U.S. patent application number 17/128570 was filed with the patent office on 2021-12-23 for t cell recruiting polypeptides based on tcr alpha/beta reactivity.
This patent application is currently assigned to Ablynx N.V.. The applicant listed for this patent is Ablynx N.V.. Invention is credited to Annelies Roobrouck, Diane Van Hoorick, Joao Vieira.
Application Number | 20210395398 17/128570 |
Document ID | / |
Family ID | 1000005825807 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210395398 |
Kind Code |
A1 |
Roobrouck; Annelies ; et
al. |
December 23, 2021 |
T CELL RECRUITING POLYPEPTIDES BASED ON TCR ALPHA/BETA
REACTIVITY
Abstract
T cell recruiting polypeptides are provided that bind the
constant domain of TCR on a T cell. The polypeptides can be used in
methods for treatment of cancers.
Inventors: |
Roobrouck; Annelies;
(Oudenaarde, BE) ; Van Hoorick; Diane; (Laarne,
BE) ; Vieira; Joao; (Didcot, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ablynx N.V. |
Ghent-Zwijnaarde |
|
BE |
|
|
Assignee: |
Ablynx N.V.
Ghent-Zwijnaarde
BE
|
Family ID: |
1000005825807 |
Appl. No.: |
17/128570 |
Filed: |
December 21, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15573288 |
Nov 10, 2017 |
10927186 |
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PCT/EP2016/060859 |
May 13, 2016 |
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17128570 |
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62319486 |
Apr 7, 2016 |
|
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62160757 |
May 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2809 20130101;
C07K 2317/565 20130101; C07K 2317/569 20130101; C07K 2317/32
20130101; C07K 2317/33 20130101; C07K 16/2863 20130101; C07K
2319/30 20130101; A61K 47/60 20170801; C07K 16/32 20130101; C07K
2317/92 20130101; C07K 2317/75 20130101; C07K 2317/22 20130101;
C07K 2317/567 20130101; C07K 2317/31 20130101; A61K 2039/505
20130101; C07K 16/3007 20130101; C07K 2317/94 20130101; C07K
16/2887 20130101; C07K 16/468 20130101; C07K 16/18 20130101; C07K
2317/24 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/28 20060101 C07K016/28; C07K 16/30 20060101
C07K016/30; C07K 16/32 20060101 C07K016/32; A61K 47/60 20060101
A61K047/60; C07K 16/18 20060101 C07K016/18 |
Claims
1.-81. (canceled)
82. A process for the production of the polypeptide, said process
comprising culturing a host cell transformed or transfected with a
nucleic acid or nucleic acid sequence encoding a polypeptide
comprising a first and a second immunoglobulin single variable
domain (ISV), wherein said first ISV specifically binds to the
constant domain of the T cell receptor (TCR) present on a T cell;
said second ISV specifically binds to a first antigen on a target
cell; wherein said first antigen is different from said TCR; and
wherein said target cell is different from said T cell under
conditions allowing the expression of the polypeptide, and
recovering the produced polypeptide from the culture.
83.-86. (canceled)
87. A method for the treatment or amelioration of a disease
selected from the group consisting of a proliferative disease, an
inflammatory disease, an infectious disease and an autoimmune
disease, comprising administering to a subject in need thereof a
polypeptide comprising a first and a second immunoglobulin single
variable domain (ISV), wherein said first ISV specifically binds to
the constant domain of the T cell receptor (TCR) present on a T
cell; said second ISV specifically binds to a first antigen on a
target cell; wherein said first antigen is different from said TCR;
and wherein said target cell is different from said T cell.
88. The method according to claim 87, wherein said proliferative
disease is cancer.
89. The method according to claim 88, wherein said cancer is chosen
from the group consisting of carcinomas, gliomas, mesotheliomas,
melanomas, lymphomas, leukemias, adenocarcinomas: breast cancer,
ovarian cancer, cervical cancer, glioblastoma, multiple myeloma
(including monoclonal gammopathy of undetermined significance,
asymptomatic and symptomatic myeloma), prostate cancer, and
Burkitt's lymphoma, head and neck cancer, colon cancer, colorectal
cancer, non-small cell lung cancer, small cell lung cancer, cancer
of the esophagus, stomach cancer, pancreatic cancer, hepatobiliary
cancer, cancer of the gallbladder, cancer of the small intestine,
rectal cancer, kidney cancer, bladder cancer, prostate cancer,
penile cancer, urethral cancer, testicular cancer, vaginal cancer,
uterine cancer, thyroid cancer, parathyroid cancer, adrenal cancer,
pancreatic endocrine cancer, carcinoid cancer, bone cancer, skin
cancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, Kaposi's sarcoma, multicentric Castleman's disease or
AIDS-associated primary effusion lymphoma, neuroectodermal tumors,
rhabdomyosarcoma; as well as any metastasis of any of the above
cancers.
90. The method according to claim 88, wherein the treatment is a
combination treatment.
91. (canceled)
92. The method according to claim 87, wherein said first ISV binds
to the constant domain of a T cell receptor .alpha. (TCR-.alpha.)
(SEQ ID NO: 348 and/or SEQ ID NO: 484) and/or the constant domain
of the T cell receptor .beta. (TCR-.beta.) (SEQ ID NO: 349 and/or
SEQ ID NO: 485), or polymorphic variants or isoforms thereof.
93. The method according to claim 87, wherein said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: (i) CDR1 is chosen from the group
consisting of: (a) SEQ ID NOs: 119-133; or (b) amino acid sequences
that have 4, 3, 2, or 1 amino acid(s) difference with the amino
acid sequence of SEQ ID NO: 123 or with any of SEQ ID NOs: 119-133;
and/or (ii) CDR2 is chosen from the group consisting of: (c) SEQ ID
NOs: 134-163; or (d) amino acid sequences that have 4, 3, 2, or 1
amino acid(s) difference with the amino acid sequence of SEQ ID NO:
153 or with any of SEQ ID NOs: 134-163; and/or (iii) CDR3 is chosen
from the group consisting of: (e) SEQ ID NOs: 164-174; or (f) amino
acid sequences that have 4, 3, 2, or 1 amino acid(s) difference
with the amino acid sequence of SEQ ID NO: 170 or with any of SEQ
ID NOs: 164-174.
94. The method according to claim 87, further comprising a third
ISV, which specifically binds to a second antigen on a target cell,
wherein said second antigen is different from said first
antigen.
95. The method according to claim 87, wherein said first antigen on
a target cell is a tumour antigen, preferably a tumour associated
antigen (TAA).
96. The method according to claim 94, wherein said second antigen
on a target cell is a tumour antigen, preferably a tumour
associated antigen (TAA).
97. The method according to claim 94, wherein said first antigen
and said second antigen are present on the same target cell.
98. The method according to claim 94, wherein said first antigen
and said second antigen are present on different target cells.
99. The method according to claim 87, further comprising a serum
protein binding moiety.
100. The method according to claim 99, wherein said serum protein
binding moiety is an ISV binding serum albumin.
101. The method according to claim 87, wherein said first ISV and
said second ISV are linked via a linker.
102. The method according to claim 101, wherein said linker is
chosen from the group consisting of linkers of 5GS, 7GS, 9GS, 10GS,
15GS, 18GS, 20GS, 25GS, 30GS and 35GS (SEQ ID NOs: 376 to 385).
103. The method according to claim 87, wherein said ISV is a
Nanobody, a V.sub.HH, a humanized VHH, or a camelized V.sub.H.
104. The method according to claim 95, wherein said TAA is chosen
from the group consisting of Melanoma-associated Chondroitin
Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor
(EGFR), Fibroblast Activation Protein (FAP), MART-1,
carcinoembryonic antigen (CEA), gp100, MAGE-1, HER-2, LewisY
antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25,
TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22,
CD33, CD36, CD45, CD52, CD147, growth factor receptors including
ErbB3 and ErbB4, Cytokine receptors including Interleukin-2
receptor gamma chain (CD132 antigen), Interleukin-10 receptor alpha
chain (IL-10R-A), Interleukin-10 receptor beta chain (IL-10R-B),
Interleukin-12 receptor beta-1 chain (IL-12R-beta1), Interleukin-12
receptor beta-2 chain (IL-12 receptor beta-2), Interleukin-13
receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen),
Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding
protein), Interleukin-17 receptor (IL-17 receptor), Interleukin-17B
receptor (IL-17B receptor), Interleukin 21 receptor precursor
(IL-21R), Interleukin-1 receptor type I (IL-1R-1) (CD121a),
Interleukin-1 receptor type II (IL-1R-beta) (CDw121b),
Interleukin-1 receptor antagonist protein (IL-1ra), Interleukin-2
receptor alpha chain (CD25 antigen), Interleukin-2 receptor beta
chain (CD122 antigen), Interleukin-3 receptor alpha chain
(IL-3R-alpha) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE,
CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6,
claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII,
AGS-16, CD27L, Nectin-4, SLITRK6, mesothelin, folate receptor,
tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1,
CD70, gastrin releasing peptide receptor, PAP, CEACAM5, CEACAM6,
CXCR7, N-cadherin, FXYD2 gamma a, CD21, CD133, Na/K-ATPase, mIgM
(membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120,
CD95, CA 195, DR5, DR6, DcR3 and CAIX, and related polymorphic
variants and isoforms.
105. The method according to claim 96, wherein said TAA is chosen
from the group consisting of Melanoma-associated Chondroitin
Sulfate Proteoglycan (MCSP), Epidermal Growth Factor Receptor
(EGFR), Fibroblast Activation Protein (FAP), MART-1,
carcinoembryonic antigen (CEA), gp100, MAGE-1, HER-2, LewisY
antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25,
TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22,
CD33, CD36, CD45, CD52, CD147, growth factor receptors including
ErbB3 and ErbB4, Cytokine receptors including Interleukin-2
receptor gamma chain (CD132 antigen), Interleukin-10 receptor alpha
chain (IL-10R-A), Interleukin-10 receptor beta chain (IL-10R-B),
Interleukin-12 receptor beta-1 chain (IL-12R-beta1), Interleukin-12
receptor beta-2 chain (IL-12 receptor beta-2), Interleukin-13
receptor alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen),
Interleukin-13 receptor alpha-2 chain (Interleukin-13 binding
protein), Interleukin-17 receptor (IL-17 receptor), Interleukin-17B
receptor (IL-17B receptor), Interleukin 21 receptor precursor
(IL-21R), Interleukin-1 receptor type I (IL-1R-1) (CD121a),
Interleukin-1 receptor type II (IL-1R-beta) (CDw121b),
Interleukin-1 receptor antagonist protein (IL-1ra), Interleukin-2
receptor alpha chain (CD25 antigen), Interleukin-2 receptor beta
chain (CD122 antigen), Interleukin-3 receptor alpha chain
(IL-3R-alpha) (CD123 antigen), CD30, IL23R, IGF-1R, IL5R, IgE,
CD248 (endosialin), CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6,
claudin 18.2, CLEC12A, CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII,
AGS-16, CD27L, Nectin-4, SLITRK6, mesothelin, folate receptor,
tissue factor, axl, glypican-3, CA9, Cripto, CD138, CD37, MUC1,
CD70, gastrin releasing peptide receptor, PAP, CEACAM5, CEACAM6,
CXCR7, N-cadherin, FXYD2 gamma a, CD21, CD133, Na/K-ATPase, mIgM
(membrane-bound IgM), mIgA (membrane-bound IgA), Mer, Tyro2, CD120,
CD95, CA 195, DR5, DR6, DcR3 and CAIX, and related polymorphic
variants and isoforms.
106. The method according to claim 94, wherein said first antigen
and said second antigen are chosen from the group consisting of:
EGFR as a first antigen and CEA as a second antigen; CD19 as a
first antigen and CD20 as a second antigen; CD19 as a first antigen
and CD22 as a second antigen; CD123 as a first antigen and Tim-3 as
a second antigen; and CD123 as a first antigen and CD69 as a second
antigen.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 15/573,288, filed Nov. 10, 2017, which is a
national stage filing under 35 U.S.C. .sctn. 371 of International
Application No. PCT/EP2016/060859, filed May 13, 2016, and claims
the benefit under 35 U.S.C. .sctn. 119(e) of U.S. provisional
application Ser. No. 62/319,486, filed Apr. 7, 2016, and of U.S.
provisional application Ser. No. 62/160,757, filed May 13, 2015,
the entire contents of each of which is incorporated by reference
herein in its entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA
EFS-WEB
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 15, 2020, is named A0848.70180US03-SEQ-JRV, and is 572,050
bytes in size.
FIELD OF THE INVENTION
[0003] The present invention provides multispecific T cell
recruiting polypeptides binding the constant domain of TCR on a T
cell and at least one antigen on a target cell. The present
invention also relates to the monovalent T cell recruiting
polypeptides for use in these multispecific polypeptides. The
invention also provides methods for treatment and kits providing
the same.
BACKGROUND
[0004] Cancer takes an enormous human toll around the world. It is
nowadays the world's leading cause of death, followed by heart
disease and stroke. Cancers figure among the leading causes of
morbidity and mortality worldwide, with approximately 14 million
new cases and 8.2 million cancer related deaths in 2012. The number
of new cases is expected to rise by about 70% over the next 2
decades (source: WHO Cancer). The total economic impact of
premature death and disability from cancer worldwide was about $900
billion in 2008, representing 1.5% of the world's gross domestic
product.
[0005] Available treatment regimens for solid tumours typically
include a combination of surgical resection chemotherapy and
radiotherapy. In 40 years of clinical experience little progress
has been achieved, especially in advanced stages of cancer.
[0006] New therapies combatting cancer are eagerly awaited.
[0007] Antibody therapy is now an important part of the physician's
armamentarium to battle diseases and especially cancer. Monoclonal
antibodies have been established as a key therapeutic approach for
a range of diseases already for several years. All of the
contemporaneously approved antibody therapies rely on monospecific
monoclonal antibodies (mAbs). Until today, most of the targets of
the mAbs require either an agonistic or an antagonistic approach.
Whereas targeting of cell-surface antigens themselves can mediate
antitumour activity through the induction of apoptosis, most
mAb-based activity against hematologic malignancies is reliant on
either Fc-mediated effector functions such as complement dependent
cytotoxicity (CDC) and antibody-dependent cell-mediated
cytotoxicity (ADCC).
[0008] Immunotherapy has emerged as a rapidly growing area of
cancer research. Immunotherapy is directing the body's immune
surveillance system, and in particular T cells, to cancer
cells.
[0009] Cytotoxic T cells (CTL) are T lymphocytes that kill cancer
cells, cells that are infected (particularly with viruses), or
cells that are damaged in other ways. T lymphocytes (or T cells)
express the T cell receptor or TCR molecule and the CD3 receptor on
the cell surface. The .alpha..beta. TCR-CD3 complex (or "TCR
complex") is composed of six different type I single-spanning
transmembrane proteins: the TCR.alpha. and TCR.beta. chains that
form the TCR heterodimer responsible for ligand recognition, and
the non-covalently associated CD3.gamma., CD3.delta., CD3.epsilon.
and .zeta. chains, which bear cytoplasmic sequence motifs that are
phosphorylated upon receptor activation and recruit a large number
of signalling components (Call et al. 2004, Molecular Immunology
40: 1295-1305).
[0010] Both .alpha. and .beta. chains of the T cell receptor
consist of a constant domain and a variable domain.
Physiologically, the .alpha..beta. chains of the T cell receptor
recognize the peptide loaded MHC complex and couple upon engagement
to the CD3 chains. These CD3 chains subsequently transduce the
engagement signal to the intracellular environment.
[0011] Considering the potential of naturally occurring cytotoxic T
lymphocytes (CTLs) to mediate cell lysis, various strategies have
been explored to recruit immune cells to mediate tumour cell
killing. Since T lymphocytes lack the expression of Fc receptors,
they are not recruited to a tumour site through the Fc tail of an
anti-tumour monoclonal. As an alternative, the patient's T cells
were modified with a second TCR of known specificity for a defined
tumour antigen. This adoptive cell transfer is by nature highly
personalized and labour intensive. However, the main problem of T
cell therapies remains the large number of immune escape mechanisms
known to occur in cancer patients (Nagorsen et al. 2012,
Pharmacology & Therapeutics 136: 334-342).
[0012] Rather than eliciting specific T cell responses, which rely
on expression by cancer cells of MHC molecules and the presence,
generation, transport and display of specific peptide antigens,
more recent developments have attempted to combine the advantages
of immunotherapy with antibody therapy by engaging all T cells of a
patient in a polyclonal fashion via recombinant antibody based
technologies: "bispecifics".
[0013] Bispecific antibodies have been engineered that have a
tumour recognition part on the one arm (target-binding arm) whereas
the other arm of the molecule has specificity for a T cell antigen
(effector-binding arm), mostly CD3. Through the simultaneous
binding of the two arms to their respective antigens, T lymphocytes
are directed towards and activated at the tumour cell where they
can exert their cytolytic function.
[0014] The concept of using bispecific antibodies to activate T
cells against tumour cells was described more than 20 years ago,
but manufacturing problems and clinical failures sent the field
into stagnation. Smaller format bispecifics were developed, which
more easily penetrate tissues and tumours than conventional
antibodies. In addition, the smaller format is better at creating
the cytolytic synapses, which kill the target cell. It was thought
that the smaller format bispecifics would be easier to manufacture
and less immunogenic than conventional antibodies. However, the
smaller bispecific BiTE molecules, consisting of two single chain
variable fragments (scFvs) joined by a 5 amino acid peptide linker,
presented a lack of stability (scFvs tend to aggregate), low
expression titres and poor solubility. Moreover, the first clinical
trials of Blinatumomab (a BiTE molecule), which recognizes CD3
chains, were prematurely stopped due to neurologic adverse events,
cytokine release syndrome and infections on the one hand and the
absence of objective clinical responses or robust signs of
biological activity on the other hand. Efficacy aside, BiTEs must
be continuously infused--probably due to the lack of an Fc
domain--which does not contribute to patient compliance. The same
problem holds true for DARTs (dual affinity retargeting molecules
developed by MacroGenics), in which the heavy chain variable domain
from one antibody (Ab) is linked with the light chain variable
domain of another Ab. MacroGenics now attempts to solve this
problem by fusing an Fc domain onto its next generation DARTs,
which makes the molecule not only bigger, but also results in
manufacturing problems and importation of other Fc functions. The
larger format with Fc is expected to have a better PK, but
re-introduces the risk of off-target activity. (Garber 2014, Nature
reviews 13: 799-801)
[0015] Hence, there remains a need for alternative bispecific
formats.
SUMMARY OF THE INVENTION
[0016] The invention solves this problem by providing multispecific
polypeptides comprising a first and at least one further
immunoglobulin single variable domain (ISV), wherein said first ISV
has a high affinity for/binds to a constant domain of the T cell
receptor (TCR); said at least one second ISV has a high affinity
for/binds to an antigen present on a target cell. In a particular
aspect, the binding of the first ISV will activate the inherent
cytolytic potential of the T cell against a target cell
independently of MHC.
[0017] Thus, in a first aspect the present invention provides a
polypeptide comprising a first and a second immunoglobulin single
variable domain (ISV), wherein [0018] said first ISV has high
affinity for/binds to the constant domain of the T cell receptor
(TCR) present on a T cell; [0019] said second ISV has high affinity
for/binds to a first antigen on a target cell; [0020] wherein said
first antigen is different from said TCR; and [0021] wherein said
target cell is different from said T cell.
[0022] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said polypeptide directs
the T cell to the target cell.
[0023] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said polypeptide induces T
cell activation.
[0024] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said T cell activation is
independent from MHC recognition.
[0025] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said T cell activation
depends on presenting said polypeptide bound to said first antigen
on a target cell to a T cell.
[0026] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said T cell activation
causes one or more cellular response of said T cell, wherein said
cellular response is selected from the group consisting of
proliferation, differentiation, cytokine secretion, cytotoxic
effector molecule release, cytotoxic activity, expression of
activation markers and redirected target cell lysis.
[0027] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said T cell activation
causes inhibition of an activity of said target cell by more than
about 10%, such as 20%, 30%, or 40% or even more than 50%, such as
more than 60%, such as 70%, 80%, or even more than 90%, such as
100%.
[0028] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first ISV binds to
the constant domain of a T cell receptor .alpha. (TCR-.alpha.) (SEQ
ID NO: 348) and/or the constant domain of the T cell receptor
.beta. (TCR-.beta.) (SEQ ID NO: 349), or polymorphic variants or
isoforms thereof.
[0029] Alternatively, the present invention provides a polypeptide
as described herein, wherein said first ISV binds to the constant
domain of a T cell receptor .alpha. (TCR-.alpha.) (SEQ ID NO: 484)
and/or the constant domain of the T cell receptor .beta.
(TCR-.beta.) (SEQ ID NO: 485), or polymorphic variants or isoforms
thereof.
[0030] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said polypeptide and/or
first ISV has an on rate constant (Kon) for binding to said TCR
selected from the group consisting of at least about 10.sup.2
M.sup.-1s.sup.-1, at least about 10.sup.3 M.sup.-1s.sup.-1, at
least about 10.sup.4 M.sup.-1s.sup.-1, at least about 10.sup.5
M.sup.-1s.sup.-1, at least about 10.sup.6 M.sup.-1s.sup.-1,
10.sup.7 M.sup.-1s.sup.-1, at least about 10.sup.8
M.sup.-1s.sup.-1, at least about 10.sup.9 M.sup.-1s.sup.-1, and at
least about 10.sup.10 M.sup.-1s.sup.-1, preferably as measured by
surface plasmon resonance or BLI.
[0031] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said polypeptide and/or
first ISV has an off rate constant (Koff) for binding to said TCR
selected from the group consisting of at most about 10.sup.-3
s.sup.-1, at most about 10.sup.-4 s.sup.-1, at most about 10.sup.-5
s.sup.-1, at most about 10.sup.-6 s.sup.-1, at most about 10.sup.-7
s.sup.-1, at most about 10.sup.-8 s.sup.-1, at most about 10.sup.-9
s.sup.-1, and at most about 10.sup.-10 s.sup.-1, preferably as
measured by surface plasmon resonance or BLI.
[0032] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first ISV binds to
said TCR with an EC50 value of between 100 nM and 1 pM, such as at
an average EC50 value of 100 nM or less, even more preferably at an
average EC50 value of 90 nM or less, such as less than 80, 70, 60,
50, 40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2,
or 1 nM or even less, such as less than 500, 400, 300, 200, 100,
90, 80, 70, 60, 50, 40, 30, 20, 10, 5 pM, or even less, such as
less than 4 pM, preferably as measured by flow cytometry.
[0033] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first ISV binds to
said TCR with an average KD value of between 100 nM and 10 pM, such
as at an average KD value of 90 nM or less, even more preferably at
an average KD value of 80 nM or less, such as less than 70, 60, 50,
40, 30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, or 1
nM, such as less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50,
40, 30, 20 pM, or even less, such as less than 10 pM. Preferably,
the KD is determined by Kinexa, BLI or SPR, for instance as
determined by Proteon.
[0034] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first ISV essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which:
[0035] (i) CDR1 is chosen from the group consisting of: [0036] (a)
SEQ ID NOs: 119-133; or (b) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 123 or with any of SEQ ID NOs: 199-133, provided that the
polypeptide comprising the CDR1 with 4, 3, 2, or 1 amino acid(s)
difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR1
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; and/or
[0037] (ii) CDR2 is chosen from the group consisting of: [0038] (c)
SEQ ID NOs: 134-163; or (d) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 153 or with any of SEQ ID NOs: 134-163, provided that the
polypeptide comprising the CDR2 with 4, 3, 2, or 1 amino acid(s)
difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR2
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; and/or
[0039] (iii) CDR3 is chosen from the group consisting of: [0040]
(e) SEQ ID NOs: 164-174; or (f) amino acid sequences that have 4,
3, 2, or 1 amino acid(s) difference with the amino acid sequence of
SEQ ID NO: 170 or with any of SEQ ID NOs: 164-174, provided that
the polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR3 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance.
[0041] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR1 is chosen from the group
consisting of [0042] (a) SEQ ID NO: 123; and [0043] (b) amino acid
sequences that have 1, 2 or 3 amino acid difference(s) with SEQ ID
NO: 123, wherein [0044] at position 2 the D has been changed into
A, S, E or G; [0045] at position 4 the H has been changed into Y;
[0046] at position 5 the K has been changed into L; [0047] at
position 6 the I has been changed into L; [0048] at position 8 the
F has been changed into I or V; and/or [0049] at position 10 the G
has been changed into S.
[0050] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR2 is chosen from the group
consisting of [0051] (a) SEQ ID NO: 153; and [0052] (b) amino acid
sequences that have 1, 2, 3, 4 or 5 amino acid difference(s) with
SEQ ID NO: 153, wherein [0053] at position 1 the H has been changed
into T or R; [0054] at position 3 the S has been changed into T or
A; [0055] at position 5 the G has been changed into S or A; [0056]
at position 7 the Q has been changed into D, E, T, A or V; [0057]
at position 8 the T has been changed into A or V; and/or [0058] at
position 9 the D has been changed into A, Q, N, V or S.
[0059] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR3 is chosen from the group
consisting of [0060] (a) SEQ ID NO: 170; and [0061] (b) amino acid
sequences that have 1, 2 or 3 amino acid difference(s) with SEQ ID
NO: 170, wherein [0062] at position 1 the F has been changed into
Y, L or G; [0063] at position 4 the I has been changed into L;
[0064] at position 5 the Y has been changed into W; and/or [0065]
at position 8 the D has been changed into N or S.
[0066] Preferably, the polypeptide comprising the one or more CDRs
with 5, 4, 3, 2, or 1 amino acid(s) difference binds TCR with about
the same or a higher affinity compared to the binding by the
polypeptide comprising the CDRs without the 5, 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance.
[0067] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which
[0068] (i) CDR1 is chosen from the group consisting of [0069] (a)
SEQ ID NO: 123; and [0070] (b) amino acid sequences that have 1, 2,
or 3 amino acid difference(s) with SEQ ID NO: 123, wherein [0071]
at position 2 the D has been changed into A, S, E or G; [0072] at
position 4 the H has been changed into Y; [0073] at position 5 the
K has been changed into L; [0074] at position 6 the I has been
changed into L; [0075] at position 8 the F has been changed into I
or V; and/or [0076] at position 10 the G has been changed into S,
provided that the polypeptide comprising the CDR1 with 3, 2, or 1
amino acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR1 without the 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance;
[0077] and in which
[0078] (ii) CDR2 is chosen from the group consisting of [0079] (a)
SEQ ID NO: 153; and [0080] (b) amino acid sequences that have 1, 2,
3, 4, or 5 amino acid difference(s) with SEQ ID NO: 153, wherein
[0081] at position 1 the H has been changed into T or R; [0082] at
position 3 the S has been changed into T or A; [0083] at position 5
the G has been changed into S or A; [0084] at position 7 the Q has
been changed into D, E, T, A or V; [0085] at position 8 the T has
been changed into A or V; and/or [0086] at position 9 the D has
been changed into A, Q, N, V or S, provided that the polypeptide
comprising the CDR2 with 5, 4, 3, 2, or 1 amino acid(s) difference
binds TCR with about the same or a higher affinity compared to the
binding by the polypeptide comprising the CDR2 without the 5, 4, 3,
2, or 1 amino acid(s) difference, said affinity as measured by
surface plasmon resonance;
[0087] and in which
[0088] (iii) CDR3 is chosen from the group consisting of [0089] (a)
SEQ ID NO: 170; and [0090] (b) amino acid sequences that have 1, 2,
or 3 amino acid difference(s) with SEQ ID NO: 170, wherein [0091]
at position 1 the F has been changed into Y, L or G; [0092] at
position 4 the I has been changed into L; [0093] at position 5 the
Y has been changed into W; and/or [0094] at position 8 the D has
been changed into N or S, provided that the polypeptide comprising
the CDR3 with 3, 2, or 1 amino acid(s) difference binds TCR with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR3 without the 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance.
[0095] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR1 is chosen from the group
consisting of [0096] (a) SEQ ID NO: 124; and [0097] (b) amino acid
sequences that have 1, or 2 amino acid difference(s) with SEQ ID
NO: 124, wherein [0098] at position 2 the E has been changed into
Q; and/or [0099] at position 6 the I has been changed into V.
[0100] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR2 is chosen from the group
consisting of [0101] (a) SEQ ID NO: 145; and [0102] (b) amino acid
sequence that has 1 amino acid difference with SEQ ID NO: 145,
wherein [0103] at position 9 the N has been changed into D.
[0104] In a further aspect the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR3 is chosen from the group
consisting of [0105] (a) SEQ ID NO: 167; and [0106] (b) amino acid
sequence that has 1 amino acid difference with SEQ ID NO: 167,
wherein [0107] at position 4 the L has been changed into 1.
[0108] Preferably, the polypeptide comprising the one or more CDRs
with 2 or 1 amino acid(s) difference binds TCR with about the same
or a higher affinity compared to the binding by the polypeptide
comprising the CDRs without the 2, or 1 amino acid(s) difference,
said affinity as measured by surface plasmon resonance.
[0109] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which
[0110] (i) CDR1 is chosen from the group consisting of [0111] (a)
SEQ ID NO: 124; and [0112] (b) amino acid sequences that have 1, or
2 amino acid difference(s) with SEQ ID NO: 124, wherein [0113] at
position 2 the E has been changed into Q; and/or [0114] at position
6 the I has been changed into V, provided that the polypeptide
comprising the CDR1 with 2, or 1 amino acid(s) difference binds TCR
with about the same or a higher affinity compared to the binding by
the polypeptide comprising the CDR1 without the 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance;
[0115] and in which
[0116] (ii) CDR2 is chosen from the group consisting of [0117] (a)
SEQ ID NO: 145; and [0118] (b) amino acid sequence that has 1 amino
acid difference with SEQ ID NO: 145, wherein [0119] at position 9
the N has been changed into D, provided that the polypeptide
comprising the CDR2 with 1 amino acid(s) difference binds TCR with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR2 without the 1 amino acid(s)
difference, said affinity as measured by surface plasmon
resonance;
[0120] and in which
[0121] (iii) CDR3 is chosen from the group consisting of [0122] (a)
SEQ ID NO: 167; and [0123] (b) amino acid sequence that has 1 amino
acid difference with SEQ ID NO: 167, wherein [0124] at position 4
the L has been changed into I, provided that the polypeptide
comprising the CDR3 with 1 amino acid(s) difference binds TCR with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR3 without the 1 amino acid(s)
difference, said affinity as measured by surface plasmon
resonance.
[0125] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR1 is SEQ ID NO: 130.
[0126] In a further, aspect the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR2 is chosen from the group
consisting of [0127] (a) SEQ ID NO: 157; and [0128] (b) amino acid
sequence that has 1 amino acid difference with SEQ ID NO: 157,
wherein [0129] at position 8 the T has been changed into I.
[0130] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which CDR3 is SEQ ID NO: 172.
[0131] Preferably, the polypeptide comprising the CDR with 1 amino
acid difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDRs
without the 1 amino acid difference, said affinity as measured by
surface plasmon resonance.
[0132] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which
[0133] (i) CDR1 is SEQ ID NO: 130;
[0134] and in which
[0135] (ii) CDR2 is chosen from the group consisting of [0136] (a)
SEQ ID NO: 157; and [0137] (b) amino acid sequence that has 1 amino
acid difference with SEQ ID NO: 157, wherein [0138] at position 8
the T has been changed into I, provided that the polypeptide
comprising the CDR2 with 1 amino acid difference binds TCR with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR2 without the 1 amino acid
difference, said affinity as measured by surface plasmon
resonance;
[0139] and in which
[0140] (iii) CDR3 is SEQ ID NO: 172.
[0141] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: [0142] (i) CDR1 is chosen from the
group consisting of: [0143] (a) SEQ ID NOs: 119-123, 125-127, 129,
132 and 133; and [0144] (b) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 123; and/or [0145] (ii) CDR2 is chosen from the group
consisting of: [0146] (c) SEQ ID NOs: 134-141, 143-144, 146-156,
159-163; and [0147] (d) amino acid sequences that have 4, 3, 2, or
1 amino acid(s) difference with the amino acid sequence of SEQ ID
NO: 153; and/or [0148] (iii) CDR3 is chosen from the group
consisting of: [0149] (e) SEQ ID NOs: 164-166, 169-171, 173-174;
and [0150] (f) amino acid sequences that have 4, 3, 2, or 1 amino
acid(s) difference with the amino acid sequence of SEQ ID NO:
170.
[0151] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: [0152] (i) CDR1 is chosen from the
group consisting of: [0153] (a) SEQ ID NOs: 119-123, 125-127, 129,
132 and 133; and [0154] (b) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 123, provided that the polypeptide comprising the CDR1 with
4, 3, 2, or 1 amino acid(s) difference binds TCR with about the
same or a higher affinity compared to the binding by the
polypeptide comprising the CDR1 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; and/or [0155] (ii) CDR2 is chosen from the group
consisting of: [0156] (c) SEQ ID NOs: 134-141, 143-144, 146-156,
159-163; and [0157] (d) amino acid sequences that have 4, 3, 2, or
1 amino acid(s) difference with the amino acid sequence of SEQ ID
NO: 153, provided that the polypeptide comprising the CDR2 with 4,
3, 2, or 1 amino acid(s) difference binds TCR with about the same
or a higher affinity compared to the binding by the polypeptide
comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
and/or [0158] (iii) CDR3 is chosen from the group consisting of:
[0159] (e) SEQ ID NOs: 164-166, 169-171, 173-174; and [0160] (f)
amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 170, provided
that the polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR3 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance.
[0161] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: CDR1 is represented by SEQ ID NO:
123, CDR2 is represented by SEQ ID NO: 153, and CDR3 is represented
by SEQ ID NO: 170.
[0162] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV is chosen
from the group consisting of SEQ ID NOs: 1-104.
[0163] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
cross-blocks the binding to the constant domain of the T cell
receptor (TCR) by at least one of the polypeptides with SEQ ID NOs:
1-104.
[0164] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV is
cross-blocked from binding to the constant domain of the T cell
receptor (TCR) by at least one of the polypeptides with SEQ ID NOs:
1-104.
[0165] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: [0166] (i) CDR1 is chosen from the
group consisting of: [0167] (a) SEQ ID NOs: 124, 128 and 131; and
[0168] (b) amino acid sequences that have 4, 3, 2, or 1 amino
acid(s) difference with the amino acid sequence of SEQ ID NO: 124;
and/or [0169] (ii) CDR2 is chosen from the group consisting of:
[0170] (c) SEQ ID NOs: 142 and 145; and [0171] (d) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 145; and/or [0172] (iii) CDR3 is
chosen from the group consisting of: [0173] (e) SEQ ID NOs: 167 and
168; and [0174] (f) amino acid sequences that have 4, 3, 2, or 1
amino acid(s) difference with the amino acid sequence of SEQ ID NO:
167.
[0175] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: [0176] (i) CDR1 is chosen from the
group consisting of: [0177] (a) SEQ ID NOs: 124, 128 and 131; and
[0178] (b) amino acid sequences that have 4, 3, 2, or 1 amino
acid(s) difference with the amino acid sequence of SEQ ID NO: 124,
provided that the polypeptide comprising the CDR1 with 4, 3, 2, or
1 amino acid(s) difference binds TCR with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR1 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
and/or [0179] (ii) CDR2 is chosen from the group consisting of:
[0180] (c) SEQ ID NOs: 142 and 145; and [0181] (d) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 145, provided that the
polypeptide comprising the CDR2 with 4, 3, 2, or 1 amino acid(s)
difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR2
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; and/or [0182] (iii) CDR3
is chosen from the group consisting of: [0183] (e) SEQ ID NOs: 167
and 168; and [0184] (f) amino acid sequences that have 4, 3, 2, or
1 amino acid(s) difference with the amino acid sequence of SEQ ID
NO: 167, provided that the polypeptide comprising the CDR3 with 4,
3, 2, or 1 amino acid(s) difference binds TCR with about the same
or a higher affinity compared to the binding by the polypeptide
comprising the CDR3 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon
resonance.
[0185] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: CDR1 is represented by SEQ ID NO:
124, CDR2 is represented by SEQ ID NO: 145, and CDR3 is represented
by SEQ ID NO: 167.
[0186] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV is chosen
from the group consisting of SEQ ID NOs: 105-115.
[0187] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
cross-blocks the binding to the constant domain of the T cell
receptor (TCR) by at least one of the polypeptides with SEQ ID NOs:
105-115.
[0188] In a further aspect, the present invention provides a
polypeptide as described herein, in which the first ISV is
cross-blocked from binding to the constant domain of the T cell
receptor (TCR) by at least one of the polypeptides with SEQ ID NOs:
105-115.
[0189] In a further aspect the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: [0190] (i) CDR1 is chosen from the
group consisting of [0191] (a) SEQ ID NO: 130; and [0192] (b) amino
acid sequences that have 4, 3, 2, or 1 amino acid(s) difference
with the amino acid sequence of SEQ ID NO: 130; and/or [0193] (ii)
CDR2 is chosen from the group consisting of: [0194] (c) SEQ ID NOs:
157-158; and [0195] (d) amino acid sequences that have 4, 3, 2, or
1 amino acid(s) difference with the amino acid sequence of SEQ ID
NO: 157; and/or [0196] (iii) CDR3 is chosen from the group
consisting of: [0197] (e) SEQ ID NO: 172; and [0198] (f) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 172.
[0199] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: [0200] (i) CDR1 is chosen from the
group consisting of [0201] (a) SEQ ID NO: 130; and [0202] (b) amino
acid sequences that have 4, 3, 2, or 1 amino acid(s) difference
with the amino acid sequence of SEQ ID NO: 130, provided that the
polypeptide comprising the CDR1 with 4, 3, 2, or 1 amino acid(s)
difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR1
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; and/or [0203] (ii) CDR2
is chosen from the group consisting of: [0204] (c) SEQ ID NOs:
157-158; and [0205] (d) amino acid sequences that have 4, 3, 2, or
1 amino acid(s) difference with the amino acid sequence of SEQ ID
NO: 157, provided that the polypeptide comprising the CDR2 with 4,
3, 2, or 1 amino acid(s) difference binds TCR with about the same
or a higher affinity compared to the binding by the polypeptide
comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
and/or [0206] (iii) CDR3 is chosen from the group consisting of:
[0207] (e) SEQ ID NO: 172; and [0208] (f) amino acid sequences that
have 4, 3, 2, or 1 amino acid(s) difference with the amino acid
sequence of SEQ ID NO: 172, provided that the polypeptide
comprising the CDR3 with 4, 3, 2, or 1 amino acid(s) difference
binds TCR with about the same or a higher affinity compared to the
binding by the polypeptide comprising the CDR3 without the 4, 3, 2,
or 1 amino acid(s) difference, said affinity as measured by surface
plasmon resonance.
[0209] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3, respectively), in which: CDR1 is represented by SEQ ID NO:
130, CDR2 is represented by SEQ ID NO: 157, and CDR3 is represented
by SEQ ID NO: 172.
[0210] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV is chosen
from the group consisting of SEQ ID NOs: 116-118.
[0211] In a further aspect, the present invention provides a
polypeptide as described herein, in which said first ISV
cross-blocks the binding to the constant domain of the T cell
receptor (TCR) by at least one of the polypeptides with SEQ ID NOs:
116-118.
[0212] In a further aspect, the present invention provides a
polypeptide as described herein in which said first ISV is
cross-blocked from binding to the constant domain of the T cell
receptor (TCR) by at least one of the polypeptides with SEQ ID NOs:
116-118.
[0213] In a further aspect, the present invention provides a
polypeptide as described herein, further comprising a third ISV,
which has high affinity for/binds to a second antigen on a target
cell, wherein said second antigen is different from said first
antigen.
[0214] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first antigen on a
target cell is a tumour antigen, preferably a tumour associated
antigen (TAA).
[0215] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said second antigen on a
target cell is a tumour antigen, preferably a tumour associated
antigen (TAA).
[0216] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first antigen and
said second antigen are present on the same target cell.
[0217] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first antigen and
said second antigen are present on different target cells.
[0218] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said TAA's are
independently chosen from the group consisting of
Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP),
Epidermal Growth Factor Receptor (EGFR), Fibroblast Activation
Protein (FAP), MART-1, carcinoembryonic antigen (CEA), gp100,
MAGE-1, HER-2, Lewis.sup.Y antigens, CD123, CD44, CLL-1, CD96,
CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2,
GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD147,
growth factor receptors including ErbB3 and ErbB4, Cytokine
receptors including Interleukin-2 receptor gamma chain (CD132
antigen), Interleukin-10 receptor alpha chain (IL-10R-A),
Interleukin-10 receptor beta chain (IL-10R-B), Interleukin-12
receptor beta-1 chain (IL-12R-beta1), Interleukin-12 receptor
beta-2 chain (IL-12 receptor beta-2), Interleukin-13 receptor
alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), Interleukin-13
receptor alpha-2 chain (Interleukin-13 binding protein),
Interleukin-17 receptor (IL-17 receptor), Interleukin-17B receptor
(IL-17B receptor), Interleukin 21 receptor precursor (IL-21R),
Interleukin-1 receptor type I (IL-1R-1) (CD121a); Interleukin-1
receptor type II (IL-1R-beta) (CDw121b), Interleukin-1 receptor
antagonist protein (IL-1ra), Interleukin-2 receptor alpha chain
(CD25 antigen, Interleukin-2 receptor beta chain (CD122 antigen),
Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen),
CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33,
Ron, Trop2, PSCA, claudin 6, claudin 18.2, CLEC12A, CD38, ephA2,
c-Met, CD56, MUC16, EGFRvIII, AGS-16, CD27L, Nectin-4, SLITRK6,
mesothelin, folate receptor, tissue factor, axl, glypican-3, CA9,
Cripto, CD138, CD37, MUC1, CD70, gastrin releasing peptide
receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2 gamma a,
CD21, CD133, Na/K-ATPase, mlgM (membrane-bound IgM), mIgA
(membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6,
DcR3 and CAIX, including related polymorphic variants and
isoforms.
[0219] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said TAA is CD20 (UniProt
11836), HER2 (Uniprot P04626), EGFR, CEA, polymorphic variants or
isoforms thereof.
[0220] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first antigen and
said second antigen are chosen from the group consisting of: [0221]
EGFR as a first antigen and CEA as a second antigen; [0222] CD19 as
a first antigen and CD20 as a second antigen; [0223] CD19 as a
first antigen and CD22 as a second antigen; [0224] CD123 as a first
antigen and Tim-3 as a second antigen; [0225] CD123 as a first
antigen and CD69 as a second antigen.
[0226] In a further aspect, the present invention provides a
polypeptide as described herein, further comprising a serum protein
binding moiety.
[0227] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said serum protein binding
moiety binds serum albumin.
[0228] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said serum protein binding
moiety is an ISV binding serum albumin.
