv (scFv) may be comprised within a
mini-immunoglobulin or small immunoprotein (SIP), e.g. as described
in (Li et al., 1997). An SIP may comprise an scFv molecule fused to
the CH4 domain of the human IgE secretory isoform IgE-S2
(.epsilon..sub.S2-CH4; Batista et al., 1996) forming an
homo-dimeric mini immunoglobulin antibody molecule.
[0111] Alternatively, a specific binding member for use in the
invention may comprise an antigen-binding site within a
non-antibody molecule, normally provided by one or more CDRs e.g. a
set of CDRs in a non-antibody protein scaffold. Specific binding
members, including non-antibody and antibody molecules, are
described in more detail elsewhere herein.
[0112] These and other aspects of the invention are described in
further detail below.
BRIEF DESCRIPTION OF THE FIGURES
[0113] FIG. 1 shows the results of immunohistochemistry on samples
from patients with endometriosis using antibodies directed to
markers of angiogenesis. Darker staining indicates strong
expression of the antigen, visualized by black arrows. Areas of
negative staining around perivascular structures are indicated
using white arrows. A, B and C show staining with F8-SIP, which is
an antibody molecule that binds ED-A, disclosed herein. D, E and F
show staining with L19-SIP, which is an antibody molecule that
binds ED-B (e.g. Pini et al. 1998). G, H and I show staining with
F16-SIP, which is an antibody molecule that binds the A1 domain of
Tenascin-C (WO2006/050834). J, K and L are negative controls in
which no primary antibody was added. A, D, G, J, B, E, H and K show
samples viewed under 10.times. magnification and C, F, I and L show
samples viewed under 5.times. magnification.
[0114] FIG. 2 shows the results of immunohistochemistry on samples
from patients with ulcerative colitis using antibodies directed to
markers of angiogenesis. The white arrows indicate areas of
negative staining around perivascular structures. FIG. 2A shows
staining with F8-SIP, which is an antibody molecule that binds
ED-A, disclosed herein. FIG. 2B shows staining with L19-SIP, which
is an antibody molecule that binds ED-B (e.g. Pini et al. 1998).
FIG. 2C shows staining with F16-SIP, which is an antibody molecule
that binds the A1 domain of Tenascin-C (WO2006/050834). FIG. 2D is
a negative control in which no primary antibody was added.
[0115] FIG. 3 shows the results of immunohistochemistry on samples
from patients with psoriatic arthritis using antibodies directed to
markers of angiogenesis. Darker staining indicates strong
expression of the antigen, visualized by black arrows. Areas of
negative staining around perivascular structures are indicated
using white arrows. FIG. 3A shows staining with F8-SIP, which is an
antibody molecule that binds ED-A, disclosed herein. FIG. 3B shows
staining with L19-SIP, which is an antibody molecule that binds
ED-B (e.g. Pini et al. 1998). FIG. 3C shows staining with F16-SIP,
which is an antibody molecule that binds the A1 domain of
Tenascin-C (WO2006/050834). FIG. 3D is a negative control in which
no primary antibody was added.
[0116] FIG. 4 shows the results of immunohistochemistry on samples
from patients with multiple sclerosis using antibodies directed to
markers of angiogenesis. The white arrows indicate areas of
negative staining around perivascular structures. FIG. 4A shows
staining with F8-SIP, which is an antibody molecule that binds
ED-A, disclosed herein. FIG. 4B shows staining with L19-SIP, which
is an antibody molecule that binds ED-B (e.g. Pini et al. 1998).
FIG. 4C shows staining with F16-SIP, which is an antibody molecule
that binds the A1 domain of Tenascin-C (WO2006/050834). FIG. 4D is
a negative control in which no primary antibody was added.
[0117] FIG. 5 shows the results of immunohistochemistry on samples
from patients with psoriasis using antibodies directed to markers
of angiogenesis. Darker staining indicates strong expression of the
antigen, visualized by black arrows. Areas of negative staining
around perivascular structures are indicated using white arrows.
FIG. 5A shows staining with F8-SIP, which is an antibody molecule
that binds ED-A, disclosed herein. FIG. 5B shows staining with
L19-SIP, which is an antibody molecule that binds ED-B (e.g. Pini
et al. 1998). FIG. 5C shows staining with F16-SIP, which is an
antibody molecule that binds the A1 domain of Tenascin-C
(WO2006/050834). FIG. 5D is a negative control in which no primary
antibody was added.
[0118] FIG. 6A shows the nucleotide sequences of the anti-ED-A
antibody F8 heavy chain (VH) (SEQ ID NO: 13) and its variant, F8
V5L VH (SEQ ID NO: 15). The nucleotide sequence of the heavy chain
CDR1 (SEQ ID NO: 41) of anti-ED-A antibody F8 is underlined. The
nucleotide sequence of the heavy chain CDR2 (SEQ ID NO: 55) of the
anti-ED-A antibody F8 is shown in italics and underlined. The
nucleotide sequence of the heavy chain CDR3 (SEQ ID NO: 59) of
anti-ED-A antibody F8 is shown in bold and underlined.
[0119] FIG. 6B shows the nucleotide sequence of the anti-ED-A
antibody F8 linker sequence (SEQ ID NO: 121).
[0120] FIG. 6C shows the nucleotide sequences of the anti-ED-A
antibody F8 light chain (VL) (SEQ ID NO: 75) and its variant, F8
K18R VL (SEQ ID NO: 77). The nucleotide sequence of the light chain
CDR1 (SEQ ID NO: 101) of anti-ED-A antibody F8 is underlined. The
nucleotide sequence of the light chain CDR2 (SEQ ID NO: 113) of the
anti-ED-A antibody F8 is shown in italics and underlined. The
nucleotide sequence of the light chain CDR3 (SEQ ID NO: 117) of
anti-ED-A antibody F8 is shown in bold and underlined.
[0121] FIG. 7A shows the amino acid sequences of the anti-ED-A
antibody F8 heavy chain (VH) (SEQ ID NO: 14), and its variant, F8
V5L VH (SEQ ID NO: 16). The amino acid sequence of the heavy chain
CDR1 (SEQ ID NO: 42) of anti-ED-A antibody F8 is underlined. The
amino acid sequence of the heavy chain CDR2 (SEQ ID NO: 56) of the
anti-ED-A antibody F8 is shown in italics and underlined. The amino
acid sequence of the heavy chain CDR3 (SEQ ID NO: 60) of anti-ED-A
antibody F8 is shown in bold and underlined.
[0122] FIG. 7B shows the amino acid sequence of the anti-ED-A
antibody F8 linker sequence (SEQ ID NO: 122).
[0123] FIG. 7C shows the amino acid sequences of the anti-ED-A
antibody F8 light chain (VL) (SEQ ID NO: 76) and its variant, F8
K18R VL (SEQ ID NO: 78). The amino acid sequence of the light chain
CDR1 (SEQ ID NO: 102) of anti-ED-A antibody F8 is underlined. The
amino acid sequence of the light chain CDR2 (SEQ ID NO: 114) of the
anti-ED-A antibody F8 is shown in italics and underlined. The amino
acid sequence of the light chain CDR3 (SEQ ID NO: 118) of anti-ED-A
antibody F8 is shown in bold and underlined.
[0124] FIG. 8A shows the nucleotide sequences of the heavy chain
(VH) of the anti-tenascin-C antibody F16 (SEQ ID NO: 25) and its
variant, 4A1-F16 (SEQ ID NO: 27). The nucleotide sequences of the
heavy chain CDR1 of anti-tenascin-C antibodies F16 and F16-4A1 are
underlined (SEQ ID NOs: 51 and 53 respectively). The nucleotide
sequence of the heavy chain CDR2 (SEQ ID NO: 57) of the
anti-tenascin-C antibodies F16 and F16-4A1 is shown in italics and
underlined. The nucleotide sequence of the heavy chain CDR3 (SEQ ID
NO: 61) of the anti-tenascin-C antibodies F16 and F16-4A1 is shown
in bold and underlined.
[0125] FIG. 8B shows the nucleotide sequence of the linker sequence
(SEQ ID NO: 121) of the anti-tenascin-C antibodies F16 and
F16-4A1.
[0126] FIG. 8C shows the nucleotide sequence of the light chain
(VL) (SEQ ID NO: 87) of the anti-tenascin-C antibodies F16 and
F16-4A1. The nucleotide sequence of the light chain CDR1 (SEQ ID
NO: 111) of the anti-tenascin-C antibodies F16 and F16-4A1 is
underlined. The nucleotide sequence of the light chain CDR2 (SEQ ID
NO: 115) of the anti-tenascin-C antibodies F16 and F16-4A1 is shown
in italics and underlined. The nucleotide sequence of the light
chain CDR3 (SEQ ID NO: 119) of anti-tenascin-C antibodies F16 and
F16-4A1 is shown in bold and underlined.
[0127] FIG. 9A shows the amino acid sequence of the heavy chain
(VH) of the anti-tenascin-C antibody F16 (SEQ ID NO: 26) and its
variant, 4A1-F16 (SEQ ID NO: 28). The amino acid sequence of the
heavy chain CDR1 of the anti-tenascin-C antibody F16 (SEQ ID NO:
52) and 4A1-F16 (SEQ ID NO: 54) is underlined. The amino acid
sequence of the heavy chain CDR2 (SEQ ID NO: 58) of the
anti-tenascin-C antibodies F16 and 4A1-F16 is shown in italics and
underlined. The amino acid sequence of the heavy chain CDR3 (SEQ ID
NO: 62) of anti-tenascin-C antibodies F16 and 4A1-F16 is shown in
bold and underlined.
[0128] FIG. 9B shows the amino acid sequence of the anti-tenascin-C
antibody F16 linker sequence (SEQ ID NO: 122).
[0129] FIG. 9C shows the amino acid sequence of the light chain
(VL) (SEQ ID NO: 88) of the anti-tenascin-C antibodies F16 and
4A1-F16. The amino acid sequence of the light chain CDR1 (SEQ ID
NO: 112) of anti-tenascin-C antibodies F16 and 4A1-F16 is
underlined. The amino acid sequence of the light chain CDR2 (SEQ ID
NO: 116) of the anti-tenascin-C antibodies F16 and 4A1-F16 is shown
in italics and underlined. The amino acid sequence of the light
chain CDR3 (SEQ ID NO: 120) of anti-tenascin-C antibodies F16 and
4A1-F16 is shown in bold and underlined.
[0130] FIG. 10 shows the results of near infrared imaging of
endometriosis lesions in mice. Areas of endometriosis in the mice
are indicated by circles. FIGS. 10 A and B show the results of near
infrared imaging using a SIP(F8)-ALEXA750 antibody molecule, which
binds ED-A. SIP(F8)-ALEXA750 was injected into mice with
endometriosis and imaged 24 h after injection. Arrows indicate
areas of positive imaging. FIG. 10C shows the results of
near-infrared imaging using a SIP(F16)-ALEXA750 antibody molecule
which recognises the human A1 domain of tenascin-C.
SIP(F16)-ALEXA750 was injected into mice with endometriosis and
imaged 24 h after injection.
[0131] FIG. 11 shows the results of ex vivo staining of murine
endometriosis lesions. FIGS. 11 A, B and C show immunofluorescent
detection of SIP(F8)-ALEXA750 using a rabbit anti-human IgE
antibody followed by goat anti-rabbit IgG. FIGS. 11 D, E and F show
detection of blood vessels using a rat anti-CD31 antibody followed
by donkey anti-rat IgG. FIGS. 11 G, H and I represent negative
controls and show the results of probing the lesions with goat
anti-rabbit IgG alone.
TERMINOLOGY
Fibronectin
[0132] Fibronectin is an antigen subject to alternative splicing,
and a number of alternative isoforms of fibronectin are known, as
described elsewhere herein. Extra Domain-A (EDA or ED-A) is also
known as ED, extra type III repeat A (EIIIA) or EDI. The sequence
of human ED-A has been published by Kornblihtt et al. (1984),
Nucleic Acids Res. 12, 5853-5868 and Paolella et al. (1988),
Nucleic Acids Res. 16, 3545-3557. The sequence of human ED-A is
also available on the SwissProt database as amino acids 1631-1720
(Fibronectin type-III 12; extra domain 2) of the amino acid
sequence deposited under accession number P02751. The sequence of
mouse ED-A is available on the SwissProt database as amino acids
1721-1810 (Fibronectin type-III 13; extra domain 2) of the amino
acid sequence deposited under accession number P11276.
[0133] The ED-A isoform of fibronectin (A-FN) contains the Extra
Domain-A (ED-A). The sequence of the human A-FN can be deduced from
the corresponding human fibronectin precursor sequence which is
available on the SwissProt database under accession number P02751.
The sequence of the mouse A-FN can be deduced from the
corresponding mouse fibronectin precursor sequence which is
available on the SwissProt database under accession number P11276.
The A-FN may be the human ED-A isoform of fibronectin. The ED-A may
be the Extra Domain-A of human fibronectin.
[0134] ED-A is a 90 amino acid sequence which is inserted into
fibronectin (FN) by alternative splicing and is located between
domain 11 and 12 of FN (Borsi et al., 1987, J. Cell Biol., 104,
595-600). ED-A is mainly absent in the plasma form of FN but is
abundant during embryogenesis, tissue remodelling, fibrosis,
cardiac transplantation and solid tumour growth.
Alternative Splicing
[0135] Alternative splicing refers to the occurrence of different
patterns of splicing of a primary RNA transcript of DNA to produce
different mRNAs. After excision of introns, selection may determine
which exons are spliced together to form the mRNA. Alternative
splicing leads to production of different isoforms containing
different exons and/or different numbers of exons. For example one
isoform may comprise an additional amino acid sequence
corresponding to one or more exons, which may comprise one or more
domains.
Tenascin-C
[0136] Tenascin-C is a large hexameric glycoprotein of the
extracellular matrix which modulates cellular adhesion. It is
involved in processes such as cell proliferation and cell migration
and is associated with changes in tissue architecture as occurring
during morphogenesis and embryogenesis as well as under
tumorigenesis or angiogenesis.
[0137] A strong over-expression of the large isoform of tenascin-C
has been reported for a number of tumors [Borsi 1992 supra], and
monoclonal antibodies specific for domains A1 and D, respectively,
have been extensively characterised in the clinic (Riva et al., Int
J Cancer (1992) 51:7-13; Riva at al., Cancer Res (1995), 55,
5952s-5956s; Paganelli et al., Eur J Nucl Med (1994) 21, 314-321.
Reardon et al., J Clin Oncol (2002), 20, 1389-1397; Bigner et al.,
J Clin Oncol (1998) 16, 2202-2212.).
[0138] Human monoclonal antibody fragments specific to tenascin-C
are described in WO2006/050834 and shown to bind preferentially to
tumor tissue relative to normal tissue. These antibodies are
useful, for example, in delivering toxins, such as cytokines,
specifically to tumour cells.