[0229] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said ISV binding serum
albumin essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which CDR1 is SFGMS (SEQ ID NO: 481), CDR2
is SISGSGSDTLYADSVKG (SEQ ID NO: 482) and CDR3 is GGSLSR (SEQ ID
NO: 475), CDR determined according to Kabat definition; and/or in
which CDR1 is GFTFSSFGMS (SEQ ID NO: 472) or GFTFRSFGMS (SEQ ID NO:
473), CDR2 is SISGSGSDTL (SEQ ID NO: 474) and CDR3 is GGSLSR (SEQ
ID NO: 475), CDR determined according to Kontermann 2010.
[0230] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said ISV binding serum
albumin is selected from Alb8, Alb23, Alb129, Alb132, Alb11, Alb11
(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG
and Alb82-GGG (SEQ ID NOs: 400 to 412).
[0231] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said ISVs are directly
linked to each other or are linked via a linker.
[0232] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first ISV and/or said
second ISV and/or possibly said third ISV and/or possibly said ISV
binding serum albumin are linked via a linker.
[0233] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said linker is chosen from
the group consisting of linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS,
20GS, 25GS, 30GS and 35GS (SEQ ID NOs: 376 to 385).
[0234] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said serum protein binding
moiety is a non-antibody based polypeptide.
[0235] In a further aspect, the present invention provides a
polypeptide as described herein, further comprising PEG.
[0236] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said ISV is a Nanobody, a
V.sub.HH, a humanized V.sub.HH, or a camelized V.sub.H.
[0237] In a further aspect, the present invention provides a
polypeptide wherein said first ISV is chosen from the group
consisting of SEQ ID NOs: 1 to 118.
[0238] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said first ISV is chosen
from the group consisting of SEQ ID NOs: 1 to 118, and wherein said
second ISV is chosen from the group consisting of SEQ ID NOs:
350-358.
[0239] In a further aspect, the present invention provides a
polypeptide chosen from the group consisting of SEQ ID NOs: 292,
295-296, 299-300, 303, 306-343, 387-388, 390, 414, 417-418,
421-422, 425, 428-464, 467-468, 470-471 and 486-487.
[0240] In a further aspect, the present invention provides a
polypeptide that specifically binds the constant domain of the T
cell receptor (TCR) and that comprises or essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which: [0241]
(i) CDR1 is chosen from the group consisting of: [0242] (a) SEQ ID
NOs: 119-133; or [0243] (b) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of any
of SEQ ID NOs: 119-133, provided that the polypeptide comprising
the CDR1 with 4, 3, 2, or 1 amino acid(s) difference binds TCR with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR1 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; and/or [0244] (ii) CDR2 is chosen from the group
consisting of: [0245] (c) SEQ ID NOs: 134-163; or [0246] (d) amino
acid sequences that have 4, 3, 2, or 1 amino acid(s) difference
with the amino acid sequence of any of SEQ ID NOs: 134-163,
provided that the polypeptide comprising the CDR2 with 4, 3, 2, or
1 amino acid(s) difference binds TCR with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
and/or [0247] (iii) CDR3 is chosen from the group consisting of:
[0248] (e) SEQ ID NOs: 164-174; or [0249] (f) amino acid sequences
that have 4, 3, 2, or 1 amino acid(s) difference with the amino
acid sequence of any of SEQ ID NOs: 164-174, provided that the
polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino acid(s)
difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR3
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance.
[0250] The present invention also provides a polypeptide as
described herein, in which: [0251] (i) CDR1 is chosen from the
group consisting of: [0252] (a) SEQ ID NOs: 119-123, 125-127, 129,
132 and 133; and [0253] (b) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 123, provided that the polypeptide comprising the CDR1 with
4, 3, 2, or 1 amino acid(s) difference binds TCR with about the
same or a higher affinity compared to the binding by the
polypeptide comprising the CDR1 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; and/or [0254] (ii) CDR2 is chosen from the group
consisting of: [0255] (c) SEQ ID NOs: 134-141, 143-144, 146-156,
159-163; and [0256] (d) amino acid sequences that have 4, 3, 2, or
1 amino acid(s) difference with the amino acid sequence of SEQ ID
NO: 153, provided that the polypeptide comprising the CDR2 with 4,
3, 2, or 1 amino acid(s) difference binds TCR with about the same
or a higher affinity compared to the binding by the polypeptide
comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
and/or [0257] (iii) CDR3 is chosen from the group consisting of:
[0258] (e) SEQ ID NOs: 164-166, 169-171, 173-174; and [0259] (f)
amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 170, provided
that the polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR3 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance.
[0260] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is chosen from the
group consisting of [0261] (a) SEQ ID NO: 123; and [0262] (b) amino
acid sequences that have 1, 2, 3 or 4 amino acid difference(s) with
SEQ ID NO: 123, wherein [0263] at position 2 the D has been changed
into A, S, E or G; [0264] at position 4 the H has been changed into
Y; [0265] at position 5 the K has been changed into L; [0266] at
position 6 the I has been changed into L; [0267] at position 8 the
F has been changed into I or V; and/or [0268] at position 10 the G
has been changed into S.
[0269] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR2 is chosen from the
group consisting of [0270] (a) SEQ ID NO: 153; and [0271] (b) amino
acid sequences that have 1, 2, 3, 4 or 5 amino acid difference(s)
with SEQ ID NO: 153, wherein [0272] at position 1 the H has been
changed into T or R; [0273] at position 3 the S has been changed
into T or A; [0274] at position 5 the G has been changed into S or
A; [0275] at position 7 the Q has been changed into D, E, T, A or
V; [0276] at position 8 the T has been changed into A or V; and/or
[0277] at position 9 the D has been changed into A, Q, N, V or
S.
[0278] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR3 is chosen from the
group consisting of [0279] (a) SEQ ID NO: 170; and [0280] (b) amino
acid sequences that have 1, 2, 3 or 4 amino acid difference(s) with
SEQ ID NO: 170, wherein [0281] at position 1 the F has been changed
into Y, L or G; [0282] at position 4 the I has been changed into L;
[0283] at position 5 the Y has been changed into W; and/or [0284]
at position 8 the D has been changed into N or S.
[0285] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is represented by
SEQ ID NO: 123, CDR2 is represented by SEQ ID NO: 153, and CDR3 is
represented by SEQ ID NO: 170.
[0286] In a further aspect, the present invention provides a
polypeptide as described herein, in which: [0287] (i) CDR1 is
chosen from the group consisting of: [0288] (a) SEQ ID NOs: 124,
128 and 131; and [0289] (b) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 124, provided that the polypeptide comprising the CDR1 with
4, 3, 2, or 1 amino acid(s) difference binds TCR with about the
same or a higher affinity compared to the binding by the
polypeptide comprising the CDR1 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; and/or [0290] (ii) CDR2 is chosen from the group
consisting of: [0291] (c) SEQ ID NOs: 142 and 145; and [0292] (d)
amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 145, provided
that the polypeptide comprising the CDR2 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR2 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance; and/or [0293]
(iii) CDR3 is chosen from the group consisting of: [0294] (e) SEQ
ID NOs: 167 and 168; and [0295] (f) amino acid sequences that have
4, 3, 2, or 1 amino acid(s) difference with the amino acid sequence
of SEQ ID NO: 167, provided that the polypeptide comprising the
CDR3 with 4, 3, 2, or 1 amino acid(s) difference binds TCR with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR3 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance.
[0296] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is chosen from the
group consisting of [0297] (a) SEQ ID NO: 124; and [0298] (b) amino
acid sequences that have 1, or 2 amino acid difference(s) with SEQ
ID NO: 124, wherein [0299] at position 2 the E has been changed
into Q; and/or [0300] at position 6 the I has been changed into
V.
[0301] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR2 is chosen from the
group consisting of [0302] (a) SEQ ID NO: 145; and [0303] (b) amino
acid sequence that has 1 amino acid difference with SEQ ID NO: 145,
wherein [0304] at position 9 the N has been changed into D.
[0305] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR3 is chosen from the
group consisting of [0306] (a) SEQ ID NO: 167; and [0307] (b) amino
acid sequence that has 1 amino acid difference with SEQ ID NO: 167,
wherein [0308] at position 4 the L has been changed into I.
[0309] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is represented by
SEQ ID NO: 124, CDR2 is represented by SEQ ID NO: 145, and CDR3 is
represented by SEQ ID NO: 167.
[0310] In a further aspect, the present invention provides a
polypeptide as described herein, in which: [0311] (i) CDR1 is
chosen from the group consisting of [0312] (a) SEQ ID NO: 130; and
[0313] (b) amino acid sequences that have 4, 3, 2, or 1 amino
acid(s) difference with the amino acid sequence of SEQ ID NO: 130,
provided that the polypeptide comprising the CDR1 with 4, 3, 2, or
1 amino acid(s) difference binds TCR with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR1 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
and/or [0314] (ii) CDR2 is chosen from the group consisting of:
[0315] (c) SEQ ID NOs: 157-158; and [0316] (d) amino acid sequences
that have 4, 3, 2, or 1 amino acid(s) difference with the amino
acid sequence of SEQ ID NO: 157, provided that the polypeptide
comprising the CDR2 with 4, 3, 2, or 1 amino acid(s) difference
binds TCR with about the same or a higher affinity compared to the
binding by the polypeptide comprising the CDR2 without the 4, 3, 2,
or 1 amino acid(s) difference, said affinity as measured by surface
plasmon resonance; and/or [0317] (iii) CDR3 is chosen from the
group consisting of: [0318] (e) SEQ ID NO: 172; and [0319] (f)
amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 172, provided
that the polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR3 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance.
[0320] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is chosen from SEQ
ID NO: 130.
[0321] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR2 is chosen from the
group consisting of [0322] (a) SEQ ID NO: 157; and [0323] (b) amino
acid sequence that has 1 amino acid difference with SEQ ID NO: 157,
wherein [0324] at position 8 the T has been changed into I.
[0325] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR3 is chosen from SEQ
ID NO: 172.
[0326] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is represented by
SEQ ID NO: 130, CDR2 is represented by SEQ ID NO: 157, and CDR3 is
represented by SEQ ID NO: 172.
[0327] In another aspect, the invention provides a polypeptide that
specifically binds carcinoembryonic antigen (CEA) and that
comprises or essentially consists of 4 framework regions (FR1 to
FR4, respectively) and 3 complementarity determining regions (CDR1
to CDR3, respectively), in which: [0328] (i) CDR1 is chosen from
the group consisting of: [0329] (a) SEQ ID NO: 361 (GDTYGSYWMG); or
[0330] (b) amino acid sequences that have 4, 3, 2, or 1 amino
acid(s) difference with the amino acid sequence of SEQ ID NO: 361,
provided that the polypeptide comprising the CDR1 with 4, 3, 2, or
1 amino acid(s) difference binds CEA with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR1 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
and/or [0331] (ii) CDR2 is chosen from the group consisting of:
[0332] (c) SEQ ID NO: 363 (AINRGGGYTV); or [0333] (d) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 363, provided that the
polypeptide comprising the CDR2 with 4, 3, 2, or 1 amino acid(s)
difference binds CEA with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR2
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; and/or [0334] (iii) CDR3
is chosen from the group consisting of: [0335] (e) SEQ ID NO: 365
(SGVLGGLHEDWFNY); or [0336] (f) amino acid sequences that have 4,
3, 2, or 1 amino acid(s) difference with the amino acid sequence of
SEQ ID NO: 365, provided that the polypeptide comprising the CDR3
with 4, 3, 2, or 1 amino acid(s) difference binds CEA with about
the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR3 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance.
[0337] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is represented by
SEQ ID NO: 361, CDR2 is represented by SEQ ID NO: 363, and CDR3 is
represented by SEQ ID NO: 365.
[0338] In another aspect, the invention provides a polypeptide that
specifically binds CD20 and that comprises or essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which: [0339] (i) CDR1 is chosen from the group consisting of:
[0340] (a) SEQ ID NO: 362 (GGTFSSYTMG); or [0341] (b) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 362, provided that the
polypeptide comprising the CDR1 with 4, 3, 2, or 1 amino acid(s)
difference binds CD20 with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR1
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; and/or [0342] (ii) CDR2
is chosen from the group consisting of: [0343] (c) SEQ ID NO: 364
(EVRWGGVTT); or [0344] (d) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 364, provided that the polypeptide comprising the CDR2 with
4, 3, 2, or 1 amino acid(s) difference binds CD20 with about the
same or a higher affinity compared to the binding by the
polypeptide comprising the CDR2 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; and/or [0345] (iii) CDR3 is chosen from the group
consisting of: [0346] (e) SEQ ID NO: 366 (VRQMYMTVVPDY); or [0347]
(f) amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 366, provided
that the polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino
acid(s) difference binds CD20 with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR3 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance.
[0348] In a further aspect, the present invention provides a
polypeptide as described herein, in which CDR1 is represented by
SEQ ID NO: 362, CDR2 is represented by SEQ ID NO: 364, and CDR3 is
represented by SEQ ID NO: 366.
[0349] In a further aspect, the present invention provides a
polypeptide as described herein, which is a Nanobody, a V.sub.HH, a
humanized V.sub.HH, or a camelized V.sub.H.
[0350] In a further aspect, the present invention provides a
polypeptide as described herein, further comprising a serum protein
binding moiety.
[0351] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said serum protein binding
moiety binds serum albumin.
[0352] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said serum protein binding
moiety is an ISV that binds serum albumin.
[0353] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said ISV that binds serum
albumin essentially consists of 4 framework regions (FR1 to FR4,
respectively) and 3 complementarity determining regions (CDR1 to
CDR3 respectively), in which CDR1 is SFGMS (SEQ ID NO: 481), CDR2
is SISGSGSDTLYADSVKG (SEQ ID NO: 482) and CDR3 is GGSLSR (SEQ ID
NO: 475), CDR as determined according to Kabat definition; and/or
in which CDR1 is GFTFSSFGMS (SEQ ID NO: 472) or GFTFRSFGMS (SEQ ID
NO: 473), CDR2 is SISGSGSDTL (SEQ ID NO: 474) and CDR3 is GGSLSR
(SEQ ID NO: 475); CDR as determined according to Kontermann
2010.
[0354] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said ISV that binds serum
albumin is selected from Alb8, Alb23, Alb129, Alb132, Alb11, Alb11
(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG,
and Alb82-GGG (SEQ ID NOs: 400 to 412).
[0355] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said ISV is directly
linked or is linked via a linker.
[0356] In a further aspect, the present invention provides a
polypeptide as described herein, wherein said linker is chosen from
the group consisting of linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS,
20GS, 25GS, 30GS and 35GS (SEQ ID NOs:376 to 385).
[0357] In a further aspect, the present invention provides a
polypeptide as described herein, further comprising a PEG
moiety.
[0358] In a further aspect, the present invention provides a
nucleic acid or nucleic acid sequence encoding a polypeptide as
defined herein.
[0359] In a further aspect, the present invention provides a vector
comprising a nucleic acid or nucleic acid sequence as defined
herein.
[0360] In a further aspect, the present invention provides a host
cell transformed or transfected with the nucleic acid or nucleic
acid sequence as defined herein or with the vector as defined
herein.
[0361] In a further aspect, the present invention provides a
process for the production of the polypeptide as described herein,
said process comprising culturing a host cell as defined herein
under conditions allowing the expression of the polypeptide as
defined herein and recovering the produced polypeptide from the
culture.
[0362] In a further aspect, the present invention provides a
pharmaceutical composition comprising the polypeptide as described
herein, or, the polypeptide produced according to the process as
described herein.
[0363] In a further aspect, the present invention provides a
polypeptide as described herein, or produced according to the
process as described herein, for use in treating a subject in need
thereof.
[0364] In a further aspect, the present invention provides a method
for delivering a prophylactic or therapeutic polypeptide to a
specific location, tissue or cell type in the body, the method
comprising the steps of administering to a subject a polypeptide as
described herein, or produced according to the process as described
herein.
[0365] In a further aspect, the present invention provides a
polypeptide as described herein, or produced according to the
process as described herein, for use in the prevention, treatment
or amelioration of a disease selected from the group consisting of
a proliferative disease, an inflammatory disease, an infectious
disease and an autoimmune disease.
[0366] In a further aspect, the present invention provides a method
for the prevention, treatment or amelioration of a disease selected
from the group consisting of a proliferative disease, an
inflammatory disease, an infectious disease and an autoimmune
disease, comprising the step of administering to a subject in need
thereof the polypeptide as described herein, or produced according
to a process as described herein.
[0367] In a further aspect, the present invention provides a
polypeptide for use in or a method for the prevention, treatment or
amelioration of a disease as described herein, wherein said
proliferative disease is cancer.
[0368] In a further aspect, the present invention provides a
polypeptide for use in or a method for the prevention, treatment or
amelioration of a disease as described herein, wherein said cancer
is chosen from the group consisting of carcinomas, gliomas,
mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas:
breast cancer, ovarian cancer, cervical cancer, glioblastoma,
multiple myeloma (including monoclonal gammopathy of undetermined
significance, asymptomatic and symptomatic myeloma), prostate
cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer,
colorectal cancer, non-small cell lung cancer, small cell lung
cancer, cancer of the esophagus, stomach cancer, pancreatic cancer,
hepatobiliary cancer, cancer of the gallbladder, cancer of the
small intestine, rectal cancer, kidney cancer, bladder cancer,
prostate cancer, penile cancer, urethral cancer, testicular cancer,
vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer,
adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone
cancer, skin cancer, retinoblastomas, Hodgkin's lymphoma,
non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's
disease or AIDS-associated primary effusion lymphoma,
neuroectodermal tumors, rhabdomyosarcoma; as well as any metastasis
of any of the above cancers, as well as non-cancer indications such
as nasal polyposis.
[0369] In a further aspect, the present invention provides a
polypeptide for use in or a method for the prevention, treatment or
amelioration of a disease as described herein, wherein the
treatment is a combination treatment.
[0370] In a further aspect, the present invention provides a kit
comprising a polypeptide as defined herein, a nucleic acid or
nucleic acid sequence as defined herein, a vector as defined
herein, or a host cell as defined herein.
FIGURE LEGENDS
[0371] FIG. 1: Assessment of the expression of human TCR/CD3 and
human CD3 on transfected CHO, HEK293 and Llana cell lines using 100
nM anti-human TCR .alpha./.beta. antibody (clone BW242/412) (black)
and 100 nM anti-human CD3 antibody (clone OKT-3) (grey). The MCF
value (mean channel fluorescence) was plotted for each cell line.
The X-axis depicts the cell type and the transfected genes; CD3
indicates transfection with the CD3 complex (epsilon, delta, gamma
and zeta chains), huTCR indicates transfection with the TCR
.alpha./.beta. chains, wherein the variable domain used is between
brackets.
[0372] FIG. 2: Quality assessment of soluble recombinant cynomolgus
TCR .alpha./.beta. proteins using anti-non-human primate/Rat
TCR.alpha./.beta. antibody clone R73; anti-human TCR .alpha./.beta.
antibodies (solid circles) and an irrelevant anti-egg lysozyme
Nanobody (cAblys) (open circles). The OD value was plotted against
the concentration of the Nanobody.
[0373] FIGS. 3A-3D: Dose dependent binding of monovalent anti-TCR
Nanobodies to human TCR/CD3 expressed on CHO-K1 cells (FIGS. 3A and
3C, panels of column labeled "CHO-K1 hu TCR(2XN9)/CD3 cells") and
to primary human T cells (FIGS. 3B and 3D, panels of column labeled
"Hu T cells"). The MCF value (mean channel fluorescence) was
plotted against the concentration of the Nanobody.
[0374] FIGS. 4A and 4B: Dose dependent binding of monovalent
anti-TCR Nanobodies to HEK293H human TCR(2IAN)/CD3 (closed circle),
HEK293H human CD3 (cross) and to HEK293H reference cell line (open
circles). The MCF value (mean channel fluorescence) was plotted
against the concentration of the Nanobody.
[0375] FIGS. 5A and 5B: Dose dependent binding of monovalent
anti-TCR Nanobodies (closed circles) and an irrelevant Nanobody
(open circles) to soluble recombinant human TCR
.alpha./.beta.(2XN9)-zipper protein. The OD at 450 nm was plotted
against the concentration of the Nanobody.
[0376] FIGS. 6A and 6B: Kinetic analysis of T01700055A02 (FIG. 6A)
and T01700056G05 (FIG. 6B) on soluble recombinant human TCR
.alpha./.beta. (2XN9)-zipper protein interaction via BioLayer
Interferometry on an Octet RED384 instrument. Applied analyte
concentrations were: 1000, 333, 111, 37, 12.3, 4.1 and 1.4 nM.
Langmuir fits to the kinetic data are indicated with the black
lines, whereas sensorgrams are presented by the grey lines.
[0377] FIGS. 7A and 7B: T cell activation data of bead coupled
monovalent anti-TCR Nanobodies (FIG. 7A). T cell activation data of
monovalent anti-TCR Nanobodies presented in solution (FIG. 7B).
Activation was measured by monitoring the CD69 upregulation on
primary human T cells. The MCF value (mean channel fluorescence)
was plotted for each Nanobody.
[0378] FIGS. 8A-8F: Binding of a dilution series of CD20xTCR (full
line) and TCRxCD20 (dotted line) multispecific polypeptides to
human TCR/CD3 expressed on CHO-K1 cells (FIGS. 8A and 8D, panels of
column labeled "CHO-K1 huTCR (2XN9)/CD3 cells"), primary human T
cells (FIGS. 8B and 8E, panels of column labeled "Human T cells")
and Ramos cells (FIGS. 8C and 8F, panels of column labeled "Ramos
cells"). The MCF value (mean channel fluorescence) was plotted
against the concentration of the polypeptides.
[0379] FIGS. 9A-9D: Dose-dependent killing effect of CD20xTCR (full
line) binding and TCRxCD20 (dotted line) binding bispecific
polypeptides in a flow cytometry based human T cell mediated Ramos
B cell killing assay. The % cell death (% of TOPRO positive cells)
is plotted against the concentration of the polypeptides (FIGS. 9A,
9C and 9D, panels of column labeled "Ramos cells"). Dose-dependent
killing effect of CD20xTCR (full line) binding bispecific
polypeptides in a flow cytometry based human T cell mediated Raji B
cell killing assay. The % cell death (% of TOPRO positive cells)
was plotted against the concentration of the polypeptides (FIG. 9B,
panels of column labeled "Raji cells").
[0380] FIG. 10: Dose-dependent binding of the anti-CD20 Nanobody on
human CD20 Ramos (open symbols) and Raji (closed symbols) cells.
The MCF value (mean channel fluorescence) was plotted against the
concentration of the Nanobody.
[0381] FIGS. 11A-11C: Dose-dependent killing effect of CD20xTCR
binding (full line) and TCRxCD20 binding (dotted line) bispecific
polypeptides in the xCELLigence based assay using CHO-K1 human CD20
transfected cells (FIGS. 11A and 11C, panels of column labeled
"CHO-K1 hu CD20 cells"). Dose-dependent killing effect of
T017000055 in the xCELLigence based assay using CHO-K1 human CD20
transfected cells (full line, closed symbol) and CHO-K1 parental
cell line (open circles) to illustrate TAA (CD20) dependent killing
(FIG. 11B, panels of column labeled "CHO-K1 hu CD20 cells and
CHO-K1 cells"). The cell index (CI) was plotted against the
concentration of polypeptides.
[0382] FIG. 12: Dose-dependent killing effect of CD20 x TCR
polypeptides using a 5GS linker (open squares), 9GS linker (open
circles) and a 35GS linker (closed symbols) in a xCELLigence based
killing assay using CHO-K1 human TCR(2XN9)/CD3 cells. The cell
index (CI) was plotted against the concentration of
polypeptides.
[0383] FIGS. 13A and 13B: Dose-dependent killing effect of
T017000055 (FIG. 13A, labeled as "T017000055") and T017000076 (FIG.
13B, labeled as "T017000076") in a flow cytometry based human T
cell mediated Ramos B cell killing assay using different effector
(E) to target (T) ratio's (E:T ratio 10:1--closed circles, E:T
ratio 5:1--open squares, E:T ratio 2:1--closed triangles and E:T
ratio 1:1--open diamonds). The % cell death (% of TOPRO positive
cells) was plotted against the concentration of the
polypeptides.
[0384] FIG. 14: Time-dependent cytolytic activity of CD20/TCR
binding polypeptides in the purified primary human T cell mediated
killing assay in xCELLigence using CHO-K1 human CD20 target cells.
The % specific lysis was plotted against the concentration of the
construct. The different curves represent the analysis time after
addition of the T cells.
[0385] FIGS. 15A-15C: Binding of a serial dilution of half-life
extended polypeptides to human TCR/CD3 expressed on CHO-K1 cells
(FIG. 15A, panels of column labeled "CHO-K1 huTCR (2XN9)/CD3
cells"), primary human T cells (FIG. 15B, panels of column labeled
"Human T cells") and Ramos cells (FIG. 15C, panels of column
labeled "Ramos cells"). The MCF value (mean channel fluorescence)
was plotted against the concentration of the polypeptides.
[0386] FIGS. 16A-16D: Dose-dependent killing effect of CD20xTCR
binding multispecific polypeptides versus CD20xTCRxALB11 binding
polypeptides in a flow cytometry based human T cell mediated Ramos
B cell killing assay (FIG. 16A, panel labeled as "T017000076
(square) vs T017000093 (triangle)"; FIG. 16C, panel labeled as
"T017000068 (square) vs T017000095 (triangle)"). Dose-dependent
killing effect of CD20xTCRxALB11 binding polypeptides in the
absence or presence of 30 .mu.M HSA in a flow cytometry based human
T cell mediated Ramos B cell killing assay (FIG. 16B, panel labeled
as "T017000093 in the absence (full line) or presence of 30 .mu.M
HSA (open symbol--dotted line)"; FIG. 16D, panel labeled as
"T017000095 in the absence (full line) or presence of 30 .mu.M HSA
(open symbol--dotted line)"). The % cell death (% of TOPRO positive
cells) was plotted against the concentration of the
polypeptides.
[0387] FIG. 17: Binding of 100 nM monovalent anti-HER2 Nanobody
(5F07) to SKBR3, MCF-7 and MDA-MB-468 cell lines in flow cytometry
to assess HER2 expression levels. The MCF value (mean channel
fluorescence) was plotted for each cell line.
[0388] FIGS. 18A-18C: Dose-dependent killing effect of
multispecific TCRxHER2 binding polypeptides (dotted line) and
multispecific HER2xTCR binding polypeptides (full line) in an
xCELLigence based human T cell mediated killing assay using SKBR-3
cells (FIG. 18A, panel labeled as "SKBR3 cells"), MCF-7 cells (FIG.
18B, panel labeled as "MCF-7 cells") and MDA-MB-468 cells (FIG.
18C, panel labeled as "-MDA-MB-468 cells"). Data were analysed
after 18 h. The cell index (CI) was plotted against the
concentration of the polypeptides.
[0389] FIG. 19: Dose-dependent INF-.gamma. production by human T
cells after incubation of HER2-positive cells with multispecific
TCRxHER2 binding polypeptides (dotted line) and multispecific
HER2xTCR binding polypeptides (full line) in an xCELLigence based
assay. Data were analysed after 72 h incubation. The OD was plotted
against the concentration of the polypeptides.
[0390] FIGS. 20A and 20B: Dose-dependent killing effect of CD20xTCR
binding (full line) and TCRxCD20 binding (dotted line)
multispecific polypeptides in a flow cytometry based cynomolgus T
cell mediated Ramos B cell killing assay. The % cell death (% of
TOPRO positive cells) was plotted against the concentration of the
polypeptides.
[0391] FIGS. 21A and 21B: Dose-dependent killing effect of CD20xTCR
binding (full line) and TCRxCD20 binding (dotted line) bispecific
polypeptides in the xCELLigence based cynomolgus T cell mediated
CHO-K1 human CD20 killing assay. The CI was plotted against the
concentration of polypeptides. FIGS. 22A-22C: Dose dependent
binding of monovalent anti-TCR Nanobodies (closed circles) and an
irrelevant Nanobody (open circles) to soluble recombinant
cynomolgus TCR .alpha./.beta.-zipper protein. The OD at 450 nm was
plotted against the concentration of the Nanobody.
[0392] FIGS. 23A and 23B: Kinetic analysis of T0170055A02 (FIG.
23A) and T0170056G05 (FIG. 23B) on soluble recombinant cynomolgus
TCR.alpha./.beta.-zipper protein interaction via BioLayer
Interferometry on an Octet RED384 instrument. Applied analyte
concentrations were: 1000, 333, 111, 37, 12.3, 4.1 and 1.4 nM.
Langmuir fits to the kinetic data are indicated with the black
lines, whereas sensorgrams are presented by the grey lines.
[0393] FIGS. 24A and 24B: Dose-dependent killing effect of
T017000076 (CD20xTCR binding multispecific construct) versus
T017000093 (CD20xTCRxALB11 binding construct) in a flow cytometry
based cynomolgus T cell mediated Ramos B cell killing assay (FIG.
24A). Dose-dependent killing of T017000093 in the presence of 30
.mu.M HSA in a flow cytometry based cynomolgus T cell mediated
Ramos B cell killing assay (FIG. 24B, panel labeled as "T017000093
in presence of 30 .mu.M HSA (open triangle)"). The % cell death (%
of TOPRO positive cells) was plotted against the concentration of
the Nanobody.
[0394] FIG. 25: Dose-dependent killing effect of CD20xTCR binding
multispecific construct versus CD20xTCRxALB11 binding construct and
versus CD20xTCRxALB11 binding construct in the presence of 30 .mu.M
HSA in a cynomolgus T cell mediated CHO-K1 human CD20 transfected
cell xCELLigence based assay. The cell index (CI) ws plotted
against the concentration of Nanobody.
[0395] FIG. 26: Dose-dependent killing effect of multispecific
TCRxHER2 binding polypeptides (dotted line) and multispecific
HER2xTCR binding polypeptides (full line) in an xCELLigence based
cynomolgus T cell mediated HER2-positive SKBR-3 tumour killing
assay. Data were analysed after 40 h incubation. The cell index
(CI) was plotted against the concentration of the Nanobody.
[0396] FIG. 27: Study design for Ramos model as described in
Example 19. Ramos cells were injected intravenously in to mice on
day 1 (D1). PBMCs were injected intraperitoneally in to animals on
day 3 (D3). Mice were treated from D3 to day 7 (D7) with T017000083
(TCR/CD20) IV once daily during 5 days (Q1Dx5) or T017000088
(irrelevant multispecific polypeptide) IV Q1Dx5.
[0397] FIGS. 28A-28D: Absolute Ramos B cell count on log scale in
bone marrow (FIG. 28A) and spleen (FIG. 28B). Absolute PBMC derived
B cell count on log scale in bone marrow (FIG. 28C) and spleen
(FIG. 28D). Individual animal results are depicted. The number of B
cells is shown in function of the different treatment groups. The
open circles on top of the graphs show which active doses were
statistically significant different from the irrelevant polypeptide
(T017000088) based on the F-tests from the mixed-effects ANOVA
analysis. All effects in the spleen are statistically significant
at the 5% level of significance. Notably, the Y-axis value of 0.001
signifies no cells counted.
[0398] FIG. 29: Study design for PBMC B cell depletion model as
described in Example 20. PBMCs were injected intraperitoneally to
animals on day 3 (D3). Mice were treated from D3 to day 7 (D7) with
T017000083 (TCR/CD20) IV once daily during 5 days (Q1Dx5) or
T017000088 (irrelevant polypeptide) IV Q1Dx5.
[0399] FIG. 30: Absolute PBMC-derived B cell count on log scale.
Individual animal results are depicted. The number of B cells is
shown in function of the different treatment groups. Notably,
Y-axis value of 0.001 signifies no cells counted.
[0400] FIGS. 31A and 31B: Determination of EGFR (FIG. 31A; with
anti-EGFR antibody; Santa Cruz, sc-120 PE) or CEACAM5 (FIG. 31B;
with anti-CEACAM antibody; Sino Biological, 11077-MM02-P)
expression level on HER14, Hela, LoVo and LS174-T cell lines in
flow cytometry. The MCF value (mean channel fluorescence) was
plotted for each cell line.
[0401] FIGS. 32A-32D: Dose-dependent binding of the monovalent
Nanobodies and multispecific polypeptides to HER14 (FIG. 32A),
LS174T (FIG. 32B), primary human T cells (FIG. 32C) and LoVo cells
(FIG. 32D). The mean channel fluorescence intensity was plotted
against the concentration.
[0402] FIG. 33: Determination of EGFR and CEACAM5 expression levels
of HER14, LoVo and LS174T cell lines using 100 nM of the EGFR and
CEACAM Nanobodies in flow cytometry. The MFI value (mean
fluorescence intensity) was plotted for each cell line.
[0403] FIGS. 34A-34E: Dose-dependent redirected LoVo (FIG. 34A;
FIG. 34B), LST174T (FIG. 34C; FIG. 34D) and HER14 (FIG. 34E) target
cell killing of multispecific polypeptides by human effector T
cells in the xCELLigence based assay using an effector to target
ratio of 15. The CI after an incubation time of 30 h-40 h (FIG.
34A, FIG. 34C) and 50 h-60 h (FIG. 34B, FIG. 34D, FIG. 34E) was
plotted against the concentration of the multispecific
polypeptides. (T017000107=circle, T017000109=square,
T017000110=triangle).
[0404] FIG. 35: Study design of the in vivo Ramos model used in
Example 26. Ramos cells were injected intravenously into mice on
day 1 (D1). PBMCs were injected intraperitoneally to animals on day
3 (D3). Mice were treated from D3 to day 7 (D7) with T017000088,
T017000106, T017000083, T017000104 or T017000105.
[0405] FIGS. 36A and 36B: Absolute Ramos B cell count on log scale
in bone marrow (FIG. 36A) and spleen (FIG. 36B). Individual animal
results are depicted. The number of Ramos B cells are shown in
function of the different treatment groups. The stars on top of the
graph show which active doses were statistically significant
different from the control Nanobody (T017000088 or T017000106)
based on the F-tests from the mixed-effects ANOVA analysis. All
effects are statistically significant at the 5% level of
significance.
[0406] FIGS. 37A and 37B: Absolute PBMC derived B cell count on log
scale in bone marrow (FIG. 37A) and spleen (FIG. 37B). Individual
animal results are depicted. The number of PBMC derived B cells are
shown in function of the different treatment groups. The stars on
top of the graph show which active doses were statistically
significant different from the control Nanobody (T017000088 or
T017000106) based on the F-tests from the mixed-effects ANOVA
analysis. All effects are statistically significant at the 5% level
of significance.
[0407] FIGS. 38A-38C: Dose-dependent redirected SKBR3 target cell
killing of T017000102 by human effector T cells isolated from donor
932 (FIG. 38A), human CD4+ T cells isolated from donor 941 (FIG.
38B) and human CD8+ T cells isolated from donor 941 (FIG. 38C) in
the xCELLigence based assay using an effector to target ratio of
15. The CI after an incubation time of 40 h was plotted against the
concentration of the multispecific polypeptide.
[0408] FIGS. 39A-39F: Dose-dependent T cell activation of human
effector T cells isolated from donor 932 (FIG. 39A; FIG. 39D),
human CD4+ T cells isolated from donor 941 (FIG. 39B; FIG. 39E) and
human CD8+ T cells isolated from donor 941 (FIG. 39C; FIG. 39F) by
T017000102 in a redirected SKBR3 target cell killing setting.
Activation was measured by monitoring the CD69 (FIG. 39A, FIG. 39B,
FIG. 39C) upregulation and CD25 (FIG. 39D, FIG. 39E, FIG. 39F)
upregulation. The mean fluorescence intensity (MFI) was plotted
against the concentration.
[0409] FIGS. 40A-40F: Dose-dependent cytokine production by human
effector T cells isolated from donor 932 (FIG. 40A; FIG. 40D),
human CD4+ T cells isolated from donor 941 (FIG. 40B; FIG. 40E) and
human CD8+ T cells isolated from donor 941 (FIG. 40C; FIG. 40F) by
T017000102 in a redirected SKBR3 target cell killing setting. Human
INF-.gamma. production (FIG. 40A, FIG. 40B, FIG. 40C) and human
IL-6 (FIG. 40D, FIG. 40E, FIG. 40F) was measured after 72 h of
incubation. The measured concentration human INF-.gamma. and human
IL-6 (in pg/ml) was plotted against the concentration of the
Nanobody.
[0410] FIGS. 41A-41D: Dose-dependent binding of HLE construct
T017000108 to HER14 (FIG. 41A), LS174T (FIG. 41B), primary human T
cells (FIG. 41C) and LoVo cells (FIG. 41D). The mean fluorescence
intensity (MFI) was plotted against the concentration.
[0411] FIG. 42: Dose-dependent redirected LS174T target cell
killing of T017000107 (solid line) and T017000108 (dotted line) by
human effector T cells in the xCELLigence based assay using an
effector to target ratio of 15. The CI after an incubation time of
48 h was plotted against the concentration of the multispecific
polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
[0412] The present inventors realized that formats bringing T cells
and tumour cells together to induce an immune response should
comply with various and frequently opposing requirements. The
format should be broadly applicable. In particular, the format
should preferably be useful in a broad range of patients and
preferably also against a broad range of tumours. The format should
preferably be safe and only target the intended cells. In addition,
the format should preferably be small enough to easily penetrate
tissues and tumours, while on the other hand the format should be
patient friendly. For instance, the format should have an extended
half-life, such that the format is not removed instantaneous upon
administration by renal clearance. However, extending the half-life
should preferably not introduce off-target activity and side
effects or limit the penetration into tissues and tumours.
Additionally, it was recognized that tumour cells often create
escape mechanisms by the down-regulation of targeted antigens
within a therapy. Accordingly, in a further preferred version, the
format should simultaneously target multiple antigens.
[0413] The present invention realizes at least one of these
requirements.
[0414] In particular, it was hypothesized that immunoglobulin
single variable domains (ISVs) would in principle be ideal
candidates, since they are small enough to easily penetrate
(tumour) tissue and can be combined with other ISVs as building
blocks. Next, ISVs directed against the constant TCR domains should
have broad applicability. In contrast to the variable TCR domains,
these constant TCR domains display less sequence variability, and
consequently should be useful in a broad range of patients.
[0415] Unexpectedly, it turned out to be extremely difficult to
generate ISVs via immunization in llamas against the constant
domains of TCR. Either no significant immune response was mounted,
or the generated ISVs were directed against the variable TCR
domains. Only by implementing a rigorously carried out immunization
and screening method using different cells and sequences for
immunization and boosting as well as using different screening
proteins, the inventors were able to isolate ISVs against the
constant TCR domains. Although only three clusters of related ISVs
were identified, these ISVs had an unexpected range of advantageous
features. First, the ISVs were unexpectedly broadly applicable,
i.e. the TCR ISVs were able to bind to T cells from different
donors with high affinity. Formatted in a multispecific
polypeptide, the TCR ISVs enabled tumour cell killing with
different tumour associated antigens. Hence, the TCR ISVs can be
used against a multitude of cancers. In addition, the multispecific
polypeptides comprising the TCR ISVs remained active when bound to
albumin. This contributes to a favourable PK profile and patient
compliance, while minimizing side effects. The polypeptides of the
invention only showed effects when bound both to the T cell and the
target cell, which is indicative of its safety.
[0416] The present inventors considered that the simultaneous
targeting of multiple antigens reduces the probability of
generating tumour escape variants, because of which the therapeutic
activity of T cell engaging strategy is improved. Multispecific
polypeptides are provided which comprise a TCR ISV combined with
immunoglobulin single variable domains against different target
antigens and/or different epitopes on a particular antigen
(biparatopic).
[0417] Immunoglobulin sequences, such as antibodies and antigen
binding fragments derived there from (e.g., immunoglobulin single
variable domains or ISVs) are used to specifically target their
respective antigens in research and therapeutic applications. The
generation of immunoglobulin single variable domains such as e.g.,
VHHs or Nanobodies may involve the immunization of an experimental
animal such as a Llama, construction of phage libraries from immune
tissue, selection of phage displaying antigen binding
immunoglobulin single variable domains and screening of said
domains and engineered constructs thereof for the desired
specificities (WO 94/04678). Alternatively, similar immunoglobulin
single variable domains such as e.g., dAbs can be generated by
selecting phage displaying antigen binding immunoglobulin single
variable domains directly from naive or synthetic libraries and
subsequent screening of said domains and engineered constructs
thereof for the desired specificities (Ward et al., Nature, 1989,
341: 544-546; Holt et al., Trends Biotechnol., 2003,
21(11):484-490; as well as for example WO 06/030220, WO 06/003388
and other published patent applications of Domantis Ltd.).
Unfortunately, the use of monoclonal and/or heavily engineered
antibodies also carries a high manufacturing cost and may result in
suboptimal tumor penetration compared to other strategies.
[0418] The present invention provides multispecific polypeptides
that specifically bind to the T cell receptor (TCR), with an
unexpected range of advantageous features. First, the polypeptides
are easy to manufacture. Moreover, the ISVs are unexpectedly
broadly applicable, i.e. the TCR ISVs were able to bind to T cells
from different donors with high affinity. Formatted in a
multispecific polypeptide, the TCR ISVs enabled tumour cell killing
with different tumour associated antigens. In contrast, no killing
was observed when the polypeptides were not bound to T cells and
target cell which underscores the safety of the polypeptides of the
invention. Hence, the TCR ISVs can be used against a multitude of
cancers. Moreover, the TCR ISVs can be considered as safe. In
addition, the multispecific polypeptides comprising the TCR ISVs
remained active when bound to albumin. This will contribute to a
favourable PK profile and patient compliance, while minimizing side
effects.
[0419] Accordingly, the present invention relates to a polypeptide
comprising a first and a second immunoglobulin single variable
domain (ISV), wherein the first ISV has high affinity for/binds to
the constant domain of the T cell receptor (TCR) and the second ISV
has high affinity for/binds to an antigen on a cell (target cell),
preferably a tumour cell. The antigen is preferably specific for
said target cell, such as e.g. a tumour associated antigen (TAA).
The multispecific polypeptide of the invention directs the T cell
to the cell, e.g. a tumour cell and induces T cell activation in
order to allow said T cell to inhibit or kill said target cell,
e.g. said tumour cell.
Definitions
[0420] a) Unless indicated or defined otherwise, all terms used
have their usual meaning in the art, which will be clear to the
skilled person. Reference is for example made to the standard
handbooks mentioned in paragraph a) on page 46 of WO 08/020079.
[0421] b) The term "immunoglobulin single variable domain",
interchangeably used with "single variable domain" and "ISV",
defines molecules wherein the antigen binding site is present on,
and formed by, a single immunoglobulin domain. This sets
immunoglobulin single variable domains apart from "conventional"
immunoglobulins or their fragments (such as Fabs, scFvs, etc.),
wherein two immunoglobulin domains, in particular two variable
domains, interact to form an antigen binding site. Typically, in
conventional immunoglobulins, a heavy chain variable domain (VH)
and a light chain variable domain (VL) interact to form an antigen
binding site. In this case, the complementarity determining regions
(CDRs) of both VH and VL will contribute to the antigen binding
site, i.e. a total of 6 CDRs will be involved in antigen binding
site formation. In contrast, the binding site of an immunoglobulin
single variable domain is formed by a single VH or VL domain.
Hence, the antigen binding site of an immunoglobulin single
variable domain is formed by no more than three CDRs. [0422] The
terms "immunoglobulin single variable domain", "single variable
domain", and "ISV" hence do not comprise conventional
immunoglobulins or their fragments which require interaction of at
least two variable domains for the formation of an antigen binding
site. However, these terms do comprise fragments of conventional
immunoglobulins wherein the antigen binding site is formed by a
single variable domain. [0423] The term "immunoglobulin single
variable domain" or "ISV" includes (without being limiting)
antigen-binding domains or fragments such as V.sub.HH domains or
V.sub.H or V.sub.L domains, respectively. The terms antigen-binding
molecules or antigen-binding protein are used interchangeably and
include also the term Nanobodies. The immunoglobulin single
variable domains can be light chain variable domain sequences
(e.g., a V.sub.L-sequence), or heavy chain variable domain
sequences (e.g., a V.sub.H-sequence); more specifically, they can
be heavy chain variable domain sequences that are derived from a
conventional four-chain antibody or heavy chain variable domain
sequences that are derived from a heavy chain antibody.
Accordingly, the immunoglobulin single variable domains can be
domain antibodies, or immunoglobulin sequences that are suitable
for use as domain antibodies, single domain antibodies, or
immunoglobulin sequences that are suitable for use as single domain
antibodies, "dAbs", or immunoglobulin sequences that are suitable
for use as dAbs, or Nanobodies, including but not limited to
V.sub.HH sequences, humanized VHH sequences or camelized VH
sequences. The invention includes immunoglobulin sequences of
different origin, comprising mouse, rat, rabbit, donkey, human and
camelid immunoglobulin sequences. The immunoglobulin single
variable domain includes fully human, humanized, otherwise sequence
optimized or chimeric immunoglobulin sequences. The immunoglobulin
single variable domain and structure of an immunoglobulin single
variable domain can be considered--without however being limited
thereto--to be comprised of four framework regions or "FR's", which
are referred to in the art and herein as "Framework region 1" or
"FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or
"FR3"; and as "Framework region 4" or "FR4", respectively; which
framework regions are interrupted by three complementary
determining regions or "CDR's", which are referred to in the art as
"Complementarity Determining Region 1" or "CDR1"; as
"Complementarity Determining Region 2" or "CDR2"; and as
"Complementarity Determining Region 3" or "CDR3", respectively. It
is noted that the terms Nanobody or Nanobodies are registered
trademarks of Ablynx N.V. and thus may also be referred to as
Nanobody or Nanobodies, respectively. [0424] c) Unless indicated
otherwise, the terms "immunoglobulin sequence", "sequence",
"nucleotide sequence" and "nucleic acid" are as described in
paragraph b) on page 46 of WO 08/020079. [0425] d) Unless indicated
otherwise, all methods, steps, techniques and manipulations that
are not specifically described in detail can be performed and have
been performed in a manner known per se, as will be clear to the
skilled person. Reference is for example again made to the standard
handbooks and the general background art mentioned herein and to
the further references cited therein; as well as to for example the
following reviews Presta 2006 (Adv. Drug Deliv. Rev. 58
(5-6):640-656), Levin and Weiss 2006 (Mol. Biosyst. 2(1):49-57),
Irving et al. 2005 (J. Immunol. Methods 248(1-2):31-45), Schmitz et
al. 2000 (Placenta 21 Suppl. A: S106-112, Gonzales et al. 2005
(Tumour Biol. 26(1):31-43), which describe techniques for protein
engineering, such as affinity maturation and other techniques for
improving the specificity and other desired properties of proteins
such as immunoglobulins. [0426] e) Amino acid residues will be
indicated according to the standard three-letter or one-letter
amino acid code. Reference is made to Table A-2 on page 48 of the
International application WO 08/020079 of Ablynx N.V. entitled
"Immunoglobulin single variable domains directed against IL-6R and
polypeptides comprising the same for the treatment of diseases and
disorders associated with 11-6 mediated signalling". [0427] f) For
the purposes of comparing two or more nucleotide sequences, the
percentage of "sequence identity" between a first nucleotide
sequence and a second nucleotide sequence may be calculated or
determined as described in paragraph e) on page 49 of WO 08/020079
(incorporated herein by reference), such as by dividing [the number
of nucleotides in the first nucleotide sequence that are identical
to the nucleotides at the corresponding positions in the second
nucleotide sequence] by [the total number of nucleotides in the
first nucleotide sequence] and multiplying by [100%], in which each
deletion, insertion, substitution or addition of a nucleotide in
the second nucleotide sequence--compared to the first nucleotide
sequence--is considered as a difference at a single nucleotide
(position); or using a suitable computer algorithm or technique,
again as described in paragraph e) on pages 49 of WO 08/020079
(incorporated herein by reference). [0428] g) For the purposes of
comparing two or more immunoglobulin single variable domains or
other amino acid sequences such e.g. the polypeptides of the
invention etc., the percentage of "sequence identity" between a
first amino acid sequence and a second amino acid sequence (also
referred to herein as "amino acid identity") may be calculated or
determined as described in paragraph f) on pages 49 and 50 of WO
08/020079 (incorporated herein by reference), such as by dividing
[the number of amino acid residues in the first amino acid sequence
that are identical to the amino acid residues at the corresponding
positions in the second amino acid sequence] by [the total number
of amino acid residues in the first amino acid sequence] and
multiplying by [100%], in which each deletion, insertion,
substitution or addition of an amino acid residue in the second
amino acid sequence--compared to the first amino acid sequence--is
considered as a difference at a single amino acid residue
(position), i.e., as an "amino acid difference" as defined herein;
or using a suitable computer algorithm or technique, again as
described in paragraph f) on pages 49 and 50 of WO 08/020079
(incorporated herein by reference). [0429] Also, in determining the
degree of sequence identity between two immunoglobulin single
variable domains, the skilled person may take into account
so-called "conservative" amino acid substitutions, as described on
page 50 of WO 08/020079. [0430] Any amino acid substitutions
applied to the polypeptides described herein may also be based on
the analysis of the frequencies of amino acid variations between
homologous proteins of different species developed by Schulz et al.
1978 (Principles of Protein Structure, Springer-Verlag), on the
analyses of structure forming potentials developed by Chou and
Fasman 1975 (Biochemistry 13: 211) and 1978 (Adv. Enzymol. 47:
45-149), and on the analysis of hydrophobicity patterns in proteins
developed by Eisenberg et al. 1984 (Proc. Natl. Acad. Sci. USA 81:
140-144), Kyte & Doolittle 1981 (J Molec. Biol. 157: 105-132),
and Goldman et al. 1986 (Ann. Rev. Biophys. Chem. 15: 321-353), all
incorporated herein in their entirety by reference. Information on
the primary, secondary and tertiary structure of Nanobodies is
given in the description herein and in the general background art
cited above. Also, for this purpose, the crystal structure of a
V.sub.HH domain from a llama is for example given by Desmyter et
al. 1996 (Nature Structural Biology, 3: 803), Spinelli et al. 1996
(Natural Structural Biology 3: 752-757), and Decanniere et al. 1999
(Structure, 7: 361). Further information about some of the amino
acid residues that in conventional V.sub.H domains form the
V.sub.H/V.sub.L interface and potential camelizing substitutions on
these positions can be found in the prior art cited above. [0431]
h) Immunoglobulin single variable domains and nucleic acid
sequences are said to be "exactly the same" if they have 100%
sequence identity (as defined herein) over their entire length.
[0432] i) When comparing two immunoglobulin single variable
domains, the term "amino acid difference" refers to an insertion,
deletion or substitution of a single amino acid residue on a
position of the first sequence, compared to the second sequence; it
being understood that two immunoglobulin single variable domains
can contain one, two or more such amino acid differences. [0433] j)
When a nucleotide sequence or amino acid sequence is said to
"comprise" another nucleotide sequence or amino acid sequence,
respectively, or to "essentially consist of" another nucleotide
sequence or amino acid sequence, this has the meaning given in
paragraph i) on pages 51-52 of WO 08/020079. [0434] k) The term "in
essentially isolated form" has the meaning given to it in paragraph
j) on pages 52 and 53 of WO 08/020079. [0435] l) The terms "domain"
and "binding domain" have the meanings given to it in paragraph k)
on page 53 of WO 08/020079. [0436] m) The terms "antigenic
determinant" and "epitope", which may also be used interchangeably
herein, have the meanings given to it in paragraph l) on page 53 of
WO 08/020079. [0437] n) As further described in paragraph m) on
page 53 of WO 08/020079, an amino acid sequence (such as an
antibody, a polypeptide of the invention, or generally an
antigen-binding protein or polypeptide or a fragment thereof) that
can (specifically) bind to, that has affinity for and/or that has
specificity for a specific antigenic determinant, epitope, antigen
or protein (or for at least one part, fragment or epitope thereof)
is said to be "against" or "directed against" said antigenic
determinant, epitope, antigen or protein. [0438] o) The term
"specificity" refers to the number of different types of antigens
or antigenic determinants to which a particular antigen-binding
molecule or antigen-binding protein (such as an ISV, Nanobody or a
polypeptide of the invention) can bind. The specificity of an
antigen-binding protein can be determined based on affinity and/or
avidity. [0439] The affinity, represented by the equilibrium
constant for the dissociation of an antigen with an antigen-binding
protein (K.sub.D or KD), is a measure for the binding strength
between an antigenic determinant, i.e. the target, and an
antigen-binding site on the antigen-binding protein, i.e. the ISV
or Nanobody: the lesser the value of the K.sub.D, the stronger the
binding strength between an antigenic determinant and the
antigen-binding molecule (alternatively, the affinity can also be
expressed as the affinity constant (K.sub.A), which is 1/K.sub.D).
As will be clear to the skilled person (for example on the basis of
the further disclosure herein), affinity can be determined in a
manner known per se, depending on the specific antigen of interest.
[0440] Avidity is the affinity of the polypeptide, i.e. the ligand
is able to bind via two (or more) pharmacophores (ISV) in which the
multiple interactions synergize to enhance the "apparent" affinity.
Avidity is the measure of the strength of binding between the
polypeptide of the invention and the pertinent antigens. The
polypeptide of the invention is able to bind via its two (or more)
building blocks, such as ISVs or Nanobodies, to the at least two
targets, in which the multiple interactions, e.g. the first
building block, ISV or Nanobody binding to the first target and the
second building block, ISV, or Nanobody binding to the second
target, synergize to enhance the "apparent" affinity. Avidity is
related to both the affinity between an antigenic determinant and
its antigen binding site on the antigen-binding molecule and the
number of pertinent binding sites present on the antigen-binding
molecules. For example, and without limitation, polypeptides that
contain two or more building blocks, such as ISVs or Nanobodies
directed against different targets on a cell may (and usually will)
bind with higher avidity than each of the individual monomers or
individual building blocks, such as, for instance, the monovalent
ISVs or Nanobodies, comprised in the polypeptides of the invention.
[0441] Any K.sub.D value greater than 10.sup.-4 mol/liter (or any
K.sub.A value lower than 10.sup.4 M.sup.-1) liters/mol is generally
considered to indicate non-specific binding. [0442] The
polypeptides of the invention comprise a first and a second
building block, e.g. a first and a second ISV, or a first and a
second Nanobody. Preferably the affinity of each building block,
e.g. ISV or Nanobody, is determined individually. In other words,
the affinity is determined for the monovalent building block, ISV
or Nanobody, independent of avidity effects due to the other
building block, ISV or Nanobody, which might or might not be
present. The affinity for a monovalent building block, ISV or
Nanobody can be determined on the monovalent building block, ISV or
Nanobody per se, i.e. when said monovalent building block, ISV or
Nanobody is not comprised in the polypeptide of the invention. In
the alternative or in addition, the affinity for a monovalent
building block, ISV or Nanobody can be determined on one target
while the other target is absent. [0443] The binding of an
antigen-binding protein to an antigen or antigenic determinant can
be determined in any suitable manner known per se, including, for
example, Scatchard analysis and/or competitive binding assays, such
as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich
competition assays, and the different variants thereof known per se
in the art; as well as the other techniques mentioned herein.
[0444] The dissociation constant may be the actual or apparent
dissociation constant, as will be clear to the skilled person.
Methods for determining the dissociation constant will be clear to
the skilled person, and for example include the techniques
mentioned herein. In this respect, it will also be clear that it
may not be possible to measure dissociation constants of more than
10.sup.-4 moles/liter or 10.sup.-3 moles/liter (e.g., of 10.sup.-2
moles/liter). Optionally, as will also be clear to the skilled
person, the (actual or apparent) dissociation constant may be
calculated on the basis of the (actual or apparent) association
constant (K.sub.A), by means of the relationship
[K.sub.D=1/K.sub.A]. [0445] The affinity denotes the strength or
stability of a molecular interaction. The affinity is commonly
given as by the K.sub.D, or dissociation constant, which has units
of mol/liter (or M). The affinity can also be expressed as an
association constant, K.sub.A, which equals 1/K.sub.D and has units
of (mol/liter).sup.-1 (or M.sup.-1). In the present specification,
the stability of the interaction between two molecules (such as an
amino acid sequence, Nanobody or polypeptide of the invention and
its intended target) will mainly be expressed in terms of the
K.sub.D value of their interaction; it being clear to the skilled
person that in view of the relation K.sub.A=1/K.sub.D, specifying
the strength of molecular interaction by its K.sub.D value can also
be used to calculate the corresponding K.sub.A value. The K.sub.D
value characterizes the strength of a molecular interaction also in
a thermodynamic sense as it is related to the free energy (DG) of
binding by the well known relation DG=RTln(K.sub.D) (equivalently
DG=-RTln(K.sub.A)), where R equals the gas constant, T equals the
absolute temperature and ln denotes the natural logarithm.
[0446] The K.sub.D for biological interactions which are considered
meaningful (e.g. specific) are typically in the range of
10.sup.-10M (0.1 nM) to 10.sup.-5M (10000 nM). The stronger an
interaction is, the lower is its K.sub.D.
[0447] The K.sub.D can also be expressed as the ratio of the
dissociation rate constant of a complex, denoted as k.sub.off, to
the rate of its association, denoted k.sub.on(so that
K.sub.D=k.sub.off/k.sub.on and K.sub.A=k.sub.on/k.sub.off). The
off-rate k.sub.off has units s.sup.-1 (where s is the SI unit
notation of second). The on-rate k.sub.on has units
M.sup.-1s.sup.-1. The on-rate may vary between 10.sup.2
M.sup.-1s.sup.-1 to about 10.sup.7 M.sup.-1s.sup.-1, approaching
the diffusion-limited association rate constant for biomolecular
interactions. The off-rate is related to the half-life of a given
molecular interaction by the relation t.sub.1/2=ln(2)/k.sub.off.
The off-rate may vary between 10.sup.-6 s.sup.-1 (near irreversible
complex with a t.sub.1/2 of multiple days) to 1 s.sup.-1
(t.sub.1/2=0.69 s).
[0448] The affinity of a molecular interaction between two
molecules can be measured via different techniques known per se,
such as the well-known surface plasmon resonance (SPR) biosensor
technique (see for example Ober et al. 2001, Intern. Immunology,
13: 1551-1559). The term "surface plasmon resonance", as used
herein, refers to an optical phenomenon that allows for the
analysis of real-time biospecific interactions by detection of
alterations in protein concentrations within a biosensor matrix,
where one molecule is immobilized on the biosensor chip and the
other molecule is passed over the immobilized molecule under flow
conditions yielding k.sub.on, k.sub.off measurements and hence
K.sub.D (or K.sub.A) values. This can for example be performed
using the well-known BIAcore.RTM. system (BIAcore International AB,
a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For
further descriptions, see Jonsson et al. 1993 (Ann. Biol. Clin. 51:
19-26), Jonsson et al. 1991 (Biotechniques 11: 620-627), Johnsson,
et al. 1995 (J. Mol. Recognit. 8: 125-131), and Johnnson, et al.
1991 (Anal. Biochem. 198: 268-277).
[0449] Another well-known biosensor technique to determine
affinities of biomolecular interactions is bio-layer interferometry
(BLI) (see for example Abdiche et al. 2008, Anal. Biochem. 377:
209-217). The term "bio-layer Interferometry" or "BLI", as used
herein, refers to a label-free optical technique that analyzes the
interference pattern of light reflected from two surfaces: an
internal reference layer (reference beam) and a layer of
immobilized protein on the biosensor tip (signal beam). A change in
the number of molecules bound to the tip of the biosensor causes a
shift in the interference pattern, reported as a wavelength shift
(nm), the magnitude of which is a direct measure of the number of
molecules bound to the biosensor tip surface. Since the
interactions can be measured in real-time, association and
dissociation rates and affinities can be determined. BLI can for
example be performed using the well-known Octet.RTM. Systems
(ForteBio, a division of Pall Life Sciences, Menlo Park, USA).
[0450] Alternatively, affinities can be measured in Kinetic
Exclusion Assay (KinExA) (see for example Drake et al. 2004, Anal.
Biochem., 328: 35-43), using the KinExA.RTM. platform (Sapidyne
Instruments Inc, Boise, USA). The term "KinExA", as used herein,
refers to a solution-based method to measure true equilibrium
binding affinity and kinetics of unmodified molecules. Equilibrated
solutions of an antibody/antigen complex are passed over a column
with beads precoated with antigen (or antibody), allowing the free
antibody (or antigen) to bind to the coated molecule. Detection of
the antibody (or antigen) thus captured is accomplished with a
fluorescently labeled protein binding the antibody (or
antigen).
[0451] It will also be clear to the skilled person that the
measured K.sub.D may correspond to the apparent K.sub.D if the
measuring process somehow influences the intrinsic binding affinity
of the implied molecules for example by artefacts related to the
coating on the biosensor of one molecule. Also, an apparent K.sub.D
may be measured if one molecule contains more than one recognition
site for the other molecule. In such situation the measured
affinity may be affected by the avidity of the interaction by the
two molecules.
[0452] Another approach that may be used to assess affinity is the
2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of
Friguet et al. 1985 (J. Immunol. Methods, 77: 305-19). This method
establishes a solution phase binding equilibrium measurement and
avoids possible artefacts relating to adsorption of one of the
molecules on a support such as plastic.
[0453] However, the accurate measurement of K.sub.D may be quite
labour-intensive, and as consequence, often apparent K.sub.D values
are determined to assess the binding strength of two molecules. It
should be noted that as long as all measurements are made in a
consistent way (e.g. keeping the assay conditions unchanged)
apparent K.sub.D measurements can be used as an approximation of
the true K.sub.D and hence, in the present document, K.sub.D and
apparent K.sub.D should be treated with equal importance or
relevance.
[0454] Finally, it should be noted that in many situations the
experienced scientist may judge it to be convenient to determine
the binding affinity relative to some reference molecule. For
example, to assess the binding strength between molecules A and B,
one may e.g. use a reference molecule C that is known to bind to B
and that is suitably labelled with a fluorophore or chromophore
group or other chemical moiety, such as biotin for easy detection
in an ELISA or FACS (Fluorescent activated cell sorting) or other
format (the fluorophore for fluorescence detection, the chromophore
for light absorption detection, the biotin for
streptavidin-mediated ELISA detection). Typically, the reference
molecule C is kept at a fixed concentration and the concentration
of A is varied for a given concentration or amount of B. As a
result an IC50 value is obtained corresponding to the concentration
of A at which the signal measured for C in absence of A is halved.
Provided K.sub.D ref, the K.sub.D of the reference molecule, is
known, as well as the total concentration c.sub.ref of the
reference molecule, the apparent K.sub.D for the interaction A-B
can be obtained from following formula:
K.sub.D=IC50/(1+c.sub.ref/K.sub.D ref). Note that if
c.sub.ref<<K.sub.D ref, K.sub.D.apprxeq.IC50. Provided the
measurement of the IC50 is performed in a consistent way (e.g.
keeping c.sub.ref fixed) for the binders that are compared, the
strength or stability of a molecular interaction can be assessed by
the IC50 and this measurement is judged as equivalent to K.sub.D or
to apparent K.sub.D throughout this text. [0455] p) The half-life
of an amino acid sequence, compound or polypeptide of the invention
can generally be defined as described in paragraph o) on page 57 of
WO 08/020079 and as mentioned therein refers to the time taken for
the serum concentration of the amino acid sequence, compound or
polypeptide to be reduced by 50%, in vivo, for example due to
degradation of the sequence or compound and/or clearance or
sequestration of the sequence or compound by natural mechanisms.
The in vivo half-life of an amino acid sequence, compound or
polypeptide of the invention can be determined in any manner known
per se, such as by pharmacokinetic analysis. Suitable techniques
will be clear to the person skilled in the art, and may for example
generally be as described in paragraph o) on page 57 of WO
08/020079. As also mentioned in paragraph o) on page 57 of WO
08/020079, the half-life can be expressed using parameters such as
the t1/2-alpha, t1/2-beta and the area under the curve (AUC).
Reference is for example made to the Experimental Part below, as
well as to the standard handbooks, such as Kenneth et al. 1996
(Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists)
and Peters et al. 1996 (Pharmacokinete analysis: A Practical
Approach). Reference is also made to Gibaldi & Perron 1982
(Pharmacokinetics, Dekker M, 2nd Rev. edition). The terms "increase
in half-life" or "increased half-life" are as defined in paragraph
o) on page 57 of WO 08/020079 and in particular refer to an
increase in the t1/2-beta, either with or without an increase in
the t1/2-alpha and/or the AUC or both. [0456] q) In respect of a
target or antigen, the term "interaction site" on the target or
antigen means a site, epitope, antigenic determinant, part, domain
or stretch of amino acid residues on the target or antigen that is
a site for binding to a ligand, receptor or other binding partner,
a catalytic site, a cleavage site, a site for allosteric
interaction, a site involved in multimerisation (such as
homomerization or heterodimerization) of the target or antigen; or
any other site, epitope, antigenic determinant, part, domain or
stretch of amino acid residues on the target or antigen that is
involved in a biological action or mechanism of the target or
antigen. More generally, an "interaction site" can be any site,
epitope, antigenic determinant, part, domain or stretch of amino
acid residues on the target or antigen to which an amino acid
sequence or polypeptide of the invention can bind such that the
target or antigen (and/or any pathway, interaction, signalling,
biological mechanism or biological effect in which the target or
antigen is involved) is modulated. [0457] r) An immunoglobulin
single variable domain or polypeptide is said to be "specific for"
a first target or antigen compared to a second target or antigen
when it binds to the first antigen with an affinity/avidity (as
described above, and suitably expressed as a K.sub.D value, K.sub.A
value, K.sub.off rate and/or K.sub.on rate) that is at least 10
times, such as at least 100 times, and preferably at least 1000
times, and up to 10.000 times or more better than the affinity with
which said amino acid sequence or polypeptide binds to the second
target or polypeptide. For example, the first antigen may bind to
the target or antigen with a K.sub.D value that is at least 10
times less, such as at least 100 times less, and preferably at
least 1000 times less, such as 10.000 times less or even less than
that, than the K.sub.D with which said amino acid sequence or
polypeptide binds to the second target or polypeptide. Preferably,
when an immunoglobulin single variable domain or polypeptide is
"specific for" a first target or antigen compared to a second
target or antigen, it is directed against (as defined herein) said
first target or antigen, but not directed against said second
target or antigen. [0458] s) The terms "cross-block",
"cross-blocked" and "cross-blocking" are used interchangeably
herein to mean the ability of an immunoglobulin single variable
domain or polypeptide to interfere with the binding of the natural
ligand to its receptor(s). The extent to which an immunoglobulin
single variable domain or polypeptide of the invention is able to
interfere with the binding of another compound such as the natural
ligand to its target and therefore whether it can be said to
cross-block according to the invention, can be determined using
competition binding assays. One particularly suitable quantitative
cross-blocking assay uses a FACS- or an ELISA-based approach or
Alphascreen to measure competition between the labelled (e.g., His
tagged or biotinylated) immunoglobulin single variable domain or
polypeptide according to the invention and the other binding agent
in terms of their binding to the target. The experimental part
generally describes suitable FACS-, ELISA- or
Alphascreen-displacement-based assays for determining whether a
binding molecule cross-blocks or is capable of cross-blocking an
immunoglobulin single variable domain or polypeptide according to
the invention. It will be appreciated that the assay can be used
with any of the immunoglobulin single variable domains or other
binding agents described herein. Thus, in general, a cross-blocking
amino acid sequence or other binding agent according to the
invention is for example one which will bind to the target in the
above cross-blocking assay such that, during the assay and in the
presence of a second amino acid sequence or other binding agent of
the invention, the recorded displacement of the immunoglobulin
single variable domain or polypeptide according to the invention is
between 60% and 100% (e.g., in ELISA/Alphascreen based competition
assay) or between 80% to 100% (e.g., in FACS based competition
assay) of the maximum theoretical displacement (e.g. displacement
by cold (e.g., unlabeled) immunoglobulin single variable domain or
polypeptide that needs to be cross-blocked) by the to be tested
potentially cross-blocking agent that is present in an amount of
0.01 mM or less (cross-blocking agent may be another conventional
monoclonal antibody such as IgG, classic monovalent antibody
fragments (Fab, scFv) and engineered variants (e.g., diabodies,
triabodies, minibodies, VHHs, dAbs, VHs, VLs)). [0459] t) An amino
acid sequence such as e.g. an immunoglobulin single variable domain
or polypeptide according to the invention is said to be a "VHH1
type immunoglobulin single variable domain" or "VHH type 1
sequence", if said VHH1 type immunoglobulin single variable domain
or VHH type 1 sequence has 85% identity (using the VHH1 consensus
sequence as the query sequence and use the blast algorithm with
standard setting, i.e., blosom62 scoring matrix) to the VHH1
consensus sequence
(QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSD
GSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA) (SEQ ID NO: 483), and
mandatorily has a cysteine in position 50, i.e., C50 (using Kabat
numbering). [0460] u) An amino acid sequence such as e.g., an
immunoglobulin single variable domain or polypeptide according to
the invention is said to be "cross-reactive" for two different
antigens or antigenic determinants (such as serum albumin from two
different species of mammal, such as human serum albumin and
cynomolgus monkey serum albumin) if it is specific for (as defined
herein) both these different antigens or antigenic determinants.
[0461] v) As further described in paragraph q) on pages 58 and 59
of WO 08/020079 (incorporated herein by reference), the amino acid
residues of an immunoglobulin single variable domain are numbered
according to the general numbering for V.sub.H domains given by
Kabat et al. ("Sequence of proteins of immunological interest", US
Public Health Services, NIH Bethesda, Md., Publication No. 91), as
applied to V.sub.HH domains from Camelids in the article of
Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2):
185-195; see for example FIG. 2 of this publication). It should be
noted that--as is well known in the art for V.sub.H domains and for
VHH domains--the total number of amino acid residues in each of the
CDRs may vary and may not correspond to the total number of amino
acid residues indicated by the Kabat numbering (that is, one or
more positions according to the Kabat numbering may not be occupied
in the actual sequence, or the actual sequence may contain more
amino acid residues than the number allowed for by the Kabat
numbering). This means that, generally, the numbering according to
Kabat may or may not correspond to the actual numbering of the
amino acid residues in the actual sequence. The total number of
amino acid residues in a VH domain and a VHH domain will usually be
in the range of from 110 to 120, often between 112 and 115. It
should however be noted that smaller and longer sequences may also
be suitable for the purposes described herein. [0462] Determination
of CDR regions may also be done according to different methods. In
the CDR determination according to Kabat, FR1 of an immunoglobulin
single variable domain comprises the amino acid residues at
positions 1-30, CDR1 of an immunoglobulin single variable domain
comprises the amino acid residues at positions 31-35, FR2 of an
immunoglobulin single variable domain comprises the amino acids at
positions 36-49, CDR2 of an immunoglobulin single variable domain
comprises the amino acid residues at positions 50-65, FR3 of an
immunoglobulin single variable domain comprises the amino acid
residues at positions 66-94, CDR3 of an immunoglobulin single
variable domain comprises the amino acid residues at positions
95-102, and FR4 of an immunoglobulin single variable domain
comprises the amino acid residues at positions 103-113. [0463] In
the present application, however, unless indicated otherwise, CDR
sequences were determined according to Kontermann and Dubel (Eds.
2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg
Berlin, Martin, Chapter 3, pp. 33-51). According to this method,
FR1 comprises the amino acid residues at positions 1-25, CDR1
comprises the amino acid residues at positions 26-35, FR2 comprises
the amino acids at positions 36-49, CDR2 comprises the amino acid
residues at positions 50-58, FR3 comprises the amino acid residues
at positions 59-94, CDR3 comprises the amino acid residues at
positions 95-102, and FR4 comprises the amino acid residues at
positions 103-113. [0464] w) The Figures, Sequence Listing and the
Experimental Part/Examples are only given to further illustrate the
invention and should not be interpreted or construed as limiting
the scope of the invention and/or of the appended claims in any
way, unless explicitly indicated otherwise herein. [0465] x) The
half maximal inhibitory concentration (IC50) is a measure of the
effectiveness of a compound in inhibiting a biological or
biochemical function, e.g. a pharmacological effect. This
quantitative measure indicates how much of the ISV or Nanobody
(inhibitor) is needed to inhibit a given biological process (or
component of a process, i.e. an enzyme, cell, cell receptor,
chemotaxis, anaplasia, metastasis, invasiveness, etc) by half. In
other words, it is the half maximal (50%) inhibitory concentration
(IC) of a substance (50% IC, or IC50). The IC50 of a drug can be
determined by constructing a dose-response curve and examining the
effect of different concentrations of antagonist such as the ISV or
Nanobody of the invention on reversing agonist activity. IC50
values can be calculated for a given antagonist such as the ISV or
Nanobody of the invention by determining the concentration needed
to inhibit half of the maximum biological response of the agonist.
[0466] The term half maximal effective concentration (EC50) refers
to the concentration of a compound which induces a response halfway
between the baseline and maximum after a specified exposure time.
In the present context it is used as a measure of a polypeptide's,
ISV's or Nanobody's potency. The EC50 of a graded dose response
curve represents the concentration of a compound where 50% of its
maximal effect is observed. Concentration is preferably expressed
in molar units. [0467] In biological systems, small changes in
ligand concentration typically result in rapid changes in response,
following a sigmoidal function. The inflection point at which the
increase in response with increasing ligand concentration begins to
slow is the EC50. This can be determined mathematically by
derivation of the best-fit line. Relying on a graph for estimation
is convenient in most cases. In case the EC50 is provided in the
examples section, the experiments were designed to reflect the KD
as accurate as possible. In other words, the EC50 values may then
be considered as KD values. The term "average KD" relates to the
average KD value obtained in at least 1, but preferably more than
1, such as at least 2 experiments. The term "average" refers to the
mathematical term "average" (sums of data divided by the number of
items in the data). [0468] It is also related to IC50 which is a
measure of a compound's inhibition (50% inhibition). For
competition binding assays and functional antagonist assays, IC50
is the most common summary measure of the dose-response curve. For
agonist/stimulator assays the most common summary measure is the
EC50. [0469] y) It must be noted that as used herein, the singular
forms
"a", "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "a
reagent" includes one or more of such different reagents and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein. [0470]
Unless otherwise indicated, the term "at least" preceding a series
of elements is to be understood to refer to every element in the
series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention. [0471] The term "and/or" wherever used herein
includes the meaning of "and", "or" and "all or any other
combination of the elements connected by said term". [0472] The
term "about" or "approximately" as used herein means within 20%,
preferably within 15%, more preferably within 10%, and most
preferably within 5% of a given value or range. [0473] Throughout
this specification and the claims which follow, unless the context
requires otherwise, the word "comprise", and variations such as
"comprises" and "comprising", will be understood to imply the
inclusion of a stated integer or step or group of integers or steps
but not the exclusion of any other integer or step or group of
integer or step. When used herein the term "comprising" can be
substituted with the term "containing" or "including" or sometimes
when used herein with the term "having".
[0474] The present invention relates to a polypeptide comprising at
least a first and at least one further immunoglobulin single
variable domain (ISV), wherein said at least first ISV has high
affinity for/binds to the constant domain of the T cell receptor
(TCR) and said at least one further ISV has high affinity for/binds
to an antigen on a target cell.
[0475] Typically, the multispecific polypeptides of the invention
combine high affinity antigen recognition on the target cell with T
cell activation, resulting in an activation that is independent of
the T cells' natural specificity. The mode of action of the binding
molecules that bind both to a cell surface molecule on a target
cell such as a tumour antigen and to the T cell TCR is commonly
known. Bringing a T cell in close vicinity to a target cell, i.e.,
engaging said T cell and clustering of the TCR complex results in
killing of the target cell by the T cell. In the present invention
this process is exploited in fighting against proliferative
disease, inflammatory disease, infectious disease and autoimmune
disease. Generally T cells are equipped with granules containing a
deadly combination of pore-forming proteins, called perforins, and
cell death-inducing proteases, called granzymes. Preferably, these
proteins are delivered into target cells via a cytolytic synapse
that forms if T cells are in close vicinity with a target cell that
is aimed to be killed. Normally, close vicinity between a T cell
and a target cell is achieved by the T cell binding to an
MHC/peptide complex using its matching T cell receptor. The
polypeptides of the invention bring a T cell into such close
vicinity to a target cell in the absence of T cell receptor/MHC
interaction.
[0476] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said polypeptide directs the T cell to
the target cell.
[0477] With one arm (first ISV), the multispecific polypeptide has
high affinity for/binds to the constant domain of the TCR subunit,
a protein component of the signal-transducing complex of the T cell
receptor on T cells. With another arm (second ISV and/or third ISV,
etc.), the multispecific polypeptide recognizes, has high affinity
for/binds an antigen(s) on target cells. Preferably, T cell
activation is only seen when the multispecific polypeptides are
presented to T cells on the surface of target cells. Antigen
dependence on target cells for activation results in a favourable
safety profile. In an embodiment, the multispecific polypeptides
transiently tether T cells and target cells. Preferably, the
multispecific polypeptide can induce resting polyclonal T cells,
such as CD4.sup.+ and/or CD8.sup.+ T cells into activation, for
highly potent redirected lysis of target cells. Preferably, the T
cell is directed to a next target cell after lysis of the first
target cell.
[0478] Proteins and polypeptides that comprise or essentially
consist of two or more immunoglobulin single variable domains (such
as at least two immunoglobulin single variable domains of the
invention) will be referred to herein as "multivalent" proteins or
polypeptides or as "multivalent constructs". Some non-limiting
examples of such multivalent constructs will become clear from the
further description herein. The polypeptides of the invention are
"multivalent", i.e. comprising two or more building blocks or ISVs
of which at least the first building block, ISV or Nanobody and the
second building block, ISV or Nanobody are different, and directed
against different targets, such as antigens or antigenic
determinants. Polypeptides of the invention that contain at least
two building blocks, ISVs or Nanobodies, in which at least one
building block, ISV or Nanobody is directed against a first antigen
(i.e., against the first target, such as e.g. the constant domain
of a TCR) and at least one building block, ISV or Nanobody is
directed against a second antigen (i.e., against the second target
which is different from the first target, such as e.g. a TAA, such
as CD20 or HER2), will also be referred to as "multispecific"
polypeptides of the invention, and the building blocks, ISVs or
Nanobodies present in such polypeptides will also be referred to
herein as being in a "multivalent format" or "multispecifc format".
Thus, for example, a "bispecific" polypeptide of the invention is a
polypeptide that comprises at least one building block, ISV or
Nanobody directed against a first target (e.g. TCR) and at least
one further building block, ISV or Nanobody directed against a
second target (i.e., directed against a second target different
from said first target, such as e.g. a TAA, e.g. CD20 or HER2),
whereas a "trispecific" polypeptide of the invention is a
polypeptide that comprises at least one building block, ISV or
Nanobody directed against a first target (e.g., TCR), a second
building block, ISV or Nanobody directed against a second target
different from said first target (e.g. a TAA, e.g. CD20 or HER2)
and at least one further building block, ISV or Nanobody directed
against a third antigen (i.e., different from both the first and
the second target, such as another TAA); etc. As will be clear from
the description, the invention is not limited to bispecific
polypeptides, in the sense that a multispecific polypeptide of the
invention may comprise at least a first building block, ISV or
Nanobody against a first target, a second building block, ISV or
Nanobody against a second target and any number of building blocks,
ISVs or Nanobodies directed against one or more targets, which may
be the same or different from the first and/or second target,
respectively. The building blocks, ISVs or Nanobodies can
optionally be linked via linker sequences.
[0479] The terms bispecific polypeptide, bispecific format,
bispecific construct, bispecific Nanobody construct, bispecific and
bispecific antibody are used interchangeably herein.
[0480] As will be clear from the further description above and
herein, the immunoglobulin single variable domains of the invention
can be used as "building blocks" to form polypeptides of the
invention, e.g., by suitably combining them with other groups,
residues, moieties or binding units, in order to form compounds or
constructs as described herein (such as, without limitations, the
bi-/tri-/tetra-/multivalent and bi-/tri-/tetra-/multispecific
polypeptides of the invention described herein) which combine
within one molecule one or more desired properties or biological
functions.
[0481] It will be appreciated (as is also demonstrated in the
Example section) that the ISV binding TCR and the ISV binding the
antigen on a target cell can be positioned in any order in the
polypeptide of the invention. More particularly, in one embodiment,
the ISV binding TCR is positioned N-terminally and the ISV binding
the antigen on a target cell is positioned C-terminally. In another
embodiment, the ISV binding the antigen on a target cell is
positioned N-terminally and the ISV binding TCR is positioned
C-terminally.
[0482] In a preferred aspect, the polypeptide of the invention
comprises at least a first, at least a second and at least a third
immunoglobulin single variable domain (ISV), wherein said at least
a first ISV has high affinity for/binds to the constant domain of
the T cell receptor (TCR); said at least a second ISV has high
affinity for/binds to a first antigen on a target cell, and said at
least a third ISV has high affinity for/binds to a second antigen
on a target cell, wherein said second antigen is different from
said first antigen. Said first antigen and said second antigen can
be on the same or on different target cells.
[0483] It will be appreciated (as is also demonstrated in the
Example section) that the ISV binding TCR and the ISVs binding the
first and second antigen on a target cell can be positioned in any
order in the polypeptide of the invention. More particularly, in
one embodiment, the ISV binding TCR is positioned N-terminally, the
ISV binding the first antigen on a target cell is positioned
centrally and the ISV binding the second antigen on a target cell
is positioned C-terminally. In another embodiment, the ISV binding
TCR is positioned N-terminally, the ISV binding the second antigen
on a target cell is positioned centrally and the ISV binding the
first antigen on a target cell is positioned C-terminally. In
another embodiment, the ISV binding the first antigen on a target
cell is positioned N-terminally, the ISV binding the second antigen
on a target cell is positioned centrally and the ISV binding the
TCR is positioned C-terminally. In another embodiment, the ISV
binding the first antigen on a target cell is positioned
N-terminally, the ISV binding the TCR is positioned centrally and
the ISV binding the second antigen on a target cell is positioned
C-terminally. In another embodiment, the ISV binding the second
antigen on a target cell is positioned N-terminally, the ISV
binding the TCR is positioned centrally and the ISV binding the
first antigen on a target cell is positioned C-terminally. In
another embodiment, the ISV binding the second antigen on a target
cell is positioned N-terminally, the ISV binding the first antigen
on a target cell is positioned centrally and the ISV binding the
TCR is positioned C-terminally.
[0484] The invention further relates to compounds or constructs,
and in particular proteins or polypeptides that comprise or
essentially consist of one or more ISVs or polypeptides of the
invention, and optionally further comprise one or more other
groups, residues, moieties or binding units. As will become clear
to the skilled person from the further disclosure herein, such
further groups, residues, moieties, binding units or amino acid
sequences may or may not provide further functionality to the
polypeptide of the invention (and/or to the compound or construct
in which it is present) and may or may not modify the properties of
the polypeptide of the invention.
[0485] The compounds, constructs or polypeptides of the invention
can generally be prepared by a method which comprises at least one
step of suitably linking the one or more immunoglobulin single
variable domains of the invention to the one or more further
groups, residues, moieties or binding units, optionally via one or
more suitable linkers, so as to provide the compound, construct or
polypeptide of the invention. Polypeptides of the invention can
also be prepared by a method which generally comprises at least the
steps of providing a nucleic acid that encodes a polypeptide of the
invention, expressing said nucleic acid in a suitable manner, and
recovering the expressed polypeptide of the invention. Such methods
can be performed in a manner known per se, which will be clear to
the skilled person, for example on the basis of the methods and
techniques further described herein.
[0486] The process of designing/selecting and/or preparing a
compound, construct or polypeptide of the invention, starting from
an amino acid sequence of the invention, is also referred to herein
as "formatting" said amino acid sequence of the invention; and an
amino acid of the invention that is made part of a compound,
construct or polypeptide of the invention is said to be "formatted"
or to be "in the format of" said compound, construct or polypeptide
of the invention. Examples of ways in which an amino acid sequence
of the invention can be formatted and examples of such formats will
be clear to the skilled person based on the disclosure herein; and
such formatted immunoglobulin single variable domains or
polypeptides form a further aspect of the invention.
[0487] For example, such further groups, residues, moieties or
binding units may be one or more additional immunoglobulin single
variable domains, such that the compound or construct is a (fusion)
protein or (fusion) polypeptide. In a preferred but non-limiting
aspect, said one or more other groups, residues, moieties or
binding units are immunoglobulin sequences. Even more preferably,
said one or more other groups, residues, moieties or binding units
are chosen from the group consisting of domain antibodies,
immunoglobulin single variable domains that are suitable for use as
a domain antibody, single domain antibodies, immunoglobulin single
variable domains (ISVs) that are suitable for use as a single
domain antibody, "dAb" 's, immunoglobulin single variable domains
that are suitable for use as a dAb, or Nanobodies. Alternatively,
such groups, residues, moieties or binding units may for example be
chemical groups, residues, moieties, which may or may not by
themselves be biologically and/or pharmacologically active. For
example, and without limitation, such groups may be linked to the
one or more immunoglobulin single variable domains or polypeptides
of the invention so as to provide a "derivative" of an ISV or
polypeptide of the invention, as further described herein.
[0488] Also within the scope of the present invention are compounds
or constructs, which comprise or essentially consist of one or more
derivatives as described herein, and optionally further comprise
one or more other groups, residues, moieties or binding units,
optionally linked via one or more linkers. Preferably, said one or
more other groups, residues, moieties or binding units are
immunoglobulin single variable domains. In the compounds or
constructs described above, the one or more immunoglobulin single
variable domains of the invention and the one or more groups,
residues, moieties or binding units may be linked directly to each
other and/or via one or more suitable linkers or spacers. For
example, when the one or more groups, residues, moieties or binding
units are immunoglobulin single variable domains, the linkers may
also be immunoglobulin single variable domains, so that the
resulting compound or construct is a fusion protein or fusion
polypeptide.