Specific Binding Member
[0139] This describes one member of a pair of molecules that bind
specifically to one another. The members of a specific binding pair
may be naturally derived or wholly or partially synthetically
produced. One member of the pair of molecules has an area on its
surface, or a cavity, which binds to and is therefore complementary
to a particular spatial and polar organization of the other member
of the pair of molecules. Examples of types of binding pairs are
antigen-antibody, biotin-avidin, hormone-hormone receptor,
receptor-ligand, enzyme-substrate. The present invention is
concerned with antigen-antibody type reactions.
[0140] A specific binding member normally comprises a molecule
having an antigen-binding site. For example, a specific binding
member may be an antibody molecule or a non-antibody protein that
comprises an antigen-binding site.
[0141] An antigen binding site may be provided by means of
arrangement of complementarity determining regions (CDRs) on
non-antibody protein scaffolds such as fibronectin or cytochrome B
etc. (Haan & Maggos, 2004; Koide 1998; Nygren 1997), or by
randomising or mutating amino acid residues of a loop within a
protein scaffold to confer binding specificity for a desired
target. Scaffolds for engineering novel binding sites in proteins
have been reviewed in detail by Nygren et al. (1997). Protein
scaffolds for antibody mimics are disclosed in WO/0034784, which is
herein incorporated by reference in its entirety, in which the
inventors describe proteins (antibody mimics) that include a
fibronectin type III domain having at least one randomised loop. A
suitable scaffold into which to graft one or more CDRs, e.g. a set
of HCDRs, may be provided by any domain member of the
immunoglobulin gene superfamily. The scaffold may be a human or
non-human protein. An advantage of a non-antibody protein scaffold
is that it may provide an antigen-binding site in a scaffold
molecule that is smaller and/or easier to manufacture than at least
some antibody molecules. Small size of a binding member may confer
useful physiological properties such as an ability to enter cells,
penetrate deep into tissues or reach targets within other
structures, or to bind within protein cavities of the target
antigen. Use of antigen binding sites in non-antibody protein
scaffolds is reviewed in Wess, 2004. Typical are proteins having a
stable backbone and one or more variable loops, in which the amino
acid sequence of the loop or loops is specifically or randomly
mutated to create an antigen-binding site that binds the target
antigen. Such proteins include the IgG-binding domains of protein A
from S. aureus, transferrin, tetranectin, fibronectin (e.g. 10th
fibronectin type III domain) and lipocalins. Other approaches
include synthetic "Microbodies" (Selecore GmbH), which are based on
cyclotides--small proteins having intra-molecular disulphide
bonds.
[0142] In addition to antibody sequences and/or an antigen-binding
site, a specific binding member for use in the present invention
may comprise other amino acids, e.g. forming a peptide or
polypeptide, such as a folded domain, or to impart to the molecule
another functional characteristic in addition to ability to bind
antigen. Binding members for use in the invention may carry a
detectable label, or may be conjugated to a toxin or a targeting
moiety or enzyme (e.g. via a peptidyl bond or linker). For example,
a binding member may comprise a catalytic site (e.g. in an enzyme
domain) as well as an antigen binding site, wherein the antigen
binding site binds to the antigen and thus targets the catalytic
site to the antigen. The catalytic site may inhibit biological
function of the antigen, e.g. by cleavage.
[0143] Although, as noted, CDRs can be carried by non-antibody
scaffolds, the structure for carrying a CDR or a set of CDRs will
generally be an antibody heavy or light chain sequence or
substantial portion thereof in which the CDR or set of CDRs is
located at a location corresponding to the CDR or set of CDRs of
naturally occurring VH and VL antibody variable domains encoded by
rearranged immunoglobulin genes. The structures and locations of
immunoglobulin variable domains may be determined by reference to
Kabat 1987, and updates thereof, now available on the Internet (at
immuno.bme.nwu.edu or find "Kabat" using any search engine).
[0144] By CDR region or CDR, it is intended to indicate the
hypervariable regions of the heavy and light chains of the
immunoglobulin as defined by Kabat et al. (1987), (Kabat 1991a, and
later editions). An antibody typically contains 3 heavy chain CDRs
and 3 light chain CDRs. The term CDR or CDRs is used here in order
to indicate, according to the case, one of these regions or
several, or even the whole, of these regions which contain the
majority of the amino acid residues responsible for the binding by
affinity of the antibody for the antigen or the epitope which it
recognizes.
[0145] Among the six short CDR sequences, the third CDR of the
heavy chain (HCDR3) has a greater size variability (greater
diversity essentially due to the mechanisms of arrangement of the
genes which give rise to it). It can be as short as 2 amino acids
although the longest size known is 26. Functionally, HCDR3 plays a
role in part in the determination of the specificity of the
antibody (Segal 1974; Amit 1986; Chothia 1987; Chothia 1989; Caton
1990; Sharon 1990a; Sharon 1990b; Kabat et al., 1991b).
Antibody Molecule
[0146] This describes an immunoglobulin whether natural or partly
or wholly synthetically produced. The term also relates to any
polypeptide or protein comprising an antibody antigen-binding site.
It must be understood here that the invention does not relate to
the antibodies in natural form, that is to say they are not in
their natural environment but that they have been able to be
isolated or obtained by purification from natural sources, or else
obtained by genetic recombination, or by chemical synthesis, and
that they can then contain unnatural amino acids as will be
described later. Antibody fragments that comprise an antibody
antigen-binding site include, but are not limited to, antibody
molecules such as Fab, Fab', Fab'-SH, scFv, Fv, dAb, Fd; and
diabodies.
[0147] It is possible to take monoclonal and other antibodies and
use techniques of recombinant DNA technology to produce other
antibodies or chimeric molecules that bind the target antigen. Such
techniques may involve introducing DNA encoding the immunoglobulin
variable region, or the CDRs, of an antibody to the constant
regions, or constant regions plus framework regions, of a different
immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or
EP-A-239400, and a large body of subsequent literature. A hybridoma
or other cell producing an antibody may be subject to genetic
mutation or other changes, which may or may not alter the binding
specificity of antibodies produced.
[0148] As antibodies can be modified in a number of ways, the term
"antibody molecule" should be construed as covering any binding
member or substance having an antibody antigen-binding site with
the required specificity and/or binding to antigen. Thus, this term
covers antibody fragments and derivatives, including any
polypeptide comprising an antibody antigen-binding site, whether
natural or wholly or partially synthetic. Chimeric molecules
comprising an antibody antigen-binding site, or equivalent, fused
to another polypeptide (e.g. derived from another species or
belonging to another antibody class or subclass) are therefore
included. Cloning and expression of chimeric antibodies are
described in EP-A-0120694 and EP-A-0125023, and a large body of
subsequent literature.
[0149] Further techniques available in the art of antibody
engineering have made it possible to isolate human and humanised
antibodies. For example, human hybridomas can be made as described
by Kontermann & Dubel (2001). Phage display, another
established technique for generating binding members has been
described in detail in many publications such as WO92/01047
(discussed further below) and U.S. Pat. Nos. 5,969,108, 5,565,332,
5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793, 5,962,255,
6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160, 6,521,404
and Kontermann & Dubel (2001). Transgenic mice in which the
mouse antibody genes are inactivated and functionally replaced with
human antibody genes while leaving intact other components of the
mouse immune system, can be used for isolating human antibodies
(Mendez 1997).
[0150] Synthetic antibody molecules may be created by expression
from genes generated by means of oligonucleotides synthesized and
assembled within suitable expression vectors, for example as
described by Knappik et al. (2000) or Krebs et al. (2001).
[0151] It has been shown that fragments of a whole antibody can
perform the function of binding antigens. Examples of binding
fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1
domains; (ii) the Fd fragment consisting of the VH and CH1 domains;
(iii) the Fv fragment consisting of the VL and VH domains of a
single antibody; (iv) the dAb fragment (Ward 1989; McCafferty 1990;
Holt 2003), which consists of a VH or a VL domain; (v) isolated CDR
regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two
linked Fab fragments (vii) single chain Fv molecules (scFv),
wherein a VH domain and a VL domain are linked by a peptide linker
which allows the two domains to associate to form an antigen
binding site (Bird 1988; Huston 1988); (viii) bispecific single
chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent
or multispecific fragments constructed by gene fusion (WO94/13804;
Holliger 1993a). Fv, scFv or diabody molecules may be stabilized by
the incorporation of disulphide bridges linking the VH and VL
domains (Reiter 1996). Minibodies comprising a scFv joined to a CH3
domain may also be made (Hu 1996). Other examples of binding
fragments are Fab', which differs from Fab fragments by the
addition of a few residues at the carboxyl terminus of the heavy
chain CH1 domain, including one or more cysteines from the antibody
hinge region, and Fab'-SH, which is a Fab' fragment in which the
cysteine residue(s) of the constant domains bear a free thiol
group.
[0152] Antibody fragments for use in the invention can be obtained
starting from any of the antibody molecules described herein, e.g.
antibody molecules comprising VH and/or VL domains or CDRs of any
of antibodies described herein, by methods such as digestion by
enzymes, such as pepsin or papain and/or by cleavage of the
disulfide bridges by chemical reduction. In another manner,
antibody fragments of the present invention may be obtained by
techniques of genetic recombination likewise well known to the
person skilled in the art or else by peptide synthesis by means of,
for example, automatic peptide synthesizers such as those supplied
by the company Applied Biosystems, etc., or by nucleic acid
synthesis and expression.
[0153] Functional antibody fragments according to the present
invention include any functional fragment whose half-life is
increased by a chemical modification, especially by PEGylation, or
by incorporation in a liposome.
[0154] A dAb (domain antibody) is a small monomeric antigen-binding
fragment of an antibody, namely the variable region of an antibody
heavy or light chain (Holt 2003). VH dAbs occur naturally in
camelids (e.g. camel, llama) and may be produced by immunizing a
camelid with a target antigen, isolating antigen-specific B cells
and directly cloning dAb genes from individual B cells. dAbs are
also producible in cell culture. Their small size, good solubility
and temperature stability makes them particularly physiologically
useful and suitable for selection and affinity maturation. A
binding member of the present invention may be a dAb comprising a
VH or VL domain substantially as set out herein, or a VH or VL
domain comprising a set of CDRs substantially as set out
herein.
[0155] As used herein, the phrase "substantially as set out" refers
to the characteristic(s) of the relevant CDRs of the VH or VL
domain of binding members described herein will be either identical
or highly similar to the specified regions of which the sequence is
set out herein. As described herein, the phrase "highly similar"
with respect to specified region(s) of one or more variable
domains, it is contemplated that from 1 to about 5, e.g. from 1 to
4, including 1 to 3, or 1 or 2, or 3 or 4, amino acid substitutions
may be made in the CDR and/or VH or VL domain.
[0156] Bispecific or bifunctional antibodies form a second
generation of monoclonal antibodies in which two different variable
regions are combined in the same molecule (Holliger 1999). Their
use has been demonstrated both in the diagnostic field and in the
therapy field from their capacity to recruit new effector functions
or to target several molecules on the surface of tumor cells. Where
bispecific antibodies are to be used, these may be conventional
bispecific antibodies, which can be manufactured in a variety of
ways (Holliger 1993b), e.g. prepared chemically or from hybrid
hybridomas, or may be any of the bispecific antibody fragments
mentioned above. These antibodies can be obtained by chemical
methods (Glennie 1987; Repp 1995) or somatic methods (Staerz 1986;
Suresh 1986) but likewise by genetic engineering techniques which
allow the heterodimerization to be forced and thus facilitate the
process of purification of the antibody sought (Merchand 1998).
Examples of bispecific antibodies include those of the BiTE.TM.
technology in which the binding domains of two antibodies with
different specificity can be used and directly linked via short
flexible peptides. This combines two antibodies on a short single
polypeptide chain. Diabodies and scFv can be constructed without an
Fc region, using only variable domains, potentially reducing the
effects of anti-idiotypic reaction.
[0157] Bispecific antibodies can be constructed as entire IgG, as
bispecific Fab'2, as Fab'PEG, as diabodies or else as bispecific
scFv. Further, two bispecific antibodies can be linked using
routine methods known in the art to form tetravalent
antibodies.
[0158] Bispecific diabodies, as opposed to bispecific whole
antibodies, may also be particularly useful because they can be
readily constructed and expressed in E. coli. Diabodies (and many
other polypeptides such as antibody fragments) of appropriate
binding specificities can be readily selected using phage display
(WO94/13804) from libraries. If one arm of the diabody is to be
kept constant, for instance, with a specificity directed against a
target antigen, then a library can be made where the other arm is
varied and an antibody of appropriate specificity selected.
Bispecific whole antibodies may be made by alternative engineering
methods as described in Ridgeway 1996.
[0159] Various methods are available in the art for obtaining
antibodies against a target antigen. The antibodies may be
monoclonal antibodies, especially of human, murine, chimeric or
humanized origin, which can be obtained according to the standard
methods well known to the person skilled in the art.
[0160] In general, for the preparation of monoclonal antibodies or
their functional fragments, especially of murine origin, it is
possible to refer to techniques which are described in particular
in the manual "Antibodies" (Harlow and Lane 1988) or to the
technique of preparation from hybridomas described by Kohler and
Milstein, 1975.
[0161] Monoclonal antibodies can be obtained, for example, from an
animal cell immunized against A-FN or tenascin-C, or one of its
fragments containing the epitope recognized by said monoclonal
antibodies, e.g. a fragment comprising or consisting of ED-A or the
A1, A2, A3, A4, B or D domain of the tenascin-C large isoform, or a
peptide fragment of ED-A or the A1, A2, A3, A4, B or D domain of
the tenascin-C large isoform. The A-FN or tenascin-C, or one of
their fragments, can especially be produced according to the usual
working methods, by genetic recombination starting with a nucleic
acid sequence contained in the cDNA sequence coding for A-FN or
tenascin-C, or fragment thereof, by peptide synthesis starting from
a sequence of amino acids comprised in the peptide sequence of the
A-FN or tenascin-C and/or fragment thereof.
[0162] Monoclonal antibodies can, for example, be purified on an
affinity column on which A-FN or tenascin-C, or one of their
fragments containing the epitope recognized by said monoclonal
antibodies, e.g. a fragment comprising or consisting of ED-A or
tenascin-C, or a peptide fragment of ED-A or tenascin-C, has
previously been immobilized. Monoclonal antibodies can be purified
by chromatography on protein A and/or G, followed or not followed
by ion-exchange chromatography aimed at eliminating the residual
protein contaminants as well as the DNA and the LPS, in itself,
followed or not followed by exclusion chromatography on Sepharose
gel in order to eliminate the potential aggregates due to the
presence of dimers or of other multimers. The whole of these
techniques may be used simultaneously or successively.
Antigen-Binding Site
[0163] This describes the part of a molecule that binds to and is
complementary to all or part of the target antigen. In an antibody
molecule it is referred to as the antibody antigen binding site,
and comprises the part of the antibody that binds to and is
complementary to all or part of the target antigen. Where an
antigen is large, an antibody may only bind to a particular part of
the antigen, which part is termed an epitope. An antibody
antigen-binding site may be provided by one or more antibody
variable domains. An antibody antigen-binding site may comprise an
antibody light chain variable region (VL) and an antibody heavy
chain variable region (VH).