[0489] In some embodiments, the polypeptides comprise at least two
or more immunoglobulin single variable domains disclosed herein. In
some embodiments, the polypeptides essentially consist of two or
more immunoglobulin single variable domains disclosed herein. A
polypeptide that "essentially consists of" two or more
immunoglobulin single variable domains, is a polypeptide that in
addition to the two or more immunoglobulin single variable domains
disclosed herein does not have additional immunoglobulin single
variable domains. For instance, a polypeptide that essentially
consists of two immunoglobulin single variable domains does not
include any additional immunoglobulin single variable domains.
However, it should be appreciated that a polypeptide that
essentially consists of two or more immunoglobulin single variable
domains may include additional functionalities, such as a label, a
toxin, one or more linkers, a binding sequence, etc. These
additional functionalities include both amino acid based and
non-amino acid based groups. In some embodiments, the polypeptides
consist of one or more immunoglobulin single variable domains
disclosed herein. It should be appreciated that the terms
"polypeptide construct" and "polypeptide" can be used
interchangeably herein (unless the context clearly dictates
otherwise).
[0490] In some embodiments, the polypeptides include multivalent or
multispecific constructs comprising immunoglobulin single variable
domains disclosed herein. In some embodiments, the polypeptides
comprise one or more antibody based-scaffolds and/or non-antibody
based scaffolds disclosed herein. In some embodiments, the
polypeptides comprise a serum binding protein moiety. In some
embodiments, the serum binding protein moiety is an immunoglobulin
single variable domain. In some embodiments, the immunoglobulin
single variable domain is a Nanobody.
[0491] It will be appreciated that the order of the building
blocks, such as e.g. a first building block, a second building
block, a third building block etc., on the polypeptide
(orientation) can be chosen according to the needs of the person
skilled in the art, as well as the relative affinities which may
depend on the location of these building blocks in the polypeptide.
Whether the polypeptide comprises a linker, is a matter of design
choice. However, some orientations, with or without linkers, may
provide preferred binding characteristics in comparison to other
orientations. For instance, the order of a first and a second
building block in the polypeptide of the invention can be (from
N-terminus to C-terminus): (i) first building block (e.g. a first
ISV such as a first Nanobody)-[linker]-second building block (e.g.
a second ISV such as a second Nanobody); or (ii) second building
block (e.g. a second ISV such as a second Nanobody)-[linker]-first
building block (e.g. a first ISV such as a first Nanobody);
(wherein the linker is optional). All orientations are encompassed
by the invention. Polypeptides that contain an orientation of
building blocks that provides desired (binding) characteristics can
be easily identified by routine screening, for instance as
exemplified in the experimental section.
[0492] The first immunoglobulin single variable domain (ISV) of the
polypeptide of the invention has high affinity for/binds to an
effector cell, preferably the TCR of said effector cell, and even
more preferably the constant domain of the T cell receptor
(TCR).
[0493] An effector cell is a cell comprising a TCR complex,
preferably an immune cell, such as a T cell, preferably a CD4.sup.+
T-helper cell (also known as CD4 cell, T-helper cell or T4 cell),
more preferably a Cytotoxic T cell (also known as Tc cell, CTL or
CD8.sup.+ T cells) or Natural Killer T cells (NKT cells). In some
embodiments, the cell is present in vivo. In some embodiments, the
cell is present in vitro. The effector cell of the invention
relates in particular to mammalian cells, preferably to primate
cells, and even more preferably to human cells.
[0494] As used herein, the terms "TCR complex" or "a TCR-CD3
complex" refers to the T cell receptor complex presented on the
surface of T cells (see Kuhns et al. 2006, Immunity 24: 133-139).
The TCR complex is composed of six different type I single-spanning
transmembrane proteins: the TCR.alpha. and TCR.beta. chains that
form the TCR heterodimer responsible for ligand recognition, and
the non-covalently associated CD3.gamma., CD3.delta., CD3.epsilon.
and .zeta. chains, which bear cytoplasmic sequence motifs that are
phosphorylated upon receptor activation and recruit a large number
of signalling components. Both .alpha. and .beta. chains of the T
cell receptor consist of a constant domain and a variable domain.
The sequences for the human CD3 and the human TCR.alpha./.beta.
constant domains are provided in Table A-6 (SEQ ID NOs: 344-349;
cf. UniProt identifiers: CD3 delta: P04234, CD3 gamma: P09693, CD3
epsilon: P07766, CD3 zeta: P20963, TCR alpha: P01848 and TCR beta:
related to P01850).
[0495] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said first ISV binds to
the constant domain of a T cell receptor .alpha. (TCR-.alpha.) (SEQ
ID NO: 348) and/or the constant domain of the T cell receptor
.beta. (TCR-.beta.) (SEQ ID NO: 349), or polymorphic variants or
isoforms thereof.
[0496] Alternatively, the present invention relates to a
polypeptide as described herein, wherein said first ISV binds to
the constant domain of a T cell receptor .alpha. (TCR-.alpha.) (SEQ
ID NO: 484) and/or the constant domain of the T cell receptor
.beta. (TCR-.beta.) (SEQ ID NO: 485), or polymorphic variants or
isoforms thereof.
[0497] Isoforms are alternative protein sequences that can be
generated from the same gene by a single or by the combination of
biological events such as alternative promoter usage, alternative
splicing, alternative initiation and ribosomal frameshifting, all
as known in the art.
[0498] "T cell activation" as used herein refers to one or more
cellular response(s) of a T cell, e.g. a cytotoxic T cell, such as
selected from: proliferation, differentiation, cytokine secretion,
cytotoxic effector molecule release, cytotoxic activity, expression
of activation markers, and redirected target cell lysis. The
polypeptides of the invention are capable of inducing T cell
activation. Suitable assays to measure T cell activation are known
in the art described herein, for instance as described in WO
99/54440 or by Schlereth et al. 2005 (Cancer Immunol. Immunother.
20: 1-12), or as exemplified in the examples or below.
[0499] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said polypeptide induces T
cell activation. Preferably, the polypeptide of the invention
induces T cell activation only when said second and/or further ISV
is bound to an antigen on a target cell.
[0500] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said T cell activation
depends on presenting said polypeptide bound to said first antigen
on a target cell to a T cell.
[0501] T cell activation by the polypeptides of the invention can
be monitored by upregulation of CD69, CD25 and various cell
adhesion molecules, de novo expression and/or release of cytokines
(e.g., IFN-.gamma., TNF-.alpha., IL-6, IL-2, IL-4 and IL-10),
upregulation of granzyme and perforin expression, and/or cell
proliferation, membrane blebbing, activation of procaspases 3
and/or 7, fragmentation of nuclear DNA and/or cleavage of caspase
substrate poly (ADPribose) polymerase. Preferably, redirected lysis
of target cells by multispecific polypeptides is independent of T
cell receptor specificity, presence of MHC class I and/or 32
microglobulin, and/or of any co-stimulatory stimuli.
[0502] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said T cell activation is
independent from MHC recognition.
[0503] The polypeptides of the invention show redirected lysis in
vitro with previously unstimulated peripheral polyclonal CD8.sup.+-
and CD4.sup.+-positive T cells. The redirected lysis of target
cells via the recruitment of T cells by the polypeptides of the
invention involves cytolytic synapse formation and delivery of
perforin and granzymes. Cell lysis by T cells has been described,
e.g. by Atkinson and Bleackley 1995 (Crit. Rev. Immunol
15(3-4):359-384). Preferably, the engaged T cells are capable of
serial target cell lysis, and are not affected by immune escape
mechanisms interfering with peptide antigen processing and
presentation, or clonal T cell differentiation (see, for example,
WO 2007/042261). In vitro, redirected lysis is seen at low
picomolar concentrations, suggesting that very low numbers of the
polypeptides of the invention need to be bound to target cells for
triggering T cells. As demonstrated in the examples, the low
effector to target ratio might be indicative for serial target cell
lysis. Accordingly, the present invention relates to potent
polypeptides. Preferably, the polypeptide of the invention mediates
killing of target cells, e.g. cancer cells, such as stimulating T
cells in pore formation and delivering pro-apoptotic components of
cytotoxic T cell granules.
[0504] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said T cell activation
causes one or more cellular response of said T cell, wherein said
cellular response is selected from the group consisting of
proliferation, differentiation, cytokine secretion, cytotoxic
effector molecule release, cytotoxic activity, expression of
activation markers and redirected target cell lysis.
[0505] As used herein, the term "potency" is a measure of the
biological activity of an agent, such as a polypeptide, ISV or
Nanobody. Potency of an agent can be determined by any suitable
method known in the art, such as for instance as described in the
experimental section. Cell culture based potency assays are often
the preferred format for determining biological activity since they
measure the physiological response elicited by the agent and can
generate results within a relatively short period of time. Various
types of cell based assays, based on the mechanism of action of the
product, can be used, including but not limited to proliferation
assays, cytotoxicity assays, cell killing assays, reporter gene
assays, cell surface receptor binding assays, and assays to measure
induction/inhibition of functionally essential proteins or other
signal molecules (such as phosphorylated proteins, enzymes,
cytokines, cAMP and the like), Ramos B cell depletion model, T cell
mediated tumour cell killing assay (for instance as set out in the
Examples section), all well known in the art. Results from cell
based potency assays can be expressed as "relative potency" as
determined by comparison of the multispecific polypeptide of the
invention to the response obtained for the corresponding reference
monovalent ISV, e.g. a polypeptide comprising only one ISV or one
Nanobody, optionally further comprising an irrelevant Nanobody (cf.
experimental section).
[0506] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said T cell activation
causes inhibition of an activity of said target cell, such as to
delay or minimize the spread of the target cell, to inhibit or
delay growth and/or proliferation of the target cell, and/or to
kill the target cell (e.g., cause regression of the disorder and/or
symptoms) by more than about 10%, such as 20%, 30%, or 40% or even
more than 50%, such as more than 60%, such as 70%, 80%, or even
more than 90%, such as 100%.
[0507] The first building block, ISV, Nanobody or VHH of the
invention has a high affinity for its--the constant domain of
TCR--target. The first building block, ISV or Nanobody of the
invention may for example be directed against an antigenic
determinant, epitope, part, domain, subunit or confirmation (where
applicable) of said first target. The first building block, e.g.
the first ISV, Nanobody or VHH, is preferably chosen for its high
affinity for its target per se, disregarding the influence of any
avidity effects.
[0508] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said first ISV binds to the constant
domain of the T cell receptor (TCR) with an average KD value of
between 100 nM and 10 .mu.M, such as at an average KD value of 90
nM or less, even more preferably at an average KD value of 80 nM or
less, such as less than 70, 60, 50, 40, 30, 20, 10, 5 nM or even
less, such as less than 4, 3, 2, or 1 nM, such as less than 500,
400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20 .mu.M, or even
less, such as less than 10 .mu.M. Preferably, the KD is determined
by Kinexa, BLI or SPR, for instance as determined by Proteon. For
instance, said KD is determined as set out in the Examples
section.
[0509] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said first ISV has a high affinity
when measured as a monovalent. Preferably said average KD is
measured by surface plasmon resonance (SPR) on recombinant
protein.
[0510] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said polypeptide has a dissociation
constant (K.sub.D) to (or for binding) said TCR selected from the
group consisting of: at most about 10.sup.-5 M, at most about
10.sup.-6 M, at most about 10.sup.-7 M, at most about 10.sup.-8 M,
at most about 10.sup.-9 M, at most about 10.sup.-10 M, at most
about 10.sup.-11 M, and at most about 10.sup.-12 M, preferably as
measured by surface plasmon resonance.
[0511] The present invention also relates to a polypeptide as
described herein, wherein said first ISV binds to said TCR with an
EC50 value of between 100 nM and 1 .mu.M, such as at an average
EC50 value of 100 nM or less, even more preferably at an average
EC50 value of 90 nM or less, such as less than 80, 70, 60, 50, 40,
30, 20, 10, 5 nM or even less, such as less than 4, 3, 2, or 1 nM
or even less, such as less than 500, 400, 300, 200, 100, 90, 80,
70, 60, 50, 40, 30, 20, 10, 5 .mu.M, or even less, such as less
than 4 .mu.M.
[0512] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said average KD is determined by FACS,
Biacore, ELISA, on a monovalent first ISV, such as a Nanobody, or a
polypeptide comprising a monovalent first ISV, such as a Nanobody,
for instance said EC50 is determined as set out in the Examples
section.
[0513] It has been shown in the examples that the K.sub.D
correlates well with the EC50.
[0514] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said polypeptide has an on
rate constant (Kon) to (or for binding) said TCR selected from the
group consisting of at least about 10.sup.2 M.sup.-1s.sup.-1, at
least about 10.sup.3 M.sup.-1s.sup.-1, at least about 10.sup.4
M.sup.-1s.sup.-1, at least about 10.sup.5 M.sup.-1s.sup.-1, at
least about 10.sup.6 M.sup.-1s.sup.-1, 10.sup.7 M.sup.-1s.sup.-1,
at least about 10.sup.8 M.sup.-1s.sup.-1, at least about 10.sup.9
M.sup.-1s.sup.-1, and at least about 10.sup.10 M.sup.-1s.sup.-1,
preferably as measured by surface plasmon resonance or as performed
in the examples section.
[0515] In an embodiment, the present invention relates to a
polypeptide as described herein, wherein said polypeptide has an
off rate constant (Koff) to (or for binding) said TCR selected from
the group consisting of at most about 10.sup.-3 s.sup.-1, at most
about 10.sup.-4 s.sup.-1, at most about 10.sup.-5 s.sup.-1, at most
about 10.sup.-6 s.sup.-1, at most about 10.sup.-7 s.sup.-1, at most
about 10.sup.-8 s.sup.-1, at most about 10.sup.-9 s.sup.-1, and at
most about 10.sup.-10 s.sup.-1, preferably as measured by surface
plasmon resonance or as performed in the examples section.
[0516] Amino acid sequence modifications of the binding molecules,
ISVs, or polypeptides described herein are contemplated. For
example, it may be desirable to improve the binding affinity and/or
other biological properties of the antibody or ISV. Amino acid
sequence variants of the binding molecules, ISVs, or polypeptides
are prepared by introducing appropriate nucleotide changes into the
binding molecules, ISVs, or polypeptides nucleic acid, or by
peptide synthesis.
[0517] Such modifications include, for example, deletions from,
and/or insertions into, and/or substitutions of, residues within
the amino acid sequences of the binding molecules, ISVs or
polypeptides. Any combination of deletion, insertion, and
substitution is made to arrive at the final construct, provided
that the final construct possesses the desired characteristics. The
amino acid changes also may alter post-translational processes of
the binding molecules, such as changing the number or position of
glycosylation sites. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
amino acids may be substituted in a CDR, while 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids
may be substituted in the framework regions (FRs). The
substitutions are preferably conservative substitutions as
described herein. Additionally or alternatively, 1, 2, 3, 4, 5, or
6 amino acids may be inserted or deleted in each of the CDRs (of
course, dependent on their length), while 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids
may be inserted or deleted in each of the FRs.
[0518] A useful method for identification of certain residues or
regions of the binding molecules, ISVs or polypeptides, that are
preferred locations for mutagenesis is called "alanine scanning
mutagenesis" as described by Cunningham and Wells 1989 (Science
244: 1081-1085). Here, a residue or group of target residues within
the binding molecule is/are identified (e.g. charged residues such
as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or
negatively charged amino acid (most preferably alanine or
polyalanine) to affect the interaction of the amino acids with the
epitope. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se needs
not to be predetermined. For example, to analyze the performance of
a mutation at a given site, ala scanning or random mutagenesis is
conducted at a target codon or region and the expressed binding
molecule variants are screened for the desired activity.
[0519] Preferably, amino acid sequence insertions include amino-
and/or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4,
5, 6, 7, 8, 9 or 10 residues to polypeptides containing a hundred
or more residues.
[0520] Another type of variant is an amino acid substitution
variant. These variants have preferably at least 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acid residues in the binding molecule, ISV or
polypeptide replaced by a different residue. The sites of greatest
interest for substitution mutagenesis include the CDRs, in
particular the hypervariable regions, but FR alterations are also
contemplated. For example, if a CDR sequence encompasses 6 amino
acids, it is envisaged that one, two or three of these amino acids
are substituted. Similarly, if a CDR sequence encompasses 15 amino
acids it is envisaged that one, two, three, four, five or six of
these amino acids are substituted.
[0521] Generally, if amino acids are substituted in one or more or
all of the CDRs, it is preferred that the then-obtained
"substituted" sequence is at least 60%, more preferably 65%, even
more preferably 70%, particularly preferably 75%, more particularly
preferably 80% or even more than 90% identical to the "original"
CDR sequence. This means that it is dependent of the length of the
CDR to which degree it is identical to the "substituted" sequence.
For example, a CDR having 5 amino acids is preferably 80% identical
to its substituted sequence in order to have at least one amino
acid substituted. Accordingly, the CDRs of the binding molecule may
have different degrees of identity to their substituted sequences,
e.g., CDR1 may have 80%, while CDR3 may have 90%.
[0522] Preferred substitutions (or replacements) are conservative
substitutions. However, any substitution (including
non-conservative substitution or one or more from the "exemplary
substitutions" listed in Table B-1 below) is envisaged as long as
the polypeptide retains its capability to bind to the constant
domain of the T cell receptor (TCR) present on a T cell via the
first ISV and to a first antigen on a target cell via the second
ISV and/or its CDRs have an identity to the then substituted
sequence (at least 60%, more preferably 65%, even more preferably
70%, particularly preferably 75%, more particularly preferably 80%
identical to the "original" CDR sequence).
[0523] Conservative substitutions are shown in Table B-1 below.
[0524] As indicated before, only after rigorous immunization and
screening and selection methods, the present inventors were able to
identify ISVs binding to the constant domains of TCR. Accordingly,
the present invention relates to polypeptides comprising a first
ISV chosen from the group consisting of SEQ ID NOs: 1-118 (cf.
Table A-4). Sequence analysis further demonstrated that all ISVs
binding to TCR comprised a very similar CDR3. Accordingly, the
present invention relates to a polypeptide according to the
invention in which said first ISV essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which CDR3 has
the amino acid sequence X.sub.1SR X.sub.2X.sub.3PYX.sub.4Y, in
which X.sub.1 is F, Y, G, L or K, X.sub.2 is I or L, X.sub.3 is Y
or W, and X.sub.4 is D, N or S.
[0525] Sequence analysis further revealed that there are only a
limited number of sequence variations in the CDRs (cf. Example 4.2
and Tables A-1 to A-3).
TABLE-US-00001 TABLE B-1 Amino Acid Substitutions Original
Exemplary Substitutions Preferred Substitutions Ala (A) val, leu,
ile val Arg (R) lys, gln, asn lys Asn (N) gln, his, asp, lys, arg
gln Asp (D) glu, asn glu Cys (C) ser, ala ser Gln (Q) asn, glu asn
Glu (E) asp, gln asp Gly (G) ala ala His (H) asn, gln, lys, arg arg
Ile (I) leu, val, met, ala, phe leu Leu (L) norleucine, ile, val,
met, ala ile Lys (K) arg, gln, asn arg Met (M) leu, phe, ile leu
Phe (F) leu, val, ile, ala, tyr tyr Pro (P) ala ala Ser (S) thr thr
Thr (T) ser ser Trp (W) tyr, phe tyr Tyr (Y) trp, phe, thr, ser phe
Val (V) ile, leu, met, phe, ala leu
[0526] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said first ISV essentially consists of
4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which: [0527] (i) CDR1 is chosen from the group consisting of:
[0528] (a) SEQ ID NOs: 119-133; or [0529] (b) amino acid sequences
that have 4, 3, 2, or 1 amino acid(s) difference with the amino
acid sequence of SEQ ID NO: 123 or with any of SEQ ID NOs: 119-133;
and/or [0530] (ii) CDR2 is chosen from the group consisting of:
[0531] (c) SEQ ID NOs: 134-163; or [0532] (d) amino acid sequences
that have 4, 3, 2, or 1 amino acid(s) difference with the amino
acid sequence of SEQ ID NO: 153 or with any of SEQ ID NOs: 134-163;
and/or [0533] (iii) CDR3 is chosen from the group consisting of:
[0534] (e) SEQ ID NOs: 164-174; or [0535] (f) amino acid sequences
that have 4, 3, 2, or 1 amino acid(s) difference with the amino
acid sequence of SEQ ID NO: 170 or with any of SEQ ID NOs:
164-174.
[0536] Further preferred CDR sequences are depicted in Table
A-4.
[0537] Generally, the combinations of CDR's listed in Table A-4
(i.e. those mentioned on the same line in Table A-4) are preferred.
Thus, it is generally preferred that, when a CDR in an ISV is a CDR
sequence mentioned in Table A-4 or suitably chosen from the group
consisting of CDR sequences that have 4, 3, 2 or only 1 amino acid
difference(s) with a CDR sequence listed in Table A-4, that at
least one and preferably both of the other CDR's are suitably
chosen from the CDR sequences that belong to the same combination
in Table A-4 (i.e. mentioned on the same line in Table A-4) or are
suitably chosen from the group consisting of CDR sequences that
have 4, 3, 2 or only 1 amino acid difference(s) with the CDR
sequence(s) belonging to the same combination.
[0538] Sequence analysis of the resulting binders further resulted
in the identification of 3 distinct clusters. Corresponding
alignments are provided (see Table A-1, Table A-2 and Table A-3).
Clustering was based on sequence similarities and differences in
CDR2 and CDR3. Cluster A is the most prominent comprising 104
clones (SEQ ID NOs: 1-104), cluster B comprises 11 clones (SEQ ID
NOs: 105-115), and cluster C is represented by only 3 clones (SEQ
ID NOs: 116-118). The clustering based on the structural
similarities and differences in the amino acid sequence translated
into functional similarities and differences as revealed by the
examples. Representatives of all clusters were isolated based on
high affinity binding to the constant domain of the TCR (Examples 3
& 4) and human T cell activation (Example 4.2). In general
cluster A representatives demonstrated the best EC50 values. In
addition, cluster A representatives were cross-reactive with the
constant domain of cynomolgus TCR (cf. Example 18). Although
cluster C representatives had somewhat less favourable EC50 values
than cluster B representatives, cluster C representatives had lower
IC50 values in a flow cytometry based T cell mediated Ramos killing
assay (cf. Example 10).
[0539] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which CDR1 is chosen from the group consisting of [0540] (a) SEQ
ID NO: 123; and [0541] (b) amino acid sequences that have 1, 2, 3
or 4 amino acid difference(s) with SEQ ID NO: 123, wherein [0542]
at position 2 the D has been changed into A, S, E or G; [0543] at
position 4 the H has been changed into Y; [0544] at position 5 the
K has been changed into L; [0545] at position 6 the I has been
changed into L; [0546] at position 8 the F has been changed into I
or V; and/or [0547] at position 10 the G has been changed into
S.
[0548] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which CDR2 is chosen from the group consisting of [0549] (a) SEQ
ID NO: 153; and [0550] (b) amino acid sequences that have 1, 2, 3
or 4 amino acid difference(s) with SEQ ID NO: 153, wherein [0551]
at position 1 the H has been changed into T or R; [0552] at
position 3 the S has been changed into T or A; [0553] at position 5
the G has been changed into S or A; [0554] at position 7 the Q has
been changed into D, E, T, A or V; [0555] at position 8 the T has
been changed into A or V; and/or [0556] at position 9 the D has
been changed into A, Q, N, V or S.
[0557] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which CDR3 is chosen from the group consisting of [0558] (a) SEQ
ID NO: 170; and [0559] (b) amino acid sequences that have 1, 2, 3
or 4 amino acid difference(s) with SEQ ID NO: 170, wherein [0560]
at position 1 the F has been changed into Y, L or G; [0561] at
position 4 the I has been changed into L; [0562] at position 5 the
Y has been changed into W; and/or [0563] at position 8 the D has
been changed into N or S.
[0564] In an embodiment, the invention relates to a polypeptide as
described herein, in which said first ISV essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which: [0565]
(i) CDR1 is chosen from the group consisting of: [0566] (a) SEQ ID
NOs: 119-123, 125-127, 129, 132 and 133; and [0567] (b) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 123; and/or [0568] (ii) CDR2 is
chosen from the group consisting of: [0569] (c) SEQ ID NOs:
134-141, 143-144, 146-156, 159-163; and [0570] (d) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 153; and/or [0571] (iii) CDR3 is
chosen from the group consisting of: [0572] (e) SEQ ID NOs:
164-166, 169-171, 173-174; and [0573] (f) amino acid sequences that
have 3, 2, or 1 amino acid(s) difference with the amino acid
sequence of SEQ ID NO: 170.
[0574] In an embodiment, the invention relates to a polypeptide as
described herein, in which said first ISV essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which: CDR1 is
represented by SEQ ID NO: 123, CDR2 is represented by SEQ ID NO:
153, and CDR3 is represented by SEQ ID NO: 170.
[0575] Nanobodies of cluster B show relatively limited sequence
variability in the CDRs.
[0576] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which CDR1 is chosen from the group consisting of [0577] (a) SEQ
ID NO: 124; and [0578] (b) amino acid sequences that have 1, or 2
amino acid difference(s) with SEQ ID NO: 124, wherein [0579] at
position 2 the E has been changed into Q; and/or [0580] at position
6 the I has been changed into V.
[0581] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which CDR2 is chosen from the group consisting of [0582] (a) SEQ
ID NO: 145; and [0583] (b) amino acid sequence that has 1 amino
acid difference with SEQ ID NO: 145, wherein [0584] at position 9
the N has been changed into D.
[0585] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which CDR3 is chosen from the group consisting of [0586] (a) SEQ
ID NO: 167; and [0587] (b) amino acid sequence that has 1 amino
acid difference with SEQ ID NO: 167, wherein [0588] at position 4
the L has been changed into I.
[0589] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which: [0590] (i) CDR1 is chosen from the group consisting of:
[0591] (a) SEQ ID NOs: 124, 128 and 131; and [0592] (b) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 124; and/or [0593] (ii) CDR2 is
chosen from the group consisting of: [0594] (c) SEQ ID NOs: 142 and
145; and [0595] (d) amino acid sequences that have 4, 3, 2, or 1
amino acid(s) difference with the amino acid sequence of SEQ ID NO:
145; and/or [0596] (iii) CDR3 is chosen from the group consisting
of: [0597] (e) SEQ ID NOs: 167 and 168; and [0598] (f) amino acid
sequences that have 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 167.
[0599] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which: CDR1 is represented by SEQ ID NO: 124, CDR2 is
represented by SEQ ID NO: 145, and CDR3 is represented by SEQ ID
NO: 167.
[0600] In Cluster C, the sequence variation is even more limited
than within the other clusters.
[0601] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which CDR2 is chosen from the group consisting of [0602] (a) SEQ
ID NO: 157; and [0603] (b) amino acid sequence that has 1 amino
acid difference with SEQ ID NO: 157, wherein [0604] at position 8
the T has been changed into I.
[0605] Accordingly, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which: [0606] (i) CDR1 is chosen from the group consisting of
[0607] (a) SEQ ID NO: 130; and [0608] (b) amino acid sequences that
have 4, 3, 2, or 1 amino acid(s) difference with the amino acid
sequence of SEQ ID NO: 130; and/or [0609] (ii) CDR2 is chosen from
the group consisting of: [0610] (c) SEQ ID NOs: 157-158; and [0611]
(d) amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 157; and/or
[0612] (iii) CDR3 is chosen from the group consisting of: [0613]
(e) SEQ ID NO: 172; and [0614] (f) amino acid sequences that have
3, 2, or 1 amino acid(s) difference with the amino acid sequence of
SEQ ID NO: 172.
[0615] In an aspect, the present invention relates to a polypeptide
as described herein, in which said first ISV essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which: CDR1 is represented by SEQ ID NO: 130, CDR2 is
represented by SEQ ID NO: 157, and CDR3 is represented by SEQ ID
NO: 172.
[0616] The second immunoglobulin single variable domain (ISV) of
the polypeptide of the invention has a high affinity for/binds to
an antigen on a target cell, preferably a cancer cell. A "target
cell" as referred to herein, is a cell that presents a particular
antigen on its surface. In a preferred aspect, the "target cell" is
a cancer cell.
[0617] The membrane (also called plasma membrane or phospholipid
bilayer) surrounds the cytoplasm of a cell, which is the outer
boundary of the cell, i.e. the membrane is the surface of the cell.
This membrane serves to separate and protect a cell from its
surrounding environment and is made mostly from a double layer of
phospholipids. Embedded within this membrane is a variety of
protein molecules, such as channels, pumps and cellular receptors.
Since the membrane is fluid, the protein molecules can travel
within the membrane. The term "antigen on a target cell" as used
herein denotes a molecule, which is displayed on the surface of a
cell. In most cases, this molecule will be located in or on the
plasma membrane of the cell such that at least part of this
molecule remains accessible from outside the cell in tertiary form.
A non-limiting example of a cell surface molecule, which is located
in the plasma membrane, is a transmembrane protein comprising, in
its tertiary conformation, regions of hydrophilicity and
hydrophobicity. Here, at least one hydrophobic region allows the
cell surface molecule to be embedded, or inserted in the
hydrophobic plasma membrane of the cell while the hydrophilic
regions extend on either side of the plasma membrane into the
cytoplasm and extracellular space, respectively.
[0618] Said antigen can be any target on a cell, e.g. a tumour
antigen. In a preferred embodiment, said antigen is specific for
said target cell, e.g. cancer cell, such as a tumour associated
antigen (TAA) on said cancer cell.
[0619] The term "tumour antigen" as used herein may be understood
as those antigens that are presented on tumour cells. These
antigens can be presented on the cell surface with an extracellular
part, which is often combined with a transmembrane and cytoplasmic
part of the molecule. These antigens can sometimes be presented
only by tumour cells and never by a normal or healthy cell. Tumour
antigens can be exclusively expressed on tumour cells or might
represent a tumour specific mutation compared to normal cells. In
this case, they are called tumour-specific antigens. However, this
will not be the case generally. More common are antigens that are
presented by tumour cells and normal cells, and they are called
"tumour-associated antigens (TAA)". These tumour-associated
antigens can be overexpressed on tumour cells compared to normal
cells or are better accessible for antibody binding in tumour cells
due to the less compact structure of the tumour tissue compared to
normal tissue. TAA are preferably antigens that are expressed on
cells of particular tumours, but that are preferably not expressed
in normal cells. Often, TAA are antigens that are normally
expressed in cells only at particular points in an organism's
development (such as during fetal development) and that are being
inappropriately expressed in the organism at the present point of
development, or are antigens not expressed in normal tissues or
cells of an organ now expressing the antigen.
[0620] In an embodiment, said first antigen on a target cell is a
tumour antigen, preferably a tumour associated antigen (TAA).
[0621] In an embodiment, said second antigen on a target cell is a
tumour antigen, preferably a tumour associated antigen (TAA).
[0622] In an embodiment, said antigen is present more abundantly on
a cancer cell than on a normal cell.
[0623] The antigen on a target cell is preferably a
tumor-associated antigen (TAA). Preferred TAAs include MART-1,
carcinoembryonic antigen ("CEA"), gp100, MAGE-1, HER-2, CD20,
Lewis' antigens, Melanoma-associated Chondroitin Sulfate
Proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR),
Fibroblast Activation Protein (FAP), CD19 and CD33.
[0624] Cell surface antigens that are preferentially expressed on
AML LSC compared with normal hematopoietic stem cells, and thus
preferred as TAA, include CD123, CD44, CLL-1, CD96, CD47, CD32,
CXCR4, Tim-3 and CD25.
[0625] Other tumor-associated antigens suitable as an antigen on a
target cell for binding by the second ISV within the polypeptides
of the invention include: TAG-72, Ep-CAM, PSMA, PSA, glycolipids
such as GD2 and GD3.
[0626] The TAA of the invention include also hematopoietic
differentiation antigens, i.e. glycoproteins usually associated
with cluster differentiation (CD) grouping, such as CD4, CD5, CD19,
CD20, CD22, CD33, CD36, CD45, CD52, CD69 and CD147; growth factor
receptors, including HER2, ErbB3 and ErbB4; Cytokine receptors,
including Interleukin-2 receptor gamma chain (CD132 antigen),
Interleukin-10 receptor alpha chain (IL-10R-A), Interleukin-10
receptor beta chain (IL-10R-B), Interleukin-12 receptor beta-1
chain (IL-12R-beta1), Interleukin-12 receptor beta-2 chain (IL-12
receptor beta-2), Interleukin-13 receptor alpha-1 chain
(IL-13R-alpha-1) (CD213a1 antigen), Interleukin-13 receptor alpha-2
chain (Interleukin-13 binding protein), Interleukin-17 receptor
(IL-17 receptor), Interleukin-17B receptor (IL-17B receptor),
Interleukin 21 receptor precursor (IL-21R), Interleukin-1 receptor
type I (IL-1R-1) (CD121a), Interleukin-1 receptor type II
(IL-1R-beta) (CDw121b), Interleukin-1 receptor antagonist protein
(IL-1ra), Interleukin-2 receptor alpha chain (CD25 antigen),
Interleukin-2 receptor beta chain (CD122 antigen), Interleukin-3
receptor alpha chain (IL-3R-alpha) (CD123 antigen); as well as
others, such as CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin),
CD44v6, gpA33, Ron, Trop2, PSCA, claudin 6, claudin 18.2, CLEC12A,
CD38, ephA2, c-Met, CD56, MUC16, EGFRvIII, AGS-16, CD27L, Nectin-4,
SLITRK6, mesothelin, folate receptor, tissue factor, axl,
glypican-3, CA9, Cripto, CD138, CD37, MUC1, CD70, gastrin releasing
peptide receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2
gamma a, CD21, CD133, Na/K-ATPase, mlgM (membrane-bound IgM), mIgA
(membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6,
DcR3 and CAIX.
[0627] Accordingly the present invention relates to a polypeptide
as described herein, wherein said TAA is chosen from the group
consisting of Melanoma-associated Chondroitin Sulfate Proteoglycan
(MCSP), Epidermal Growth Factor Receptor (EGFR), Fibroblast
Activation Protein (FAP), MART-1, carcinoembryonic antigen ("CEA"),
gp100, MAGE-1, HER-2, Lewis' antigens, CD123, CD44, CLL-1, CD96,
CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2,
GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, CD147;
growth factor receptors, including ErbB3 and ErbB4; Cytokine
receptors, including Interleukin-2 receptor gamma chain (CD132
antigen), Interleukin-10 receptor alpha chain (IL-10R-A),
Interleukin-10 receptor beta chain (IL-10R-B), Interleukin-12
receptor beta-1 chain (IL-12R-beta1), Interleukin-12 receptor
beta-2 chain (IL-12 receptor beta-2), Interleukin-13 receptor
alpha-1 chain (IL-13R-alpha-1) (CD213a1 antigen), Interleukin-13
receptor alpha-2 chain (Interleukin-13 binding protein),
Interleukin-17 receptor (IL-17 receptor), Interleukin-17B receptor
(IL-17B receptor), Interleukin 21 receptor precursor (IL-21R),
Interleukin-1 receptor type I (IL-1R-1) (CD121a), Interleukin-1
receptor type II (IL-1R-beta) (CDw121b), Interleukin-1 receptor
antagonist protein (IL-1ra), Interleukin-2 receptor alpha chain
(CD25 antigen), Interleukin-2 receptor beta chain (CD122 antigen),
Interleukin-3 receptor alpha chain (IL-3R-alpha) (CD123 antigen),
CD30, IL23R, IGF-1R, IL5R, IgE, CD248 (endosialin), CD44v6, gpA33,
Ron, Trop2, PSCA, claudin 6, claudin 18.2, CLEC12A, CD38, ephA2,
c-Met, CD56, MUC16, EGFRvIII, AGS-16, CD27L, Nectin-4, SLITRK6,
mesothelin, folate receptor, tissue factor, axl, glypican-3, CA9,
Cripto, CD138, CD37, MUC1, CD70, gastrin releasing peptide
receptor, PAP, CEACAM5, CEACAM6, CXCR7, N-cadherin, FXYD2 gamma a,
CD21, CD133, Na/K-ATPase, mlgM (membrane-bound IgM), mIgA
(membrane-bound IgA), Mer, Tyro2, CD120, CD95, CA 195, DR5, DR6,
DcR3 and CAIX, and related polymorphic variants and isoforms,
preferably said TAA is CD20 (UniProt 11836), HER2 (Uniprot P04626),
EGFR, or CEACAM, polymorphic variants and/or isoforms thereof.
[0628] The second building block, ISV, Nanobody or VHH of the
invention has a high affinity for its antigen. The second building
block, ISV or Nanobody of the invention may, for example, be
directed against an antigenic determinant, epitope, part, domain,
subunit or confirmation (where applicable) of said antigen on a
target cell.
[0629] The target cell of the invention relates in particular to
mammalian cells, preferably to primate cells, and even more
preferably to human cells. The target cell is preferably a
hyperproliferative cell such as e.g. a cancer cell.
[0630] The present invention relates to a polypeptide as described
herein, wherein said second or further ISV binds to an antigen on a
target cell with an average KD value of between 100 nM and 10
.mu.M, such as at an average KD value of 90 nM or less, even more
preferably at an average KD value of 80 nM or less, such as less
than 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such as less
than 4, 3, 2, or 1 nM, such as less than 500, 400, 300, 200, 100,
90, 80, 70, 60, 50, 40, 30, 20 .mu.M, or even less, such as less
than 10 .mu.M. Preferably, the KD is determined by Kinexa, BLI or
SPR, for instance as determined by a Proteon.
[0631] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said second or further ISV has a high
affinity for its antigen when measured as a monovalent.
[0632] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said average KD is measured by surface
plasmon resonance (SPR) and/or KinExA or Proteon, for instance on
recombinant protein, such as described in the Examples section.
[0633] The present invention also relates to a polypeptide as
described herein, wherein said second or further ISV binds to an
antigen on a target cell with an EC50 value of between 100 nM and 1
.mu.M, such as at an average EC50 value of 100 nM or less, even
more preferably at an average EC50 value of 90 nM or less, such as
less than 80, 70, 60, 50, 40, 30, 20, 10, 5 nM or even less, such
as less than 4, 3, 2, or 1 nM or even less, such as less than 500,
400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 .mu.M, or
even less, such as less than 4 .mu.M.
[0634] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said average EC50 is determined by
FACS or ELISA, on a monovalent second ISV, such as a Nanobody, or a
polypeptide comprising a monovalent second ISV, such as a
Nanobody.
[0635] It has been shown in the examples that the KD correlates
well with the EC50.
[0636] Simultaneous targeting of multiple antigens can reduce the
probability of generating tumour escape variants, because of which
the therapeutic activity of T cell engaging strategy is improved.
The present invention provides multispecific polypeptides which
comprise a TCR ISV combined with immunoglobulin single variable
domains against different (target) antigens (on a target cell).
[0637] Preferred combinations of first and second antigens are
provided below (it will be appreciated that the ISVs binding said
antigens can be positioned in any order in the polypeptide of the
invention):
TABLE-US-00002 first antigen second antigen EGFR (OMIM: 131550)
CD20 (OMIM: 112210) EGFR (OMIM: 131550) CEA (OMIM: 114890) EGFR
(OMIM: 131550) HER2 (OMIM: 164870) HER2 (OMIM: 164870) CD20 (OMIM:
112210) HER2 (OMIM: 164870) CEA (OMIM: 114890) CD20 (OMIM: 112210)
CEA (OMIM: 114890)
[0638] Similarly, simultaneous targeting of multiple epitopes,
antigenic determinants, parts, domains, subunits or conformations
of a protein or antigen on a target cell can reduce the probability
of generating tumour escape variants, because of which the
therapeutic activity of T cell engaging strategy is improved (cf.
Example 22). The present invention provides polypeptides which
comprise an anti-TCR ISV combined with immunoglobulin single
variable domains against different epitopes, antigenic
determinants, parts, domains, subunits or conformations of an
antigen on a target cell (also referred to as biparatopic
constructs). Preferred combinations of first and second TAA ISVs
are provided below (it will be appreciated that the ISVs binding
said antigens can be positioned in any order in the polypeptide of
the invention):
TABLE-US-00003 SEQ SEQ TAA1 ISV name ID NO: TAA2 ISV name ID NO:
EGFR-1 7D12 355 EGFR-2 9G08 352 HER2-1 5F07 350 HER2-2 47D05 351
CEA-1 CEA#1 353 CEA-2 CEA#5 354
[0639] The polypeptides and compositions of the present invention
can be used for the prevention and/or treatment of diseases and
disorders of the present invention (herein also "diseases and
disorders of the present invention") which include, but are not
limited to cancer. The term "cancer" refers to the pathological
condition in mammals that is typically characterized by
dysregulated cellular proliferation or survival. Examples of cancer
include, but are not limited to, carcinomas, gliomas,
mesotheliomas, melanomas, lymphomas, leukemias, adenocarcinomas:
breast cancer, ovarian cancer, cervical cancer, glioblastoma,
multiple myeloma (including monoclonal gammopathy of undetermined
significance, asymptomatic and symptomatic myeloma), prostate
cancer, and Burkitt's lymphoma, head and neck cancer, colon cancer,
colorectal cancer, non-small cell lung cancer, small cell lung
cancer, cancer of the esophagus, stomach cancer, pancreatic cancer,
hepatobiliary cancer, cancer of the gallbladder, cancer of the
small intestine, rectal cancer, kidney cancer, bladder cancer,
prostate cancer, penile cancer, urethral cancer, testicular cancer,
vaginal cancer, uterine cancer, thyroid cancer, parathyroid cancer,
adrenal cancer, pancreatic endocrine cancer, carcinoid cancer, bone
cancer, skin cancer, retinoblastomas, Hodgkin's lymphoma,
non-Hodgkin's lymphoma, Kaposi's sarcoma, multicentric Castleman's
disease or AIDS-associated primary effusion lymphoma,
neuroectodermal tumors, rhabdomyosarcoma (see e.g., Cancer,
Principles and practice (DeVita et al. eds 1997) for additional
cancers); as well as any metastasis of any of the above cancers, as
well as non-cancer indications such as nasal polyposis, as well as
other disorders and diseases described herein.
[0640] For a general description of immunoglobulin single variable
domains, reference is made to the further description below, as
well as to the prior art cited herein. In this respect, it should
however be noted that this description and the prior art mainly
describes immunoglobulin single variable domains of the so-called
"V.sub.H3 class" (i.e., immunoglobulin single variable domains with
a high degree of sequence homology to human germline sequences of
the V.sub.H3 class such as DP-47, DP-51 or DP-29), which form a
preferred aspect of this invention. It should, however, be noted
that the invention in its broadest sense generally covers any type
of immunoglobulin single variable domains and for example also
covers the immunoglobulin single variable domains belonging to the
so-called "V.sub.H4 class" (i.e., immunoglobulin single variable
domains with a high degree of sequence homology to human germline
sequences of the V.sub.H4 class such as DP-78), as for example
described in WO 07/118670.
[0641] Generally, immunoglobulin single variable domains (in
particular V.sub.HH sequences and sequence optimized immunoglobulin
single variable domains) can in particular be characterized by the
presence of one or more "Hallmark residues" (as described for
example in Table B2) in one or more of the framework sequences
(again as further described herein).
TABLE-US-00004 TABLE B-2 Hallmark Residues in VHHs Position Human
V.sub.H3 Hallmark Residues 11 L, V; L, S, V, M, W, F, T, Q, E,
predominantly L A, R, G, K, Y, N, P, I; preferably L 37 V, I, F;
F.sup.(1), Y, V, L, A, H, S, usually V I, W, C, N, G, D, T, P,
preferably F.sup.(1) or Y 44.sup.(8) G E.sup.(3), Q.sup.(3),
G.sup.(2), D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably
G.sup.(2), E.sup.(3) or Q.sup.(3); most preferably G.sup.(2) or
Q.sup.(3). 45.sup.(8) L L.sup.(2), R.sup.(3), P, H, F, G, Q, S, E,
T, Y, C, I, D, V; preferably L.sup.(2) or R.sup.(3) 47.sup.(8) W, Y
F.sup.(1), L.sup.(1) or W.sup.(2) G, I, S, A, V, M, R, Y, E, P, T,
C, H, K, Q, N, D; preferably W.sup.(2), L.sup.(1) or F.sup.(1) 83 R
or K; R, K.sup.(5), T, E.sup.(5), Q, N, S, usually R I, V, G, M, L,
A, D, Y, H; preferably K or R; most preferably K 84 A, T, D;
P.sup.(5), S, H, L, A, V, I, T, predominantly A F, D, R, Y, N, Q,
G, E; preferably P 103 W W.sup.(4), R.sup.(6), G, S, K, A, M, Y, L,
F, T, N, V, Q, P.sup.(6), E, C; preferably W 104 G G, A, S, T, D,
P, N, E, C, L; preferably G 108 L, M or T; Q, L.sup.(7), R, P, E,
K, S, predominantly L T, M, A, H; preferably Q or L.sup.(7) Notes:
.sup.(1)In particular, but not exclusively, in combination with
KERE or KQRE at positions 43-46. .sup.(2)Usually as GLEW at
positions 44-47. .sup.(3)Usually as KERE or KQRE at positions
43-46, e.g. as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at
positions 43-47. Alternatively, also sequences such as TERE (for
example TEREL), TQRE (for example TQREL), KECE (for example KECEL
or KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE
(for example RQREL, RQREF or RQREW), QERE (for example QEREG),
QQRE, (for example QQREW, QQREL or QQREF), KGRE (for example
KGREG), KDRE (for example KDREV) are possible. Some other possible,
but less preferred sequences include for example DECKL and NVCEL.
.sup.(4)With both GLEW at positions 44-47 and KERE or KQRE at
positions 43-46. .sup.(5)Often as KP or EP at positions 83-84 of
naturally occurring V.sub.HH domains. .sup.(6)In particular, but
not exclusively, in combination with GLEW at positions 44-47.
.sup.(7)With the proviso that when positions 44-47 are GLEW,
position 108 is always Q in (non- humanized) V.sub.HH sequences
that also contain a W at 103. .sup.(8)The GLEW group also contains
GLEW-like sequences at positions 44-47, such as for example GVEW,
EPEW, GLER, DQ.EW, DLEW, GIEW, ELEW, GPEW, EWLP, and GPER.
[0642] The immunoglobulins of the invention may also contain a
C-terminal extension (X)n (in which n is 1 to 10, preferably 1 to
5, such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1); and
each X is an (preferably naturally occurring) amino acid residue
that is independently chosen, and preferably independently chosen
from the group consisting of alanine (A), glycine (G), valine (V),
leucine (L) or isoleucine (I)), for which reference is made to WO
12/175741 and WO 15/060643.
[0643] Apart from this and/or in addition, the immunoglobulin of
the invention may have certain preferred amino acid residues at
positions 11, 89, 110 and/or 112 as is described in further detail
in WO 15/060643 (which is incorporated herein as reference).
[0644] Again, such immunoglobulin single variable domains may be
derived in any suitable manner and from any suitable source, and
may for example be naturally occurring V.sub.HH sequences (i.e.,
from a suitable species of Camelid, e.g., llama) or synthetic or
semi-synthetic VHs or VLs (e.g., from human). Such immunoglobulin
single variable domains may include "humanized" or otherwise
"sequence optimized" VHHs, "camelized" immunoglobulin sequences
(and in particular camelized heavy chain variable domain sequences,
i.e., camelized VHs), as well as human VHs, human VLs, camelid VHHs
that have been altered by techniques such as affinity maturation
(for example, starting from synthetic, random or naturally
occurring immunoglobulin sequences), CDR grafting, veneering,
combining fragments derived from different immunoglobulin
sequences, PCR assembly using overlapping primers, and similar
techniques for engineering immunoglobulin sequences well known to
the skilled person; or any suitable combination of any of the
foregoing as further described herein. As mentioned herein, a
particularly preferred class of immunoglobulin single variable
domains of the invention comprises immunoglobulin single variable
domains with an amino acid sequence that corresponds to the amino
acid sequence of a naturally occurring V.sub.HH domain, but that
has been "humanized", i.e. by replacing one or more amino acid
residues in the amino acid sequence of said naturally occurring
V.sub.HH sequence (and in particular in the framework sequences) by
one or more of the amino acid residues that occur at the
corresponding position(s) in a V.sub.H domain from a conventional
4-chain antibody from a human being (e.g. indicated above). This
can be performed in a manner known per se, which will be clear to
the skilled person, for example on the basis of the further
description herein and the prior art on humanization referred to
herein. Again, it should be noted that such humanized
immunoglobulin single variable domains of the invention can be
obtained in any suitable manner known per se and thus are not
strictly limited to polypeptides that have been obtained using a
polypeptide that comprises a naturally occurring V.sub.HH domain as
a starting material.
[0645] Another particularly preferred class of immunoglobulin
single variable domains of the invention comprises immunoglobulin
single variable domains with an amino acid sequence that
corresponds to the amino acid sequence of a naturally occurring
V.sub.H domain, but that has been "camelized", i.e. by replacing
one or more amino acid residues in the amino acid sequence of a
naturally occurring V.sub.H domain from a conventional 4-chain
antibody by one or more of the amino acid residues that occur at
the corresponding position(s) in a V.sub.HH domain of a heavy chain
antibody. This can be performed in a manner known per se, which
will be clear to the skilled person, for example on the basis of
the description herein. Such "camelizing" substitutions are
preferably inserted at amino acid positions that form and/or are
present at the V.sub.H--V.sub.L interface, and/or at the so-called
Camelidae hallmark residues, as defined herein (see also for
example WO 94/04678 and Davies and Riechmann 1994 (FEBS letters
339: 285-290) and 1996 (Protein Engineering 9: 531-537)).
Preferably, the V.sub.H sequence that is used as a starting
material or starting point for generating or designing the
camelized immunoglobulin single variable domains is preferably a
V.sub.H sequence from a mammal, more preferably the V.sub.H
sequence of a human being, such as a V.sub.H3 sequence. However, it
should be noted that such camelized immunoglobulin single variable
domains of the invention can be obtained in any suitable manner
known per se and thus are not strictly limited to polypeptides that
have been obtained using a polypeptide that comprises a naturally
occurring V.sub.H domain as a starting material.
[0646] For example, again as further described herein, both
"humanization" and "camelization" can be performed by providing a
nucleotide sequence that encodes a naturally occurring V.sub.HH
domain or V.sub.H domain, respectively, and then changing, in a
manner known per se, one or more codons in said nucleotide sequence
in such a way that the new nucleotide sequence encodes a
"humanized" or "camelized" immunoglobulin single variable domain of
the invention, respectively. This nucleic acid can then be
expressed in a manner known per se, so as to provide the desired
immunoglobulin single variable domains of the invention.
Alternatively, based on the amino acid sequence of a naturally
occurring V.sub.HH domain or V.sub.H domain, respectively, the
amino acid sequence of the desired humanized or camelized
immunoglobulin single variable domains of the invention,
respectively, can be designed and then synthesized de novo using
techniques for peptide synthesis known per se. Also, based on the
amino acid sequence or nucleotide sequence of a naturally occurring
V.sub.HH domain or V.sub.H domain, respectively, a nucleotide
sequence encoding the desired humanized or camelized immunoglobulin
single variable domains of the invention, respectively, can be
designed and then synthesized de novo using techniques for nucleic
acid synthesis known per se, after which the nucleic acid thus
obtained can be expressed in a manner known per se, so as to
provide the desired immunoglobulin single variable domains of the
invention.
[0647] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said ISV is a Nanobody, a V.sub.HH, a
humanized V.sub.HH, or a camelized V.sub.H.
[0648] Generally, proteins or polypeptides that comprise or
essentially consist of a single building block, single
immunoglobulin single variable domain or single Nanobody will be
referred to herein as "monovalent" proteins or polypeptides, as
"monovalent constructs", as "monovalent building block", as
"monovalent immunoglobulin single variable domain", or as
"monovalent Nanobody", respectively.
[0649] In this respect, the present invention also relates to the
monovalent building blocks that make up the polypeptides of the
invention.
[0650] Accordingly, the present invention relates to an ISV or
polypeptide that specifically binds the constant domain of the T
cell receptor (TCR) and that comprises or essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which: [0651]
(i) CDR1 is chosen from the group consisting of: [0652] (a) SEQ ID
NOs: 119-133; or [0653] (b) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of any
of SEQ ID NOs: 119-133, provided that the polypeptide comprising
the CDR1 with 4, 3, 2, or 1 amino acid(s) difference binds TCR with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR1 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; [0654] and/or [0655] (ii) CDR2 is chosen from the group
consisting of: [0656] (c) SEQ ID NOs: 134-163; or [0657] (d) amino
acid sequences that have 4, 3, 2, or 1 amino acid(s) difference
with the amino acid sequence of any of SEQ ID NOs: 134-163,
provided that the polypeptide comprising the CDR2 with 4, 3, 2, or
1 amino acid(s) difference binds TCR with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
[0658] and/or [0659] (iii) CDR3 is chosen from the group consisting
of: [0660] (e) SEQ ID NOs: 164-174; or [0661] (f) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of any of SEQ ID NOs: 164-174, provided that
the polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR3 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance.
[0662] As discussed above, ISVs were isolated that belong to
different clusters, based on structural similarities and
differences in CDR2 and CDR3.
[0663] Immunoglobulin single variable domains belonging to cluster
A are represented by polypeptides according in which: [0664] (i)
CDR1 is chosen from the group consisting of: [0665] (a) SEQ ID NOs:
119-123, 125-127, 129, 132 and 133; and [0666] (b) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 123, provided that the
polypeptide comprising the CDR1 with 4, 3, 2, or 1 amino acid(s)
difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR1
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance;
[0667] and/or [0668] (ii) CDR2 is chosen from the group consisting
of: [0669] (c) SEQ ID NOs: 134-141, 143-144, 146-156, 159-163; and
[0670] (d) amino acid sequences that have 4, 3, 2, or 1 amino
acid(s) difference with the amino acid sequence of SEQ ID NO: 153,
provided that the polypeptide comprising the CDR2 with 4, 3, 2, or
1 amino acid(s) difference binds TCR with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR2 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon
resonance;
[0671] and/or [0672] (iii) CDR3 is chosen from the group consisting
of: [0673] (e) SEQ ID NOs: 164-166, 169-171, 173-174; and [0674]
(f) amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 170, provided
that the polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR3 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance.
[0675] In another aspect, in the polypeptides belonging to cluster
A, CDR1 is chosen from the group consisting of [0676] (a) SEQ ID
NO: 123; and [0677] (b) amino acid sequences that have 1, 2, or 3
amino acid difference(s) with SEQ ID NO: 123, wherein [0678] at
position 2 the D has been changed into A, S, E or G; [0679] at
position 4 the H has been changed into Y; [0680] at position 5 the
K has been changed into L; [0681] at position 6 the I has been
changed into L; [0682] at position 8 the F has been changed into I
or V; and/or [0683] at position 10 the G has been changed into
S.
[0684] In another aspect, in the polypeptides belonging to cluster
A, CDR2 is chosen from the group consisting of [0685] (a) SEQ ID
NO: 153; and [0686] (b) amino acid sequences that have 1, 2, 3, 4
or 5 amino acid difference(s) with SEQ ID NO: 153, wherein [0687]
at position 1 the H has been changed into T or R; [0688] at
position 3 the S has been changed into T or A; [0689] at position 5
the G has been changed into S or A; [0690] at position 7 the Q has
been changed into D, E, T, A or V; [0691] at position 8 the T has
been changed into A or V; and/or [0692] at position 9 the D has
been changed into A, Q, N, V or S.
[0693] In another aspect, in the polypeptides belonging to cluster
A, CDR3 is chosen from the group consisting of [0694] (a) SEQ ID
NO: 170; and [0695] (b) amino acid sequences that have 1, 2, or 3
amino acid difference(s) with SEQ ID NO: 170, wherein [0696] at
position 1 the F has been changed into Y, L or G; [0697] at
position 4 the I has been changed into L; [0698] at position 5 the
Y has been changed into W; and/or [0699] at position 8 the D has
been changed into N or S.
[0700] Accordingly, the present invention relates to an ISV or
polypeptide that specifically binds the constant domain of the T
cell receptor (TCR) and that comprises or essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which: [0701]
(i) CDR1 is chosen from the group consisting of: [0702] (a) SEQ ID
NO: 123; and [0703] (b) amino acid sequences that have 1, 2, or 3
amino acid difference(s) with SEQ ID NO: 123, wherein [0704] at
position 2 the D has been changed into A, S, E or G; [0705] at
position 4 the H has been changed into Y; [0706] at position 5 the
K has been changed into L; [0707] at position 6 the I has been
changed into L; [0708] at position 8 the F has been changed into I
or V; and/or [0709] at position 10 the G has been changed into S;
[0710] and [0711] (ii) CDR2 is chosen from the group consisting of:
[0712] (a) SEQ ID NO: 153; and [0713] (b) amino acid sequences that
have 1, 2, 3, 4 or 5 amino acid difference(s) with SEQ ID NO: 153,
wherein [0714] at position 1 the H has been changed into T or R;
[0715] at position 3 the S has been changed into T or A; [0716] at
position 5 the G has been changed into S or A; [0717] at position 7
the Q has been changed into D, E, T, A or V; [0718] at position 8
the T has been changed into A or V; and/or [0719] at position 9 the
D has been changed into A, Q, N, V or S; and [0720] (iii) CDR3 is
chosen from the group consisting of: [0721] (a) SEQ ID NO: 170; and
[0722] (b) amino acid sequences that have 1, 2, or 3 amino acid
difference(s) with SEQ ID NO: 170, wherein [0723] at position 1 the
F has been changed into Y, L or G; [0724] at position 4 the I has
been changed into L; [0725] at position 5 the Y has been changed
into W; and/or [0726] at position 8 the D has been changed into N
or S.
[0727] In another aspect, the invention relates to a polypeptide in
which CDR1 is represented by SEQ ID NO: 123, CDR2 is represented by
SEQ ID NO: 153, and CDR3 is represented by SEQ ID NO: 170.
Preferably the polypeptide is selected from any of SEQ ID NOs: 1 to
104.
[0728] Immunoglobulin single variable domains belonging to cluster
B are represented by polypeptides according in which: [0729] (i)
CDR1 is chosen from the group consisting of: [0730] (a) SEQ ID NOs:
124, 128 and 131; and [0731] (b) amino acid sequences that have 4,
3, 2, or 1 amino acid(s) difference with the amino acid sequence of
SEQ ID NO: 124, provided that the polypeptide comprising the CDR1
with 4, 3, 2, or 1 amino acid(s) difference binds TCR with about
the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR1 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; [0732] and/or [0733] (ii) CDR2 is chosen from the group
consisting of: [0734] (c) SEQ ID NOs: 142 and 145; and [0735] (d)
amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of SEQ ID NO: 145, provided
that the polypeptide comprising the CDR2 with 4, 3, 2, or 1 amino
acid(s) difference binds TCR with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR2 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance; [0736] and/or
[0737] (iii) CDR3 is chosen from the group consisting of: [0738]
(e) SEQ ID NOs: 167 and 168; and [0739] (f) amino acid sequences
that have 4, 3, 2, or 1 amino acid(s) difference with the amino
acid sequence of SEQ ID NO: 167, provided that the polypeptide
comprising the CDR3 with 4, 3, 2, or 1 amino acid(s) difference
binds TCR with about the same or a higher affinity compared to the
binding by the polypeptide comprising the CDR3 without the 4, 3, 2,
or 1 amino acid(s) difference, said affinity as measured by surface
plasmon resonance.
[0740] In another aspect, in the polypeptides belonging to cluster
B, CDR1 is chosen from the group consisting of [0741] (a) SEQ ID
NO: 124; and [0742] (b) amino acid sequences that have 1, or 2
amino acid difference(s) with SEQ ID NO: 124, wherein [0743] at
position 2 the E has been changed into Q; and/or [0744] at position
6 the I has been changed into V.
[0745] In another aspect, in the polypeptides belonging to cluster
B, CDR2 is chosen from the group consisting of [0746] (a) SEQ ID
NO: 145; and [0747] (b) amino acid sequence that has 1 amino acid
difference with SEQ ID NO: 145, wherein [0748] at position 9 the N
has been changed into D.
[0749] In another aspect, in the polypeptides belonging to cluster
B, CDR3 is chosen from the group consisting of [0750] (a) SEQ ID
NO: 167; and [0751] (b) amino acid sequence that has 1 amino acid
difference with SEQ ID NO: 167, wherein [0752] at position 4 the L
has been changed into 1.
[0753] Accordingly, the present invention relates to an ISV or
polypeptide that specifically binds the constant domain of the T
cell receptor (TCR) and that comprises or essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which: [0754]
(i) CDR1 is chosen from the group consisting of: [0755] (a) SEQ ID
NO: 124; and [0756] (b) amino acid sequences that have 1, or 2
amino acid difference(s) with SEQ ID NO: 124, wherein [0757] at
position 2 the E has been changed into Q; and/or [0758] at position
6 the I has been changed into V; and [0759] (ii) CDR2 is chosen
from the group consisting of: [0760] (a) SEQ ID NO: 145; and [0761]
(b) amino acid sequence that has 1 amino acid difference with SEQ
ID NO: 145, wherein [0762] at position 9 the N has been changed
into D; and (iii) CDR3 is chosen from the group consisting of:
[0763] (a) SEQ ID NO: 167; and [0764] (b) amino acid sequence that
has 1 amino acid difference with SEQ ID NO: 167, wherein [0765] at
position 4 the L has been changed into 1.
[0766] In another aspect, the invention relates to a polypeptide in
which CDR1 is represented by SEQ ID NO: 124, CDR2 is represented by
SEQ ID NO: 145, and CDR3 is represented by SEQ ID NO: 167.
Preferably the polypeptide is selected from any of SEQ ID NOs:
105-115.
[0767] Immunoglobulin single variable domains belonging to cluster
C are represented by polypeptides according in which: [0768] (i)
CDR1 is chosen from the group consisting of: [0769] (a) SEQ ID NO:
130; and [0770] (b) amino acid sequences that have 4, 3, 2, or 1
amino acid(s) difference with the amino acid sequence of SEQ ID NO:
130, provided that the polypeptide comprising the CDR1 with 4, 3,
2, or 1 amino acid(s) difference binds TCR with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR1 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
[0771] and/or [0772] (ii) CDR2 is chosen from the group consisting
of: [0773] (c) SEQ ID NOs: 157-158; and [0774] (d) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 157, provided that the
polypeptide comprising the CDR2 with 4, 3, 2, or 1 amino acid(s)
difference binds TCR with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR2
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; [0775] and/or [0776]
(iii) CDR3 is chosen from the group consisting of: [0777] (e) SEQ
ID NO: 172; and [0778] (f) amino acid sequences that have 4, 3, 2,
or 1 amino acid(s) difference with the amino acid sequence of SEQ
ID NO: 172, provided that the polypeptide comprising the CDR3 with
4, 3, 2, or 1 amino acid(s) difference binds TCR with about the
same or a higher affinity compared to the binding by the
polypeptide comprising the CDR3 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance.
[0779] In another aspect, in the polypeptides belonging to cluster
C, CDR1 is chosen from SEQ ID NO: 130.
[0780] In another aspect, in the polypeptides belonging to cluster
C, CDR2 is chosen from the group consisting of [0781] (a) SEQ ID
NO: 157; and [0782] (b) amino acid sequence that has 1 amino acid
difference with SEQ ID NO: 157, wherein [0783] at position 8 the T
has been changed into I.
[0784] In another aspect, in the polypeptides belonging to cluster
C, CDR3 is chosen from SEQ ID NO: 172.
[0785] Accordingly, the present invention relates to an ISV or
polypeptide that specifically binds the constant domain of the T
cell receptor (TCR) and that comprises or essentially consists of 4
framework regions (FR1 to FR4, respectively) and 3 complementarity
determining regions (CDR1 to CDR3, respectively), in which: [0786]
(i) CDR1 is chosen from SEQ ID NO: 130; [0787] and [0788] (ii) CDR2
is chosen from the group consisting of: [0789] (a) SEQ ID NO: 157;
and [0790] (b) amino acid sequence that has 1 amino acid difference
with SEQ ID NO: 157, wherein [0791] at position 8 the T has been
changed into I; and [0792] (iii) CDR3 is chosen from SEQ ID NO:
172.
[0793] In another aspect, the invention relates to a polypeptide in
which CDR1 is represented by SEQ ID NO: 130, CDR2 is represented by
SEQ ID NO: 157, and CDR3 is represented by SEQ ID NO: 172.
Preferably the polypeptide is selected from any of SEQ ID NOs:
116-118.
[0794] In a further aspect, the invention relates to polypeptides
that cross-block the binding to the constant domain of the T cell
receptor (TCR) by at least one of the ISVs or polypeptides
belonging to Cluster A, B or C.
[0795] Accordingly, the present invention relates to polypeptides
that cross-block the binding to the constant domain of the T cell
receptor (TCR) by at least one of the ISVs or polypeptides with SEQ
ID NOs: 1-104.
[0796] In another aspect, the present invention relates to ISVs or
polypeptides that cross-block the binding to the constant domain of
the T cell receptor (TCR) by at least one of the ISVs or
polypeptides with SEQ ID NOs: 105-115.
[0797] In yet another aspect, the present invention relates to ISVs
or polypeptides that cross-block the binding to the constant domain
of the T cell receptor (TCR) by at least one of the ISVs or
polypeptides with SEQ ID NOs: 116-118.
[0798] In a further aspect, the invention relates to polypeptides
that are cross-blocked from binding to the constant domain of the T
cell receptor (TCR) by at least one of the ISVs or polypeptides
belonging to Cluster A, B or C.
[0799] Accordingly, the present invention relates to polypeptides
that are cross-blocked from binding to the constant domain of the T
cell receptor (TCR) by at least one of the ISVs or polypeptides
belonging to SEQ ID NOs: 1-104.
[0800] In another aspect, the present invention relates to
polypeptides that are cross-blocked from binding to the constant
domain of the T cell receptor (TCR) by at least one of the ISVs or
polypeptides belonging to SEQ ID NOs: 105-115.
[0801] In another aspect, the present invention relates to
polypeptides that are cross-blocked from binding to the constant
domain of the T cell receptor (TCR) by at least one of the ISVs or
polypeptides belonging to SEQ ID NOs: 116-118.
[0802] The present invention also relates to an ISV or polypeptide
that specifically binds carcinoembryonic antigen (CEA) and that
comprises or essentially consists of 4 framework regions (FR1 to
FR4, respectively) and 3 complementarity determining regions (CDR1
to CDR3, respectively), in which: [0803] (i) CDR1 is chosen from
the group consisting of: [0804] (a) SEQ ID NO: 361 (GDTYGSYWMG); or
[0805] (b) amino acid sequences that have 4, 3, 2, or 1 amino
acid(s) difference with the amino acid sequence of SEQ ID NO: 361,
provided that the polypeptide comprising the CDR1 with 4, 3, 2, or
1 amino acid(s) difference binds CEA with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR1 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon resonance;
[0806] and/or [0807] (ii) CDR2 is chosen from the group consisting
of: [0808] (c) SEQ ID NO: 363 (AINRGGGYTV); or [0809] (d) amino
acid sequences that have 4, 3, 2, or 1 amino acid(s) difference
with the amino acid sequence of any of SEQ ID NO: 363, provided
that the polypeptide comprising the CDR2 with 4, 3, 2, or 1 amino
acid(s) difference binds CEA with about the same or a higher
affinity compared to the binding by the polypeptide comprising the
CDR2 without the 4, 3, 2, or 1 amino acid(s) difference, said
affinity as measured by surface plasmon resonance; [0810] and/or
[0811] (iii) CDR3 is chosen from the group consisting of: [0812]
(e) SEQ ID NO: 365 (SGVLGGLHEDWFNY); or [0813] (f) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of any of SEQ ID NO: 365, provided that the
polypeptide comprising the CDR3 with 4, 3, 2, or 1 amino acid(s)
difference binds CEA with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR3
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance.
[0814] In another aspect, the invention relates to a polypeptide in
which CDR1 is represented by SEQ ID NO: 361, CDR2 is represented by
SEQ ID NO: 363, and CDR3 is represented by SEQ ID NO: 365.
Preferred polypeptides include SEQ ID NOs: 353 and 354.
[0815] In a further aspect, the invention relates to polypeptides
that cross-block the binding to carcinoembryonic antigen (CEA) by
at least one of the ISVs or polypeptides with SEQ ID NOs: 353 or
354.
[0816] In a further aspect, the invention relates to polypeptides
that are cross-blocked from binding to carcinoembryonic antigen
(CEA) by at least one of the ISVs or polypeptides with SEQ ID NOs:
353 or 354.
[0817] The present invention also relates to an ISV or polypeptide
that specifically binds CD20 and that comprises or essentially
consists of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3, respectively),
in which: [0818] (i) CDR1 is chosen from the group consisting of:
[0819] (a) SEQ ID NO: 362 (GGTFSSYTMG); or [0820] (b) amino acid
sequences that have 4, 3, 2, or 1 amino acid(s) difference with the
amino acid sequence of SEQ ID NO: 362, provided that the
polypeptide comprising the CDR1 with 4, 3, 2, or 1 amino acid(s)
difference binds CD20 with about the same or a higher affinity
compared to the binding by the polypeptide comprising the CDR1
without the 4, 3, 2, or 1 amino acid(s) difference, said affinity
as measured by surface plasmon resonance; [0821] and/or [0822] (ii)
CDR2 is chosen from the group consisting of: [0823] (c) SEQ ID NO:
364 (EVRWGGVTT); or [0824] (d) amino acid sequences that have 4, 3,
2, or 1 amino acid(s) difference with the amino acid sequence of
any of SEQ ID NO: 364, provided that the polypeptide comprising the
CDR2 with 4, 3, 2, or 1 amino acid(s) difference binds CD20 with
about the same or a higher affinity compared to the binding by the
polypeptide comprising the CDR2 without the 4, 3, 2, or 1 amino
acid(s) difference, said affinity as measured by surface plasmon
resonance; [0825] and/or [0826] (iii) CDR3 is chosen from the group
consisting of: [0827] (e) SEQ ID NO: 366 (VRQMYMTVVPDY); or [0828]
(f) amino acid sequences that have 4, 3, 2, or 1 amino acid(s)
difference with the amino acid sequence of any of SEQ ID NO: 366,
provided that the polypeptide comprising the CDR3 with 4, 3, 2, or
1 amino acid(s) difference binds CD20 with about the same or a
higher affinity compared to the binding by the polypeptide
comprising the CDR3 without the 4, 3, 2, or 1 amino acid(s)
difference, said affinity as measured by surface plasmon
resonance.
[0829] In another aspect, the invention relates to a polypeptide in
which CDR1 is represented by SEQ ID NO: 362, CDR2 is represented by
SEQ ID NO: 364, and CDR3 is represented by SEQ ID NO: 366. A
preferred polypeptide includes SEQ ID NO: 357.
[0830] In a further aspect, the invention relates to polypeptides
that cross-block the binding to CD20 by the ISV or polypeptide with
SEQ ID NO: 357.
[0831] In a further aspect, the invention relates to polypeptides
that are cross-blocked from binding to CD20 by the ISV or
polypeptide with SEQ ID NO: 357.
[0832] The invention further relates to compounds or constructs,
and in particular proteins or polypeptides that comprise or
essentially consist of one or more ISVs or polypeptides of the
invention, and optionally further comprise one or more other
groups, residues, moieties or binding units. As will become clear
to the skilled person from the further disclosure herein, such
further groups, residues, moieties, binding units or amino acid
sequences may or may not provide further functionality to the
polypeptide of the invention (and/or to the compound or construct
in which it is present) and may or may not modify the properties of
the polypeptide of the invention.
[0833] In a specific, but non-limiting aspect of the invention,
which will be further described herein, the ISVs and polypeptides
of the invention may have an increased half-life in serum (as
further described herein) compared to the immunoglobulin single
variable domain or polypeptide from which they have been derived.
For example, an immunoglobulin single variable domain or
polypeptide of the invention may be linked (chemically or
otherwise) to one or more groups or moieties that extend the
half-life (such as PEG), so as to provide a derivative of the ISV
or polypeptide of the invention with increased half-life.
[0834] In a specific aspect of the invention, a compound or
construct of the invention or a polypeptide of the invention may
have an increased half-life, compared to the corresponding ISV or
polypeptide of the invention. Some preferred, but non-limiting
examples of such compounds, constructs and polypeptides will become
clear to the skilled person based on the further disclosure herein,
and for example comprise immunoglobulin single variable domains or
polypeptides of the invention that have been chemically modified to
increase the half-life thereof (for example, by means of
pegylation); immunoglobulin single variable domains or polypeptides
of the invention that comprise at least one additional binding site
for binding to a serum protein (such as serum albumin); or
constructs or polypeptides of the invention which comprise at least
ISV or polypeptide of the invention that is linked to at least one
moiety (and in particular at least one amino acid sequence) which
increases the half-life of the ISV or polypeptide of the invention.
Examples of ISVs or polypeptides of the invention which comprise
such half-life extending moieties or immunoglobulin single variable
domains will become clear to the skilled person based on the
further disclosure herein; and for example include, without
limitation, polypeptides in which the one or more immunoglobulin
single variable domains or polypeptide of the invention are
suitably linked to one or more serum proteins or fragments thereof
(such as (human) serum albumin or suitable fragments thereof) or to
one or more binding units that can bind to serum proteins (such as,
for example, domain antibodies, immunoglobulin single variable
domains that are suitable for use as a domain antibody, single
domain antibodies, immunoglobulin single variable domains that are
suitable for use as a single domain antibody, "dAb" 's,
immunoglobulin single variable domains that are suitable for use as
a dAb, or Nanobodies that can bind to serum proteins such as serum
albumin (such as human serum albumin), serum immunoglobulins such
as IgG, or transferrin; reference is made to the further
description and references mentioned herein); ISVs or polypeptides
in which an ISV or polypeptide of the invention is linked to an Fc
portion (such as a human Fc) or a suitable part or fragment
thereof; or polypeptides in which the one or more immunoglobulin
single variable domains or polypeptide of the invention are
suitable linked to one or more small proteins or peptides that can
bind to serum proteins, such as, without limitation, the proteins
and peptides described in WO 91/01743, WO 01/45746, WO 02/076489,
WO 08/068280, WO 09/127691 and WO 11/095545.
[0835] Generally, the compounds, constructs or polypeptides of the
invention with increased half-life preferably have a half-life that
is at least 1.5 times, preferably at least 2 times, such as at
least 5 times, for example at least 10 times or more than 20 times,
greater than the half-life of the corresponding ISV or polypeptide
of the invention per se. For example, the compounds, constructs or
polypeptides of the invention with increased half-life may have a
half-life e.g., in humans that is increased with more than 1 hours,
preferably more than 2 hours, more preferably more than 6 hours,
such as more than 12 hours, or even more than 24, 48 or 72 hours,
compared to the corresponding ISV or polypeptide of the invention
per se.
[0836] In a preferred, but non-limiting aspect of the invention,
such compounds, constructs or polypeptides of the invention have a
serum half-life e.g. in humans that is increased with more than 1
hours, preferably more than 2 hours, more preferably more than 6
hours, such as more than 12 hours, or even more than 24, 48 or 72
hours, compared to the corresponding ISV or polypeptide of the
invention per se.
[0837] In another preferred, but non-limiting aspect of the
invention, such compounds, constructs or polypeptides of the
invention exhibit a serum half-life in human of at least about 12
hours, preferably at least 24 hours, more preferably at least 48
hours, even more preferably at least 72 hours or more. For example,
compounds, constructs or polypeptides of the invention may have a
half-life of at least 5 days (such as about 5 to 10 days),
preferably at least 9 days (such as about 9 to 14 days), more
preferably at least about 10 days (such as about 10 to 15 days), or
at least about 11 days (such as about 11 to 16 days), more
preferably at least about 12 days (such as about 12 to 18 days or
more), or more than 14 days (such as about 14 to 19 days).
[0838] In the present invention, it was demonstrated that the
inclusion of an albumin targeting binding unit in the construct as
such did not have an essential impact on the obtained potency or
efficacy. Although a minor loss of efficacy/potency was observed in
the presence of HSA, the half-life extended TCR binding
multispecific polypeptides were still potent in tumour cell
killing. Albumin-based drug delivery has been demonstrated to be
useful for achieving improved cancer therapy, largely due to its
passive target toward tumour via the enhanced permeability and
retention effect and the increased demand for albumin by tumour
cells as source of energy and amino acids. However, albumin lacks
not only the active mechanism to overcome the cell membrane
barrier, but also the ability to penetrate into tumour tissues
(Qianqian Guo et al. 2013, Polym. Chem. 4: 4584-4587).
[0839] In a particularly preferred but non-limiting aspect of the
invention, the invention provides a polypeptide of the invention
comprising a first and a second immunoglobulin single variable
domain (ISV); and further comprising one or more (preferably one)
serum albumin binding immunoglobulin single variable domain as
described herein, e.g. the serum albumin binding immunoglobulin
single variable domain referred to as Alb8, Alb23, Alb129, Alb132,
Alb11, Alb11 (S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA,
Alb82-G, Alb82-GG, Alb82-GGG (Table B-3).
TABLE-US-00005 TABLE B-3 Immunoglobulin single variable domains for
use in HLE of the ISVs and polypeptides of the invention SEQ ID ISV
Sequence NO Alb8
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 400
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23
EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDT 401
LYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129
EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 402
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132
EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD 403
TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 404
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11
EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 405
(S112K)-A
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82
EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 406
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A
EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 407
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA
EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 408
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA
Alb82-AAA
EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 409
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA
Alb82-G EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT
410 LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSG
Alb82-GG EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT
411 LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG
Alb82-GGG
EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT 412
LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG
[0840] Accordingly, the present invention relates to a polypeptide
as described herein, further comprising a serum protein binding
moiety.
[0841] The present invention relates to a polypeptide as described
herein, wherein said serum protein binding moiety binds serum
albumin.
[0842] The present invention relates to a polypeptide as described
herein, wherein said serum protein binding moiety is an
immunoglobulin single variable domain binding serum albumin.
[0843] The present invention relates to a polypeptide as described
herein, wherein said ISV binding serum albumin essentially consists
of 4 framework regions (FR1 to FR4, respectively) and 3
complementarity determining regions (CDR1 to CDR3 respectively), in
which CDR1 is SFGMS (SEQ ID NO: 481), CDR2 is SISGSGSDTLYADSVKG
(SEQ ID NO: 482), and in which CDR3 is GGSLSR (SEQ ID NO: 475), CDR
determined according to Kabat definition; and/or in which CDR1 is
GFTFSSFGMS (SEQ ID NO: 472) or GFTFRSFGMS (SEQ ID NO: 473), CDR2 is
SISGSGSDTL (SEQ ID NO: 474) and CDR3 is GGSLSR (SEQ ID NO: 475),
CDR determined according to Kontermann 2010.
[0844] The present invention relates to a polypeptide as described
herein, wherein said ISV binding serum albumin comprises AMb,
Alb23, Alb129, Alb132, Alb11, Alb11 (S112K)-A, Alb82, Alb82-A,
Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (Table-1B3).
[0845] In the polypeptides of the invention, the two or more
building blocks, ISVs or Nanobodies and the optionally one or more
polypeptides, one or more other groups, drugs, agents, residues,
moieties or binding units may be directly linked to each other (as
for example described in WO 99/23221) and/or may be linked to each
other via one or more suitable spacers or linkers, or any
combination thereof.
[0846] Suitable spacers or linkers for use in multivalent and
multispecific polypeptides will be clear to the skilled person, and
may generally be any linker or spacer used in the art to link amino
acid sequences. Preferably, said linker or spacer is suitable for
use in constructing proteins or polypeptides that are intended for
pharmaceutical use.
[0847] Some particularly preferred spacers include the spacers and
linkers that are used in the art to link antibody fragments or
antibody domains. These include the linkers mentioned in the
general background art cited above, as well as for example linkers
that are used in the art to construct diabodies or ScFv fragments
(in this respect, however, it should be noted that, whereas in
diabodies and in ScFv fragments, the linker sequence used should
have a length, a degree of flexibility and other properties that
allow the pertinent V.sub.H and V.sub.L domains to come together to
form the complete antigen-binding site, there is no particular
limitation on the length or the flexibility of the linker used in
the polypeptide of the invention, since each ISV or Nanobody by
itself forms a complete antigen-binding site).
[0848] For example, a linker may be a suitable amino acid sequence,
and in particular amino acid sequences of between 1 and 50,
preferably between 1 and 30, such as between 1 and 10 amino acid
residues.
[0849] Some preferred examples of such amino acid sequences include
gly-ser linkers, for example of the type
(gly.sub.xser.sub.y).sub.z, such as (for example
(gly.sub.4ser).sub.3 or (gly.sub.3ser.sub.2).sub.3, as described in
WO 99/42077, and the GS30, GS15, GS9 and GS7 linkers described in
the applications by Ablynx mentioned herein (see for example WO
06/040153 and WO 06/122825), as well as hinge-like regions, such as
the hinge regions of naturally occurring heavy chain antibodies or
similar sequences (such as described in WO 94/04678). Preferred
linkers are depicted in Table B-4.
TABLE-US-00006 TABLE B-4 Linkers SEQ ID Linker Sequence NO 5GS
GGGGS 376 7GS SGGSGGS 377 9GS GGGGSGGGS 378 10GS GGGGSGGGGS 379
15GS GGGGSGGGGSGGGGS 380 18GS GGGGSGGGGSGGGGGGGS 381 20GS
GGGGSGGGGSGGGGSGGGGS 382 25GS GGGGSGGGGSGGGGSGGGGSGGGGS 383 30GS
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 384 35GS
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 385 Poly-A AAA 386
[0850] Some other particularly preferred linkers are poly-alanine
(such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO
06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).
[0851] Other suitable linkers generally comprise organic compounds
or polymers, in particular those suitable for use in proteins for
pharmaceutical use. For instance, poly(ethyleneglycol) moieties
have been used to link antibody domains, see for example WO
04/081026.
[0852] It is encompassed within the scope of the invention that the
length, the degree of flexibility and/or other properties of the
linker(s) used (although not critical, as it usually is for linkers
used in ScFv fragments) may have some influence on the properties
of the final polypeptide of the invention, including but not
limited to the affinity, specificity or avidity for the TCR, or for
one or more of the other antigens. Based on the disclosure herein,
the skilled person will be able to determine the optimal linker(s)
for use in a specific polypeptide of the invention, optionally
after some limited routine experiments.
[0853] For example, in multivalent polypeptides of the invention
that comprise building blocks, ISVs or Nanobodies directed against
a first and second target, the length and flexibility of the linker
are preferably such that it allows each building block, ISV or
Nanobody of the invention present in the polypeptide to bind to its
cognate target, e.g. the antigenic determinant on each of the
targets. Again, based on the disclosure herein, the skilled person
will be able to determine the optimal linker(s) for use in a
specific polypeptide of the invention, optionally after some
limited routine experiments.
[0854] It is also within the scope of the invention that the
linker(s) used confer one or more other favourable properties or
functionality to the polypeptides of the invention, and/or provide
one or more sites for the formation of derivatives and/or for the
attachment of functional groups (e.g. as described herein for the
derivatives of the ISVs, Nanobodies, or polypeptides of the
invention). For example, linkers containing one or more charged
amino acid residues can provide improved hydrophilic properties,
whereas linkers that form or contain small epitopes or tags can be
used for the purposes of detection, identification and/or
purification. Again, based on the disclosure herein, the skilled
person will be able to determine the optimal linkers for use in a
specific polypeptide of the invention, optionally after some
limited routine experiments.
[0855] Finally, when two or more linkers are used in the
polypeptides of the invention, these linkers may be the same or
different. Again, based on the disclosure herein, the skilled
person will be able to determine the optimal linkers for use in a
specific polypeptide of the invention, optionally after some
limited routine experiments.
[0856] Usually, for ease of expression and production, a
polypeptide of the invention will be a linear polypeptide. However,
the invention in its broadest sense is not limited thereto. For
example, when a polypeptide of the invention comprises three or
more building blocks, ISVs or Nanobodies, it is possible to link
them by use of a linker with three or more "arms", with each "arm"
being linked to a building block, ISV or Nanobody, so as to provide
a "star-shaped" construct. It is also possible, although usually
less preferred, to use circular constructs.
[0857] Accordingly, the present invention relates to a polypeptide
as described herein, wherein said first ISV and said second ISV and
possibly said third ISV and/or said ISV binding serum albumin are
directly linked to each other or are linked via a linker.
[0858] The present invention relates to a polypeptide as described
herein, wherein said linker is chosen from the group consisting of
linkers of 5GS, 7GS, 9GS, 10GS, 15GS, 18GS, 20GS, 25GS, 30GS and
35GS.
[0859] The present invention relates to a polypeptide as described
herein, wherein said serum protein binding moiety is a non-antibody
based polypeptide (e.g. PEG).
[0860] The invention also relates to methods for preparing the
ISVs, polypeptides and constructs described herein. The ISVs,
polypeptides and constructs of the invention can be prepared in a
manner known per se, as will be clear to the skilled person from
the further description herein. For example, the ISVs, polypeptides
and constructs of the invention can be prepared in any manner known
per se for the preparation of antibodies and in particular for the
preparation of antibody fragments (including but not limited to
(single) domain antibodies and ScFv fragments). Some preferred, but
non-limiting methods for preparing the polypeptides and constructs
include the methods and techniques described herein.