Isolated
[0164] This refers to the state in which specific binding members
for use in the invention or nucleic acid encoding such specific
binding members, will generally be in accordance with the present
invention. Thus, specific binding members, VH and/or VL domains of
the present invention may be provided isolated and/or purified,
e.g. from their natural environment, in substantially pure or
homogeneous form, or, in the case of nucleic acid, free or
substantially free of nucleic acid or genes of origin other than
the sequence encoding a polypeptide with the required function.
Isolated members and isolated nucleic acid will be free or
substantially free of material with which they are naturally
associated such as other polypeptides or nucleic acids with which
they are found in their natural environment, or the environment in
which they are prepared (e.g. cell culture) when such preparation
is by recombinant DNA technology practised in vitro or in vivo.
Specific binding members and nucleic acid may be formulated with
diluents or adjuvants and still for practical purposes be
isolated--for example the members will normally be mixed with
gelatin or other carriers if used to coat microtitre plates for use
in immunoassays, or will be mixed with pharmaceutically acceptable
carriers or diluents when used in diagnosis or therapy. Specific
binding members may be glycosylated, either naturally or by systems
of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC 85110503)
cells, or they may be (for example if produced by expression in a
prokaryotic cell) unglycosylated.
[0165] Heterogeneous preparations comprising antibody molecules may
also be used in the invention. For example, such preparations may
be mixtures of antibodies with full-length heavy chains and heavy
chains lacking the C-terminal lysine, with various degrees of
glycosylation and/or with derivatized amino acids, such as
cyclization of an N-terminal glutamic acid to form a pyroglutamic
acid residue.
[0166] One or more specific binding members for an antigen, e.g.
the A-FN, the tenascin-C, the ED-A of fibronectin, or the A1, A2,
A3, A4, B or D domain of the tenascin-C large isoform may be
obtained by bringing into contact a library of specific binding
members according to the invention and the antigen or a fragment
thereof, e.g. a fragment comprising or consisting of ED-A or
tenascin-C, or a peptide fragment of ED-A or tenascin-C and
selecting one or more specific binding members of the library able
to bind the antigen.
[0167] An antibody library may be screened using Iterative Colony
Filter Screening (ICFS). In ICFS, bacteria containing the DNA
encoding several binding specificities are grown in a liquid medium
and, once the stage of exponential growth has been reached, some
billions of them are distributed onto a growth support consisting
of a suitably pre-treated membrane filter which is incubated until
completely confluent bacterial colonies appear. A second trap
substrate consists of another membrane filter, pre-humidified and
covered with the desired antigen.
[0168] The trap membrane filter is then placed onto a plate
containing a suitable culture medium and covered with the growth
filter with the surface covered with bacterial colonies pointing
upwards. The sandwich thus obtained is incubated at room
temperature for about 16 h. It is thus possible to obtain the
expression of the genes encoding antibody fragments scFv having a
spreading action, so that those fragments binding specifically with
the antigen which is present on the trap membrane are trapped. The
trap membrane is then treated to point out bound antibody fragments
scFv with colorimetric techniques commonly used to this
purpose.
[0169] The position of the coloured spots on the trap filter allows
to go back to the corresponding bacterial colonies which are
present on the growth membrane and produced the antibody fragments
trapped. Such colonies are gathered and grown and the bacteria--a
few millions of them are distributed onto a new culture membrane
repeating the procedures described above. Analogous cycles are then
carried out until the positive signals on the trap membrane
correspond to single positive colonies, each of which represents a
potential source of monoclonal antibody fragments directed against
the antigen used in the selection. ICFS is described in e.g.
WO0246455, which is incorporated herein by reference. A library may
also be displayed on particles or molecular complexes, e.g.
replicable genetic packages such bacteriophage (e.g. T7) particles,
or other in vitro display systems, each particle or molecular
complex containing nucleic acid encoding the antibody VH variable
domain displayed on it, and optionally also a displayed VL domain
if present. Phage display is described in WO92/01047 and e.g. U.S.
Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907,
5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447,
6,291,650, 6,492,160 and 6,521,404, each of which is herein
incorporated by reference in its entirety.
[0170] Following selection of binding members able to bind the
antigen and displayed on bacteriophage or other library particles
or molecular complexes, nucleic acid may be taken from a
bacteriophage or other particle or molecular complex displaying a
said selected binding member. Such nucleic acid may be used in
subsequent production of a binding member or an antibody VH or VL
variable domain by expression from nucleic acid with the sequence
of nucleic acid taken from a bacteriophage or other particle or
molecular complex displaying a said selected binding member.
[0171] An antibody VH variable domain with the amino acid sequence
of an antibody VH variable domain of a said selected binding member
may be provided in isolated form, as may a binding member
comprising such a VH domain.
[0172] Ability to bind the A-FN or the tenascin-C, or the ED-A of
fibronectin or the A1, A2, A3, A4, B or D domain of the tenascin-C
large isoform, or other target antigen or isoform may be further
tested, e.g. ability to compete with e.g. any one of anti-ED-A
antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8 or G9 for binding
to the A-FN or a fragment of the A-FN, e.g. the ED-A of
fibronectin, or an anti-tenascin-C antibody, such as F16 or
4A1-F16, for binding to the tenascin-C or a fragment of tenascin-C,
e.g. the A1, A2, A3, A4, B or D domain of the tenascin-C large
isoform.
[0173] A specific binding member for use in the invention may bind
the A-FN and/or the ED-A of fibronectin, or tenascin-C and/or the
A1, A2, A3, A4, B or D domain of the tenascin-C isoform
specifically. A specific binding member of the present invention
may bind the A-FN and/or the ED-A of fibronectin, or the
tenascin-C, and/or the A1 domain of the tenascin-C large isoform,
with the same affinity as an anti-ED-A antibody H1, B2, C5, D5, E5,
C8, F8, F1, B7, E8 or G9, e.g. in scFv format, or as the
anti-tenascin-C antibodies F16 or 4A1-F16, respectively, or with an
affinity that is better. A specific binding member for use in the
invention may bind the A-FN and/or the ED-A of fibronectin, or the
tenascin-C and/or the A1, A2, A3, A4, B or D domain of the
tenascin-C large isoform, with a K.sub.D of 3.times.10.sup.-8 M or
an affinity that is better. Preferably, a specific binding member
for use in the invention binds the A-FN and/or the ED-A of
fibronectin, or the tenascin-C and/or the A1, A2, A3, A4, B or D
domain of the tenascin-C large isoform, with a K.sub.D of
2.times.10.sup.-8 M or an affinity that is better. More preferably,
a specific binding member for use in the invention binds the A-FN
and/or the ED-A of fibronectin, or the tenascin-C and/or the A1,
A2, A3, A4, B or D domain of the tenascin-C large isoform, with a
K.sub.D of 1.7.times.10.sup.-8 M or an affinity that is better. Yet
more preferably, a specific binding member for use in the invention
binds the A-FN and/or the ED-A of fibronectin, or the tenascin-C
and/or the A1, A2, A3, A4, B or D domain of the tenascin-C large
isoform, with a K.sub.D of 1.4.times.10.sup.-8 M or an affinity
that is better. Most preferably, a specific binding member for use
in the invention binds the A-FN and/or the ED-A of fibronectin, or
the tenascin-C and/or the A1, A2, A3, A4, B or D domain of the
tenascin-C large isoform, with a K.sub.D of 3.times.10.sup.-9 M or
an affinity that is better.
[0174] A specific binding member of the present invention may bind
to the same epitope on A-FN and/or the ED-A of fibronectin as one
of the anti-ED-A antibodies H1, B2, C5, D5, E5, C8, F8, F1, B7, E8
or G9, or to the same epitope on tenascin-C, and/or the A1 domain
of the tenascin-C large isoform, as the anti-tenascin-C antibody
F16 or 4A1-F16.
[0175] A specific binding member for use in the invention may not
show any significant binding to molecules other than to the A-FN
and/or the ED-A of fibronectin, or to the tenascin-C and/or the A1,
A2, A3, A4, B or D domain of the tenascin-C large isoform. In
particular, the specific binding member may not bind other isoforms
of fibronectin, for example the ED-B isoform and/or the IIICS
isoform of fibronectin, or other isoforms of tenascin-C, for
example the tenascin-C small isoform.
[0176] Variants of antibody molecules disclosed herein may be
produced and used in the present invention. The techniques required
to make substitutions within amino acid sequences of CDRs, antibody
VH or VL domains and binding members generally are available in the
art. Variant sequences may be made, with substitutions that may or
may not be predicted to have a minimal or beneficial effect on
activity, and tested for ability to bind A-FN and/or the ED-A of
fibronectin, or tenascin-C and/or the A1, A2, A3, A4, B or D domain
of the tenascin-C large isoform, and/or for any other desired
property.
[0177] Variable domain amino acid sequence variants of any of the
VH and VL domains whose sequences are specifically disclosed herein
may be employed in accordance with the present invention, as
discussed. Particular variants may include one or more amino acid
sequence alterations (addition, deletion, substitution and/or
insertion of an amino acid residue), may be less than about 20
alterations, less than about 15 alterations, less than about 10
alterations or less than about 5 alterations, maybe 5, 4, 3, 2 or
1. Alterations may be made in one or more framework regions and/or
one or more CDRs. The alterations normally do not result in loss of
function, so a specific binding member comprising a thus-altered
amino acid sequence may retain an ability to bind A-FN and/or the
ED-A of fibronectin, or tenascin C and/or the A1, A2, A3, A4, B or
D domain of the tenascin-C large isoform. For example, it may
retain the same quantitative binding as a specific binding member
in which the alteration is not made, e.g. as measured in an assay
described herein. The specific binding member comprising a
thus-altered amino acid sequence may have an improved ability to
bind A-FN and/or the ED-A of fibronectin, or tenascin-C and/or the
A1, A2, A3, A4, B or D domain of the tenascin-C large isoform.
[0178] Novel VH or VL regions carrying CDR-derived sequences for
use in the invention may be generated using random mutagenesis of
one or more selected VH and/or VL genes to generate mutations
within the entire variable domain. In some embodiments one or two
amino acid substitutions are made within an entire variable domain
or set of CDRs. Another method that may be used is to direct
mutagenesis to CDR regions of VH or VL genes.
[0179] As noted above, a CDR amino acid sequence substantially as
set out herein may be carried as a CDR in a human antibody variable
domain or a substantial portion thereof. The HCDR3 sequences
substantially as set out herein represent embodiments of the
present invention and for example each of these may be carried as a
HCDR3 in a human heavy chain variable domain or a substantial
portion thereof.
[0180] Variable domains employed in the invention may be obtained
or derived from any germ-line or rearranged human variable domain,
or may be a synthetic variable domain based on consensus or actual
sequences of known human variable domains. A variable domain can be
derived from a non-human antibody. A CDR sequence for use in the
invention (e.g. CDR3) may be introduced into a repertoire of
variable domains lacking a CDR (e.g. CDR3), using recombinant DNA
technology. For example, Marks et al. (1992) describe methods of
producing repertoires of antibody variable domains in which
consensus primers directed at or adjacent to the 5' end of the
variable domain area are used in conjunction with consensus primers
to the third framework region of human VH genes to provide a
repertoire of VH variable domains lacking a CDR3. Marks et al.
further describe how this repertoire may be combined with a CDR3 of
a particular antibody. Using analogous techniques, the CDR3-derived
sequences of the present invention may be shuffled with repertoires
of VH or VL domains lacking a CDR3, and the shuffled complete VH or
VL domains combined with a cognate VL or VH domain to provide
binding members for use in the invention. The repertoire may then
be displayed in a suitable host system such as the phage display
system of WO92/01047, which is herein incorporated by reference in
its entirety, or any of a subsequent large body of literature,
including Kay, Winter & McCafferty (1996), so that suitable
binding members may be selected. A repertoire may consist of from
anything from 10.sup.4 individual members upwards, for example at
least 10.sup.5, at least 10.sup.6, at least 10.sup.7, at least
10.sup.8, at least 10.sup.9 or at least 10.sup.10 members.
[0181] Similarly, one or more, or all three CDRs may be grafted
into a repertoire of VH or VL domains that are then screened for a
binding member or binding members for the A-FN and/or the ED-A of
fibronectin, or the tenascin-C and/or the A1, A2, A3, A4, B or D
domain of the tenascin-C large isoform.
[0182] One or more of the HCDR1, HCDR2 and HCDR3 of antibody H1,
B2, C5, D5, E5, C8, F8, F1, B7, E8, G9, F16, or 4A1-F16, or the set
of HCDRs of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8, G9,
F16 or 4A1-F16 may be employed, and/or one or more of the LCDR1,
LCDR2 and LCDR3 of antibody H1, B2, C5, D5, E5, C8, F8, F1, B7, E8,
G9, F16, or 4A1-F16, or the set of LCDRs of antibody H1, B2, C5,
D5, E5, C8, F8, F1, B7, E8, G9, F16 or 4A1-F16 may be employed.
[0183] Similarly, other VH and VL domains, sets of CDRs and sets of
HCDRs and/or sets of LCDRs disclosed herein may be employed.
[0184] The A-FN and/or the ED-A of fibronectin, or the tenascin-C
and/or the A1, A2, A3, A4, B or D domain of the tenascin-C large
isoform, may be used in a screen for specific binding members, e.g.
antibody molecules, for use in the preparation of a medicament for
the treatment of endometriosis, psoriasis, or psoriatic arthritis.
The screen may a screen of a repertoire as disclosed elsewhere
herein.
[0185] A substantial portion of an immunoglobulin variable domain
may comprise at least the three CDR regions, together with their
intervening framework regions. The portion may also include at
least about 50% of either or both of the first and fourth framework
regions, the 50% being the C-terminal 50% of the first framework
region and the N-terminal 50% of the fourth framework region.
Additional residues at the N-terminal or C-terminal end of the
substantial part of the variable domain may be those not normally
associated with naturally occurring variable domain regions. For
example, construction of specific binding members of the present
invention made by recombinant DNA techniques may result in the
introduction of N- or C-terminal residues encoded by linkers
introduced to facilitate cloning or other manipulation steps. Other
manipulation steps include the introduction of linkers to join
variable domains disclosed elsewhere herein to further protein
sequences including antibody constant regions, other variable
domains (for example in the production of diabodies) or
detectable/functional labels as discussed in more detail elsewhere
herein.
[0186] Although specific binding members may comprise a pair of VH
and VL domains, single binding domains based on either VH or VL
domain sequences may also be used in the invention. It is known
that single immunoglobulin domains, especially VH domains, are
capable of binding target antigens in a specific manner. For
example, see the discussion of dAbs above.