[0861] The method for producing an ISV, polypeptide or protein
construct of the invention may comprise the following steps: [0862]
the expression, in a suitable host cell or host organism (also
referred to herein as a "host of the invention") or in another
suitable expression system of a nucleic acid that encodes said ISV,
polypeptide or protein construct of the invention, [0863]
optionally followed by: [0864] isolating and/or purifying the ISV,
polypeptide or protein construct of the invention thus
obtained.
[0865] In particular, such a method may comprise the steps of:
[0866] cultivating and/or maintaining a host of the invention under
conditions that are such that said host of the invention expresses
and/or produces at least one ISV, polypeptide or protein construct
of the invention; optionally followed by: [0867] isolating and/or
purifying the ISV, polypeptide or protein construct of the
invention thus obtained.
[0868] Accordingly, the present invention also relates to a nucleic
acid or nucleotide sequence that encodes an ISV, polypeptide or
protein construct of the invention (also referred to as "nucleic
acid of the invention" or "nucleotide sequence of the invention").
A nucleic acid of the invention can be in the form of single or
double stranded DNA or RNA, and is preferably in the form of double
stranded DNA. For example, the nucleotide sequences of the
invention may be genomic DNA, cDNA or synthetic DNA (such as DNA
with a codon usage that has been specifically adapted for
expression in the intended host cell or host organism).
[0869] According to one embodiment of the invention, the nucleic
acid of the invention is in essentially isolated from, as defined
herein. The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a vector, such as for example a
plasmid, cosmid or YAC, which again may be in essentially isolated
form.
[0870] The nucleic acids of the invention can be prepared or
obtained in a manner known per se, based on the information on the
polypeptides or protein constructs of the invention given herein,
and/or can be isolated from a suitable natural source. Also, as
will be clear to the skilled person, to prepare a nucleic acid of
the invention, also several nucleotide sequences, such as at least
one nucleotide sequence encoding an immunoglobulin single variable
domain of the invention and for example nucleic acids encoding one
or more linkers can be linked together in a suitable manner.
[0871] Techniques for generating the nucleic acids of the invention
will be clear to the skilled person and may for instance include,
but are not limited to, automated DNA synthesis; site-directed
mutagenesis; combining two or more naturally occurring and/or
synthetic sequences (or two or more parts thereof), introduction of
mutations that lead to the expression of a truncated expression
product; introduction of one or more restriction sites (e.g. to
create cassettes and/or regions that may easily be digested and/or
ligated using suitable restriction enzymes), and/or the
introduction of mutations by means of a PCR reaction using one or
more "mismatched" primers. These and other techniques will be clear
to the skilled person, and reference is again made to the standard
handbooks, such as Sambrook et al. and Ausubel et al., mentioned
herein, as well as the Examples below.
[0872] The nucleic acid of the invention may also be in the form
of, be present in and/or be part of a genetic construct, as will be
clear to the person skilled in the art. Such genetic constructs
generally comprise at least one nucleic acid of the invention that
is optionally linked to one or more elements of genetic constructs
known per se, such as for example one or more suitable regulatory
elements (such as a suitable promoter(s), enhancer(s),
terminator(s), etc.) and the further elements of genetic constructs
referred to herein. Such genetic constructs comprising at least one
nucleic acid of the invention will also be referred to herein as
"genetic constructs of the invention".
[0873] The genetic constructs of the invention may be DNA or RNA,
and are preferably double-stranded DNA. The genetic constructs of
the invention may also be in a form suitable for transformation of
the intended host cell or host organism, in a form suitable for
integration into the genomic DNA of the intended host cell or in a
form suitable for independent replication, maintenance and/or
inheritance in the intended host organism. For instance, the
genetic constructs of the invention may be in the form of a vector,
such as for example a plasmid, cosmid, YAC, a viral vector or
transposon. In particular, the vector may be an expression vector,
i.e. a vector that can provide for expression in vitro and/or in
vivo (e.g. in a suitable host cell, host organism and/or expression
system).
[0874] In a preferred but non-limiting embodiment, a genetic
construct of the invention comprises [0875] a) at least one nucleic
acid of the invention; operably connected to [0876] b) one or more
regulatory elements, such as a promoter and optionally a suitable
terminator;
[0877] and optionally also [0878] c) one or more further elements
of genetic constructs known per se; in which the terms "regulatory
element", "promoter", "terminator" and "operably connected" have
their usual meaning in the art (as further described herein); and
in which said "further elements" present in the genetic constructs
may for example be 3'- or 5'-UTR sequences, leader sequences,
selection markers, expression markers/reporter genes, and/or
elements that may facilitate or increase (the efficiency of)
transformation or integration. These and other suitable elements
for such genetic constructs will be clear to the skilled person,
and may for instance depend upon the type of construct used; the
intended host cell or host organism; the manner in which the
nucleotide sequences of the invention of interest are to be
expressed (e.g. via constitutive, transient or inducible
expression); and/or the transformation technique to be used. For
example, regulatory sequences, promoters and terminators known per
se for the expression and production of antibodies and antibody
fragments (including but not limited to (single) domain antibodies
and ScFv fragments) may be used in an essentially analogous
manner.
[0879] Preferably, in the genetic constructs of the invention, said
at least one nucleic acid of the invention and said regulatory
elements, and optionally said one or more further elements, are
"operably linked" to each other, by which is generally meant that
they are in a functional relationship with each other. For
instance, a promoter is considered "operably linked" to a coding
sequence if said promoter is able to initiate or otherwise
control/regulate the transcription and/or the expression of a
coding sequence (in which said coding sequence should be understood
as being "under the control of" said promoter). Generally, when two
nucleotide sequences are operably linked, they will be in the same
orientation and usually also in the same reading frame. They will
usually also be essentially contiguous, although this may also not
be required.
[0880] The nucleic acids of the invention and/or the genetic
constructs of the invention may be used to transform a host cell or
host organism, i.e. for expression and/or production of the
polypeptide or protein construct of the invention. The host is
preferably a non-human host. Suitable hosts or host cells will be
clear to the skilled person, and may for example be any suitable
fungal, prokaryotic or eukaryotic cell or cell line or any suitable
fungal, prokaryotic or eukaryotic organism, for example: [0881] a
bacterial strain, including but not limited to gram-negative
strains such as strains of Escherichia coli; of Proteus, for
example of Proteus mirabilis; of Pseudomonas, for example of
Pseudomonas fluorescens; and gram-positive strains such as strains
of Bacillus, for example of Bacillus subtilis or of Bacillus
brevis; of Streptomyces, for example of Streptomyces lividans; of
Staphylococcus, for example of Staphylococcus carnosus; and of
Lactococcus, for example of Lactococcus lactis; [0882] a fungal
cell, including but not limited to cells from species of
Trichoderma, for example from Trichoderma reesei; of Neurospora,
for example from Neurospora crassa; of Sordaria, for example from
Sordaria macrospora; of Aspergillus, for example from Aspergillus
niger or from Aspergillus sojae; or from other filamentous fungi;
[0883] a yeast cell, including but not limited to cells from
species of Saccharomyces, for example of Saccharomyces cerevisiae;
of Schizosaccharomyces, for example of Schizosaccharomyces pombe;
of Pichia, for example of Pichia pastoris or of Pichia methanolica;
of Hansenula, for example of Hansenula polymorpha; of
Kluyveromyces, for example of Kluyveromyces lactis; of Arxula, for
example of Arxula adeninivorans; of Yarrowia, for example of
Yarrowia lipolytica; [0884] an amphibian cell or cell line, such as
Xenopus oocytes; [0885] an insect-derived cell or cell line, such
as cells/cell lines derived from lepidoptera, including but not
limited to Spodoptera SF9 and Sf21 cells or cells/cell lines
derived from Drosophila, such as Schneider and Kc cells; [0886] a
plant or plant cell, for example in tobacco plants; and/or [0887] a
mammalian cell or cell line, for example a cell or cell line
derived from a human, a cell or a cell line from mammals including
but not limited to CHO-cells, BHK-cells (for example BHK-21 cells)
and human cells or cell lines such as HeLa, COS (for example COS-7)
and PER.C6 cells;
[0888] as well as all other hosts or host cells known per se for
the expression and production of antibodies and antibody fragments
(including but not limited to (single) domain antibodies and ScFv
fragments), which will be clear to the skilled person. Reference is
also made to the general background art cited hereinabove, as well
as to for example WO 94/29457; WO 96/34103; WO 99/42077; Frenken et
al. 1998 (Res. Immunol. 149: 589-99); Riechmann and Muyldermans
1999 (J. Immunol. Met. 231: 25-38); van der Linden 2000 (J.
Biotechnol. 80: 261-70); Joosten et al. 2003 (Microb. Cell Fact. 2:
1); Joosten et al. 2005 (Appl. Microbiol. Biotechnol. 66: 384-92);
and the further references cited herein.
[0889] For expression of the ISVs, polypeptides or constructs in a
cell, they may also be expressed as so-called "intrabodies", as for
example described in WO 94/02610, WO 95/22618 and U.S. Pat. No.
7,004,940; WO 03/014960; in Cattaneo and Biocca 1997 (Intracellular
Antibodies: Development and Applications. Landes and
Springer-Verlag); and in Kontermann 2004 (Methods 34: 163-170).
[0890] According to one preferred, but non-limiting embodiment of
the invention, the ISV, polypeptide or protein construct of the
invention is produced in a bacterial cell, in particular a
bacterial cell suitable for large scale pharmaceutical production,
such as cells of the strains mentioned above.
[0891] According to another preferred, but non-limiting embodiment
of the invention, the ISV, polypeptide or protein construct of the
invention is produced in a yeast cell, in particular a yeast cell
suitable for large scale pharmaceutical production, such as cells
of the species mentioned above.
[0892] According to yet another preferred, but non-limiting
embodiment of the invention, the ISV, polypeptide or construct of
the invention is produced in a mammalian cell, in particular in a
human cell or in a cell of a human cell line, and more in
particular in a human cell or in a cell of a human cell line that
is suitable for large scale pharmaceutical production, such as the
cell lines mentioned hereinabove.
[0893] Suitable techniques for transforming a host or host cell of
the invention will be clear to the skilled person and may depend on
the intended host cell/host organism and the genetic construct to
be used. Reference is again made to the handbooks and patent
applications mentioned above.
[0894] After transformation, a step for detecting and selecting
those host cells or host organisms that have been successfully
transformed with the nucleotide sequence/genetic construct of the
invention may be performed. This may for instance be a selection
step based on a selectable marker present in the genetic construct
of the invention or a step involving the detection of the
polypeptide of the invention, e.g. using specific antibodies.
[0895] The transformed host cell (which may be in the form or a
stable cell line) or host organisms (which may be in the form of a
stable mutant line or strain) form further aspects of the present
invention.
[0896] Preferably, these host cells or host organisms are such that
they express, or are (at least) capable of expressing (e.g. under
suitable conditions), an ISV, polypeptide or protein construct of
the invention (and in case of a host organism: in at least one
cell, part, tissue or organ thereof). The invention also includes
further generations, progeny and/or offspring of the host cell or
host organism of the invention, for instance obtained by cell
division or by sexual or asexual reproduction.
[0897] Accordingly, in another aspect, the invention relates to a
host or host cell that expresses (or that under suitable
circumstances is capable of expressing) an ISV, polypeptide or
protein construct of the invention; and/or that contains a nucleic
acid encoding the same. Some preferred but non-limiting examples of
such hosts or host cells can be as generally described in WO
04/041867, WO 04/041865 or WO 09/068627. For example, ISVs,
polypeptides and protein constructs of the invention may with
advantage be expressed, produced or manufactured in a yeast strain,
such as a strain of Pichia pastoris. Reference is also made to WO
04/25591, WO 10/125187, WO 11/003622, and WO 12/056000 which also
describes the expression/production in Pichia and other hosts/host
cells of immunoglobulin single variable domains and polypeptides
comprising the same.
[0898] To produce/obtain expression of the ISVs, polypeptides or
protein constructs of the invention, the transformed host cell or
transformed host organism may generally be kept, maintained and/or
cultured under conditions such that the (desired) ISV, polypeptide
or protein construct of the invention is expressed/produced.
Suitable conditions will be clear to the skilled person and will
usually depend upon the host cell/host organism used, as well as on
the regulatory elements that control the expression of the
(relevant) nucleotide sequence of the invention. Again, reference
is made to the handbooks and patent applications mentioned above in
the paragraphs on the genetic constructs of the invention.
[0899] Generally, suitable conditions may include the use of a
suitable medium, the presence of a suitable source of food and/or
suitable nutrients, the use of a suitable temperature, and
optionally the presence of a suitable inducing factor or compound
(e.g. when the nucleotide sequences of the invention are under the
control of an inducible promoter); all of which may be selected by
the skilled person. Again, under such conditions, the ISVs,
polypeptides or protein constructs of the invention may be
expressed in a constitutive manner, in a transient manner, or only
when suitably induced.
[0900] It will also be clear to the skilled person that the ISV,
polypeptide or protein construct of the invention may (first) be
generated in an immature form (as mentioned above), which may then
be subjected to post-translational modification, depending on the
host cell/host organism used. Also, the ISV, polypeptide or protein
construct of the invention may be glycosylated, again depending on
the host cell/host organism used.
[0901] The ISV, polypeptide or protein construct of the invention
may then be isolated from the host cell/host organism and/or from
the medium in which said host cell or host organism was cultivated,
using protein isolation and/or purification techniques known per
se, such as (preparative) chromatography and/or electrophoresis
techniques, differential precipitation techniques, affinity
techniques (e.g. using a specific, cleavable amino acid sequence
fused with the polypeptide or construct of the invention) and/or
preparative immunological techniques (i.e. using antibodies against
the amino acid sequence to be isolated).
[0902] The constructs of the invention can generally be prepared by
a method which comprises at least the step of suitably linking ISVs
or polypeptides of the invention to the one or more further groups,
residues, moieties or binding units, optionally via the one or more
suitable linkers, so as to provide the constructs of the invention.
The ISVs, polypeptides and constructs of the invention can then
further be modified, and in particular by chemical and/or
biological (e.g. enzymatical) modification, of one or more of the
amino acid residues that form the polypeptides or constructs of the
invention, to obtain derivatives of the polypeptides or constructs
of the invention.
[0903] The invention also relates to a pharmaceutical composition
comprising the ISV, polypeptide, compound or construct of the
invention.
[0904] In the above methods, the amino acid sequences, ISVs,
Nanobodies, polypeptides, compounds or constructs of the invention
and/or the compositions comprising the same can be administered in
any suitable manner, depending on the specific pharmaceutical
formulation or composition to be used. Thus, the amino acid
sequences, ISVs, Nanobodies, polypeptides, compounds or constructs
of the invention and/or the compositions comprising the same can
for example be administered orally, intraperitoneally (e.g.
intravenously, subcutaneously, intramuscularly, or via any other
route of administration that circumvents the gastrointestinal
tract), intranasally, transdermally, topically, by means of a
suppository, by inhalation, again depending on the specific
pharmaceutical formulation or composition to be used. The clinician
will be able to select a suitable route of administration and a
suitable pharmaceutical formulation or composition to be used in
such administration, depending on the disease or disorder to be
prevented or treated and other factors well known to the
clinician.
[0905] As used herein, the term "therapeutic agent" refers to any
agent that can be used in the treatment and/or management of a
hyperproliferative cell disorder, e.g., cancer, or one or more
symptoms thereof. In certain embodiments, the term "therapeutic
agent" refers to a multispecific polypeptide of the invention.
Preferably, a therapeutic agent is an agent which is known to be
useful for, or has been or is currently being used for the
treatment, prevention and/or management of a hyperproliferative
cell disorder, e.g., cancer, or one or more symptoms thereof.
[0906] As used herein, a "therapeutically effective amount" in the
context of cancer refers to the amount of a therapy alone, or in
combination with other therapies, that provides a therapeutic
benefit in the treatment and/or management of cancers. In one
aspect, a therapeutically effective amount refers to the amount of
a therapy sufficient to destroy, modify, control or remove primary,
regional or metastatic cancer tissue. In another aspect, a
therapeutically effective amount refers to the amount of a therapy
sufficient to reduce the symptoms of a cancer. In another aspect, a
therapeutically effective amount refers to the amount of a therapy
sufficient to delay or minimize the spread of cancer. In a specific
embodiment, a therapeutically effective amount of a therapy is an
amount of a therapy sufficient to inhibit growth or proliferation
of cancer cells, kill existing cancer cells (e.g., cause regression
of the cancer), and/or prevent the spread of cancer cells to other
tissues or areas (e.g., prevent metastasis). In another specific
embodiment, a therapeutically effective amount of a therapy is the
amount of a therapy sufficient to inhibit the growth of a tumor by
at least 5%, preferably at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, or at least 100% as measured by a standard method
known in the art. Used in connection with an amount of a
multispecific polypeptide of the invention, the term can encompass
an amount that improves overall therapy, reduces or avoids unwanted
effects, or enhances the therapeutic efficacy of or synergies with
another therapy. In one embodiment, a therapeutically effective
amount of a therapy reduces or avoids unwanted effects, or enhances
the therapeutic efficacy of or synergies with another therapy by at
least 5%, preferably at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 100% relative to a control (e.g., a negative
control such as phosphate buffered saline) in an assay known in the
art or described herein.
[0907] As used herein, a "therapeutically effective amount" in the
context of a non-cancer hyperproliferative cell disorder refers to
the amount of a therapy alone, or in combination with other
therapies, that provides a therapeutic benefit in the treatment
and/or management of said disorder. In one aspect, a
therapeutically effective amount refers to the amount of a therapy
sufficient to destroy, modify, control or remove cells affected by
a non-cancer hyperproliferative cell disorder. In another aspect, a
therapeutically effective amount refers to the amount of a therapy
sufficient to reduce the symptoms of a non-cancer
hyperproliferative cell disorder. In another aspect, a
therapeutically effective amount refers to the amount of a therapy
sufficient to delay or minimize the spread of the non-cancer
hyperproliferative cell disorder. In a specific embodiment, a
therapeutically effective amount of a therapy is an amount of a
therapy sufficient to inhibit growth or proliferation of the
non-cancer hyperproliferative cell disorder, kill existing
non-cancer hyperproliferative cells (e.g., cause regression of the
disorder). In another specific embodiment, a therapeutically
effective amount of a therapy is the amount of a therapy sufficient
to inhibit the growth of the non-cancer hyperproliferative cells by
at least 5%, preferably at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, or at least 100% as measured by a standard method
known in the art. Used in connection with an amount of a
multispecific polypeptide of the invention, the term can encompass
an amount that improves overall therapy, reduces or avoids unwanted
effects, or enhances the therapeutic efficacy of or synergies with
another therapy. In one embodiment, a therapeutically effective
amount of a therapy reduces or avoids unwanted effects, or enhances
the therapeutic efficacy of or synergies with another therapy by at
least 5%, preferably at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 100% relative to a control (e.g., a negative
control such as phosphate buffered saline) in an assay known in the
art.
[0908] As used herein, the term "therapy" refers to any protocol,
method and/or agent that can be used in the treatment, prevention
and/or management of a hyperproliferative cell disorder, e.g.,
cancer. In certain embodiments, the terms "therapies" and "therapy"
refer to a biological therapy, supportive therapy, and/or other
therapies useful in the treatment, prevention and/or management of
a hyperproliferative cell disorder, e.g., cancer, or one or more
symptoms thereof known to one of skill in the art such as medical
personnel.
[0909] As used herein, the terms "treat", "treatment" and
"treating" in the context of administering (a) therapy(ies) to a
subject refer to the reduction or amelioration of the progression,
severity, and/or duration of a disorder associated with a
hyperproliferative cell disorder, e.g., cancer, and/or the
amelioration of one or more symptoms thereof resulting from the
administration of one or more therapies (including, but not limited
to, the administration of one or more prophylactic or therapeutic
agents). In specific embodiments, the terms "treat", "treatment"
and "treating" in the context of administering (a) therapy(ies) to
a subject refer to the reduction or amelioration of the
progression, severity, and/or duration of a hyperproliferative cell
disorder, e.g., cancer, refers to a reduction in cancer cells by at
least 5%, preferably at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at least 55%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 100% relative to a control (e.g., a negative
control such as phosphate buffered saline). In other embodiments,
the terms "treat", "treatment" and "treating" in the context of
administering (a) therapy(ies) to a subject refer to the reduction
or amelioration of the progression, severity, and/or duration of a
hyperproliferative cell disorder, e.g., cancer, refers to no change
in cancer cell number, a reduction in hospitalization time, a
reduction in mortality, or an increase in survival time of the
subject with cancer.
[0910] The amino acid sequences, ISVs, Nanobodies, polypeptides,
compounds and/or constructs of the invention and/or the
compositions comprising the same are administered according to a
regime of treatment that is suitable for preventing and/or treating
the hyperproliferative cell disorder, e.g., cancer, to be prevented
or treated. The clinician will generally be able to determine a
suitable treatment regimen, depending on factors such as the stage
of the hyperproliferative cell disorder, e.g., cancer, to be
treated, the severity of the hyperproliferative cell disorder,
e.g., cancer, to be treated and/or the severity of the symptoms
thereof, the specific amino acid sequence, ISV, Nanobody,
polypeptide, compound and/or construct of the invention to be used,
the specific route of administration and pharmaceutical formulation
or composition to be used, the age, gender, weight, diet, general
condition of the patient, and similar factors well known to the
clinician.
[0911] Generally, the treatment regimen will comprise the
administration of one or more amino acid sequences, ISVs,
Nanobodies, polypeptides, compounds and/or constructs of the
invention, or of one or more compositions comprising the same, in
one or more pharmaceutically effective amounts or doses. The
specific amount(s) or doses to be administered can be determined by
the clinician, again based on the factors cited above.
[0912] Generally, for the prevention and/or treatment of a
hyperproliferative cell disorder, e.g., cancer, mentioned herein
and depending on the type of hyperproliferative cell disorder,
e.g., cancer, and stage of the disease to be treated, the potency
of the specific amino acid sequence, ISV, Nanobody, polypeptide,
compound or construct of the invention to be used, the specific
route of administration and the specific pharmaceutical formulation
or composition used, the amino acid sequences, ISVs, Nanobodies,
polypeptides, compounds or constructs of the invention will
generally be administered in an amount between 1 gram and 0.01
milligram per kg body weight per day, preferably between 0.1 gram
and 0.01 milligram per kg body weight per day, such as about 0.1,
1, 10, 100 or 1000 milligram per kg body weight per day, e.g. from
0.1 mg per kg to 25 mg per kg of the subject's body weight; either
continuously (e.g. by infusion), as a single daily dose or as
multiple divided doses during the day. The clinician will generally
be able to determine a suitable daily dose, depending on the
factors mentioned herein. It will also be clear that in specific
cases, the clinician may choose to deviate from these amounts, for
example on the basis of the factors cited above and his expert
judgment. Generally, some guidance on the amounts to be
administered can be obtained from the amounts usually administered
for comparable conventional antibodies or antibody fragments
against the same target administered via essentially the same
route, taking into account however differences in affinity/avidity,
efficacy, biodistribution, half-life and similar factors well known
to the skilled person.
[0913] Usually, in the above method, a single amino acid sequence,
ISV, Nanobody, polypeptide, compound or construct of the invention
will be used. It is however within the scope of the invention to
use two or more amino acid sequences, ISVs, Nanobodies,
polypeptides compounds and/or constructs of the invention in
combination.
[0914] The ISVs, Nanobodies, amino acid sequences, polypeptides,
compounds and/or constructs of the invention may also be used in
combination with one or more further pharmaceutically active
compounds or principles, i.e. as a combined treatment regimen,
which may or may not lead to a synergistic effect. Again, the
clinician will be able to select such further compounds or
principles, as well as a suitable combined treatment regimen, based
on the factors cited above and his expert judgement.
[0915] In particular, the amino acid sequences, ISVs, Nanobodies,
polypeptides, compounds and/or constructs of the invention may be
used in combination with other pharmaceutically active compounds or
principles that are or can be used for the prevention and/or
treatment of the hyperproliferative cell disorder, e.g., cancer,
disease and/or disorder cited herein, as a result of which a
synergistic effect may or may not be obtained. Examples of such
compounds and principles, as well as routes, methods and
pharmaceutical formulations or compositions for administering them
will be clear to the clinician.
[0916] When two or more substances or principles are to be used as
part of a combined treatment regimen, they can be administered via
the same route of administration or via different routes of
administration, at essentially the same time or at different times
(e.g. essentially simultaneously, consecutively, or according to an
alternating regime). When the substances or principles are to be
administered simultaneously via the same route of administration,
they may be administered as different pharmaceutical formulations
or compositions or part of a combined pharmaceutical formulation or
composition, as will be clear to the skilled person.
[0917] In one aspect, the disclosure provides methods for the
administration of immunoglobulin single variable domains and
polypeptide constructs thereof comprising one or more
immunoglobulin single variable domains, polypeptides, compounds
and/or constructs. In some embodiments, the immunoglobulin single
variable domain, polypeptide, compound and/or construct is
administered as a pharmaceutical composition. The pharmaceutical
composition, in addition to the immunoglobulin single variable
domains and polypeptide constructs thereof includes a
pharmaceutically-acceptable carrier.
[0918] As described in detail, the pharmaceutical compositions of
the disclosure may be specially formulated for administration in
solid or liquid form, including those adapted for the following:
oral administration, for example, drenches (aqueous or non-aqueous
solutions or suspensions), tablets, e.g., those targeted for
buccal, sublingual, and systemic absorption, boluses, powders,
granules, pastes for application to the tongue; parenteral
administration, for example, by subcutaneous, intramuscular,
intravenous or epidural injection as, for example, a sterile
solution or suspension, or sustained-release formulation; topical
application, for example, as a cream, ointment, or a
controlled-release patch or spray applied to the skin, lungs, or
oral cavity; intravaginally or intrarectally, for example, as a
pessary, cream or foam; sublingually; ocularly; transdermally; or
nasally, pulmonary and to other mucosal surfaces.
[0919] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0920] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient, or
solvent encapsulating material, involved in carrying or
transporting the subject compound from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; pH buffered
solutions; polyesters, polycarbonates and/or polyanhydrides; and
other non-toxic compatible substances employed in pharmaceutical
formulations.
[0921] Formulations of the disclosure include those suitable for
oral, nasal, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient (e.g., immunoglobulin single variable domain or
polypeptide constructs thereof) which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the host being treated, and the particular mode of
administration. The amount of active ingredient that can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the compound which produces a
therapeutic effect. Generally, this amount will range from about 1%
to about 99% of active ingredient, preferably from about 5% to
about 70%, most preferably from about 10% to about 30%.
[0922] In certain embodiments, a formulation comprises an excipient
selected from the group consisting of cyclodextrins, liposomes,
micelle forming agents, e.g., bile acids, and polymeric carriers,
e.g., polyesters and polyanhydrides. In certain embodiments, an
aforementioned formulation renders orally bioavailable an
immunoglobulin single variable domain or polypeptide construct.
[0923] Methods of preparing these formulations or compositions
include the step of bringing into association an immunoglobulin
single variable domain or polypeptide construct with the carrier
and, optionally, one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association an immunoglobulin single variable domain or polypeptide
construct with liquid carriers, or finely divided solid carriers,
or both, and then, if necessary, shaping the product.
[0924] Formulations suitable for oral administration may be in the
form of capsules, cachets, pills, tablets, lozenges (using a
flavored basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of an immunoglobulin single variable domain or polypeptide
construct as an active ingredient. An immunoglobulin single
variable domain or polypeptide construct invention may also be
administered as a bolus, electuary or paste.
[0925] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like), the
active ingredient is mixed with one or more
pharmaceutically-acceptable carriers, such as sodium citrate or
dicalcium phosphate, and/or any of the following: fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; humectants, such as glycerol; disintegrating
agents, such as agar-agar, calcium carbonate, potato or tapioca
starch, alginic acid, certain silicates, and sodium carbonate;
solution retarding agents, such as paraffin; absorption
accelerators, such as quaternary ammonium compounds; wetting
agents, such as, for example, cetyl alcohol, glycerol monostearate,
and non-ionic surfactants; absorbents, such as kaolin and bentonite
clay; lubricants, such as talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, and
mixtures thereof; and coloring agents. In the case of capsules,
tablets and pills, the pharmaceutical compositions may also
comprise buffering agents. Solid compositions of a similar type may
also be employed as fillers in soft and hard-shelled gelatin
capsules using such excipients as lactose or milk sugars, as well
as high molecular weight polyethylene glycols and the like.
[0926] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxy-propylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made in a suitable machine in which a mixture
of the powdered compound is moistened with an inert liquid
diluent.
[0927] The tablets, and other solid dosage forms of the
pharmaceutical compositions, such as dragees, capsules, pills and
granules, may optionally be scored or prepared with coatings and
shells, such as enteric coatings and other coatings well known in
the pharmaceutical-formulating art. They may also be formulated so
as to provide slow or controlled release of the active ingredient
therein using, for example, hydroxypropylmethyl cellulose in
varying proportions to provide the desired release profile, other
polymer matrices, liposomes and/or microspheres. They may be
formulated for rapid release, e.g., freeze-dried. They may be
sterilized by, for example, filtration through a bacteria-retaining
filter, or by incorporating sterilizing agents in the form of
sterile solid compositions that can be dissolved in sterile water,
or some other sterile injectable medium immediately before use.
These compositions may also optionally contain opacifying agents
and may be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain portion of the
gastrointestinal tract, optionally, in a delayed manner. Examples
of embedding compositions that can be used include polymeric
substances and waxes. The active ingredient can also be in
micro-encapsulated form, if appropriate, with one or more of the
above-described excipients.
[0928] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as, for example, water or other
solvents, solubilizing agents and emulsifiers, such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
oils (in particular, cottonseed, groundnut, corn, germ, olive,
castor and sesame oils), glycerol, tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof.
[0929] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0930] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0931] Formulations of the pharmaceutical compositions for rectal
or vaginal administration may be presented as a suppository, which
may be prepared by mixing an immunoglobulin single variable domain
or polypeptide construct with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound.
[0932] Formulations suitable for vaginal administration also
include pessaries, tampons, creams, gels, pastes, foams or spray
formulations containing such carriers as are known in the art to be
appropriate.
[0933] Dosage forms for the topical or transdermal administration
of an immunoglobulin single variable domain or polypeptide
construct include powders, sprays, ointments, pastes, creams,
lotions, gels, solutions, patches and inhalants. The active
compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0934] The ointments, pastes, creams and gels may contain,
excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc and zinc oxide,
or mixtures thereof.
[0935] Powders and sprays can contain excipients such as lactose,
talc, silicic acid, aluminum hydroxide, calcium silicates and
polyamide powder, or mixtures of these substances. Sprays can
additionally contain customary propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,
such as butane and propane.
[0936] Transdermal patches have the added advantage of providing
controlled delivery of an immunoglobulin single variable domain or
polypeptide construct to the body. Dissolving or dispersing the
compound in the proper medium can make such dosage forms.
Absorption enhancers can also be used to increase the flux of the
compound across the skin. Either providing a rate controlling
membrane or dispersing the compound in a polymer matrix or gel can
control the rate of such flux.
[0937] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this disclosure.
[0938] Pharmaceutical compositions suitable for parenteral
administration comprise one or more an immunoglobulin single
variable domains or polypeptide constructs in combination with one
or more pharmaceutically-acceptable sterile isotonic aqueous or
non-aqueous solutions, dispersions, suspensions or emulsions, or
sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
sugars, alcohols, antioxidants, buffers, bacteriostats, solutes
which render the formulation isotonic with the blood of the
intended recipient or suspending or thickening agents.
[0939] Examples of suitable aqueous and non-aqueous carriers, which
may be employed in the pharmaceutical compositions include water,
ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and the like), and suitable mixtures thereof, vegetable
oils, such as olive oil, and injectable organic esters, such as
ethyl oleate. Proper fluidity can be maintained, for example, by
the use of coating materials, such as lecithin, by the maintenance
of the required particle size in the case of dispersions, and by
the use of surfactants.
[0940] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms upon the subject
compounds may be ensured by the inclusion of various antibacterial
and antifungal agents, for example, paraben, chlorobutanol, phenol
sorbic acid, and the like. It may also be desirable to include
isotonic agents, such as sugars, sodium chloride, and the like into
the compositions. In addition, prolonged absorption of the
injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption such as aluminum
monostearate and gelatin.
[0941] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0942] Injectable depot forms are made by forming microencapsule
matrices of the subject compounds in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly-(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions, which are
compatible with body tissue.
[0943] In another aspect, kits are provided comprising a binding
molecule of the invention, a nucleic acid molecule of the
invention, a vector of the invention, or a host cell of the
invention. The kit may comprise one or more vials containing the
binding molecule and instructions for use. The kit may also contain
means for administering the binding molecule of the present
invention such as a syringe, pump, infuser or the like.
[0944] The embodiments illustrated and discussed in this
specification are intended only to teach those skilled in the art
the best way known to the inventors to make and use the invention.
Modifications and variation of the above-described embodiments of
the invention are possible without departing from the invention, as
appreciated by those skilled in the art in light of the above
teachings. It is therefore understood that, within the scope of the
claims and their equivalents, the invention may be practiced
otherwise than as specifically described.
[0945] The invention will now be further described by means of the
following non-limiting preferred aspects, examples and figures.
[0946] The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by reference, in
particular for the teaching that is referenced hereinabove.
EXAMPLES
Example 1: Material and Methods
[0947] 1.1 TCR .alpha..alpha./CD3 Transfected Cell Lines
[0948] Transient and stable CHO-K1 (ATCC: CCL-61), HEK293H (Life
technologies 11631-017), Llana (Fibroblast cells from llama Navel
cord cells) cell lines with recombinant overexpression of all 6
chains of the full human T cell Receptor complex were generated.
For this, the coding sequences of the TCR alpha (a) and TCR beta
(P) chain were cloned in a pcDNA3.1-derived vector, downstream of a
CMV promotor and a 2A-like viral peptide sequence was inserted
between both chains to induce ribosomal skipping during translation
of the polyprotein. In the same vector, the coding sequences of the
epsilon, delta, gamma and zeta chains of the CD3 complex were
cloned downstream of an additional CMV promotor, also using 2A-like
viral peptide sequences between the respective chains. In addition,
a stable HEK293H clone with recombinant overexpression of the 4
chains of the human CD3 was generated as described above using a
single gene vector.
[0949] The used sequences for the human CD3 and the human
TCR.alpha./.beta. constant domains were derived from UniProtKB (CD3
delta: P04234, CD3 gamma: P09693, CD3 epsilon: P07766, CD3 zeta:
P20963, TCR .alpha.: P01848 and TCR .beta.: P01850; SEQ ID NOs: 344
to 349, respectively). The sequences for the human
TCR.alpha./.beta. variable domains were derived from crystal
structure sequences (PDB codes: 2IAN, 2XN9 and 3TOE) (human TCR
.alpha. variable domains derived from 2IAN, 2XN9 and 3TOE with SEQ
ID NOs: 393 to 395, respectively; human TCR 1 variable domains
derived from 2IAN, 2XN9 and 3TOE with SEQ ID NOs: 476 to 478,
respectively).
[0950] The cell surface expression of the human T cell receptor
complex was confirmed by flow cytometry using a functional mouse
IgG2b anti-human TCR.alpha./.beta. antibody, clone BW242/412
(Miltenyi 130-098-219) and a functional mouse IgG2a anti-CD3 PE
labelled antibody, clone OKT-3 (eBioscience 12-0037) (FIG. 1).
[0951] 1.2 Soluble Recombinant TCR .alpha./.beta. Proteins
[0952] Soluble human and cynomolgus/rhesus monkey TCR
.alpha./.beta. proteins were generated in house. The sequences for
the extracellular part of the human TCR.alpha./.beta. constant
domain were derived from UniProtKB (TCR .alpha.: P01848 and TCR 1:
P01850; SEQ ID NOs: 479 and 480, respectively). The human TCR
.alpha./.beta. variable domains were derived from crystal structure
sequence (PDB code: 2XN9, SEQ ID NOs: 394 and 477, respectively for
.alpha. and .beta. chain).
[0953] The sequences for the extracellular part of the
cynomolgus/rhesus monkey TCR .alpha./.beta. constant domains were
derived from GenBank files EHH63463 and AEA41868 respectively (SEQ
ID NOs: 396 and 397). The sequences for the cynomolgus/rhesus
monkey TCR .alpha./.beta. variable domains were derived from
AEA41865 and AEA41866 (SEQ ID NOs: 398 and 399, respectively for
.alpha. and .beta. chain).
[0954] The extracellular domains of human TCR .alpha./.beta. (2XN9)
or cynomolgus/rhesus monkey TCR .alpha./.beta. were fused to a
zipper protein coding sequence (O'Shea et al. 1993 Curr. Biol.
3(10): 658-667), produced by CHOK1SV cells (Lonza) using Lonza's GS
Gene Expression System.TM. and subsequently purified.
[0955] Quality of the TCR .alpha./.beta. zipper proteins was
assessed in an ELISA binding assay. Maxisorp 96-well ELISA plates
(Nunc) were coated with 2 .mu.g/mL soluble recombinant human TCR
.alpha./.beta. (2XN9)-zipper protein or soluble recombinant
cynomolgus TCR .alpha./.beta.-zipper protein. After an overnight
incubation, plates were washed and blocked with PBS+1% casein for 1
h at room temperature. Next, plates were incubated with serial
dilutions of either a functional flag tagged Nanobody or the
functional mouse IgG anti-non-human primate/Rat TCR.alpha./.beta.
antibody, clone R73 (eBioscience 16-5960) for 1 h at room
temperature while shaking, washed again and incubated with mouse
anti-flag-HRP (Sigma, #A8592) respectively rabbit anti-mouse-HRP
(Dako, #P0260). After 1 h, TMB One Solution (Promega #G7431) was
added. The reaction was stopped with 2M H.sub.2SO.sub.4 and the
dose dependent binding was determined by measuring the OD at 450 nm
using the Tecan sunrise 4 (FIG. 2).
Example 2: Immunization of Llamas with TCR/CD3, Cloning of the
Heavy Chain-Only Antibody Fragment Repertoires and Preparation of
Phages
[0956] 2.1 Immunization
[0957] It was set out to generate heavy chain only antibodies in
camelidae (e.g. llama and alpaca) against T cell receptor (TCR)
.alpha. and/or .beta. constant chains. Although the native T cell
receptor complex consists of both CD3 (gamma, delta, epsilon and
zeta) chains, as well as TCR .alpha.- and .beta.-chains, it was
hypothesized that the absence of CD3 chains would facilitate access
to the constant domains of the TCR. Especially since the CD3 chains
laterally surround, and limit access to the constant domains of the
TCR .alpha.- and .beta.-chains. Contrary to our experience with
other targets, the obtaining of an immune response against TCR
.alpha.- or .beta.-chains was not as straight forward as
expected.
[0958] In a final approach, after approval of the Ethical Committee
(CRIA, LA1400575, Belgium--EC2012#1), the inventors attempted a
complex immunization protocol with DNA encoding for T cell complex.
In short, 3 additional llamas were immunized with a pVAX1-human
TCR(2IAN)/CD3 (described in Example 1.2) plasmid vector
(Invitrogen, Carlsbad, Calif., USA) and with a pVAX1-human
TCR(2XN9)/CD3 (described in Example 1.2) plasmid vector
(Invitrogen, Carlsbad, Calif., USA) according to standard
protocols. Two llamas received additionally 1 subcutaneous
injection of primary human T cells. Human T cells were collected
from Buffy Coat blood, from healthy volunteers (Blood bank Gent)
using RosetteSep (StemCell Technologies, #15061) followed by
enriching on Ficoll-Paque.TM. PLUS (GE Healthcare #17-1440-03)
according to manufactures instructions and stored in liquid
nitrogen. After thawing, cells were washed, and re-suspended in
D-PBS from Gibco and kept on ice prior to injection.
[0959] 2.2 Cloning of the Heavy Chain-Only Antibody Fragment
Repertoires and Preparation of Phages
[0960] Per animal, blood samples were collected after the injection
of one type of immunization antigen. From these blood samples, PBMC
were prepared using Ficoll-Hypaque according to the manufacturer's
instructions (Amersham Biosciences, Piscataway, N.J., USA). For
each immunized llama, libraries were constructed by pooling the
total RNA isolated from samples originating from a certain subset
of the immunization schedule, i.e. after one type of immunization
antigen.
[0961] In short, the PCR-amplified VHH repertoire was cloned via
specific restriction sites into a vector designed to facilitate
phage display of the VHH library. The vector was derived from
pUC119. In frame with the VHH coding sequence, the vector encodes a
C-terminal 3.times.FLAG and His6 tag. Phages were prepared
according to standard protocols (see for example WO 04/041865, WO
04/041863, WO 04/062551, WO 05/044858 and other prior art and
applications filed by Ablynx N.V. cited herein).
Example 3: Selection of TCR/CD3 Specific VHHs Via Phage Display
[0962] The vast majority of selected VHHs were directed against the
variable regions of either the TCR .alpha. or TCR .beta. chain.
Therefore different selection and counter-selection strategies had
to be devised by the inventors.
[0963] In short, VHH repertoires obtained from all llamas and
cloned as phage library were used in different selection
strategies, applying a multiplicity of selection conditions.
Selections using human TCR/CD3 transfected cell lines with the same
variable domain as used during immunization resulted in only
variable domain binders. Therefore, tools containing a different
variable TCR.alpha./.beta. domain (transfected cells (described in
Example 1.1), soluble protein (described in Example 1.2), or human
primary T cells (isolated as described in Example 2.1)) were used
during selections and proved to be crucial in identification of
constant domain binders. Additional variables during selections
included the antigen presentation method (in solution when using
cells or coated onto plates when proteins), the antigen
concentration, the orthologue used (human or cynomolgus recombinant
TCR .alpha./P protein), and the number of selection rounds. All
solid coated phase selections were done in Maxisorp 96-well plates
(Nunc, Wiesbaden, Germany).
[0964] Selections were performed as follows: TCR.alpha./.beta.-CD3
antigen preparations for solid and solution phase selection formats
were presented as described above at multiple concentrations. After
2 h incubation with the phage libraries, followed by extensive
washing, bound phages were eluted with trypsin (1 mg/mL) for 15
minutes. The trypsin protease activity was immediately neutralized
by applying 0.8 mM protease inhibitor ABSF. As control, selections
without antigen were performed in parallel.
[0965] Phage outputs were used to infect E. coli for analysis of
individual VHH clones. Periplasmic extracts were prepared according
to standard protocols (see for example WO 03/035694, WO 04/041865,
WO 04/041863, WO 04/062551 and other prior art and applications
filed by Ablynx N.V. cited herein).
Example 4: Screening, Sequence Analysis and Purification
[0966] 4.1 Screening for TCR/CD3 Binding Nanobodies in a Flow
Cytometry Assay
[0967] Periplasmic extracts were screened for cell expressed
TCR/CD3 binding using human TCR/CD3 transfected CHO-K1 or HEK293H
cells and the respective CHO-K1 or HEK293H reference cell line in a
mixed cell line setup. To this end, a large batch of the reference
cell lines were labelled with 8 .mu.M PKH26 and frozen.
5.times.10.sup.4 PKH labelled reference cells were mixed with
5.times.10.sup.4 target cells and incubated with periplasmic
extracts for 30 min at 4.degree. C., and washed 3 times. Next,
cells were incubated with 1 .mu.g/ml monoclonal ANTI-FLAG.RTM. M2
antibody (Sigma-Aldrich, cat #F1804) for 30 min at 4.degree. C.,
washed again, and incubated for 30 min at 4.degree. C. with goat
anti-mouse APC labelled antibody (Jackson Immunoresearch
115-135-164, 1:100). Samples were washed, resuspended in FACS
Buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium
azide from Merck) and then analysed via a BD FACSArray. First a P1
population which represented more than 80% of the total cell
population was selected based on FSC-SSC distribution. In this
gate, 20,000 cells were counted during acquisition. Based on
PKH26-SSC distribution, the PKH labelled parental population and
the human TCR/CD3 unlabeled target population was selected. For
these 2 populations the mean APC value was calculated.
[0968] 4.2 Screening for TCR/CD3 Binding Nanobodies in a Human T
Cell Activation Assay
[0969] After several attempts, it turned out that activation of
purified human T cells by antibodies or Nanobodies according to
standard protocols, i.e. coated onto a 96 well plate, was not
sensitive enough (data not shown).
[0970] In order to assess activity, a different assay was
developed, based on bead coupled T cell activation. In short, goat
anti-mouse IgG dynabeads (Life technologies #11033) were coated
with mouse anti-flag IgG antibodies (Sigma F1804), (15 .mu.g/1E7
beads). After an incubation period of 2 h at 4.degree. C., beads
were washed and incubated with 80 .mu.l periplasmic extract for 20
min at 4.degree. C. while shaking. Non-coupled Nanobodies were
washed away before adding the bead complex together with soluble
mouse anti-CD28 antibody (Pelicluster CD28--Sanquin #M1650) to
purified primary human T cells (isolated as described in Example
2.1). As control condition, non-stimulated human T cells were used.
In brief, goat anti-mouse IgG dynabeads coupled to mouse anti-flag
IgG were incubated in 80 .mu.l periplasmic extract containing
irrelevant Nanobodies. After removal of the non-coupled Nanobodies
during a wash step, the irrelevant Nanobody-bead complex was added
to purified primary human T cells.
[0971] After an incubation of 24 h at 37.degree. and 5% CO.sub.2
the activation status of the human T cells was determined by
measuring the CD69 expression level in flow cytometry using
monoclonal mouse anti-human CD69PE (BD Biosciences #557050).
[0972] 4.3 Sequence Analysis of the Obtained Nanobodies
[0973] Nanobodies which scored positive in the flow cytometric
binding screen and the T cell activation assay were sequenced.
[0974] The sequence analysis demonstrated that all anti-TCR ISVs
comprised a very similar CDR3. In particular, the CDR3 has the
amino acid sequence X.sub.1SR X.sub.2X.sub.3PYX.sub.4Y, in which
X.sub.1 is F, Y, G, L or K, X.sub.2 is I or L, X.sub.3 is Y or W,
and X.sub.4 is D, N or S.
[0975] The sequence analysis further resulted in the identification
of 3 distinct clusters. Corresponding alignments are provided
(Table A-1, Table A-2, Table A-3). Clustering was based on sequence
similarities and differences in CDR2 and CDR3. Cluster A is the
most prominent comprising 104 clones (SEQ ID NOs: 1-104), cluster B
comprises 11 clones (SEQ ID NOs: 105-115), and cluster C is
represented by only 3 clones (SEQ ID NOs: 116-118).
[0976] Sequence variability of the CDRs was determined for the
different clusters. For cluster A, the amino acid sequence of the
CDRs of clone 56G05 was used as a reference, against which the CDRs
of all other cluster A clones were compared. The sequence
variability against 56G05 is depicted in the tables below.
TABLE-US-00007 56G05 CDR1 Kabat 26 27 28 29 30 31 32 33 34 35
numbering absolute 1 2 3 4 5* 6 7 8 9 10 numbering 56G05 G D V H K
I N F L G sequence variations A Y L L I S variations S V variations
E variations G *in case position 5 is an L, then position 6 is also
L
TABLE-US-00008 56G05 CDR2 Kabat 50 51 52 53 54 55 56 57 58
numbering absolute 1 2 3 4 5 6 7 8 9 numbering 56G05 H I S I G D Q
T D sequence variations T T S D V A variations R A A E A Q
variations T N variations A V variations V S
TABLE-US-00009 56G05 CDR3 Kabat 95 96 97 98 99 100 100a 101 102
numbering absolute 1 2 3 4 5 6 7 8 9 numbering 56G05 F S R I Y P Y
D Y sequence variations Y L W N variations G S variations L
[0977] For cluster B, the amino acid sequence of the CDRs of clone
55C07 was used as a reference, against which the CDRs of all other
cluster B clones were compared. The sequence variability against
55C07 is depicted in the tables below.
TABLE-US-00010 55C07 CDR1 Kabat 26 27 28 29 30 31 32 33 34 35
numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering 55C07 G E T F K I
N I W G sequence variations Q V
TABLE-US-00011 55C07 CDR2 Kabat 50 51 52 53 54 55 56 57 58
numbering absolute 1 2 3 4 5 6 7 8 9 numbering 55C07 S L T I G G A
T N sequence variations D
TABLE-US-00012 55C07 CDR3 Kabat 95 96 97 98 99 100 100a 101 102
numbering absolute 1 2 3 4 5 6 7 8 9 numbering 55C07 K S R L Y P Y
D Y sequence variations I
[0978] For cluster C, the amino acid sequence of the CDRs of clone
61G01 was used as a reference, against which the CDRs of all other
cluster C clones were compared. The sequence variability against
61G01 is depicted in the tables below.
TABLE-US-00013 61G01 CDR1 Kabat 26 27 28 29 30 31 32 33 34 35
numbering absolute 1 2 3 4 5 6 7 8 9 10 numbering 61G01 G E I G R I
N F Y R sequence variations
TABLE-US-00014 61G01 CDR2 Kabat 50 51 52 53 54 55 56 57 58
numbering absolute 1 2 3 4 5 6 7 8 9 numbering 61G01 T I T I A D K
T D sequence variations I
TABLE-US-00015 61G01 CDR3 Kabat 95 96 97 98 99 100 100a 101 102
numbering absolute 1 2 3 4 5 6 7 8 9 numbering 61G01 G S R L Y P Y
D Y sequence variations
[0979] The clustering based on the sequence transmuted into
functional differences (see infra).
[0980] 4.4 Purification of Monovalent Nanobodies
[0981] Representative Nanobodies for each cluster were selected and
expressed in E. coli TG1 as triple Flag, His6-tagged proteins.
Expression was induced by addition of 1 mM IPTG and allowed to
continue for 4 hours at 37.degree. C. After spinning the cell
cultures, periplasmic extracts were prepared by freeze-thawing the
pellets. These extracts were used as starting material and
Nanobodies were purified via IMAC and size exclusion chromatography
(SEC).
[0982] The Nanobodies were purified to 95% purity as assessed via
SDS-PAGE (data not shown).
Example 5: Binding of Anti-TCR Nanobodies to Human TCR/CD3
Expressed on CHO-K1 Cells and to Purified Primary Human T Cells
[0983] Binding of purified monovalent anti-TCR Nanobodies to human
TCR(2XN9)/CD3 expressed on CHO-K1 cells and to purified primary
human T cells was evaluated in flow cytometry as outlined in
Example 4.1. Dilution series of Nanobodies 55A02 (cluster A), 56G05
(cluster A), 68G05 (cluster B) and 61G01 (cluster C) starting from
1 .mu.M were applied to the cells.
[0984] The results are shown in FIG. 3.
[0985] Nanobodies clearly bound to human TCR/CD3 expressed on
CHO-K1 cells. The cluster A representatives showed the best
affinity, followed by the cluster B representative and the cluster
C representative. Nanobodies bound to purified primary human T
cells, although with slightly lower potency compared to the CHO-K1
human TCR(2XN9)/CD3 cells. The representatives of cluster A showed
the best affinity for binding human primary T cells, in line with
the data on the CHO-K1 (2XN9)/CD3. The EC50 values obtained from
the dose response curve are represented in Table C-1.
TABLE-US-00016 TABLE C-1 EC50 (M) of anti-TCR monovalent Nanobodies
for binding CHO-K1 human TCR(2XN9)/CD3 cells and for binding
purified primary T cells as determined in flow cytometry.
CHO-K1TCR(2XN9)/CD3 Primary human T cells Cluster sample ID EC50
(M) 95% LCI 95% UCI EC50 (M) 95% LCI 95% UCI A T0170055A02 8.4E-09
7.2E-09 9.7E-09 9.1E-08 8.1E-08 1.0E-07 A T0170056G05 8.9E-09
8.3E-09 9.4E-09 9.1E-08 8.3E-08 9.9E-08 B T0170068G05 1.2E-08
1.0E-08 1.3E-08 >1E-07 / / C T0170061G01 3.1E-08 2.8E-08 3.4E-08
>1E-07 / /
Example 6: Determination of Binding Epitope
[0986] Binding of purified monovalent anti-TCR Nanobodies to human
TCR(2IAN)/CD3 expressed on HEK293H cells was evaluated and compared
with the binding to HEK293H cells transfected with human CD3 in
flow cytometry, as outlined in Example 5. Dilution series of
anti-TCR Nanobodies starting from 1 .mu.M were applied to the
cells. The parental HEK293H cell line was included as TCR/CD3
negative cell line.
[0987] The results are shown in FIG. 4.
[0988] Nanobodies clearly bound to human TCR(2IAN)/CD3 expressed on
HEK293H but not to the HEK293H cells transfected with human CD3
only, nor to the HEK293H parental cell line. The EC50 values
obtained from the dose response curve are depicted in Table
C-2.
TABLE-US-00017 TABLE C-2 EC50 (M) of anti-TCR monovalent Nanobodies
for binding human TCR(2IAN)/CD3 or human CD3 expressed on HEK293H
cells, as determined in flow cytometry. HEK293H wt HEK293H CD3
HEK293H TCR/CD3 cluster Sample ID EC50 MCF at 1 .mu.M EC50 MCF at 1
.mu.M EC50 MCF at 1 .mu.M A T0170055A02 No fit 246 No fit 1194
5.5E-08 91229 A T0170056G05 No fit 299 No fit 352 8.4E-08 86510 B
T0170068G05 No fit 206 No fit 240 >1E-07 31202 C T0170061G01 No
fit 374 No fit 495 >1E-07 10032
[0989] In conclusion, the clones were specific for binding to human
TCR .alpha./p. No binding was observed to human CD3.
Example 7: Binding of Anti-TCR Nanobodies to Soluble Recombinant
Human TCR .alpha./.beta. Protein
[0990] 7.1 Binding of Anti-TCR Nanobodies to Human T Cell Receptor
Protein in ELISA
[0991] Binding of purified monovalent TCR Nanobodies to soluble
recombinant human TCR .alpha./.beta. protein was evaluated in ELISA
(as described in Example 1.2) using 2 .mu.g/ml directly coated
soluble recombinant human TCR .alpha./P protein.
[0992] The results are shown in FIG. 5. The EC50 values obtained
from the dose response curve are depicted in Table C-3.
TABLE-US-00018 TABLE C-3 EC50 (M) of anti-TCR monovalent Nanobodies
for binding soluble recombinant human TCR(2XN9) protein, as
determined in ELISA. Cluster sample ID EC50 (M) 95% LCI 95% UCI A
T0170055A02 1.9E-09 1.7E-09 2.2E-09 A T0170056G05 4.0E-09 3.5E-09
4.6E-09 B T0170068G05 1.6E-08 1.3E-08 1.9E-08 C T0170061G01 5.2E-08
4.2E-08 6.5E-08
[0993] In conclusion, representative clones of all clusters bind to
soluble recombinant human TCR .alpha./P protein.
[0994] 7.2 Binding of Anti-TCR Nanobodies to Human T Cell Receptor
Protein in BLI
[0995] Binding affinities were measured using Bio-Layer
Interferometry (BLI) on an Octet RED384 instrument (Pall ForteBio
Corp.). Recombinant human soluble TCR(2XN9)-zipper protein was
covalently immobilized on amine-reactive sensors (ForteBio) via
NHS/EDC coupling chemistry. For kinetic analysis, sensors were
first dipped into running buffer (10 mM Hepes, 150 mM NaCl, 0.05%
p20, pH7.4 from GE Healthcare Life Sciences) to determine baseline
setting. Subsequently, sensors were dipped into wells containing
different concentrations of purified Nanobodies (range between 1.4
nM and 1 mM) for the association step (180 s) and transferred to
wells containing running buffer for the dissociation (15 min) step.
Affinity constants (KD) were calculated applying a 1:1 interaction
model using the ForteBio Data Analysis software.
[0996] The results are depicted in FIG. 6. The binding
characteristics are listed in Table C-4.
TABLE-US-00019 TABLE C-4 Kinetic analysis of anti-TCR monovalent
Nanobodies for binding soluble recombinant human TCR(2XN9) protein
as determined with the Octet RED384 instrument. Human sTCR
(2XN9)-zipper Cluster sample ID kon(1/Ms) koff(1/s) KD (M) A
T0170055A02 4.9E+04 8.4E-04 1.7E-08 A T0170056G05 5.0E+04 1.2E-03
2.4E-08
[0997] In conclusion, the binding affinity for cluster A
representatives determined using BLI on human soluble 2XN9 showed
correlation with the affinities determined on CHO-K1(2XN9)/CD3
cells in flow cytometry (cf. Example 5).
Example 8: Determination of Purified Primary Human T Cell
Activation Capacity
[0998] Functionality of purified monovalent anti-TCR Nanobodies was
evaluated in the human T cell activation assay. Goat anti-mouse IgG
dynabeads (Life technologies #11033) were coated with mouse
anti-Flag IgG antibodies (Sigma F1804), (15 .mu.g/1E7 beads). After
an incubation period of 2 h at 4.degree. C., beads were washed and
incubated with a fixed (1 .mu.g) amount of purified Flag tagged
Nanobody for 20 min at 4.degree. C. while shaking. Non-coupled
Nanobodies were washed away before adding the bead complex together
with soluble mouse anti-CD28 antibody (Pelicluster CD28--Sanquin
#M1650) to purified primary human T cells isolated (isolated as
described in Example 2.1) from distinct healthy donors.
[0999] In addition, the effect of monovalent TCR binding by the
Nanobodies was evaluated by the incubation of the Nanobody with the
purified primary human T cells without prior capture onto
anti-mouse IgG dynabeads, in the presence of anti-CD28
antibody.
[1000] The activation status of the purified primary human T cells
was monitored by measuring the CD69 expression in flow cytometry
using monoclonal mouse anti-human CD69PE (BD Biosciences #557050)
after an incubation of 24 h at 37.degree. C. and 5% CO.sub.2, as
described in Example 4.2.
[1001] In conclusion, anti-TCR Nanobodies of all clusters showed
clear CD69 upregulation after capturing onto anti-mouse IgG
dynabeads. The irrelevant Nanobody did not show any CD69
upregulation (FIG. 7A). In addition, none of the Nanobodies
presented in solution were able to activate purified primary human
T cells as measured by increased expression of CD69 (FIG. 7B).
Example 9: Binding of Multispecific TCR Binding Polypeptides to
Human T Cell Receptor Complex Expressed on Cells
[1002] To demonstrate that redirection of engaged T cells to tumour
cells is possible by the Nanobodies, the CD20 antigen was chosen as
exemplary tumour target.
[1003] Different TCR binding building blocks (i.e. Nanobodies) were
formatted into a multispecific construct with a human CD20
targeting Nanobody (see Table C-5). The effector and tumour
Nanobodies were genetically linked with a 35GS linker and
subsequently expressed in the yeast Pichia according to standard
protocols (multispecific polypeptides).
[1004] Irrelevant constructs were generated by replacing the
effector or tumour Nanobody with an irrelevant anti-egg lysozyme
(cAblys) Nanobody (Table C-5)
TABLE-US-00020 TABLE C-5 Sample ID and description of multispecific
constructs. Cluster Sample ID SEQ ID NO Description Target Nanobody
.times. Effector Nanobody A T017000014 300
20CD019C07-35GS-T0170028B01-FLAG3-HIS6 A T017000015 301
cAbLys3(D1E)-35GS-T0170028B01-FLAG3-HIS6 ctrl T017000018 302
20CD019C07-35GS-cAbLys3-FLAG3-HIS6 A T017000054 321
20CD019C07-35GS-T0170055A03-FLAG3-HIS6 A T017000055 322
20CD019C07-35GS-T0170055A02-FLAG3-HIS6 A T017000058 323
20CD019C07-35GS-T0170040C01-FLAG3-HIS6 A T017000060 324
20CD019C07-35GS-T0170028B01-FLAG3-HIS6 A T017000076 334
20CD019C07-35GS-T0170056G05-FLAG3-HIS6 A T017000063 325
20CD019C07-35GS-T0170069B08-FLAG3-HIS6 A T017000064 326
20CD019C07-35GS-T0170068E08-FLAG3-HIS6 B T017000068 328
20CD019C07-35GS-T0170055C07-FLAG3-HIS6 B T017000070 330
20CD019C07-35GS-T0170055B06-FLAG3-HIS6 A T017000069 329
20CD019C07-35GS-T0170055B11-FLAG3-HIS6 A T017000050 319
20CD019C07-35GS-T0170069C08-FLAG3-HIS6 A T017000065 327
20CD019C07-35GS-T0170067E06-FLAG3-HIS6 A T017000078 336
20CD019C07-35GS-T0170069F05-FLAG3-HIS6 A T017000079 337
20CD019C07-35GS-T0170067D01-FLAG3-HIS6 C T017000051 320
20CD019C07-35GS-T0170061G01-FLAG3-HIS6 A T017000075 333
20CD019C07-35GS-T0170067E03-FLAG3-HIS6 Effector Nb .times. Target
Nb A T017000019 303 T0170028B01-35GS-20CD019C07-FLAG3-HIS6 A
T017000025 305 T0170028B01-35GS-cAbLys3-FLAG3-HIS6 ctrl T017000023
304 cAbLys3(D1E)-35GS-20CD019C07-FLAG3-HIS6 A T017000041 314
T0170055A03-35GS-20CD019C07-FLAG3-HIS6 A T017000042 315
T0170055A02-35GS-20CD019C07-FLAG3-HIS6 A T017000044 316
T0170040C01-35GS-20CD019C07-FLAG3-HIS6 A T017000046 317
T0170028B01-35GS-20CD019C07-FLAG3-HIS6 A T017000074 332
T0170056G05-35GS-20CD019C07-FLAG3-HIS6 A T017000029 306
T0170069B08-35GS-20CD019C07-FLAG3-HIS6 A T017000035 311
T0170068E08-35GS-20CD019C07-FLAG3-HIS6 B T017000031 308
T0170055C07-35GS-20CD019C07-FLAG3-HIS6 B T017000033 310
T0170055B06-35GS-20CD019C07-FLAG3-HIS6 A T017000032 309
T0170055B11-35GS-20CD019C07-FLAG3-HIS6 A T017000037 312
T0170069C08-35GS-20CD019C07-FLAG3-HIS6 A T017000077 335
T0170067E06-35GS-20CD019C07-FLAG3-HIS6 A T017000049 318
T0170069F05-35GS-20CD019C07-FLAG3-HIS6 A T017000030 307
T0170067D01-35GS-20CD019C07-FLAG3-HIS6 C T017000038 313
T0170061G01-35GS-20CD019C07-FLAG3-HIS6 A T017000073 331
T0170067E03-35GS-20CD019C07-FLAG3-HIS6
[1005] Binding of the multispecific constructs to human TCR/CD3
expressed on CHO-K1 cells, purified primary human T cells and CD20
positive Ramos cells (ATCC: CRL-1596) was evaluated in flow
cytometry as outlined in Example 5. The results are presented in
FIG. 8.
[1006] The EC50 values obtained from the dose response curve are
depicted in Table C-6.
[1007] The data indicate similar binding of the TCRxCD20
multispecific polypeptides compared to their monovalent
counterparts. However, a reduced binding of the CD20XTCR
multispecific polypeptides to CHO-K1 human TCR(2XN9)/CD3 cells and
purified primary human T cells was detected compared to their
monovalent counterparts. On the human CD20 positive Ramos cell
line, the multispecific polypeptide with the CD20 at the C terminus
showed reduced binding in comparison to the polypeptides with the
CD20 at the N terminus.
Example 10: Functional Characterization of Multispecific CD20xTCR
Binding Polypeptides in a Flow Cytometry Based Killing Assay
[1008] In order to assess whether multispecific polypeptides were
able to kill tumour cells, cytotoxicity assays were performed with
isolated human T cells as effector cells.
[1009] Human T cells were isolated as described in Example 2.1. The
quality and purity of the purified human T cells was checked with
anti-CD3 (eBioscience #12-0037-73), anti-CD8 (BD Biosciences
#345775), anti-CD4 (BD Biosciences #345771), anti-CD45RO (BD
Biosciences #555493), anti-CD45RA (BD Biosciences #550855),
anti-CD19 (BD Biosciences #555413), anti-CD25 (BD Pharmigen
#557138) and anti-CD69 (BD Pharmigen #557050) fluorescently
labelled antibodies in a flow cytometric assay. Human CD20
expressing Ramos cells and human CD20 expressing Raji cells (ECACC:
85011429), labelled with the PKH-26 membrane dye as described above
were used as target cells. 2.5.times.10.sup.5 effector and
2.5.times.10.sup.4 target cells were co-incubated in 96-well
V-bottom plates at an effector versus target ratio of 10:1. For
measurement of the concentration-dependent cell lysis, serial
dilutions of multispecific polypeptides (Table C-5) were added to
the samples and incubated for 18 h in a 5% CO.sub.2 atmosphere at
37.degree. C. After incubation, cells were pelleted by
centrifugation and washed with FACS buffer. Subsequently, cells
were resuspended in FACS buffer supplemented with 5 nM TOPRO3
(Molecular Probes cat #T3605) to distinguish live from dead cells.
Cells were analysed using a FACS Array flow cytometer (BD
Biosciences). Per sample, a total sample volume of 80 .mu.l was
acquired. Gating was set on PKH26 positive cells, and within this
population the TOPRO3 positive cells were determined.
[1010] The CD20xTCR binding multispecific polypeptides showed dose
dependent killing of the Ramos cells (FIG. 9A). T017000014 (cluster
A, 20CD019C07-35GS-T0170028B01-FLAG3-HIS6) showed a dose dependent
killing on both Ramos (FIG. 9A) and Raji (FIG. 9B) cells confirming
that the observed cytotoxic effect was not restricted to a single
tumour cell line. The expression level of the tumour antigen CD20
was determined for both cell lines (FIG. 10).
[1011] The IC50 values and the % lysis obtained from the dose
response curve are depicted in Table C-7 (% lysis=% death cells at
500 nM of Nanobody minus % dead cells of the no Nanobody
control).
[1012] These results demonstrate that the TCR multispecific
polypeptides can induce T cell mediated killing of tumour target
positive cell lines. When either the targeting Nanobody or the
effector Nanobody was replaced by an irrelevant Nanobody, no effect
on the viability of the Ramos cells could be observed. There was no
clear preference of the orientation between the individual binding
blocks in the multispecific polypeptide.
Example 11: Functional Characterization of Multispecific CD20xTCR
Binding Polypeptides in an xCELLigence Based Killing Assay
[1013] The TCR binding multispecific polypeptides were also tested
for their cell toxicity on human CD20 transfected adherent target
cells in the presence of human effector T cells using real-time
electrical impedance based technique. Here, fluctuations in
impedance induced by the adherence of cells to the surface of an
electrode were measured. T cells are non-adherent and therefore do
not impact the impedance measurements.
[1014] In brief, the xCELLigence station was placed in a 37.degree.
C. incubator at 5% CO.sub.2. 50 .mu.l of assay medium was added to
each well of E-plate 96 (ACEA Biosciences; cat #05 232 368 001) and
a blank reading on the xCELLigence system was performed to measure
background impedance in absence of cells. Subsequently, human CD20
transfected CHO-K1 or CHO-K1 reference cells (1.times.10.sup.4)
were seeded onto the E-plates 96, and 50 .mu.l of a serial dilution
of multispecific polypeptide was added. After 30 min at RT, 50
.mu.l of human T cells were added per well (3.times.10.sup.5) to
have an effector to target ratio of 30:1. The plate was placed in
the xCELLigence station and impedance was measured every 15 min
during 3 days. The data were analysed using a fixed time point
indicated in the results.
[1015] The IC50 values are depicted in Table C-8.
TABLE-US-00021 TABLE C-8 IC50 (M) of the multispecific polypeptides
in the xCELLigence based human T cell mediated CHO-K1 CD20 killing
assay using an effector to target ratio of 30 to 1, analysed at 44
h after seeding. ID monovalent sample ID IC50 sample ID IC50
Cluster Nanobody (CD20 .times. TCR) n (M) (TCR .times. CD20) n (M)
A T0170055A02 T017000055 2 1.4E-09 T017000042 2 4.2E-09 A
T0170056G05 T017000076 3 3.6E-10 T017000074 3 1.5E-09 B T0170055C07
T017000068 1 1.3E-08 T017000031 1 2.1E-09 C T0170061G01 T017000051
1 2.7E-08 T017000038 1 4.1E-09
[1016] The multispecific polypeptides showed tumour antigen
dependent killing. The multispecific polypeptides were not able to
induce T cell mediated killing of CHO-K1 reference cells, but
induced dose dependent human T cell mediated killing of the CD20
transfected CHO-K1 cells. An example is shown in FIG. 11.
[1017] These results confirm the outcome obtained in the flow
cytometry based killing assay of Example 10. In addition, only when
the tumour target antigen is present T cell mediated killing was
observed, indicating that the multispecific polypeptides are
critically dependent on their target for induction of
cytotoxicity.
Example 12: Linker Length Evaluation of the Multispecific
Polypeptides
[1018] To evaluate the impact of the linker length used in the
CD20/TCR binding multispecific polypeptides on the cytotoxic
capacity, the effector and tumour building blocks were genetically
linked with a 5GS (SEQ ID NO: 376), 9GS (SEQ ID NO: 378) or 35GS
(SEQ ID NO: 385) linker and subsequently expressed in Pichia
according to standard protocols (see Table C-9).
TABLE-US-00022 TABLE C-9 Sample ID and description of multispecific
construct to evaluate impact of linker length. Cluster Sample ID
SEQ ID NO Description Target Nanobody .times. Effector Nanobody A
T017000002 292 20CD019C07-5GS-T0170028B01-FLAG3-HIS6 A T017000008
296 20CD019C07-9GS-T0170028B01-FLAG3-HIS6 A T017000014 300
20CD019C07-35GS-T0170028B01-FLAG3-HIS6 A T017000060 324
20CD019C07-35GS-T0170028B01-FLAG3-HIS6 Effector Nanobody .times.
Target Nanobody A T017000013 299
T0170028B01-9GS-20CD019C07-FLAG3-HIS6 A T017000019 303
T0170028B01-35GS-20CD019C07-FLAG3-HIS6 A T017000046 317
T0170028B01-35GS-20CD019C07-FLAG3-HIS6 Control Polypeptides ctrl
T017000003 293 cAbLys3(D1E)-5GS-T0170028B01-FLAG3-HIS6 ctrl
T017000006 294 20CD019C07-5GS-cAbLys3-FLAG3-HIS6 ctrl T017000009
297 cAbLys3(D1E)-9GS-T0170028B01-FLAG3-HIS6 ctrl T017000012 298
20CD019C07-9GS-cAbLys3-FLAG3-HIS6 ctrl T017000015 301
cAbLys3(D1E)-35GS-T0170028B01-FLAG3-HIS6 ctrl T017000018 302
20CD019C07-35GS-cAbLys3-FLAG3-HIS6 ctrl T017000023 304
cAbLys3(D1E)-35GS-20CD019C07-FLAG3-HIS6 ctrl T017000025 305
T0170028B01-35GS-cAbLys3-FLAG3-HIS6
[1019] The impact of the linker length used in the CD20/TCR binding
multispecific polypeptides on the human primary effector T cell
induced cellular toxicity on the adherent CHO-K1 human CD20
transfected target cells was evaluated using real-time electrical
impedance based technique as described Example 11.
[1020] The results are summarized in FIG. 12.
[1021] All multispecific polypeptides, i.e. all linker lengths
demonstrated specific cell killing. Unexpectedly, the TCR
multispecific polypeptides with the longest linker (35GS linker)
showed the best potency. In view of these results, further
experiments were performed with multispecific polypeptides
comprising the 35GS linker.
Example 13: Influence of Effector to Target Ratio on the Killing
Effect of the Multispecific Polypeptides
[1022] To evaluate the effect of different effector to target (E:T)
ratios on the killing properties of the polypeptides, CD20xTCR
binding multispecific polypeptides were incubated with
2.5.times.10.sup.4 PKH labelled Ramos cells in the presence of
respectively 2.5.times.10.sup.5 (E:T=10:1), 1.25.times.10.sup.5
(E:T=5:1), 5.times.10.sup.4 (E:T=2:1) and 2.5.times.10.sup.4
(E:T=1:1) human primary T cells as described in Example 10.
[1023] Exemplary results are shown in FIG. 13. The IC50 values are
depicted in Table C-10.
TABLE-US-00023 TABLE C-10 IC50 (M) of the multispecific
polypeptides in the flow cytometry based T cell mediated Ramos
killing assay using different effector to target ratios. ID
monovalent sample ID IC50 95% 95% % Cluster Nanobody (CD20 .times.
TCR) E:T n (M) LCI UCI lysis A T0170056G05 T017000076 10 1 7.5E-10
5.2E-10 1.1E-09 25 A T0170056G05 T017000076 5 1 8.9E-10 5.2E-10
1.5E-09 17 A T0170056G05 T017000076 2 1 1.6E-09 6.1E-10 4.1E-09 10
A T0170056G05 T017000076 1 1 4.6E-09 3.0E-10 6.9E-08 4 A
T0170055A02 T017000055 10 1 4.1E-09 3.1E-09 5.3E-09 28 A
T0170055A02 T017000055 5 1 2.5E-09 1.6E-09 4.0E-09 15 A T0170055A02
T017000055 2 1 7.4E-10 3.4E-10 1.6E-09 9 A T0170055A02 T017000055 1
1 3.4E-09 1.8E-10 6.4E-08 3
[1024] Both constructs were able to kill the human CD20 target
cells at different E:T ratios, even at a ratio of 1:1, after an
incubation time of 18 h with little difference in potency. Although
there was an impact of the E:T ratio on the % lysis, this might
also be linked to the incubation time (see below).
Example 14: Time Dependent Cytolytic Activity of CD20/TCR Binding
Multispecific Constructs in the Purified Primary Human T Cell
Mediated Assay in xCELLigence
[1025] To evaluate the impact of incubation time on the killing
properties of the CD20xTCR binding multispecific constructs,
specific lysis of target cells was calculated for different
time-points in xCELLigence. In brief, the xCELLigence station was
placed in a 37.degree. C. incubator at 5% CO.sub.2. 50 .mu.l of
assay medium was added to each well of E-pate 96 (ACEA Biosciences;
cat #05 232 368 001) and a blank reading on the xCELLigence system
was performed to measure background impedance in absence of cells.
Subsequently, human CD20 transfected CHO-K1 or CHO-K1 reference
cells (1.times.10.sup.4) were seeded onto the E-plates 96. After 20
h, 3.times.10.sup.5 purified primary human T cells (described
supra) and 100 nM or 1.5 nM multispecific constructs were added,
respectively. The cell index (CI) was measured every 15 min during
5 days. Using the normalized CI (the normalized cell index--NCI, is
calculated by dividing the cell index value at a particular time
point by the cell index value of the time-point when purified
primary human T cells were added) specific lysis at different time
points of the condition with constructs was calculated in relation
to the condition lacking construct. (% specific lysis=((NCI.sub.no
construct-NCI.sub.with construct)/NCI.sub.no
construct)).times.100.
[1026] The results are depicted in FIG. 14.
[1027] Already one hour after the addition of human primary T cells
and the multispecific construct, an increase of cell lysis can be
observed which clearly increased further upon longer incubation
times. The maximal effect was clearly dependent on the incubation
time but the obtained IC50 value did not change with increased
incubation times. The irrelevant construct did not show any killing
of the human CD20 transfected CHO-K1 cells.
Example 15: Exploration of Half-Life Extension (HLE)
[1028] It was hypothesized that HLE via albumin binding might be
suitable to comply with various requirements, including (i)
half-life extension (HLE) of the moiety; and (ii) efficacy of the
multispecific polypeptide. Preferably, the HLE function would not
impair the penetration of tumours and tissues.
[1029] Alb11 (SEQ ID NO: 404), a Nanobody binding to human serum
albumin (HSA) was linked to the multispecific CD20xTCR binding
polypeptides to increase the in vivo half-life of the formatted
molecules (WO 06/122787). A number of formats were generated based
on the CD20 tumour targeting building block at the N-terminus, the
TCR.alpha./.beta. recruiting building blocks in the middle and the
albumin targeting Nanobody at the C-terminus using a 35GS linker
and expressed as indicated above. An overview of the explored
formats is shown in Table C-11.
TABLE-US-00024 TABLE C-11 Sample ID and description of HLE
constructs. Cluster Sample ID SEQ ID NO Description A T017000093
340 20CD019C07-35GS- T0170056G05-35GS- ALB11-FLAG3-HIS6 B
T017000095 341 20CD019C07-35GS- T0170055C07-35GS-
ALB11-FLAG3-HIS6
[1030] As the addition of the Alb11 Nanobody might influence the
affinity or potency of the construct and the binding of HSA to the
Alb11 Nanobody might have an impact on the affinity or potency of
the half-life extended constructs, the half-life extended
constructs were characterized for binding to TCR overexpressing
CHO-K1 and primary human T cells. In addition, the potency in the
functional T cell dependent Ramos B cell killing assay was
evaluated in the presence and absence of HSA (described in 15.1 and
15.2 below).
[1031] 15.1 Impact of Alb11 Building Block on the Binding
Properties
[1032] Analogous to the experiments described in Example 5, binding
of half-life extended anti-TCR polypeptides to CHO-K1 human
TCR(2XN9)/CD3 cells, primary human T cells and Ramos cells was
evaluated in a flow cytometric assay in the absence of HSA.
[1033] The results are provided in FIG. 15. The EC50 values
obtained in this assay are listed in Table C-12. Comparison of the
CD20-35GS-TCR HLE construct with the non-HLE constructs showed
similar binding on all three cell lines tested. The data presented
showed that coupling of the Alb11 building block did not influence
the binding properties.
[1034] 15.2 Impact of Human Serum Albumin on Potency in Human T
Cell Mediated B Cell Killing Assay
[1035] The functionality of half-life extended anti-TCR
polypeptides was evaluated in the human T cell mediated Ramos
killing assay as described in Example 10 in the presence and
absence of 301M HSA and compared with the functionality of the
non-HLE multispecific constructs.
[1036] The results are depicted in FIG. 16. The IC50 values
obtained in this assay are listed in Table C-13.
TABLE-US-00025 TABLE C-13 IC50 (M) of and % lysis by the HLE
polypeptides in the T cell dependent B cell (Ramos) killing assay
to evaluate the effect of HLE. sample IC50 95% 95% % Cluster ID (M)
LCI UCI lysis A T017000076 1.1E-10 7.6E-11 1.5E-10 25 A T017000093
3.4E-10 2.4E-10 4.9E-10 28 A T017000093 2.7E-10 2.1E-10 3.5E-10 26
A T017000093 +HSA 1.6E-09 1.1E-09 2.2E-09 21 B T017000068 5.6E-10
2.7E-10 1.2E-09 12 B T017000095 3.3E-09 1.8E-09 5.8E-09 16 B
T017000095 1.1E-09 6.5E-10 2.0E-09 16 B T017000095 +HSA 5.7E-09
2.0E-09 1.6E-08 9
[1037] The results indicate that the inclusion of the albumin
targeting Nanobody in the construct as such did not have an
essential impact on the obtained potency or efficacy. Although a
minor loss of efficacy/potency was observed in the presence of HSA,
the half-life extended TCR multispecific polypeptides were still
potent in tumour cell killing.
Example 16: Functional Characterization of Multispecific
Polypeptides in an xCELLigence Based Human T Cell Mediated
HER2-Positive Tumour Killing Assay
[1038] In order to assess the general applicability of the TCR
building blocks in directing T cells to tumour cells, TCR binding
building blocks were combined with building block that binds a
different TAA, in this case a Nanobody binding to HER2.
[1039] The anti-TCR building block was combined with a Nanobody
that binds the HER2 solid tumour antigen in two orientations (Table
C-14) and characterized in the xCELLigence based human T cell
mediated HER2-positive tumour killing assay as described in Example
11 using two HER2 expressing cell lines (SKBR3 (ATCC: HTB-30),
MCF-7 (ATCC: HTB-22)) and a HER2 negative reference cell line
(MDA-MB-468 (ATCC HTB-132)) as target cell population. Human HER2
expression levels were confirmed using 100 nM of the monovalent
anti-HER2 Nanobody HER2005F07 (SEQ ID NO: 350) in flow cytometry as
described in Example 5. Results are shown in FIG. 17.
TABLE-US-00026 TABLE C-14 Sample ID and description of HER2/TCR
binding polypeptides. Cluster Sample ID SEQ ID NO Description
Target Nb .times. Effector Nb A T017000102 342 HER2005F07(Q108L)-
35GS-T0170056G05- FLAG3-HIS6 Effector Nb .times. Target Nb A
T017000103 343 T0170056G05-35GS- HER2005F07(Q108L)- FLAG3-HIS6
[1040] In brief, SKBR3 (4.times.10.sup.4 cells/well), MDA-MB-468
(4.times.10.sup.4 cells/well) or MCF-7 (2.times.10.sup.4
cells/well) were seeded in 96 well E-plates and incubated with
6.times.10.sup.5 cells or 3.times.10.sup.5 cells human primary T
cells (effector target ratio of 15 to 1) in the presence or absence
of the multispecific constructs and followed over time. Data were
analysed after 18 h and are shown in FIG. 18.
[1041] The IC50 values obtained in this assay are listed in Table
C-15.
[1042] The data indicate specific killing of HER2-positive tumour
cell lines by directing human primary T cells to the tumour cells
via the anti-TCR Nanobody. Hence, the TCR binding building blocks
are broadly applicable for directing cytotoxic T cells to tumours.
Despite the large difference in tumour antigen density on SKBR3 and
MCF-7 cells, both were efficiently killed by the addition of
multispecific polypeptide constructs.
Example 17: Effect of HER2/TCR Binding Polypeptides on IFN-.gamma.
Release by Human T Cells in the HER2-Positive Tumour Cell Killing
Assay
[1043] To further evaluate the broad applicability of the TCR
binding building blocks, the induction of cytokine release was
monitored during the human T cell mediated SKBR3 killing assay
based on xCELLigence. The release of the cytokine IFN-7 was
measured by ELISA. Briefly, SKBR3 cells were seeded in 96 E-plate
in the presence of purified human primary T cells with or without
multispecific HER2/TCR binding or irrelevant polypeptides as
described in Example 16. 72 h after the addition of the human
primary T cells/polypeptides to the E-plates, IFN-7 production by
the human primary T cells was measured. Maxisorp 96-well ELISA
plates (Nunc) were coated with anti-human IFN-7 antibody (BD
Biosciences #551221). After overnight incubation, plates were
washed and blocked with PBS+2% BSA for 1 h at room temperature.
Next, plates were incubated with 1001 of the supernatants (2 fold
diluted) and 1 .mu.g/ml biotinylated anti-human IFN-7 antibody (BD
Biosciences, #554550) for 2 h 30 min while shaking, washed again
and incubated with streptavidin-HRP (Dakocytomation #P0397). After
30 min, TMB One Solution (Promega #G7431) was added. The reaction
was stopped with 2M H.sub.2SO.sub.4 and the polypeptide dose
dependent production of IFN-7 was determined by measurement of the
OD at 405 nm using the Tecan sunrise 4.
[1044] The results are shown in FIG. 19. The EC50 values obtained
in this assay are listed in Table C-16.
[1045] The multispecific HER2/TCR binding polypeptides induced a
dose dependent production of the cytokine IFN-7, indicating that
the human T cells were activated only in presence of the relevant
polypeptide.
Example 18: Cynomolgus Cross-Reactivity of Anti-TCR Nanobodies
[1046] The cross-reactivity of the TCR binding building blocks with
cynomolgus monkey TCR was evaluated.
[1047] 18.1 Functional Characterization of the Multispecific
Polypeptides in a Cynomolgus T Cell Mediated Ramos CD20 Positive
Tumour Killing Assay
[1048] In a first experiment, a flow cytometric killing assay was
set up, essentially as described in Example 10, using
2.5.times.10.sup.5 primary cynomolgus T cells (isolated using Pan T
Cell Isolation Kit MACS #130-091-993) as effector cells and
2.5.times.10.sup.4 human CD20 positive Ramos cells as target
cells.
[1049] The IC50 values and the % lysis obtained from the dose
response curve are depicted in Table C-17. The results are shown in
FIG. 20.
[1050] The TCR binding multispecific polypeptides that contained a
TCR binding building block belonging to cluster A showed dose
dependent killing of the Ramos cells using cynomolgus T cells.
[1051] 18.2 Functional Characterization of the Multispecific
Polypeptides in a Cynomolgus T Cell Mediated CHO-K1 Human CD20
Positive Cell Killing Assay
[1052] To further assess the cross-reactivity of the TCR binding
building blocks in the TCR/CD20 binding multispecific constructs,
the xCELLigence based killing assay using purified primary
cynomolgus T cells essentially as described in Example 11 was
used.
[1053] The assay used an effector to target ratio of 30 to 1, i.e.
3.times.10.sup.5 effector cynomolgus T cells (isolated using Pan T
Cell Isolation Kit MACS #130-091-993) and 1.times.10.sup.4 target
CHO-K1 human CD20 cells.
[1054] The IC50 values obtained in this assay are listed in Table
C-18. The results are summarized in FIG. 21.
[1055] It can be concluded that the TCR binding multispecific
polypeptides that contain a TCR binding building block belonging to
cluster A showed dose dependent killing of the CHO-K1 CD20
transfected cells using cynomolgus T cells. Hence, cluster A
Nanobodies cross-react with primary cynomolgus T cells and can
elicit potent killing based on these cynomolgus T cells.