[0187] In the case of either of the single binding domains, these
domains may be used to screen for complementary domains capable of
forming a two-domain binding member able to bind A-FN and/or the
ED-A of fibronectin, or tenascin-C, and/or the A1, A2, A3, A4, B or
D domain of the tenascin-C large isoform. This may be achieved by
phage display screening methods using the so-called hierarchical
dual combinatorial approach as disclosed in WO92/01047, herein
incorporated by reference in its entirety, in which an individual
colony containing either an H or L chain clone is used to infect a
complete library of clones encoding the other chain (L or H) and
the resulting two-chain binding member is selected in accordance
with phage display techniques such as those described in that
reference. This technique is also disclosed in Marks 1992.
[0188] Specific binding members for use in the present invention
may further comprise antibody constant regions or parts thereof,
e.g. human antibody constant regions or parts thereof. For example,
a VL domain may be attached at its C-terminal end to antibody light
chain constant domains including human C.kappa. or C.lamda. chains,
e.g. C.lamda.. Similarly, a specific binding member based on a VH
domain may be attached at its C-terminal end to all or part (e.g. a
CH1 domain) of an immunoglobulin heavy chain derived from any
antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype
sub-classes, particularly IgG1 and IgG4. Any synthetic or other
constant region variant that has these properties and stabilizes
variable regions is also useful in embodiments of the present
invention.
[0189] Specific binding members for use in the invention may be
labelled with a detectable or functional label. A label can be any
molecule that produces or can be induced to produce a signal,
including but not limited to fluorescers, radiolabels, enzymes,
chemiluminescers or photosensitizers. Thus, binding may be detected
and/or measured by detecting fluorescence or luminescence,
radioactivity, enzyme activity or light absorbance. Detectable
labels may be attached to antibodies for use in the invention using
conventional chemistry known in the art.
[0190] There are numerous methods by which the label can produce a
signal detectable by external means, for example, by visual
examination, electromagnetic radiation, heat, and chemical
reagents. The label can also be bound to another specific binding
member that binds the antibody for use in the invention, or to a
support.
[0191] Labelled specific binding members, e.g. scFv labelled with a
detectable label, may be used diagnostically in vivo, ex vivo or in
vitro, and/or therapeutically.
[0192] For example, radiolabelled binding members (e.g. binding
members conjugated to a radioisotope) may be used in radiodiagnosis
and radiotherapy. Radioisotopes which may be conjugated to a
binding member for use in the invention include isotopes such as
.sup.94mTc, .sup.99mTc, .sup.186Re, .sup.188Re, .sup.203Pb,
.sup.67Ga, .sup.68Ga, .sup.47Sc, .sup.111In, .sup.97Ru, .sup.62Cu,
.sup.64Cu, .sup.86Y, .sup.88Y, .sup.90Y, .sup.121Sn, .sup.161Tb,
.sup.153Sm, .sup.166Ho, .sup.105Rh, .sup.177Lu, .sup.123I,
.sup.124I, .sup.125I, .sup.131I, .sup.18F, .sup.211At and
.sup.225Ac. Preferably, positron emitters, such as .sup.18F and
.sup.124I, or gamma emitters, such as .sup.99mTc, .sup.111In and
.sup.123I, are used for diagnostic applications (e.g. for PET),
while beta-emitters, such as .sup.131I, .sup.90Y and .sup.177Lu,
are preferably used for therapeutic applications. Alpha-emitters,
such as .sup.211At and .sup.225Ac may also be used for therapy.
[0193] For example, a specific binding member for use in the
invention labelled with a detectable label may be used to detect,
diagnose or monitor endometriosis, psoriasis, or psoriatic
arthritis in a human or animal.
[0194] A specific binding member of the present invention may be
used for the manufacture of a diagnostic product for use in
diagnosing endometriosis, psoriasis, or psoriatic arthritis.
[0195] The present invention provides a method of detecting or
diagnosing endometriosis, psoriasis, or psoriatic arthritis in a
human or animal comprising: [0196] (a) administering to the human
or animal a specific binding member of the present invention, for
example labelled with a detectable label, which binds the ED-A
isoform of fibronectin and/or the ED-A of fibronectin, or
tenascin-C and/or the A1, A2, A3, A4, B and/or D domain of the
tenascin-C large isoform, and [0197] (b) determining the presence
or absence of the specific binding member in sites of
endometriosis, psoriasis, or psoriatic arthritis of the human or
animal body; wherein localisation of the specific binding member to
sites of endometriosis, psoriasis, or psoriatic arthritis indicates
the presence of endometriosis, psoriasis, or psoriatic
arthritis.
[0198] Where the binding member is labelled with a detectable
label, the presence or absence of the detectable label may be
determined by detecting the label.
[0199] A conjugate or fusion between a binding member for use in
the invention and a molecule that exerts a biocidal, cytotoxic
immunosuppressive or anti-inflammatory effect on target cells in
the lesions and an antibody directed against an extracellular
matrix component which is present in such lesions may be employed
in the present invention. For example, the conjugated molecule may
be inter alia interleukin-10, TGF-.beta., IL-2, IL-12, IL-15,
IL-21, IL-24, IL-33, tumour necrosis factor (TNF), or
interferon-.alpha., -.beta. or -.gamma., an anti-inflammatory or
other drug, a photosensitizer or a radionuclide. Such conjugates
may be used therapeutically, e.g. for treatment of endometriosis,
psoriasis, or psoriatic arthritis as referred to herein.
[0200] Production and use of fusions or conjugates of specific
binding members with biocidal or cytotoxic molecules is described
for example in WO01/62298, which is incorporated by reference
herein.
[0201] The invention provides a method of treating endometriosis,
psoriasis, or psoriatic arthritis, the method comprising
administering to an individual a therapeutically effective amount
of a medicament comprising a specific binding member for use in the
invention.
[0202] The specific binding member for use in the invention may be
a conjugate of (i) a molecule which exerts an anti-inflammatory
effect on target cells by cellular interaction, an
anti-inflammatory molecule, a cytokine e.g. IL-10, TGF-.beta.,
IL-2, IL-12, IL-15, IL-21, IL-24, IL-33, tumour necrosis factor
(TNF), interferon-.alpha., -.beta. or -.gamma., or other drug, and
(ii) a specific binding member for the ED-A isoform of fibronectin
and/or the ED-A of fibronectin, or tenascin-C, and/or the A1, A2,
A3, A4, B and/or D domain of the tenascin-C large isoform.
[0203] The specific binding member for use in the invention may be
a conjugate of (i) a molecule which exerts an immunosuppressive or
anti-inflammatory effect and (ii) a specific binding member for the
ED-A isoform of fibronectin and/or the ED-A of fibronectin, or
tenascin-C, and/or the A1, A2, A3, A4, B and/or D domain of the
tenascin-C large isoform.
[0204] The specific binding member for use in the invention may be
a conjugate of (i) interleukin-10 (IL10) or TGF beta and (ii) a
specific binding member for the ED-A isoform of fibronectin and/or
the ED-A of fibronectin, or tenascin-C, and/or the A1, A2, A3, A4,
B and/or D domain of the tenascin-C large isoform. Such a specific
binding member is useful in aspects of the invention disclosed
herein relating to treatment of endometriosis, psoriasis and
psoriatic arthritis.
[0205] The invention provides the use of a specific binding member
as described herein for the preparation of a medicament for the
treatment of endometriosis, psoriasis and psoriatic arthritis.
[0206] The specific binding member for use in the invention may be
a conjugated or fused to a molecule that exerts a biocidal,
cytotoxic, immunosuppressive or anti-inflammatory effect as
described herein. The specific binding member for use in the
invention may be a conjugate of (i) a molecule which exerts a
biocidal or cytotoxic effect on target cells by cellular
interaction or has an immunosuppressive or anti-inflammatory effect
and (ii) a specific binding member for the ED-A isoform of
fibronectin and/or the ED-A of fibronectin, or tenascin-C, and/or
the A1, A2, A3, A4, B and/or D domain of the tenascin-C large
isoform.
[0207] Also described herein is a conjugate of (i) a molecule which
exerts a biocidal or cytotoxic effect on target cells by cellular
interaction, or an immunosuppressive or anti-inflammatory effect
and (ii) a binding member for the ED-A isoform of fibronectin
and/or the ED-A of fibronectin, or tenascin-C, and/or the A1, A2,
A3, A4, Band/or D domain of the tenascin-C large isoform. Such a
conjugate preferably comprises a fusion protein comprising the
biocidal, cytotoxic, immunosuppressive or anti-inflammatory
molecule and a said binding member, or, where the binding member is
two-chain or multi-chain, a fusion protein comprising the biocidal,
cytotoxic, immunosuppressive or anti-inflammatory molecule and a
polypeptide chain component of said binding member. Preferably the
binding member is a single-chain polypeptide, e.g. a single-chain
antibody molecule, such as scFv.
[0208] A fusion protein comprising the immunosuppressive or
anti-inflammatory molecule and a single-chain Fv antibody molecule
may be used in the invention.
[0209] The immunosuppressive or anti-inflammatory molecule that
exerts its effect on target cells by cellular interaction, may
interact directly with the target cells, may interact with a
membrane-bound receptor on the target cell or perturb the
electrochemical potential of the cell membrane. Preferably, the
molecule is IL-10 or TGF-.beta..
[0210] Examples of other molecules which can be conjugated to the
specific binding member include IL-2, IL-12, IL-15, IL-21, IL-24,
IL-33, tumour necrosis factor (TNF), or interferon-.alpha., -.beta.
or -.gamma..
[0211] As discussed further below, the specific binding member for
use in the invention is preferably an antibody molecule or
comprises an antibody antigen-binding site. Conveniently, the
specific, binding member may be a single-chain polypeptide, such as
a single-chain antibody. This allows for convenient production of a
fusion protein comprising single-chain antibody and, for example,
immunosuppressive or anti-inflammatory molecule (e.g.
interleukin-10 or TGF beta). An antibody antigen-binding site may
be provided by means of association of an antibody VH domain and an
antibody VL domain in separate polypeptides, e.g. in a complete
antibody or in an antibody fragment such as Fab or diabody. Where
the specific binding member is a two-chain or multi-chain molecule
(e.g. Fab or whole antibody, respectively), an immunosuppressive or
anti-inflammatory molecule, for example, may be conjugated as a
fusion polypeptide with one or more polypeptide chains in the
specific binding member.
[0212] The specific binding member may be conjugated with the
immunosuppressive or anti-inflammatory molecule by means of a
peptide bond, i.e. within a fusion polypeptide comprising said
molecule and the specific binding member or a polypeptide chain
component thereof (see e.g. Trachsel et al.). Other means for
conjugation include chemical conjugation, especially cross-linking
using a bifunctional reagent (e.g. employing DOUBLE-REAGENTS.TM.
Cross-linking Reagents Selection Guide, Pierce).
[0213] Also described herein is isolated nucleic acid encoding a
specific binding member for use in the present invention. Nucleic
acid may include DNA and/or RNA. A nucleic acid may code for a CDR
or set of CDRs or VH domain or VL domain or antibody
antigen-binding site or antibody molecule, e.g. scFv or IgG, e.g.
IgG1, as defined above. The nucleotide sequences may encode the VH
and/or VL domains disclosed herein.
[0214] Further described herein are constructs in the form of
plasmids, vectors, transcription or expression cassettes which
comprise at least one polynucleotide as described above.
[0215] A recombinant host cell that comprises one or more
constructs as above are also described. A nucleic acid encoding any
CDR or set of CDRs or VH domain or VL domain or antibody
antigen-binding site or antibody molecule, e.g. scFv or IgG1 or
IgG4 as provided, is described, as is a method of production of the
encoded product, which method comprises expression from encoding
nucleic acid. Expression may conveniently be achieved by culturing
under appropriate conditions recombinant host cells containing the
nucleic acid. Following production by expression a VH or VL domain,
or specific binding member may be isolated and/or purified using
any suitable technique, then used as appropriate.
[0216] A nucleic acid may comprise DNA or RNA and may be wholly or
partially synthetic. Reference to a nucleotide sequence as set out
herein encompasses a DNA molecule with the specified sequence, and
encompasses a RNA molecule with the specified sequence in which U
is substituted for T, unless context requires otherwise.
[0217] A method of production of an antibody VH variable domain,
the method including causing expression from encoding nucleic acid
is also described. Such a method may comprise culturing host cells
under conditions for production of said antibody VH variable
domain.
[0218] A method of production may comprise a step of isolation
and/or purification of the product. A method of production may
comprise formulating the product into a composition including at
least one additional component, such as a pharmaceutically
acceptable excipient.
[0219] Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host cells
include bacteria, mammalian cells, plant cells, filamentous fungi,
yeast and baculovirus systems and transgenic plants and animals.
The expression of antibodies and antibody fragments in prokaryotic
cells is well established in the art. For a review, see for example
Pluckthun 1991. A common bacterial host is E. coli.
[0220] Expression in eukaryotic cells in culture is also available
to those skilled in the art as an option for production of a
specific binding member for example Chadd & Chamow (2001),
Andersen & Krummen (2002), Larrick & Thomas (2001).
Mammalian cell lines available in the art for expression of a
heterologous polypeptide include Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells,
YB2/0 rat myeloma cells, human embryonic kidney cells, human
embryonic retina cells and many others.
[0221] Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator sequences, polyadenylation sequences, enhancer
sequences, marker genes and other sequences as appropriate. Vectors
may be plasmids e.g. phagemid, or viral e.g. `phage, as
appropriate. For further details see, for example, Sambrook &
Russell (2001). Many known techniques and protocols for
manipulation of nucleic acid, for example in preparation of nucleic
acid constructs, mutagenesis, sequencing, introduction of DNA into
cells and gene expression, and analysis of proteins, are described
in detail in Ausubel 1999.
[0222] A host cell may contain a nucleic acid as described herein.
Such a host cell may be in vitro and may be in culture. Such a host
cell may be in vivo. In vivo presence of the host cell may allow
intracellular expression of a binding member for use in the present
invention as "intrabodies" or intracellular antibodies. Intrabodies
may be used for gene therapy.
[0223] A method comprising introducing a nucleic acid disclosed
herein into a host cell is also described. The introduction may
employ any available technique. For eukaryotic cells, suitable
techniques may include calcium phosphate transfection,
DEAE-Dextran, electroporation, liposome-mediated transfection and
transduction using retrovirus or other virus, e.g. vaccinia or, for
insect cells, baculovirus. Introducing nucleic acid in the host
cell, in particular a eukaryotic cell may use a viral or a plasmid
based system. The plasmid system may be maintained episomally or
may be incorporated into the host cell or into an artificial
chromosome. Incorporation may be either by random or targeted
integration of one or more copies at single or multiple loci. For
bacterial cells, suitable techniques may include calcium chloride
transformation, electroporation and transfection using
bacteriophage.
[0224] The introduction may be followed by causing or allowing
expression from the nucleic acid, e.g. by culturing host cells
under conditions for expression of the gene. The purification of
the expressed product may be achieved by methods known to one of
skill in the art.
[0225] The nucleic acid may be integrated into the genome (e.g.
chromosome) of the host cell. Integration may be promoted by
inclusion of sequences that promote recombination with the genome,
in accordance with standard techniques.
[0226] A method that comprises using a construct as stated above in
an expression system in order to express a specific binding member
or polypeptide as above is also described.
[0227] Specific binding members for use in the present invention
are designed to be used in methods of diagnosis or treatment in
human or animal subjects, e.g. human. Specific binding members for
use in the invention may be used in diagnosis or treatment of
endometriosis, psoriasis and psoriatic arthritis.
[0228] Accordingly, the invention provides methods of treatment
comprising administration of a specific binding member as
described, pharmaceutical compositions comprising such a specific
binding member, and use of such a specific binding member in the
manufacture of a medicament for administration, for example in a
method of making a medicament or pharmaceutical composition
comprising formulating the specific binding member with a
pharmaceutically acceptable excipient. Pharmaceutically acceptable
vehicles are well known and will be adapted by the person skilled
in the art as a function of the nature and of the mode of
administration of the active compound(s) chosen.
[0229] Specific binding members for use in the present invention
will usually be administered in the form of a pharmaceutical
composition, which may comprise at least one component in addition
to the specific binding member. Thus, pharmaceutical compositions
described herein, and for use in accordance with the present
invention, may comprise, in addition to active ingredient, a
pharmaceutically acceptable excipient, carrier, buffer, stabilizer
or other materials well known to those skilled in the art. Such
materials should be non-toxic and should not interfere with the
efficacy of the active ingredient. The precise nature of the
carrier or other material will depend on the route of
administration, which may be oral, inhaled or by injection, e.g.
intravenous.
[0230] Pharmaceutical compositions for oral administration such as
for example nanobodies etc are also envisaged in the present
invention. Such oral formulations may be in tablet, capsule,
powder, liquid or semi-solid form. A tablet may comprise a solid
carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally comprise a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included.
[0231] For intravenous injection, or injection at the site of
affliction, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and
has suitable pH, isotonicity and stability. Those of relevant skill
in the art are well able to prepare suitable solutions using, for
example, isotonic vehicles such as Sodium Chloride Injection,
Ringer's Injection, Lactated Ringer's Injection. Preservatives,
stabilizers, buffers, antioxidants and/or other additives may be
employed, as required. Many methods for the preparation of
pharmaceutical formulations are known to those skilled in the art.
See e.g. Robinson, 1978.
[0232] A composition may be administered alone or in combination
with other treatments, concurrently or sequentially or as a
combined preparation with another therapeutic agent or agents,
dependent upon the condition to be treated.
[0233] A specific binding member for use in the present invention
may be used as part of a combination therapy in conjunction with an
additional medicinal component. Combination treatments may be used
to provide significant synergistic effects, particularly the
combination of a specific binding member for use in the present
invention with one or more other drugs. A specific binding member
for use in the present invention may be administered concurrently
or sequentially or as a combined preparation with another
therapeutic agent or agents, for the treatment of one or more of
the conditions listed herein.
[0234] For example, a specific binding member for use in the
invention may be used in combination with an existing therapeutic
agent for the treatment of endometriosis, psoriasis or psoriatic
arthritis.
[0235] A specific binding member for use in the invention and one
or more of the above additional medicinal components may be used in
the manufacture of a medicament. The medicament may be for separate
or combined administration to an individual, and accordingly may
comprise the specific binding member and the additional component
as a combined preparation or as separate preparations. Separate
preparations may be used to facilitate separate and sequential or
simultaneous administration, and allow administration of the
components by different routes e.g. oral and parenteral
administration.
[0236] In accordance with the present invention, compositions
provided may be administered to mammals. Administration may be in a
"therapeutically effective amount", this being sufficient to show
benefit to a patient. Such benefit may be at least amelioration of
at least one symptom. Thus "treatment of endometriosis, psoriasis
or psoriatic arthritis" refers to amelioration of at least one
symptom. The actual amount administered, and rate and time-course
of administration, will depend on the nature and severity of what
is being treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the
site of delivery of the composition, the type of specific binding
member, the method of administration, the scheduling of
administration and other factors known to medical practitioners.
Prescription of treatment, e.g. decisions on dosage etc, is within
the responsibility of general practitioners and other medical
doctors, and may depend on the severity of the symptoms and/or
progression of a disease being treated. Appropriate doses of
antibody are well known in the art (Ledermann 1991 and Bagshawe
1991. Specific dosages indicated herein, or in the Physician's Desk
Reference (2003) as appropriate for the type of medicament being
administered, may be used. A therapeutically effective amount or
suitable dose of a specific binding member for use in the invention
can be determined by comparing its in vitro activity and in vivo
activity in an animal model. Methods for extrapolation of effective
dosages in mice and other test animals to humans are known. The
precise dose will depend upon a number of factors, including
whether the antibody is for diagnosis, prevention or for treatment,
the size and location of the area to be treated, the precise nature
of the antibody (e.g. whole antibody, fragment or diabody), and the
nature of any detectable label or other molecule attached to the
antibody. A typical antibody dose will be in the range 100 .mu.g to
1 g for systemic applications, and 1 .mu.g to 1 mg for topical
applications. An initial higher loading dose, followed by one or
more lower doses, may be administered. An antibody may be a whole
antibody, e.g. the IgG1 or IgG4 isotype. This is a dose for a
single treatment of an adult patient, which may be proportionally
adjusted for children and infants, and also adjusted for other
antibody formats in proportion to molecular weight. Treatments may
be repeated at daily, twice-weekly, weekly or monthly intervals, at
the discretion of the physician. Treatments may be every two to
four weeks for subcutaneous administration and every four to eight
weeks for intravenous administration. In some embodiments of the
present invention, treatment is periodic, and the period between
administrations is about two weeks or more, e.g. about three weeks
or more, about four weeks or more, or about once a month. In other
embodiments of the invention, treatment may be given before, and/or
after surgery, and may be administered or applied directly at the
anatomical site of surgical treatment. Further aspects and
embodiments of the invention will be apparent to those skilled in
the art given the present disclosure including the following
experimental exemplification.
EXPERIMENTAL
Results
Histochemical Analysis of Human Endometriotic Specimens
[0237] Expression of fibronectin domains ED-A and ED-B and the A1
domain of the tenascin-C large isoform was investigated by
immunohistochemistry on human endometriotic specimens using
biotinylated F8-, L19- and F16-SIP antibodies respectively. The
results of the immunohistochemical analysis are shown in FIG.
1.
[0238] In FIG. 1 darker staining indicates expression of the
respective antigens (indicated with black arrows).
[0239] Both the ED-A of fibronectin (recognised by the biotinylated
F8-SIP antibody) and the A1 domain of tenascin-C (recognised by the
biotinylated F16-SIP antibody) were strongly expressed around the
perivascular structures of biopsies of human endometriotic
specimens (see FIGS. 1 A,B&C for staining with F8-SIP and FIGS.
1 G,H&I for staining with F16-SIP). The intense staining of
vascular structures with F16-SIP was stronger than observed with
F8-SIP. No staining is visible for the negative control, i.e. the
same type of endometriotic specimen incubated with the streptavidin
reagent, but without any primary antibody (see FIGS. 1
J,K&L).
[0240] The ED-B domain of fibronectin (recognised by the
biotinylated L19-SIP antibody) was only weakly positive (see FIGS.
1 D,E&F).
Histochemical Analysis of Human Ulcerative Colitic Specimens
[0241] Expression of fibronectin domains ED-A and ED-B, and the A1
domain of the tenascin-C large isoform were investigated by
immunohistochemistry on human ulcerative colitic specimens using
biotinylated F8-, L19- and F16-SIP antibodies respectively. The
results of the immunohistochemical analysis are shown in FIG.
2.
[0242] In FIG. 2, areas of negative staining around perivascular
structures are indicated using white arrows.
[0243] Immunohistochemical analysis of human ulcerative colitic
samples revealed virtually negative staining for all three
antibodies, F8, L19 and F16 (see FIGS. 2A,B&C respectively).
Only a weak positivity was observed with F8-SIP in some
specimens.
Histochemical Analysis of Human Psoriatic Arthritic Specimens
[0244] Expression of fibronectin domains ED-A and ED-B, and the A1
domain of the tenascin-C large isoform were investigated by
immunohistochemistry on human psoriatic arthritic specimens using
biotinylated F8-, L19- and F16-SIP antibodies respectively. The
results of the immunohistochemical analysis are shown in FIG.
3.
[0245] In FIG. 3, darker staining indicates expression of the
respective antigens (indicated with black arrows).
[0246] The A1 domain of tenascin-C (recognised by the biotinylated
F16-SIP antibody) was strongly expressed around the perivascular
structures of biopsies of human psoriatic arthritic specimens, i.e.
very intense staining was observed (see FIG. 3C). No staining was
visible for the negative control nor for the ED-B domain of
fibronectin (recognised by the biotinylated L19-SIP antibody) or
the ED-A domain of fibronectin (recognised by the biotinylated
F8-SIP antibody) (see FIGS. 3 A, B and D respectively).
Histochemical Analysis of Samples from Patients with Multiple
Sclerosis
[0247] Expression of fibronectin domains ED-A and ED-B, and the A1
domain of the tenascin-C large isoform were investigated by
immunohistochemistry on samples from patients with multiple
sclerosis using biotinylated F8-, L19- and F16-SIP antibodies
respectively. The results of the immunohistochemical analysis are
shown in FIG. 4.
[0248] In FIG. 4, areas of negative staining around perivascular
structures are indicated using white arrows.
[0249] Immunohistochemical analysis of tissue sections from
pathological specimens from patients with multiple sclerosis
revealed only extremely weak positivity at vascular structures for
L19, F8 and F16 (see FIGS. 4 A,B&C).
Histochemical Analysis of Samples from Patients with Psoriasis
[0250] Expression of fibronectin domains ED-A and ED-B, and the A1
domain of the tenascin-C large isoform were investigated by
immunohistochemistry on samples from patients with psoriasis using
biotinylated F8-, L19- and F16-SIP antibodies respectively. The
results of the immunohistochemical analysis are shown in FIG.
5.
[0251] In FIG. 5, darker staining indicates expression of the
respective antigens (indicated with black arrows).
[0252] The A1 domain of tenascin-C (recognised by the biotinylated
F16-SIP antibody) was strongly expressed around the perivascular
structures of biopsies of human psoriatic specimens, i.e. very
intense staining was observed (see FIG. 5C). No staining was
visible for the negative control nor for the ED-B domain of
fibronectin (recognised by the biotinylated L19-SIP antibody) or
the ED-A domain of fibronectin (recognised by the biotinylated
F8-SIP antibody) (see FIGS. 5 D, B and A respectively).
[0253] Based on the findings described above, expression of ED-A
and the tenascin-C large isoform is not found in all
angiogenesis-related diseases. Endometriosis is particularly suited
for pharmacodelivery using anti-ED-A antibody molecules, while
endometriosis, psoriasis and psoriatic arthritis are particularly
suited for pharmacodelivery using anti-tenascin-C antibody
molecules.
Near Infrared Imaging of Endometriotic Lesions in Mice
[0254] A mouse model of endometriosis was generated as described
below. The selective accumulation of F8-SIP in mice with
endometriosis was tested by near infrared imaging analysis, as
described by Birchler et al. [Birchler et al., J Immunological
Methods 1999, 231, 239-248]. F8-SIP and F16-SIP were labeled using
Alexa750 (Molecular Probes), according to the manufacturer's
recommendations, and injected into the tail vein of endometriosis
mice. Mice were sacrificed and imaged in a near infrared mouse
imager 24 hours after injection. F16-SIP was used as a negative
control as it recognises the human A1 domain of tenascin-C but not
the murine antigen.
[0255] As shown in FIGS. 10 A and B, SIP(F8)-ALEXA750 accumulates
on the endometriotic lesions (indicated by arrows), whereas
SIP(F16)-ALEXA750 does not (FIG. 10C), thus confirming the in vivo
specificity of F8-SIP.
Ex Vivo Detection of SIP(F8)-ALEXA750
[0256] As soon as the near infrared imaging was completed,
endometriotic lesions were removed from sacrificed animals,
embedded in cryoembedding compound (Microm, Walldorf, Germany) and
stored at -80.degree. C. Sections (10 .mu.m) were then cut and
fixed in acetone. SIP(F8)-ALEXA750 was detected using a rabbit
anti-human IgE antibody (Dako, Glostrup, Denmark), followed by
Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Leiden, The
Netherlands). For the detection of blood vessels, double staining
with a rat anti-CD31 antibody followed by Alexa Fluor 594 donkey
anti-rat IgG was performed. As a negative control, the specimen was
probed with Alexa Fluor 488 goat anti-rabbit IgG minus the human
IgE antibody.
[0257] The results clearly show correspondence between the blood
vessels (see brighter areas in FIGS. 11 D, E and F) and staining
with SIP(F8)-ALEXA750 (see brighter areas in FIGS. 11 A, B and C).
These results confirm that SIP(F8)-ALEXA750 had accumulated around
vascular structures during the imaging studies.
[0258] These data show the first successful attempt at in vivo
imaging of endometriosis without the need for laparoscopy, or other
form of surgery.
Materials and Methods
[0259] Immunohistochemistry with Biotinylated SIP Antibodies
[0260] The tissue sections were flied in cold acetone (-20.degree.
C.) for 10 minutes and the slides were dried at room temperature
for 30 minutes. Silicon was applied using a pen and the slides were
then immersed in TBS (50 mM Tris, 100 mM NaCl, pH 7.4, 0.01%
aprotinin) for 5 minutes. The slides were dried with paper without
touching the sections. The sections were blocked with 20% fetal
calf serum (FCS) in TBS for 30 minutes. The blocking solution was
then removed and the slides were submerged in TBS for 5 minutes.
The primary biotinylated antibody in SIP format was diluted in
TBS/3% BSA to a final concentration of 1.6 .mu.g/ml and applied to
the sections for 60 minutes at room temperature. The slides were
washed twice with TBS+2 mM MgCl.sub.2 (406 mg/l) (2.times.5
minutes). The back of the slides were dried with paper and
SAP-complex 1:150 (Biopsa F014-62) in TBS+2 mM MgCl.sub.2/3% BSA
for 60 minutes at room temperature. The sections were washed three
times with TBS+2 mM MgCl.sub.2 (3.times.5 minutes). The substrate
(made up by dissolving 1 Tris and 1 FastRed tablet per 1 ml MilliQ
water) was added and incubated on the sections for 10 minutes. The
sections were washed twice with deionised water (2.times.2 minutes)
and transferred to Gill's hematoxylin solution no. 2 for 2 minutes.
The slides were quickly transferred to de-ionised water and rinsed
with water for 5 minutes. The slides were allowed to dry and
mounted with glycerol and visualised with an optical microscope
(Zeiss Axiovert 5100 TV).
Mouse Model of Endometriosis
[0261] 6-8 week old C57BL/6 mice were subjected to ovariectomy 7
days prior to induction of endometriosis. Mice were anesthesized by
isoflurane in combination with carprofen (Rimadyl). After
ovariectomy, mice were oestrogen-treated (3.times./week 4 ug
estradiol/mouse in a volume of 100 ul arachis oil subcutaneously
injected, starting at the day of transplantation). Ovariectomy plus
oestrogen supplementation was done in order to abrogate differences
related to the stage of the oestrous cycle. At day 0, mice were
split into 2 groups: donor mice (33%) and recipient mice (66%).
Donor mice were killed and both uterine horns were removed and
subsequently placed in a sterile Petri dish containing sterile
saline. Endometrium was detached from the uterine muscle and finley
chopped using a scalpel. Endometrial fragments were suspended in
saline and injected into the peritoneal cavity of the recipient
mice. 4 weeks after transplantation, mice were used for near
infrared imaging.
TABLE-US-00001 TABLE 1 Nucleotide sequences of the heavy chains
(VH) of anit-ED-A and anti-tenascin C antibodies. VH CDR1 sequences
are underlined; VH CDR2 sequences are in italics and underlined; VH
CDR 3 sequences are in bold and underlined. Antibody VH domain H1
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCGCGGAGGATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 1) B2
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGCGGCTAAGATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 3) C5
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCCGATTACTATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 5) D5
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGTGATGAAGATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 7) E5
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCACTGGTTCTATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 9) C8
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTTCAGACTATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 11) F8 VH
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 13) F8 VH V5L
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 15) F1
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTAGGCGCGTATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 17) B7
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCATTTTGATATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 19) E8
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCGATATGCATATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 21) G9
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCATATGCAGATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (SEQ ID NO: 23) F16
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGGTATGGTATGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAGCGCATAATGCTTTTGACTACTGGGGCCAGGGAACC
CTGGTCACCGTGTCGAGA (SEQ ID NO: 25) 4A1-F16
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA
GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCCGGTATGGTGCGAGCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT
GGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGC
CGTATATTACTGTGCGAAAGCGCATAATGCTTTTGACTACTGGGGCCAGGGAACC
CTGGTCACCGTGTCGAGA (SEQ ID NO: 27)
TABLE-US-00002 TABLE 2 Amino acid sequences of the heavy chains
(VH) of anti-ED-A and anti-tenascin C antibodies. VH CDR1 sequences
are underlined; VH CDR2 sequences are in italics and underlined; VH
CDR 3 sequences are in bold and underlined. Antibody VH domain H1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSPRRMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 2) B2
EVQLVESGGGLVQPGGSLRLSCAASGFTFSAAKMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 4) C5
EVQLVESGGGLVQPGGSLRLSCAASGFTFSPITMSWVRQAPGKGLEWVSAISGS
GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYW GQGTLVTVSS
(SEQ ID NO: 6) D5
EVQLVESGGGLVQPGGSLRLSCAASGFTFSVMKMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDW GQGTLVTVSS
(SEQ ID NO: 8) E5
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTGSMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDW GQGTLVTVSS
(SEQ ID NO: 10) C8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSLQTMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 12) F8 VH
EVQLVESGGGLVQPGGSLRLSCAASGFTFSLFTMSINVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 14) F8 VH V5L
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 16) F1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSQARMSWVRQAPGKGLEWVSAISGS
GGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYW GQGTLVTVSS
(SEQ ID NO: 18) B7
EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFDMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 20) E8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDMHMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 22) G9
EVQLVESGGGLVQPGGSLRLSCAASGFTFSHMQMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDY WGQGTLVTVSS
(SEQ ID NO: 24) F16
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWG QGTLVTVSR
(SEQ ID NO: 26) 5A1-F16
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGASVVVRQAPGKGLEWVSAISG
SGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWG QGTLVTVSR
(SEQ ID NO: 28)
TABLE-US-00003 TABLE 3 Nucleotide sequences of the light chains
(VL) of anti-ED-A and anti-tenascin C antibodies. VL CDR1 sequences
are underlined; VL CDR2 sequences are in italics and underlined; VL
CDR 3 sequences are in bold and underlined. Antibody VL domain H1
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTCTGCGTGGTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 63) B2
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGTGGCTTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 65) C5
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTTGCATTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 67) D5
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAATGCTTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 69) E5
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTTGCGCATTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 71) C8
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTTCCTTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 73) F8 VL
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCATGCCGTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 75) F8 VL
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA K18R
GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCATGCCGTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 77) F1
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCGCGCCTTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 79) B7
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCCTGGCTTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 81) E8
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCTCGTCTTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 83) G9
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAA
AAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCACTGCTTTTTTAGC
CTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCA
TCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGA
CAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTA
TTACTGTCAGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAG GTGGAAATCAAA
(SEQ ID NO: 85) F16 and
TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAG 4A1-F16
TCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTA
CCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAAC
CGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAG
CTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTG
TAACTCCTCTGTTTATACTATGCCGCCCGTGGTATTCGGCGGAGGGACCAAG CTGACCGTCCTA
(SEQ ID NO: 87)
TABLE-US-00004 TABLE 4 Amino acid sequences of the light chains
(VL) of anti-ED-A and anti-tenascin C antibodies. VL CDR1 sequences
are underlined; VH CDR2 sequences are in italics and underlined; VH
CDR 3 sequences are in bold and underlined. Antibody VL domain H1
EIVLTQSPGTLSLSPGEKATLSCRASQSVSSAWLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
64) B2 EIVLTQSPGTLSLSPGEKATLSCRASQSVSVAFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
66) C5 EIVLTQSPGTLSLSPGEKATLSCRASQSVSLHFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
68) D5 EIVLTQSPGTLSLSPGEKATLSCRASQSVSNAFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
70) E5 EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAHLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
72) C8 EIVLTQSPGTLSLSPGEKATLSCRASQSVSLPFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
74) F8 VL EIVLTQSPGTLSLSPGEKATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
76) F8 VL EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASS
K18R RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID
NO: 78) F1 EIVLTQSPGTLSLSPGEKATLSCRASQSVSAPFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
80) B7 EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
82) E8 EIVLTQSPGTLSLSPGEKATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
84) G9 EIVLTQSPGTLSLSPGEKATLSCRASQSVSTAFLAWYQQKPGQAPRLLIYGASS
RATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (SEQ ID NO:
86) F16 and SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRP
4A1-F16 SGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPVVFGGGTKLTVL (SEQ
ID NO: 88)
Sequences Disclosed in Application
TABLE-US-00005 [0262] (H1 VH domain) SEQ ID NO: 1
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCCCGCGGAGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC
CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC
GAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (H1 VH domain) SEQ ID NO: 2
EVQLVESGGGLVQPGGSLRLSCAASGFTFSPRRMSWVRQAPGKGLEWVSAISGSGGSTYYADSVK
GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (B2 VH
domain) SEQ ID NO: 3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCGCGGCTAAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC
CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC
GAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (B2 VH domain) SEQ ID NO: 4
EVQLVESGGGLVQPGGSLRLSCAASGFTFSAAKMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C5 VH domain)
SEQ ID NO: 5
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCCCGATTACTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCC
GGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCG
AGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGG
GAACCCTGGTCACCGTCTCGAGT (C5 VH domain) SEQ ID NO: 6
EVQLVESGGGLVQPGGSLRLSCAASGFTFSPITMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (D5 VH domain)
SEQ ID NO: 7
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCGTGATGAAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC
CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC
GAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (D5 VH domain) SEQ ID NO: 8
EVQLVESGGGLVQPGGSLRLSCAASGFTFSVMKMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVIVSS (E5 VH domain)
SEQ ID NO: 9
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCACTGGTTCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC
CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC
GAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (E5 VH domain) SEQ ID NO: 10
EVQLVESGGGLVQPGGSLRLSCAASGFTFSTGSMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C8 VH domain)
SEQ ID NO: 11
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCCTTCAGACTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC
CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC
GAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (C8 VH domain) SEQ ID NO: 12
EVQLVESGGGLVQPGGSLRLSCAASGFTFSLQTMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVIVSS (F8 VH domain)
SEQ ID NO: 13
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGT
GCAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG
CTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC
CGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCC
GAGGACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCGAGT (F8 VH domain) SEQ ID NO: 14
EVQLVESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F8 VH V5L
domain) SEQ ID NO: 15
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCCTGTTTACGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT
GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG
TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGT (F8 VH V5L domain) SEQ ID NO: 16
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLFTMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR
FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F1 VH domain)
SEQ ID NO: 17
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCTAGGCGCGTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT
GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG
TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGT (F1 VH domain) SEQ ID NO: 18
EVQLVESGGGLVQPGGSLRLSCAASGFTFSQARMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR
FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGOGTLVTVSS (B7 VH domain)
SEQ ID NO: 19
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCCATTTTGATATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT
GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG
TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGT (B7 VH domain) SEQ ID NO: 20
EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFDMSWVRQAPGKGLBANSAISGSGGSTYYADSVKGR
FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (E8 VH domain)
SEQ ID NO: 21
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCGATATGCATATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT
GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG
TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGT (E8 VH domain) SEQ ID NO: 22
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDMHMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR
FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (G9 VH domain)
SEQ ID NO: 23
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCCATATGCAGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT
GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG
TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCCGTATATTACTGTGCGAAAAGTACTCATTTGTATCTTTTTGACTACTGGGGCCAGGGAAC
CCTGGTCACCGTCTCGAGT (G9 VH domain) SEQ ID NO: 24
EVQLVESGGGLVQPGGSLRLSCAASGFTFSHMQMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR
FTISRDNSKNTLYLCIMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F16 VH
domain) SEQ ID NO: 25
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCCGGTATGGTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT
GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG
TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCCGTATATTACTGTGCGAAAGCGCATAATGCTTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTGTCGAGA (F16 VH domain) SEQ ID NO: 26
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSINVRQAPGKGLEWVSAISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSR (4A1-F16 VH
domain) SEQ ID NO: 27
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTG
CAGCCTCTGGATTCACCTTTAGCCGGTATGGTGCGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCT
GGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGG
TTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGG
ACACGGCCGTATATTACTGTGCGAAAGCGCATAATGCTTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTGTCGAGA (4A1-F16 VH domain) SEQ ID NO: 28
EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGASWVRQAPGKGLEWVSAISGSGGSTYYADSVKGR
FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAHNAFDYWGQGTLVTVSR (H1 VH CDR1) SEQ
ID NO: 29 CCGCGGAGG (H1 VH CDR1) SEQ ID NO: 30 PRR (B2 VH CDR1) SEQ
ID NO: 31 GCGGCTAAG (B2 VH CDR1) SEQ ID NO: 32 AAK (C5 VH CDR1) SEQ
ID NO: 33 CCGATTACT (C5 VH CDR1) SEQ ID NO: 34 PIT (D5 VH CDR1) SEQ
ID NO: 35 GTGATGAAG (D5 VH CDR1) SEQ ID NO: 36 VMK (E5 VH CDR1) SEQ
ID NO: 37 ACTGGTTCT (E5 VH CDR1) SEQ ID NO: 38 TGS (C8 VH CDR1) SEQ
ID NO: 39 CTTCAGACT (C8 VH CDR1) SEQ ID NO: 40 LQT (F8 VH and F8 VH
V5L CDR1) SEQ ID NO: 41 CTGTTTACG (F8 VH and F8 VH VSL CDR1) SEQ ID
NO: 42
LFT (F1 VH CDR1) SEQ ID NO: 43 TAGGCGCGT (F1 VH CDR1) SEQ ID NO: 44
QAR (B7 VH CDR1) SEQ ID NO: 45 CATTTTGAT (B7 VH CDR1) SEQ ID NO: 46
HFD (ES VH CDR1) SEQ ID NO: 47 GATATGCAT (B8 VH CDR1) SEQ ID NO: 48
DMH (G9 VH CDR1) SEQ ID NO: 49 CATATGCAG (G9 VH CDR1) SEQ ID NO: 50
HMQ (F16 VH CDR1) SEQ ID NO: 51 CGGTATGGTATGAGC (F16 VH CDR1) SEQ
ID NO: 52 RYGMS (4A1-F16 VH CDR1) SEQ ID NO: 53 CGGTATGGTGCGAGC
(4A1-F16 VH CDR1) SEQ ID NO: 54 RYGAS (H1, B2, C5, D5, E5, C8, F8,
F8 V5L, F1, 87, E8 and G9 VH CDR2) SEQ ID NO: 55 AGTGGTAGTGGTGGTAGC
(H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1, B7, ES and G9 VH CDR2) SEQ
ID NO: 56 SGSGGS (F16 and 4A1-F16 VH CDR2) SEQ ID NO: 57
GCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGC (F16 and
4A1-F16 VH CDR2) SEQ ID NO: 58 AISGSGGSTYYADSVKG (H1, B2, C5, D5,
E5, C8, F8, F8 V5L, F1, B7, ES and G9 VH CDR3 SEQ ID NO: 59
AGTACTCATTTGTATCTT (H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1, B7, E8
and G9 VH CDR3 SEQ ID NO: 60 STHLYL (F16 and 4A1-F16 VH CDR3) SEQ
ID NO: 61 GCGCATAATGCTTTTGACTAC (F16 and 4A1-F16 VH CDR3) SEQ ID
NO: 62 AHNAFDY (H1 VL domain) SEQ ID NO: 63
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCTCTGCGTGGTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (H1 VL
domain) SEQ ID NO: 64
EIVLTQSPGTLSLSPGEKATLSCRASQSVSSAWLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B2 VL domain) SEQ ID NO:
65
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCGTGGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (B2 VL
domain) SEQ ID NO: 66
EIVLTQSPGTLSLSPGEKATLSCRASQSVSVAFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C5 VL domain) SEQ ID NO:
67
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCTTGCATTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (C5 VL
domain) SEQ ID NO: 68
EIVLTQSPGTLSLSPGEKATLSCRASQSVSLHFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (D5 VL domain) SEQ ID NO:
69
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCAATGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (D5 VL
domain) SEQ ID NO: 70
EIVLTQSPGTLSLSPGEKATLSCRASQSVSNAFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSG
TDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E5 VL domain) SEQ ID NO:
71
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCCTTGCGCATTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (E5 VL
domain) SEQ ID NO: 72
EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAHLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C8 VL domain) SEQ ID NO:
73
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCCTTCCTTTTTTAGCCTGGTACCAGCAGAAACCMGCCAGGCTCCC
AGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGT
CTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCA
GCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (C8 VL
domain) SEQ ID NO: 74
EIVLTQSPGTLSLSPGEKATLSCRASQSVSLPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F8 VL domain) SEQ ID NO:
75
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCATGCCGTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (F8 VL
domain) SEQ ID NO: 76
EIVLTQSPGTLSLSPGEKATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F8 VL K18R domain) SEQ
ID NO: 77
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCATGCCGTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (F8 VL
K18R domain) SEQ ID NO: 78
EIVLTQSPGTLSLSPGERATLSCRASQSVSMPFLAWYQQKPGQAPRLLIYGASSSRATGIPDRFSGS
GSGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F1 VL domain) SEQ ID
NO: 79
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCGCGCCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (F1 VL
domain) SEQ ID NO: 80
EIVLTQSPGTLSLSPGEKATLSCRASQSVSAPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B7 VL domain) SEQ ID NO:
81
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCCTGGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (B7 VL
domain) SEQ ID NO: 82
EIVLTQSPGTLSLSPGEKATLSCRASQSVSLAFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E8 VL domain) SEQ ID NO:
83
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GAGGGCCAGTCAGAGTGTTAGCTCGTCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCC
AGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGT
CTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCA
GCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (E8 VL
domain) SEQ ID NO: 84
EIVLTQSPGTLSLSPGEKATLSCRASQSVSSSFLAWYQQKPGQAPRLLIVGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (G9 VL domain) SEQ ID NO:
85
GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAAAGCCACCCTCTCCT
GCAGGGCCAGTCAGAGTGTTAGCACTGCTTTTTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCC
CAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGG
TCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTC
AGCAGATGCGTGGTCGGCCGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA (G9 VL
domain) SEQ ID NO: 86
EIVLTQSPGTLSLSPGEKATLSCRASQSVSTAFLAWYQQKPGQAPRLLIVGASSRATGIPDRFSGSG
SGTDFTLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F16 and 4A1-F16 VL
domain) SEQ ID NO: 87
TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCA
AGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTG
TCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAAC
ACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCTCTGT
TTATACTATGCCGCCCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (F16 and
4A1-F16 VL domain)
SEQ ID NO: 88
SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGN
TASLTITGAQAEDEADYYCNSSVYTMPPVVFGGGTKLTVL (H1 VL CDR1) SEQ ID NO: 89
TCTGCGTGG (H1 VL CDR1) SEQ ID NO: 90 SAW (B2 VL CDR1) SEQ ID NO: 91
GTGGCTTTT (B2 VL CDR1) SEQ ID NO: 92 VAF (C5 VL CDR1) SEQ ID NO: 93
TTGCATTTT (C5 VL CDR1) SEQ ID NO: 94 LHF (D5 VL CDR1) SEQ ID NO: 95
AATGCTTTT (D5 VL CDR1) SEQ ID NO: 96 NAF (E5 VL CDR1) SEQ ID NO: 97
CTTGCGCAT (E5 VL CDR1) SEQ ID NO: 98 LAH (C8 VL CDR1) SEQ ID NO: 99
CTTCCTTTT (C8 VL CDR1) SEQ ID NO: 100 LPF (F8 VL and F8 VL K18R
CDR1) SEQ ID NO: 101 ATGCCGTTT (F8 VL and F8 VL K18R CDR1) SEQ ID
NO: 102 MPF (F1 VL CDR1) SEQ ID NO: 103 GCGCCTTTT (F1 VL CDR1) SEQ
ID NO: 104 APF (B7 VL CDR1) SEQ ID NO: 105 CTGGCTTTT (B7 VL CDR1)
SEQ ID NO: 106 LAF (E8 VL CDR1) SEQ ID NO: 107 TCGTCTTTT (E8 VL
CDR1) SEQ ID NO: 108 SSF (G9 VL CDR1) SEQ ID NO: 109 ACTGCTTTT (G9
VL CDR1) SEQ ID NO: 110 TAF (F16 and 4A1-F16 VL CDR1) SEQ ID NO:
111 CAAGGAGACAGCCTCAGAAGCTATTATGCAAGC (F16 and 4A1-F16 VL CDR1) SEQ
ID NO: 112 QGDSLRSYYAS (H1, B2, C5, D5, E5, C8, F8, F8 K18R, F1,
B7, ES and G9 VL CDR2) SEQ ID NO: 113 GGTGCATCCAGCAGGGCCACT (H1,
B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR2) SEQ ID
NO: 114 GASSRAT (F16 and 4A1-F16 VL CDR2) SEQ ID NO: 115
GGTAAAAACAACCGGCCCTCA (F16 and 4A1-F16 VZ CDR2) SEQ ID NO: 116
GKNNRPS (H1, B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, ES and G9 VL
CDR3) SEQ ID NO: 117 ATGCGTGGTCGGCCGCCG (H1, B2, C5, D5, E5, C8,
F8, F8 K18R, F1, B7, ES and G9 VL CDR3) SEQ ID NO: 118 MRGRPP (F16
and 4A1-F16 VL CDR3) SEQ ID NO: 119
AACTCCTCTGTTTATACTATGCCGCCCGTGGTA (F16 and 4A1-F16 VL CDR3) SEQ ID
NO: 120 NSSVYTMPPVV (F8, F16 and 4A1-F16 linker) SEQ ID NO: 121
GGCGGTAGCGGAGGG (F8, F16 and 4A1-F16 linker) SEQ ID NO: 122 GGSGG
(H1 VH V5L domain) SEQ ID NO: 123
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPRRMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (B2 VH V5L domain)
SEQ ID NO: 124
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAAKMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C5 VH V5L domain)
SEQ ID NO: 125
EVQLLESGGGLVQPGGSLRLSCAASGFTFSPITMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (D5 VH V5L domain)
SEQ ID NO: 126
EVQLLESGGGLVQPGGSLRLSCAASGFTFSVMKMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (E5 VH V5L domain)
SEQ ID NO: 127
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTGSMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (C8 VH V5L domain)
SEQ ID NO: 128
EVQLLESGGGLVQPGGSLRLSCAASGFTFSLQTMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (F1 VH V5L domain)
SEQ ID NO: 129
EVQLLESGGGLVQPGGSLRLSCAASGFTFSQARMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (B7 VH V5L domain)
SEQ ID NO: 130
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHFDMSWVRQAPGKGLEWVSAISGSGGSTYVADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (E8 VH V5L domain)
SEQ ID NO: 131
EVQLLESGGGLVOIDGGSLRLSCAASGFTFSDMHMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTIS
RDNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (G9 VH V5L domain)
SEQ ID NO: 132
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHMQMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCAKSTHLYLFDYWGQGTLVTVSS (H1 VL K18R domain)
SEQ ID NO: 133
EIVLTQSPGTLSLSPGERATLSCRASQSVSSAWLAVVYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFT
LTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B2 VL K18R domain) SEQ ID NO:
134
EIVLTQSPGTLSLSPGERATLSCRASQSVSVAFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFTL
TISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C5 VL K18R domain) SEQ ID NO:
135
EIVLTQSPGTLSLSPGERATLSCRASQSVSLHFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFTL
TISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (D5 VL K18R domain) SEQ ID NO:
136
EIVLTQSPGTLSLSPGERATLSCRASQSVSNAFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFTL
TISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E5 VL K18R domain) SEQ ID NO:
137
EIVLTQSPGTLSLSPGEFtATLSCRASQSVSLAHLAVVYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDF
TLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (C8 VL K18R domain) SEQ ID NO:
138
EIVLTQSPGTLSLSPGERATLSCRASQSVSLPFLAWYQQKPGQAPRWYGASSRATGIPDRFSGSGSGTDFTL
TISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (F1 VL K18R domain) SEQ ID NO:
139
EIVLTQSPGTLSLSPGERATLSCRASQSVSAPFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDF
TLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (B7 VL K18R domain) SEQ ID NO:
140
EIVLTQSPGTLSLSPGERATLSCRASQSVSLAFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDF
TLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (E8 VL K18R domain) SEQ ID NO:
141
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYGASSRATGIPORFSGSGSGTDF
TLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK (G9 VL K18R domain) SEQ ID NO:
142
EIVLTQSPGILSLSPGERATLSCRASQSVSTAFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDF
TLTISRLEPEDFAVYYCQQMRGRPPTFGQGTKVEIK
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2004.
Sequence CWU 1
1
1421354DNAArtificial sequenceSynthetic sequence H1 VH domain
1gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagc ccgcggagga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaaagtact 300catttgtatc tttttgacta
ctggggccag ggaaccctgg tcaccgtctc gagt 3542118PRTArtificial
sequenceSynthetic sequence H1 VH domain 2Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Arg 20 25 30Arg Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 1153354DNAArtificial
sequenceSynthetic sequence B2 VH domain 3gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc gcggctaaga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaaagtact 300catttgtatc tttttgacta ctggggccag
ggaaccctgg tcaccgtctc gagt 3544118PRTArtificial sequenceSynthetic
sequence B2 VH domain 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ala Ala 20 25 30Lys Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr
His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val
Thr Val Ser Ser 1155354DNAArtificial sequenceSynthetic sequence C5
VH domain 5gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt cacctttagc ccgattacta tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta
gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaaagtact 300catttgtatc
tttttgacta ctggggccag ggaaccctgg tcaccgtctc gagt
3546118PRTArtificial sequenceSynthetic sequence C5 VH domain 6Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Ile
20 25 30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
1157354DNAArtificial sequenceSynthetic sequence D5 VH domain
7gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagc gtgatgaaga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaaagtact 300catttgtatc tttttgacta
ctggggccag ggaaccctgg tcaccgtctc gagt 3548118PRTArtificial
sequenceSynthetic sequence D5 VH domain 8Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Val Met 20 25 30Lys Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 1159354DNAArtificial
sequenceSynthetic sequence E5 VH domain 9gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc actggttcta tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaaagtact 300catttgtatc tttttgacta ctggggccag
ggaaccctgg tcaccgtctc gagt 35410118PRTArtificial sequenceSynthetic
sequence E5 VH domain 10Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Thr Gly 20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr
His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val
Thr Val Ser Ser 11511354DNAArtificial sequenceSynthetic sequence C8
VH domain 11gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt cacctttagc cttcagacta tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta
gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaaagtact 300catttgtatc
tttttgacta ctggggccag ggaaccctgg tcaccgtctc gagt
35412118PRTArtificial sequenceSynthetic sequence C8 VH domain 12Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Gln
20 25 30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11513354DNAArtificial sequenceSynthetic sequence F8 VH domain
13gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagc ctgtttacga tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaaagtact 300catttgtatc tttttgacta
ctggggccag ggaaccctgg tcaccgtctc gagt 35414118PRTArtificial
sequenceSynthetic sequence F8 VH domain 14Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Phe 20 25 30Thr Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 11515354DNAArtificial
sequenceSynthetic sequence F8 VH V5L domain 15gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc ctgtttacga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaaagtact 300catttgtatc tttttgacta ctggggccag
ggaaccctgg tcaccgtctc gagt 35416118PRTArtificial sequenceSynthetic
sequence F8 VH V5L domain 16Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Leu Phe 20 25 30Thr Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser
Thr His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu
Val Thr Val Ser Ser 11517354DNAArtificial sequenceSynthetic
sequence F1 VH domain 17gaggtgcagc tggtggagtc tgggggaggc ttggtacagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagc taggcgcgta
tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct
attagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccg
gttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcaaatga
acagcctgag agccgaggac acggccgtat attactgtgc gaaaagtact
300catttgtatc tttttgacta ctggggccag ggaaccctgg tcaccgtctc gagt
35418118PRTArtificial sequenceSynthetic sequence F1 VH domain 18Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Ala
20 25 30Arg Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11519354DNAArtificial sequenceSynthetic sequence B7 VH domain
19gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagc cattttgata tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaaagtact 300catttgtatc tttttgacta
ctggggccag ggaaccctgg tcaccgtctc gagt 35420118PRTArtificial
sequenceSynthetic sequence B7 VH domain 20Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser His Phe 20 25 30Asp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 11521354DNAArtificial
sequenceSynthetic sequence E8 VH domain 21gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc gatatgcata tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gaaaagtact 300catttgtatc tttttgacta ctggggccag
ggaaccctgg tcaccgtctc gagt 35422118PRTArtificial sequenceSynthetic
sequence E8 VH domain 22Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Asp Met 20 25 30His Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly
Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr
His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val
Thr Val Ser Ser 11523354DNAArtificial sequenceSynthetic sequence G9
VH domain 23gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc
cctgagactc 60tcctgtgcag cctctggatt cacctttagc catatgcaga tgagctgggt
ccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggta
gtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc gaaaagtact 300catttgtatc
tttttgacta ctggggccag ggaaccctgg tcaccgtctc gagt
35424118PRTArtificial sequenceSynthetic sequence G9 VH domain 24Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Met
20 25 30Gln Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
11525348DNAArtificial sequenceSynthetic sequence F16 VH domain
25gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc
60tcctgtgcag cctctggatt cacctttagc cggtatggta tgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagcgcat 300aatgcttttg actactgggg
ccagggaacc ctggtcaccg tgtcgaga 34826116PRTArtificial
sequenceSynthetic sequence F16 VH domain 26Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Lys Ala His Asn Ala Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val 100 105 110Thr Val Ser Arg 11527348DNAArtificial
sequenceSynthetic sequence 4A1-F16 VH domain 27gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggatt cacctttagc cggtatggtg cgagctgggt ccgccaggct
120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag
cacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagcgcat 300aatgcttttg actactgggg
ccagggaacc ctggtcaccg tgtcgaga 34828116PRTArtificial
sequenceSynthetic sequence 4A1-F16 VH domain 28Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30Gly Ala Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ala His Asn Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Arg 115299DNAArtificial
sequenceSynthetic sequence H1 VH CDR1 29ccgcggagg 9303PRTArtificial
sequenceSynthetic sequence H1 VH CDR1 30Pro Arg
Arg1319DNAArtificial sequenceSynthetic sequence B2 VH CDR1
31gcggctaag 9323PRTArtificial sequenceSynthetic sequence B2 VH CDR1
32Ala Ala Lys1339DNAArtificial sequenceSynthetic sequence C5 VH
CDR1 33ccgattact 9343PRTArtificial sequenceSynthetic sequence C5 VH
CDR1 34Pro Ile Thr1359DNAArtificial sequenceSynthetic sequence D5
VH CDR1 35gtgatgaag 9363PRTArtificial sequenceSynthetic sequence D5
VH CDR1 36Val Met Lys1379DNAArtificial sequenceSynthetic sequence
E5 VH CDR1 37actggttct 9383PRTArtificial sequenceSynthetic sequence
E5 VH CDR1 38Thr Gly Ser1399DNAArtificial sequenceSynthetic
sequence C8 VH CDR1 39cttcagact 9403PRTArtificial sequenceSynthetic
sequence C8 VH CDR1 40Leu Gln Thr1419DNAArtificial
sequenceSynthetic sequence F8 VH and F8 VH V5L CDR1 41ctgtttacg
9423PRTArtificial sequenceSynthetic sequence F8 VH and F8 VH V5L
CDR1 42Leu Phe Thr1439DNAArtificial sequenceSynthetic sequence F1
VH CDR1 43taggcgcgt 9443PRTArtificial sequenceSynthetic sequence F1
VH CDR1 44Gln Ala Arg1459DNAArtificial sequenceSynthetic sequence
B7 VH CDR1 45cattttgat 9463PRTArtificial sequenceSynthetic sequence
B7 VH CDR1 46His Phe Asp1479DNAArtificial sequenceSynthetic
sequence E8 VH CDR1 47gatatgcat 9483PRTArtificial sequenceSynthetic
sequence E8 VH CDR1 48Asp Met His1499DNAArtificial
sequenceSynthetic sequence G9 VH CDR1 49catatgcag 9503PRTArtificial
sequenceSynthetic sequence G9 VH CDR1 50His Met
Gln15115DNAArtificial sequenceSynthetic sequence F16 VH CDR1
51cggtatggta tgagc 15525PRTArtificial sequenceSynthetic sequence
F16 VH CDR1 52Arg Tyr Gly Met Ser1 55315DNAArtificial
sequenceSynthetic sequence 4A1-F16 VH CDR1 53cggtatggtg cgagc
15545PRTArtificial sequenceSynthetic sequence 4A1-F16 VH CDR1 54Arg
Tyr Gly Ala Ser1 55518DNAArtificial sequenceSynthetic sequence H1,
B2, C5, D5, E5, C8, F8, F8 V5L, F1, B7, E8 and G9 VH CDR2
55agtggtagtg gtggtagc 18566PRTArtificial sequenceSynthetic sequence
H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1, B7, E8 and G9 VH CDR2 56Ser
Gly Ser Gly Gly Ser1 55751DNAArtificial sequenceSynthetic sequence
F16 and 4A1-F16 VH CDR2 57gctattagtg gtagtggtgg tagcacatac
tacgcagact ccgtgaaggg c 515817PRTArtificial sequenceSynthetic
sequence F16 and 4A1-F16 VH CDR2 58Ala Ile Ser Gly Ser Gly Gly Ser
Thr Tyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly5918DNAArtificial
sequenceSynthetic sequence H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1,
B7, E8 and G9 VH CDR3 59agtactcatt tgtatctt 18606PRTArtificial
sequenceSynthetic sequence H1, B2, C5, D5, E5, C8, F8, F8 V5L, F1,
B7, E8 and G9 VH CDR3 60Ser Thr His Leu Tyr Leu1 56121DNAArtificial
sequenceSynthetic sequence F16 and 4A1-F16 VH CDR3 61gcgcataatg
cttttgacta c 21627PRTArtificial sequenceSynthetic sequence F16 and
4A1-F16 VH CDR3 62Ala His Asn Ala Phe Asp Tyr1 563324DNAArtificial
sequenceSynthetic sequence H1 VL domain 63gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aaaagccacc 60ctctcctgca gggccagtca
gagtgttagc tctgcgtggt tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca
180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag
cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagatgcgtg
gtcggccgcc gacgttcggc 300caagggacca aggtggaaat caaa
32464108PRTArtificial sequenceSynthetic sequence H1 VL domain 64Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ala
20 25 30Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10565324DNAArtificial sequenceSynthetic sequence B2 VL
domain 65gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aaaagccacc 60ctctcctgca gggccagtca gagtgttagc gtggcttttt tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagatgcgtg gtcggccgcc gacgttcggc 300caagggacca
aggtggaaat caaa 32466108PRTArtificial sequenceSynthetic sequence B2
VL domain 66Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Val Ala 20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10567324DNAArtificial sequenceSynthetic
sequence C5 VL domain 67gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aaaagccacc 60ctctcctgca gggccagtca gagtgttagc ttgcattttt
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagatgcgtg gtcggccgcc gacgttcggc
300caagggacca aggtggaaat caaa 32468108PRTArtificial
sequenceSynthetic sequence C5 VL domain 68Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Lys Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Leu His 20 25 30Phe Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly
Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10569324DNAArtificial sequenceSynthetic sequence D5 VL domain
69gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aaaagccacc
60ctctcctgca gggccagtca gagtgttagc aatgcttttt tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagatgcgtg gtcggccgcc gacgttcggc 300caagggacca aggtggaaat caaa
32470108PRTArtificial sequenceSynthetic sequence D5 VL domain 70Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asn Ala
20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10571324DNAArtificial sequenceSynthetic sequence E5 VL
domain 71gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aaaagccacc 60ctctcctgca gggccagtca gagtgttagc cttgcgcatt tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagatgcgtg gtcggccgcc gacgttcggc 300caagggacca
aggtggaaat caaa 32472108PRTArtificial sequenceSynthetic sequence E5
VL domain 72Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Leu Ala 20 25 30His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10573324DNAArtificial sequenceSynthetic
sequence C8 VL domain 73gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aaaagccacc 60ctctcctgca gggccagtca gagtgttagc cttccttttt
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagatgcgtg gtcggccgcc gacgttcggc
300caagggacca aggtggaaat caaa 32474108PRTArtificial
sequenceSynthetic sequence C8 VL domain 74Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Lys Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Leu Pro 20 25 30Phe Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly
Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10575324DNAArtificial sequenceSynthetic sequence F8 VL domain
75gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aaaagccacc
60ctctcctgca gggccagtca gagtgttagc atgccgtttt tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagatgcgtg gtcggccgcc gacgttcggc 300caagggacca aggtggaaat caaa
32476108PRTArtificial sequenceSynthetic sequence F8 VL domain 76Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Met Pro
20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10577324DNAArtificial sequenceSynthetic sequence F8 VL
K18R domain 77gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc atgccgtttt
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagatgcgtg gtcggccgcc gacgttcggc
300caagggacca aggtggaaat caaa 32478108PRTArtificial
sequenceSynthetic sequence F8 VL K18R domain 78Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Met Pro 20 25 30Phe Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10579324DNAArtificial sequenceSynthetic sequence F1 VL domain
79gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aaaagccacc
60ctctcctgca gggccagtca gagtgttagc gcgccttttt tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagtgta ttactgtcag
cagatgcgtg gtcggccgcc gacgttcggc 300caagggacca aggtggaaat caaa
32480108PRTArtificial sequenceSynthetic sequence F1 VL domain 80Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ala Pro
20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10581324DNAArtificial sequenceSynthetic sequence B7 VL
domain 81gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aaaagccacc 60ctctcctgca gggccagtca
gagtgttagc ctggcttttt tagcctggta ccagcagaaa 120cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tggcatccca
180gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag
cagactggag 240cctgaagatt ttgcagtgta ttactgtcag cagatgcgtg
gtcggccgcc gacgttcggc 300caagggacca aggtggaaat caaa
32482108PRTArtificial sequenceSynthetic sequence B7 VL domain 82Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Leu Ala
20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 10583324DNAArtificial sequenceSynthetic sequence E8 VL
domain 83gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga
aaaagccacc 60ctctcctgca gggccagtca gagtgttagc tcgtcttttt tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtgcatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgg gtctgggaca
gacttcactc tcaccatcag cagactggag 240cctgaagatt ttgcagtgta
ttactgtcag cagatgcgtg gtcggccgcc gacgttcggc 300caagggacca
aggtggaaat caaa 32484108PRTArtificial sequenceSynthetic sequence E8
VL domain 84Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Lys Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Ser 20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 10585324DNAArtificial sequenceSynthetic
sequence G9 VL domain 85gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aaaagccacc 60ctctcctgca gggccagtca gagtgttagc actgcttttt
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagtgta ttactgtcag cagatgcgtg gtcggccgcc gacgttcggc
300caagggacca aggtggaaat caaa 32486108PRTArtificial
sequenceSynthetic sequence G9 VL domain 86Glu Ile Val Leu Thr Gln
Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Lys Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ala 20 25 30Phe Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly
Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
10587324DNAArtificial sequenceSynthetic sequence F16 and 4A1-F16 VL
domain 87tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac
agtcaggatc 60acatgccaag gagacagcct cagaagctat tatgcaagct ggtaccagca
gaagccagga 120caggcccctg tacttgtcat ctatggtaaa aacaaccggc
cctcagggat cccagaccga 180ttctctggct ccagctcagg aaacacagct
tccttgacca tcactggggc tcaggcggaa 240gatgaggctg actattactg
taactcctct gtttatacta tgccgcccgt ggtattcggc 300ggagggacca
agctgaccgt ccta 32488108PRTArtificial sequenceSynthetic sequence
F16 and 4A1-F16 VL domain 88Ser Ser Glu Leu Thr Gln Asp Pro Ala Val
Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp
Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser
Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala
Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp
Tyr Tyr Cys Asn Ser Ser Val Tyr Thr Met Pro Pro 85 90 95Val Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105899DNAArtificial
sequenceSynthetic sequence H1 VL CDR1 89tctgcgtgg 9903PRTArtificial
sequenceSynthetic sequence H1 VL CDR1 90Ser Ala
Trp1919DNAArtificial sequenceSynthetic sequence B2 VL CDR1
91gtggctttt 9923PRTArtificial sequenceSynthetic sequence B2 VL CDR1
92Val Ala Phe1939DNAArtificial sequenceSynthetic sequence C5 VL
CDR1 93ttgcatttt 9943PRTArtificial sequenceSynthetic sequence C5 VL
CDR1 94Leu His Phe1959DNAArtificial sequenceSynthetic sequence D5
VL CDR1 95aatgctttt 9963PRTArtificial sequenceSynthetic sequence D5
VL CDR1 96Asn Ala Phe1979DNAArtificial sequenceSynthetic sequence
E5 VL CDR1 97cttgcgcat 9983PRTArtificial sequenceSynthetic sequence
E5 VL CDR1 98Leu Ala His1999DNAArtificial sequenceSynthetic
sequence C8 VL CDR1 99cttcctttt 91003PRTArtificial
sequenceSynthetic sequence C8 VL CDR1 100Leu Pro
Phe11019DNAArtificial sequenceSynthetic sequence F8 VL and F8 VL
K18R CDR1 101atgccgttt 91023PRTArtificial sequenceSynthetic
sequence F8 VL and F8 VL K18R CDR1 102Met Pro Phe11039DNAArtificial
sequenceSynthetic sequence F1 VL CDR1 103gcgcctttt
91043PRTArtificial sequenceSynthetic sequence F1 VL CDR1 104Ala Pro
Phe11059DNAArtificial sequenceSynthetic sequence B7 VL CDR1
105ctggctttt 91063PRTArtificial sequenceSynthetic sequence B7 VL
CDR1 106Leu Ala Phe11079DNAArtificial sequenceSynthetic sequence E8
VL CDR1 107tcgtctttt 91083PRTArtificial sequenceSynthetic sequence
E8 VL CDR1 108Ser Ser Phe11099DNAArtificial sequenceSynthetic
sequence G9 VL CDR1 109actgctttt 91103PRTArtificial
sequenceSynthetic sequence G9 VL CDR1 110Thr Ala
Phe111133DNAArtificial sequenceSynthetic sequence F16 and 4A1-F16
VL CDR1 111caaggagaca gcctcagaag ctattatgca agc
3311211PRTArtificial sequenceSynthetic sequence F16 and 4A1-F16 VL
CDR1 112Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser1 5
1011321DNAArtificial sequenceSynthetic sequence H1, B2, C5, D5, E5,
C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR2 113ggtgcatcca gcagggccac
t 211147PRTArtificial sequenceSynthetic sequence H1, B2, C5, D5,
E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR2 114Gly Ala Ser Ser
Arg Ala Thr1 511521DNAArtificial sequenceSynthetic sequence F16 and
4A1-F16 VL CDR2 115ggtaaaaaca accggccctc a 211167PRTArtificial
sequenceSynthetic sequence F16 and 4A1-F16 VL CDR2 116Gly Lys Asn
Asn Arg Pro Ser1 511718DNAArtificial sequenceSynthetic sequence H1,
B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL CDR3
117atgcgtggtc ggccgccg 181186PRTArtificial sequenceSynthetic
sequence H1, B2, C5, D5, E5, C8, F8, F8 K18R, F1, B7, E8 and G9 VL
CDR3 118Met Arg Gly Arg Pro Pro1 511933DNAArtificial
sequenceSynthetic sequence F16 and 4A1-F16 VL CDR3 119aactcctctg
tttatactat gccgcccgtg gta 3312011PRTArtificial sequenceSynthetic
sequence F16 and 4A1-F16 VL CDR3 120Asn Ser Ser Val Tyr Thr Met Pro
Pro Val Val1 5 1012115DNAArtificial sequenceSynthetic sequence F8,
F16 and 4A1-F16 linker 121ggcggtagcg gaggg 151225PRTArtificial
sequenceSynthetic sequence F8, F16 and 4A1-F16 linker 122Gly Gly
Ser Gly Gly1 5123118PRTArtificial sequenceSynthetic sequence H1 VH
V5L domain 123Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Pro Arg 20 25 30Arg Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr
Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu
Tyr Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val
Ser Ser 115124118PRTArtificial sequenceSynthetic sequence B2 VH V5L
domain 124Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ala Ala 20 25 30Lys Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115125118PRTArtificial sequenceSynthetic sequence C5 VH V5L
domain 125Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Pro Ile 20 25 30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115126118PRTArtificial sequenceSynthetic sequence D5 VH V5L
domain 126Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Val Met 20 25 30Lys Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115127118PRTArtificial sequenceSynthetic sequence E5 VH V5L
domain 127Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Thr Gly 20 25 30Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115128118PRTArtificial sequenceSynthetic sequence C8 VH V5L
domain 128Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Leu Gln 20 25 30Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115129118PRTArtificial sequenceSynthetic sequence F1 VH V5L
domain 129Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Gln Ala 20 25 30Arg Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115130118PRTArtificial sequenceSynthetic sequence B7 VH V5L
domain 130Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser His Phe 20 25 30Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115131118PRTArtificial sequenceSynthetic sequence E8 VH V5L
domain 131Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Asp Met 20 25 30His Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Ser Thr His Leu Tyr
Leu Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser
Ser 115132118PRTArtificial sequenceSynthetic sequence G9 VH V5L
domain 132Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser His Met 20 25 30Gln Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Lys Ser Thr His Leu Tyr Leu Phe Asp Tyr Trp Gly Gln Gly
Thr 100 105 110Leu Val Thr Val Ser Ser 115133108PRTArtificial
sequenceSynthetic sequence H1 VL K18R domain 133Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ala 20 25 30Trp Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105134108PRTArtificial sequenceSynthetic sequence B2 VL K18R domain
134Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Val
Ala 20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105135108PRTArtificial sequenceSynthetic sequence
C5 VL K18R domain 135Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Leu His 20 25 30 Phe Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg
Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe
Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105136108PRTArtificial
sequenceSynthetic sequence D5 VL K18R domain 136Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Asn Ala 20 25 30Phe Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105137108PRTArtificial sequenceSynthetic sequence E5 VL K18R domain
137Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Leu
Ala 20 25 30His Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105138108PRTArtificial sequenceSynthetic sequence
C8 VL K18R domain 138Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Leu Pro 20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105139108PRTArtificial
sequenceSynthetic sequence F1 VL K18R domain 139Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ala Pro 20 25 30Phe Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105140108PRTArtificial sequenceSynthetic sequence B7 VL K18R domain
140Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1
5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Leu
Ala 20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp
Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105141108PRTArtificial sequenceSynthetic sequence
E8 VL K18R domain 141Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Ser 20 25 30Phe Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro 85 90 95Pro Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105142108PRTArtificial
sequenceSynthetic sequence G9 VL K18R domain 142Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ala 20 25 30Phe Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr
Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75
80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Met Arg Gly Arg Pro
85 90 95Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
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