[1056] 18.3 Binding of Anti-TCR Nanobodies to Cynomolgus T Cell
Receptor Protein (ELISA)
[1057] Binding of purified monovalent anti-TCR Nanobodies to
soluble recombinant cynomolgus TCR .alpha./P protein was evaluated
in ELISA (as described in Example 1.2) using 2 .mu.g/ml directly
coated recombinant soluble cynomolgus TCR-.alpha./.beta. zipper
protein.
[1058] The EC50 values obtained from the dose response curve are
depicted in Table C-19.
[1059] An exemplary result is shown in FIG. 22.
TABLE-US-00027 TABLE C-19 EC50 (M) of anti-TCR monovalent
Nanobodies for binding to soluble recombinant cynomolgus TCR/CD3
protein as determined in ELISA. sample EC50 95% 95% Cluster ID (M)
LCI UCI A T0170055A02 1.6E-07 1.5E-07 1.7E-07 A T0170056G05 7.7E-08
6.6E-08 9.1E-08 B T0170068G05 8.7E-08 8.5E-08 8.9E-08 C T0170061G01
>1E7.sup.
[1060] The results indicate that the anti-TCR Nanobodies from
cluster A and cluster B bind to the recombinant soluble cynomolgus
TCR-.alpha./.beta. zipper protein.
[1061] 18.4 Evaluation of Cynomolgus Cross-Reactivity in Bio-Layer
Interferometry
[1062] Binding affinities of the monovalent anti-TCR Nanobodies
were measured using Bio-Layer Interferometry (BLI) on an Octet
RED384 instrument (Pall ForteBio Corp.) essentially as described in
Example 7.1 using cynomolgus TCR .alpha./.beta. zipper. The results
are depicted in FIG. 23, the binding characteristics of the
anti-TCR Nanobodies are listed in Table C-20.
TABLE-US-00028 TABLE C-20 Binding characteristics of monovalent
anti-TCR Nanobodies determined in Octet using directly coated
cynoTCR-zipper protein. Cluster sample ID kon(1/Ms) koff(1/s) KD
(M) A T0170055A02 1.1E+05 2.4E-02 2.1E-07 A T0170056G05 1.1E+05
1.6E-02 1.5E-07
[1063] The cluster A Nanobodies bind to the soluble recombinant
cynomolgus TCR .alpha./.beta. zipper with a 10 fold lower affinity
compared to soluble recombinant human TCR .alpha./.beta.
zipper.
[1064] 18.5 Functional Characterization of Half-Life Extended
Multispecific Polypeptides in a Cynomolgus T Cell Mediated Ramos
CD20 Positive Tumour Killing Assay
[1065] Analogous to the set up described in Example 18.1, the
half-life extended TCR binding polypeptides were evaluated in a
cynomolgus T cell mediated Ramos killing assay.
[1066] The IC50 values obtained in this assay are listed in Table
C-21. The results are depicted in FIG. 24.
TABLE-US-00029 TABLE C-21 IC50 of and % lysis by HLE multispecific
polypeptides in the cynomolgus T cell dependent B cell (Ramos)
killing assay to evaluate the effect of HLE. sample IC50 95% 95% %
Cluster ID (M) LCI UCI lysis A T017000076 5.2E-10 2.9E-10 9.5E-10
29 A T017000093 1.0E-09 5.5E-10 2.0E-09 28 A T017000093 +HSA
8.2E-10 4.7E-10 1.4E-09 18
[1067] The HLE extended TCR binding multispecific polypeptides that
contain a TCR binding building block belonging to cluster A showed
dose dependent killing of the Ramos cells using purified primary
cynomolgus T cells. The inclusion of the ALB11 in the construct as
such did not impact the potency (overlapping CI). Upon addition of
HSA, a small drop in efficacy was observed while the potency was
not affected.
[1068] 18.6 Functional Characterization of Half-Life Extended
CD20xTCR Binding Multispecific Polypeptides in a Cynomolgus T Cell
Mediated CHO-CD20 Positive Tumour Cell Killing Assay
[1069] To confirm the data in the flow cytometry based assay, the
HLE constructs were tested in the xCELLigence based CHO-K1 human
CD20 killing using purified primary cynomolgus T cells as described
in Example 11.
[1070] The results are shown in FIG. 25. The IC50 values obtained
from the dose response curve are depicted in Table C-22.
TABLE-US-00030 TABLE C-22 IC50 (M) of the TCR/CD20 binding
multispecific and HLE constructs in the cynomolgus T cell mediated
CHO-K1 human CD20 tumour killing assay to evaluate the effect of
ALB11 and HSA. sample ID IC50 (M) 95% LCI 95% UCI T017000076
1.4E-10 9.5E-11 2.1E-10 T017000093 1.9E-10 1.3E-10 2.8E-10
T017000093 +HSA 7.4E-10 5.0E-10 1.1E-09
[1071] The HLE TCR binding multispecific polypeptides showed dose
dependent killing of CHO-K1 human CD20 transfected cells using
cynomolgus T cells, confirming that the ALB11 has no impact on the
cynomolgus cross-reactivity of the TCR binding building block.
[1072] 18.7 Functional Characterization of Multispecific
Polypeptides in a Cynomolgus T Cell Mediated HER2 Positive Tumour
Cell Killing Assay
[1073] The multispecific polypeptides were functionally
characterized in a cynomolgus T cell mediated HER2 positive tumour
cell killing assay. In short, the TCR/HER2 binding multispecific
polypeptides were evaluated in a xCELLigence based killing assay
essentially as described in Example 16, using 6.times.10.sup.5
cynomolgus T cells as effector cells and 4.times.10.sup.4 SKBR3 as
target cells (effector to target of 30 to 1). Data were analysed
after 18 h.
[1074] The results are depicted in FIG. 26. The IC50 values
obtained in this assay are listed in Table C-23.
[1075] The cynomolgus cross-reactivity of the TCR binding building
block was confirmed using the HER2/TCR binding multispecific
constructs.
Example 19: In Vivo Proof-of-Concept in a Ramos B Cell Depletion
Model
[1076] In this B cell depletion model, Ramos cells (a Burkitt's
lymphoma cell line) and human PBMC were injected respectively
intravenously and intraperitoneally in to NOG mice. Ramos B cell
and PBMC-derived B cell killing by Nanobody-mediated recruitment of
T cells present in the PBMC population was evaluated reflecting the
potential of multispecific polypeptides to activate T cells by
direct linkage of T cells via TCR to target B cells via CD20,
resulting in target cell killing.
[1077] The in vivo efficacy of the bi-specific polypeptide
T017000083 (CD20xTCR binding) on B cell depletion in a Ramos NOG
mouse model was evaluated and compared with the irrelevant
multispecific polypeptide T017000088 (irrelevant Nanobody+TCR
binding Nanobody). The study demonstrated a statistically
significant effect in bone marrow and spleen on Ramos B cell
depletion and on PBMC derived B cell depletion in spleen.
[1078] In detail, the B cell depletion was evaluated in mice,
intravenously injected with 10.sup.6 Ramos cells in 200 .mu.L of
Roswell Park Memorial Institute (RPMI) medium 1640 at day one (D1).
This injection took place 24 hours after a whole body irradiation
of mice with a .gamma.-source (1.44 Gy, 60Co) (D0). 10.sup.7 PBMCs
(500 .mu.L in PBS) were injected on D3 (i.e. two days after tumor
cell injection) after randomization of the mice into groups each of
24 animals. The treatment started on D3 one hour after PBMC
injection and was repeated for 5 consecutive days in total until D7
(FIG. 27). Three dose levels of the TCR/CD20 binding polypeptides
were tested (0.5 mg/kg, 5 mg/kg and 23 mg/kg).
[1079] On D20 or on D21, mice were sacrificed and spleen and bone
marrow (femur) were collected for FACS analysis (mCD45, hCD45,
hCD19, hCD20, hCD10) to analyze and quantify the presence of Ramos
B cells (hCD19+ hCD20+ hCD45+ mCD45- hCD10+) and PBMC-derived B
cells (hCD19+ hCD20+ hCD45+ mCD45- hCD10-).
[1080] Results for Ramos B cell depletion are represented in FIG.
28A and FIG. 28B. Mice treated with an irrelevant multispecific
polypeptide were considered as control group for analyses. From
FIG. 28A, a dose response pattern was seen in the bone marrow for
T017000083 (CD20/TCR) versus the irrelevant multispecific
polypeptide for reducing Ramos cell numbers. The 2 highest dose
levels for the CD20/TCR binding multispecific polypeptide were
statistically significantly different from the irrelevant
multispecific polypeptide. Statistical analysis has been performed
with F-tests from the mixed-effects ANOVA analysis. For the spleen,
all tested dose levels of T017000083 were statistically
significantly different from the irrelevant multispecific
polypeptide, as depicted in FIG. 28B. The dose response pattern in
spleen was less pronounced since all doses were close to or
estimated to be on the maximum effect and thus very similar.
[1081] Results for PBMC-derived B cell depletion are represented in
FIG. 28C and FIG. 28D. In bone marrow, a dose response pattern was
seen for T017000083 as depicted in FIG. 28C. The estimated
difference in human B cell numbers for T017000083 versus the
irrelevant multispecific polypeptide for the 2 highest dose levels
was statistically significantly different at the 5% level of
significance. In the spleen, B cell counts were statistically
significantly different from the irrelevant multispecific
polypeptide-treated group in all the groups treated with
T017000083, and this at all tested dose levels. The dose response
pattern was less pronounced since all doses were close to or
estimated to be on the maximum effect and thus very similar,
although an increase in estimated difference with increasing dose
was observed.
[1082] In conclusion, these results demonstrate that CD20/TCR
multispecific polypeptides are able to significantly decrease Ramos
B cells and PBMC-derived B cells in spleen and Ramos B cells in
bone marrow in this model. This confirms the polypeptide-induced T
cell activation by cross-linking T cells to target B cells and
killing of the latter.
Example 20: In Vivo Proof-of-Concept in a PBMC B Cell Depletion
Model
[1083] In this B cell depletion model, human PBMC were injected
intraperitoneally in to NOG mice. PBMC-derived B cell killing by
polypeptide-mediated recruitment of T cells present in the PBMC
population was evaluated reflecting the potential of the
polypeptides of the invention to activate T cells by direct linkage
of T cells via TCR to target B cells via CD20, resulting in target
cell killing.
[1084] The in vivo efficacy of the multispecific polypeptide
T017000083 (CD20xTCR binding) on B cell depletion in a PBMC NOG
mouse model was evaluated and compared with the irrelevant
polypeptide T017000088. The study demonstrated a clear effect on
PBMC derived B cell depletion in spleen.
[1085] In detail, the B cell depletion was evaluated in mice,
intraperitoneally injected with 3.times.10.sup.7 PBMCs in 500 .mu.L
of PBS at day three (D3) after a whole body irradiation of mice
with a .gamma.-source (1.44 Gy, 60Co) (D0) and randomization of the
mice into groups each of 12 animals. The treatment started on D3
one hour after PBMC injection and was repeated for 5 consecutive
days, in total until day 7 (D7) (FIG. 29). Three dose levels of the
CD20/TCR binding polypeptide were tested (0.5 mg/kg, 5 mg/kg and 23
mg/kg).
[1086] On day 18 (D18), mice were sacrificed and the spleen was
collected for FACS analysis (mCD45, hCD45, hCD19, hCD20) to analyze
and quantify the presence of PBMC-derived human B cells (hCD19+
hCD20+ hCD45+ mCD45-).
[1087] Results for PBMC-derived B cell depletion are represented in
FIG. 30. In the spleen, B cell counts were clearly different from
the irrelevant polypeptide-treated group in all the groups treated
with T017000083 and this at all tested dose levels. The dose
response pattern is not pronounced since all doses are close to or
estimated to be on the maximum effect and thus very similar.
[1088] In conclusion, these results demonstrate that a CD20/TCR
binding multispecific polypeptide is able to significantly decrease
PBMC-derived B cells in spleen in this model. This confirms the
polypeptide-induced T cell activation by cross-linking T cells to
target B cells and killing of the latter.
Example 21: Targeting of Tumour Cells with Multispecific T Cell
Engaging Polypeptides
[1089] The therapeutic activity of T cell engaging strategy can be
improved by the simultaneous targeting of multiple tumour
associated antigens. Often tumour cells create an escape mechanism
by the down-regulation of targeted antigens within a therapy. The
simultaneous targeting of multiple antigens is likely to reduce the
probability of generating tumour escape variants. The individual
affinity of the respective tumour targeting Nanobodies may be
varied such that preferable binding to either a single marker or
simultaneous binding to both tumour markers is achieved. Antigens
present on different cell populations can be combined or even
soluble proteins can be targeted in combination with a tumour
associated antigen.
[1090] As the Nanobody platform is ideally suited to combine
different specificities into a multispecific format, the anti-TCR
Nanobodies of the invention are combined into formats illustrating
these concepts, i.e. with different tumour antigen binding
Nanobodies in a multispecific polypeptide.
[1091] For the double tumour antigen targeting concept, a Nanobody
reactive towards a first tumour antigen (TA1, e.g. CEA) is linked
to a second Nanobody with different specificity (TA2, e.g. EGFR),
different from TA1, in combination with a TCR reactive Nanobody.
The specific order of the building blocks is varied within the
format as well as the applied linker lengths in between the
different building blocks. Combinations of TA1 and TA2 which are
tested are depicted in Table C-24.
TABLE-US-00031 TABLE C-24 Combination of TCR, TA1, TA2 and Alb
binding building blocks in multispecific polypeptides. T cell ISV
TA1 ISV TA2 ISV ALB-ISV TCR CEA Irr + TCR CEA Irr - TCR CEA EGFR +
TCR CEA EGFR - TCR Irr EGFR + TCR Irr EGFR -
[1092] In order to test half-life extension, an Alb Nanobody is
included as well in the polypeptides as set out in Table C-24.
[1093] To demonstrate the specific killing, a mixed cell culture
assay system is used where TA1 single positive (e.g. MC38-huCEA or
MKN45) and TA2 single positive tumour cells (e.g. Hela or Her14)
are co-incubated. The expression level of the respective tumour
antigens was determined in different cell lines and is represented
in FIG. 31. Upon addition of the polypeptides of the invention,
primary human T cells and albumin if required, the T cell mediated
cytotoxicity is monitored based on a cytometric read out. A
comparison is made with respect to double negative cells or formats
containing one or more irrelevant Nanobodies.
[1094] In order to verify the specific killing, the induced killing
of double positive tumour (for TA1 and TA2, e.g. LS174T or LoVo)
cells is compared with the induced killing of single positive
tumour cells. For this, a T cell mediated cytoxicity assay is used
as described above with a single type of tumour cells positive for
both markers (cf. Example 19).
Example 22: Targeting of Tumour Cells with Multispecific T Cell
Engaging Polypeptides
[1095] As mentioned above, the therapeutic activity of T cell
engaging strategy can be improved by the simultaneous targeting of
multiple tumour associated antigens. Not only do tumour cells
create an escape mechanism by the down-regulation of targeted
antigens within a therapy, but also by introducing
(point-)mutations. Also in this case, simultaneous targeting of
multiple epitopes on an antigen is likely to reduce the probability
of generating tumour escape variants. Moreover, targeting multiple
epitopes on a single antigen can increase the affinity of binding
(avidity effect).
[1096] As the Nanobody platform is ideally suited to combine
different specificities into a multivalent format, the anti-TCR
Nanobodies of the invention are combined into formats illustrating
these concepts, i.e. with different tumour antigen binding
Nanobodies in a multispecific polypeptide.
[1097] For the multivalent tumour antigen targeting concept, two
Nanobodies reactive towards an antigen are linked (TA1 and TA2,
respectively), followed by a TCR reactive Nanobody. The specific
order of the building blocks is varied within the format as well as
the applied linker lengths in between the different building
blocks. Combinations of TA1 and TA2 which are tested are depicted
in Table C-25.
TABLE-US-00032 TABLE C-25 Combination of TCR, TA1, TA2 and Alb
binding building blocks in multispecific polypeptides. T cell ISV
TA1 ISV TA2 ISV ALB-ISV TCR EGFR-1 (7D12) EGFR-2 (9G08) + TCR
EGFR-1 (7D12) EGFR-2 (9G08) - TCR Her2-1 (5F07) Her2-2 (47D05) +
TCR Her2-1 (5F07) Her2-2 (47D05) -
[1098] In order to test half-life extension, an albumin binding
Nanobody is included as well in the polypeptides as set out in
Table C-25.
[1099] The potency and efficacy of these multivalent formats is
evaluated and compared with the respective bispecific formats in an
in vitro tumour cell killing assay comparable to the assay
described in Example 10 but with the relevant cell lines (e.g.
Hela, Her14, Ls174T, SKBR3, MCF7). Additionally, the
effector-target ratio is varied such that an estimate is made
whether a multivalent/multispecific polypeptide has a higher
efficacy with lower effector target ratios.
Example 23: Binding of Monovalent Nanobodies and Multispecific
Polypeptides to Cells in Flow Cytometry
[1100] As described earlier, the therapeutic activity of T cell
engaging strategy can be improved by the simultaneous targeting of
multiple tumour associated antigens, as tumour cells often create
an escape mechanism by the down-regulation of targeted antigens
within a therapy. The simultaneous targeting of multiple antigens
is likely to reduce the generation of tumour escape variants.
[1101] For this double tumour antigen targeting concept, a Nanobody
reactive towards a first tumour antigen (EGFR) was linked to a
second Nanobody with different specificity (CEACAM5), in
combination with a TCR reactive Nanobody. The specific order of the
building blocks was varied within the format. The effector and
tumour Nanobodies were genetically linked with 35GS linker and
subsequently expressed in the yeast Pichia according to standard
protocols. Irrelevant constructs were generated by replacing the
tumour Nanobody with an irrelevant anti-egg lysozyme (cAblys)
Nanobody (Table C-26).
TABLE-US-00033 TABLE C-26 Sample ID and description of
multispecific polypeptides. Sample ID SEQ ID NO TAA1 TAA2 T cell
ISV Description T017000107 390 EGFR CEACAM5 TCR EGFR038G07-35GS-
NbCEA5-35GS- T0170056G05-FLAG3-HIS6 T017000109 391 Irrelevant
CEACAM5 TCR cAbLys3(D1E, Q5V, A6E, Q108L)-35GS-NbCEA5-
35GS-T0170056G05- FLAG3-HIS6 T017000110 392 EGFR Irr TCR
EGFR038G07-35GS- cAbLys3(D1E, Q5V, A6E, Q108L)-35GS-
T0170056G05-FLAG3-HIS6
[1102] Dose-dependent binding of the monovalent Nanobodies and
multispecific polypeptides to cancer cell lines expressing CEACAM5
and EGFR (LoVo; ATCC CCL-229 and LS174T; ECACC 87060401), a cell
line expressing EGFR (HER14; NIH3T3 (ATCC CRL-1658) transfected
with EGFR), and to purified primary human T cells (isolated as
described in Example 2.1) was evaluated in flow cytometry as
outlined in Example 5. The results are presented in FIG. 32.
[1103] The expression level of the respective tumour antigens on
the different cells was determined in flow cytometry using 100 nM
of a monovalent anti-EGFR Nanobody (EGFR038G07) and a monovalent
anti-CEA Nanobody, as described in Example 5. Results are shown in
FIG. 33.
[1104] The EC50 values obtained from the dose response curve for
binding HER14 cells are depicted in Table C-27. The EC50 values
obtained from the dose response curve for binding LS174T and LoVo
cells are depicted in Table C-28.
TABLE-US-00034 TABLE C-27 EC50 (M) of monovalent Nanobodies and
multispecific polypeptides for binding HER14 cells as determined in
flow cytometry. HER14 sample ID EC50 (M) 95% LCI 95% UCI Top
T017000107 1.6E-09 1.4E-09 1.9E-09 146111 T017000109 / / / /
T017000110 7.3E-09 6.4E-09 8.3E-09 164582 EGFR038G07 1.8E-09
1.6E-09 2.1E-09 166237 NbCEA5 / / /
TABLE-US-00035 TABLE C-28 EC50 (M) of monovalent Nanobodies and
multispecific polypeptides for binding LoVo and LS174T cells as
determined in flow cytometry. LS174T LoVo sample ID EC50 (M) 95%
LCI 95% UCI Top EC50 (M) 95% LCI 95% UCI Top T017000107 1.1E-08
9.7E-09 1.3E-08 64605 3.2E-09 2.7E-09 3.7E-09 88604 T017000109
2.5E-08 2.2E-08 2.9E-08 50982 3.6E-08 1.4E-08 9.1E-08 18514
T017000110 6.3E-09 5.5E-09 7.2E-09 40779 2.4E-09 2.1E-09 2.8E-09
95866 EGFR038G07 9.3E-10 8.1E-10 1.1E-09 32191 5.4E-10 4.8E-10
6.2E-10 52268 NbCEA5 5.9E-10 5.3E-10 6.6E-10 42978 1.2E-09 6.4E-10
2.4E-09 11238
[1105] The data showed binding of the EGFR monovalent Nanobody and
of the multispecific polypeptides containing the EGFR building
block to the HER14 cells, expressing only EGFR. No binding of the
CEACAM monovalent Nanobody and the multispecific polypeptides
containing only the CEACAM5 tumour anchor building block was
observed. All monovalent and multispecific polypeptides showed
binding to the EGFR, CEACAM5 double positive cell lines LS174T and
LoVo, as expected. There was also binding of the multispecific
polypeptides to the human primary T cells. A drop in affinity of
the multispecific polypeptides versus the monovalent TCR building
block was observed due to the C-terminal position of the TCR
building block.
Example 24: Binding of Monovalent Nanobodies to Human EGFR and
CEACAM5 Protein (SPR)
[1106] Binding affinity of the purified EGFR monovalent Nanobody
was evaluated by means of a surface plasmon resonance (SPR) based
affinity determination on a Biacore T100 instrument. Thereto, hEGFR
(Sino Biological, #10001-H08H) was immobilized onto a CM5 chip via
amine coupling, using EDC and NHS chemistry. Purified Nanobodies
were injected for 2 minutes at different concentrations (between
1.37 and 3000 nM) and allowed to dissociate for 15 min at a flow
rate of 45 .mu.l/min. In between sample injections, the surfaces
were regenerated with 50 mM NaOH. HBS-EP+ (Hepes buffer pH7.4) was
used as running buffer.
[1107] Binding affinities of the purified CEACAM5 monovalent
Nanobody was evaluated by means of an SPR based affinity
determination on a Biacore T100 instrument. Thereto, hCEACAM-5
(R&D Systems, #4128-CM) was immobilized onto a CM5 chip via
amine coupling, using EDC and NHS chemistry. Purified Nanobodies
were injected for 2 minutes at different concentrations (between
0.31 and 2000 nM) and allowed to dissociate for 15 min at a flow
rate of 45 .mu.l/min. In between sample injections, the surfaces
were regenerated with 10 mM Glycine pH 1.5. HBS-EP+(Hepes buffer
pH7.4) was used as running buffer.
[1108] The kinetic constants were calculated from the sensorgrams
using the BIAEvaluation software (1:1 interaction). The affinity
constants (KD) were calculated from resulting association and
dissociation rate constants kon and koff, and are shown in Table
C-29.
TABLE-US-00036 TABLE C-29 Affinity constant of monovalent
Nanobodies for binding hEGFR and hCEACAM5, determined in Biacore
using directly coated proteins. hCEACAM5 hEGFR ECD kon (1/Ms) Koff
(1/s) KD (M) kon (1/Ms) koff (1/s) KD (M) EGFR038G07 / / / 5.58E+05
4.26E-04 7.63E-10 NbCEA5 9.9E+05 5.1E-04 5.1E-10 / / /
Example 25: Redirected Cell Killing of Multispecific Polypeptides
by Human Effector T Cells in the xCELLigence Based Assay
[1109] The multispecific polypeptides were functionally
characterized in a human T cell mediated EGFR/CEACAM positive
tumour cell killing assay. In short, the multispecific constructs
were evaluated in a xCELLigence based killing assay essentially as
described in Example 16, using 6.times.10.sup.5 human T cells as
effector cells and 4.times.10.sup.4 LS174T cells (ECACC 87060401)
or LoVo cells (ATCC CCL-229) respectively as target cells (effector
to target of 15 to 1). Data were analysed after 30-40 h and after
50-60 h.
[1110] The results are depicted in FIG. 34. The IC50 values
obtained in this assay are listed in Tables 30 and 31.
TABLE-US-00037 TABLE 30 IC50 (M) of the multispecific polypeptides
in the human T cell mediated xCELLigence based killing assay using
an effector to target ratio of 15. Data were analysed after 30-40
h. LoVo LS174T ID construct n IC50 (M) 95% LCI 95% UCI n IC50 (M)
95% LCI 95% UCI T017000107 1 3.0E-10 2.6E-10 3.6E-10 1 1.0E-08
6.1E-09 1.7E-08 T017000109 1 9.3E-09 6.7E-09 1.3E-08 1 1.0E-07
4.9E-08 2.2E-07 T017000110 1 1.5E-09 1.3E-09 1.9E-09 1 2.6E-08
1.1E-08 6.0E-08
[1111] The data on the EGFR+/CEA+ LoVo cells showed a .about.28
fold difference in potency between the CEACAM5 only and the
EGFR-CEA multispecific constructs and a .about.8 fold difference in
potency between the EGFR only constructs and the EGFR-CEA
multispecific constructs. On EGFR+/CEA+ LS174T cells, a .about.7
fold difference in potency between the CEACAM5 only and the
EGFR-CEA multispecific constructs was observed and .about.2 fold
difference between the EGFR only constructs and the EGFR-CEA
multispecific constructs. These results showed that potency
enhancements were obtained with multispecific constructs reactive
against two different antigens present on a cell.
Example 26: In Vivo B Cell Depletion by Half-Life Extended (HLE)
Polypeptides in a Ramos B Cell Depletion Model
[1112] In this B cell depletion model, Burkitt's lymphoma Ramos
cells and human PBMC were injected respectively intravenously and
intraperitoneally in to mice after which Ramos B cell and
PBMC-derived B cell killing by Nanobody-mediated recruitment of T
cells present in PBMC population was evaluated: i.e. the potential
of Nanobodies, HLE and non-HLE (NHLE), to activate T cells by
direct linking of T cells via TCR to target B cells via CD20,
resulting in target cell killing.
[1113] The polypeptides used in this study are described in Table
32:
TABLE-US-00038 TABLE 32 Sample ID and description of multispecific
polypeptides used in in vivo study. Sample ID SEQ ID NO TAA TCR ISV
HLE Description T017000083 338 CD20 TCR NHLE 20CD019C07(E1D)-
35GS-T0170055A02-A T017000088 339 Irr TCR NHLE RSV007B02(E1D)-
35GS-T0170055A02-A T017000104 387 CD20 TCR HLE 20CD019C07(E1D)-
35GS-T0170055A02- 35GS-ALB11-A T017000105 388 CD20 TCR NHLE
20CD019C07(E1D)- 35GS-T0170056G05-A T017000106 389 Irr TCR HLE
RSV007B02(E1D)- 35GS-T0170055A02- 35GS-ALB11-A
[1114] The in vivo efficacy of a HLE bispecific Nanobody,
T017000104 (TCR and CD20-specific coupled to an albumin targeting
building block) in a Ramos NOG mouse model was evaluated and
compared with the irrelevant Nanobody T017000106. The non-half-life
extended (NHLE) Nanobody T017000105 (TCR and CD20-specific) and
T0107000083 (TCR and CD20-specific) were compared to the NHLE
irrelevant Nanobody T017000088 (irrelevant and TCR-specific). The
study demonstrated a statistically significant effect in bone
marrow and spleen on PBMC derived B cell depletion for T017000104
and T017000105 compared to their respective irrelevant control NBs.
The Ramos cells were significantly reduced in T017000104 and
T017000105 treated mice, both in spleen and bone marrow.
[1115] In detail, the B cell depletion was evaluated in the mice,
intravenously injected with Ramos cells at day one (D1) and
intraperitoneally with PBMCs at D3. Mice were treated from D3 to D7
(FIG. 35). Tumors were induced by intravenous injection of 10'
Ramos cells in 200 .mu.L of Roswell Park Memorial Institute (RPMI)
1640 medium (D1) 24 hours after a whole body irradiation of mice
with a .gamma.-source (1.44 Gy, 60Co) (D0). PBMCs were injected on
D3 (i.e. two days after tumor cell injection) after randomization
of the mice into groups each of 12 animals (11 animals for group 6)
based on body weight. The animals received one single
intraperitoneal (IP) injection of 10.sup.7 PBMCs (500 .mu.L in
PBS). The treatment started on D3 one hour after PBMC injection and
was repeated for 5 consecutive days in total for groups 1-4 and 6,
whereas group 5 received only two injections (D3 and D5).
[1116] On D20 or on D21, the mice were sacrificed and spleen and
bone marrow (femur) were collected for FACS analysis (mCD45, hCD45,
hCD19, hCD20, hCD10) and analyzed for presence of Ramos B cells and
PBMC-derived B cells.
[1117] Results for Ramos B cell depletion are represented in FIG.
36. Test items T017000083 and T017000105, and T017000104 were
compared to their respective control groups (groups 1 and 2) based
on presence/absence of HLE. In bone marrow, a statistically
significant difference in Ramos B cell number between the control
Nanobodies and the NHLE Nanobodies T017000083 and T017000105 was
observed (FIG. 36A). In spleen the difference was significant only
for T017000105 (FIG. 36B). The HLE Nanobody T017000104
significantly reduced the Ramos cells in spleen and bone marrow
irrespective of the dosing frequency applied (FIGS. 36A and B).
Statistical analysis has been performed with F-tests from the
mixed-effects ANOVA analysis.
[1118] Results for PBMC-derived B cell depletion are represented in
FIG. 37. In bone marrow, a reduction was observed in absolute B
cell count in all treatment groups, compared to the irrelevant
control Nanobody and this effect was significant for Nanobodies
T017000083 and T017000104 (FIG. 37 A). In the spleen, the effect
was more pronounced and significant for all treatment groups (FIG.
37 B).
[1119] In conclusion, these results demonstrate that both the
anti-CD20/anti-TCR polypeptide and the HLE anti-CD20/anti-TCR
polypeptide are able to significantly decrease Ramos B cells and
PBMC-derived B cells in spleen and bone marrow in this B cell
depletion model. This confirms the Nanobody-induced T cell
activation by cross-linking T cells to target B cells and killing
of the latter.
Example 27: Role of Human Effector CD4+ and CD8+ T Cells in T Cell
Activation
[1120] To investigate the role of CD4+, respectively CD8+ human T
cells in the redirected killing, targeting of tumour cells with
multispecific T cell engaging polypeptides was performed using T
cell subsets. In this case, the HER2-positive cell line SKBR3 and
Nanobody T017000102 (targeting HER2 and TCR) were used.
[1121] 27.1 Redirected Cell Killing of Multispecific Polypeptides
by Human Effector CD4+ and CD8.sup.+ T Cells in the xCELLigence
Based Assay
[1122] Human T cells were collected as described in Example 2.1.
After thawing, human CD4+, respectively CD8+ T cells were isolated
using the CD4+(Miltenyi Biotec #130-096-533), respectively
CD8+(Miltenyi Biotech #130-096-495) T cell isolation kit. SKBR3
cells, were seeded in 96 well E-plates (2.times.10.sup.4
cells/well) and incubated with 3.times.10.sup.5 human primary
effector T cells, human primary effector CD4+ T cells or human
primary effector CD8+ T cells (effector target ratio of 15:1) in
the presence or absence of multispecific constructs and followed
over time, as described in Example 16. Data were analysed after 40
h.
[1123] Results are shown in FIG. 38. The IC50 values are depicted
in Table 33.
TABLE-US-00039 TABLE 33 IC50 (M) of the multispecific polypeptides
in the xCELLigence based human T cell mediated SKBR3 killing assay
using an effector to target ratio of 15 to 1. IC50 (M) 95% LCI 95%
UCI T cells 2.4E-11 1.9E-11 3.0E-11 CD8+ T cells 2.9E-11 2.1E-11
4.1E-11 CD4+ T cells 1.2E-11 1.1E-11 1.3E-11
[1124] The data showed dose dependent specific killing of
HER2-positive tumour cell lines by directing human primary T cells,
human CD4+ or human CD8+ T cells to the tumour cells via the TCR
polypeptide.
[1125] 27.2 Effect of HER2/TCR Binding Polypeptides on CD69 and
CD25 Expression on Human Effector CD4+ and CD8+ T Cells in a
HER2-Positive Tumour Cell Killing Assay
[1126] Primary human T cells and CD4+ and CD8+ T cell
subpopulations were isolated and a redirected HER2-positive tumour
cell killing assay using SKBR3 cells was performed as described in
Example 27.1. T cell activation was determined by measuring CD69
and CD25 upregulation after 24 h and 72 h of incubation
respectively on the human primary T cells and on the CD4+ and CD8+
human T cell populations. CD69 and CD25 expression was measured in
flow cytometry, using monoclonal mouse anti-human CD69PE (BD
Biosciences #557050) and mouse anti-human CD25PE (BD Pharmigen
#557138) antibody, for CD69 and CD25 measurement respectively.
[1127] Exemplary results are shown in FIG. 39.
[1128] The data showed dose dependent upregulation of CD69 and CD25
on human primary T cells, human CD4+ and human CD8+ T cells.
[1129] 27.3 Effect of HER2/TCR Binding Polypeptides on IFN-.gamma.
and IL-6 Release by Human Effector CD4+ and CD8+ T Cells in a
HER2-Positive Tumour Cell Killing Assay
[1130] Primary human T cells and CD4+ and CD8+ T cell
subpopulations were isolated and a redirected HER2-positive tumour
cell killing assay using SKBR3 cells was performed as described in
Example 27.1. The release of the cytokine IFN-7 was measured by
ELISA as described in Example 17 and the release of IL-6 was
measured in ELISA using the human IL-6 Quantikine ELISA Kit
(R&D Systems, #D6050) according to manufactures
instructions.
[1131] Exemplary results are shown in FIG. 40.
[1132] The data showed dose dependent IFN-.gamma. and IL-6 release
by human primary T cells, human CD4+ and human CD8+ T cells.
Example 28: Exploration of Half-Life Extension (HLE)
[1133] It was hypothesized that HLE via albumin binding might be
suitable to comply with various requirements, including (i)
half-life extension (HLE) of the moiety; and (ii) efficacy of the
multispecific polypeptide. Preferably, the HLE function would not
impair the penetration of tumours and tissues.
[1134] Alb11 (SEQ ID NO: 404), a Nanobody binding to human serum
albumin (HSA) was linked to the multispecific EGFRxCEAxTCR binding
polypeptide to increase the in vivo half-life of the formatted
molecule (WO 06/122787). A format was generated with the albumin
targeting Nanobody at the C-terminus using a 35GS linker and
expressed as indicated above. The explored format is shown in Table
34.
TABLE-US-00040 TABLE 34 Sample ID and description of the
multispecific HLE polypeptide. Sample ID SEQ ID NO Description
T017000108 486 EGFR038G07-35GS-NbCEA5-35GS-
T0170056G05-35GS-ALB11-FLAG3-HIS6
[1135] As the addition of the Alb11 Nanobody might influence the
affinity or potency of the construct, the half-life extended
multispecific polypeptide was characterized for binding to EGFR and
CEA expressing cell lines and primary human T cells. In addition,
the potency in the functional T cell dependent LS174T killing assay
was evaluated (described in 28.1 and 28.2 below).
[1136] 28.1 Impact of the Alb11 Building Block on the Binding
Properties
[1137] Dose-dependent binding of the HLE multispecific polypeptide
to cancer cell lines expressing CEACAM5 and EGFR (LS174T and LoVo),
a cell line expressing EGFR (HER14; NIH3T3 transfected with EGFR),
and to purified primary human T cells (isolated as described in
Example 2.1) was evaluated in flow cytometry as outlined in Example
5, in the absence of HSA.
[1138] The results are presented in FIG. 41. The EC50 value
obtained from the dose response curve for binding to HER14 cells is
depicted in Table 35. The EC50 values obtained from the dose
response curves for binding to LS174T and LoVo cells are depicted
in Table 36.
TABLE-US-00041 TABLE 35 EC50 (M) of T017000108 for binding to HER14
cells as determined in flow cytometry. HER14 sample ID EC50 (M) 95%
LCI 95% UCI Top T017000108 1.9E-09 1.7E-09 2.2E-09 158884
TABLE-US-00042 TABLE 36 EC50 (M) of T017000108 for binding to LoVo
and LS174T as determined in flow cytometry. LS174T LoVo sample ID
EC50 (M) 95% LCI 95% UCI Top EC50 (M) 95% LCI 95% UCI Top
T017000108 1.4E-08 1.2E-08 1.6E-08 70202 3.3E-09 2.9E-09 3.9E-09
92787
[1139] Comparison of the HLE construct with the non-HLE construct
showed similar binding to all three cell lines tested. The data
presented showed that coupling of the Alb11 building block did not
influence the binding properties.
[1140] 28.2 Impact of the Alb11 Nanobody in the Redirected Cell
Killing by Human Effector T Cells in the xCELLigence Based
Assay
[1141] The functionality of the half-life extended multispecific
polypeptide was evaluated in the absence of HSA in the human T cell
mediated LS174T killing assay as described in Example 10 and
compared with the functionality of the non-HLE multispecific
constructs.
[1142] The results are depicted in FIG. 42. The obtained IC50
values are listed in Table 37.
TABLE-US-00043 TABLE 37 IC50 (M) of the multispecific polypeptides
in the human T cell mediated xCELLigence based killing assay using
an effector to target ratio of 15. Data were analysed after 40-50
h. sample ID IC50 (M) 95% LCI 95% UCI T017000107 7.9E-10 5.9E-10
1.1E-09 T017000108 5.5E-09 4.1E-09 7.3E-09
[1143] The results indicate that the inclusion of the albumin
targeting Nanobody in the construct as such did not have an
essential impact on the obtained potency or efficacy.
TABLE-US-00044 TABLE A-1 Sequence alignment of TCR cluster A
binders-part 1 1 10 20 30 40 50 60 70 80 Kabat # | | | | | | | | |
SEQ ID NO: 50 T0170PMP056G05:
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMV SEQ ID NO: 8 T0170PMP053D01:
..................K...A...A............T.E....M..T.T...DV................-
A.... SEQ ID NO: 60 T0170PMP067D01:
..................K...A...A............T.E....M..T.T...EV................-
A.... SEQ ID NO: 47 T0170PMP056F01:
..................K.P.A...A............T.E....M..T.T...EV................-
A.... SEQ ID NO: 76 T0170PMP069A06:
..................K...A...A............T.E....M..T.T...EV..E.............-
A.... SEQ ID NO: 65 T0170PMP067F02:
..................K...A...A............T.E....M..T.T...EV...H............-
A.... SEQ ID NO: 66 T0170PMP068C03:
..................K...A...A............T.E....M..T.T...EVA...............-
A.... SEQ ID NO: 26 T0170PMP055E05:
..................K...A...A..............E....M..T.T...EV................-
A.... SEQ ID NO: 19 T0170PMP055C02:
..................K...A...A............T.E....M....T...EV................-
A.... SEQ ID NO: 61 T0170PMP067D06:
..................K...A...A..............E....M..T.T...EV................-
AT... SEQ ID NO: 50 T0170PMP069C08:
.........................................................................-
..... SEQ ID NO: 28 T0170PMP055F03:
................................I....................S......E............-
..... SEQ ID NO: 54 T0170PMP061A02:
................................I..................A.S......E............-
..... SEQ ID NO: 45 T0170PMP056D11:
................................I................R...S......E............-
..... SEQ ID NO: 77 T0170PMP069B02:
............R...................I....................S......E............-
..... SEQ ID NO: 96 T0170PMP070G02:
................................I....................S......E............-
..... SEQ ID NO: 2 T0170PMP028B01:
......................A...S..................GL..T.T...T.....Y...........-
AR... SEQ ID NO: 4 T0170PMP028G06:
......................A...S...................L..T.T...T.....Y...........-
AR... SEQ ID NO: 29 T0170PMP055F06:
......................A...S...................L..T.T...A.....Y...........-
AR... SEQ ID NO: 1 T0170PMP027A05:
......................A...S...................L..T.T...T.................-
A.... SEQ ID NO: 6 T0170PMP040C01:
......................A...S...................L..T.T...T.................-
A.... SEQ ID NO: 3 T0170PMP028F10:
................R...P.A...S...................M..T.T...A.................-
A.... SEQ ID NO: 5 T0170PMP029F08:
......................A...S...................M..T.T...A.................-
A.... SEQ ID NO: 13 T0170PMP055A08:
..........S...........A...S...................M..T.T...A.................-
A.... SEQ ID NO: 32 T0170PMP055G09:
..........S...........A...S...................V..T.T...A.................-
A.... SEQ ID NO: 69 T0170PMP068D05:
..........S........P..A...S...................M..T.T...A.................-
A.... SEQ ID NO: 75 T0170PMP068F08:
..........S...........A...S..................GM..T.T...A.................-
A.... SEQ ID NO: 95 T0170PMP070F11:
..........S...........A...S..........C........M..T.T...A.................-
A.... SEQ ID NO: 87 T0170PMP069E11:
......................A...E.....I......C.....DM..T.T...A.................-
A.... SEQ ID NO: 99 T0170PMP084B07:
......................A...E.....I............DM..T.T...E.Q...............-
A.... SEQ ID NO: 10 T0170PMP055A01:
............R.........A.........I........A....M....T...A.................-
A.... SEQ ID NO: 11 T0170PMP055A02:
......................A.........I........A....M....T...A.................-
A.... SEQ ID NO: 22 T0170PMP055D03:
......................A.........I........A....M....T...A.N...............-
A.... SEQ ID NO: 24 T0170PMP055D10:
......................A.........I........A....M....T...A...G.............-
A....
TABLE-US-00045 TABLE A-2 Sequence alignment of TCR cluster A
binders-part 1 continued 79 90 100 110 Kabat # | ab | |a | SEQ ID
NO: 50 T0170PMP056G05: YLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS SEQ
ID NO: 8 T0170PMP053D01: ....T..........V...G..L...N............
SEQ ID NO: 60 T0170PMP067D01:
....T..........V...G..L...N............ SEQ ID NO: 47
T0170PMP056F01: ....T..........V...G..L...N............ SEQ ID NO:
76 T0170PMP069A06: ....TG.........V...G..L...N............ SEQ ID
NO: 65 T0170PMP067F02: ....T..........V...G..L...N......Q..... SEQ
ID NO: 66 T0170PMP068C03: ....T..........V...G..L...N......Q.....
SEQ ID NO: 26 T0170PMP055E05:
....T.......T..V...G..L...N......Q..... SEQ ID NO: 19
T0170PMP055C02: ....T..T.......V...G..L...N......Q..... SEQ ID NO:
61 T0170PMP067D06: ....T..............G......N......Q..... SEQ ID
NO: 50 T0170PMP069C08: ....................................... SEQ
ID NO: 28 T0170PMP055F03: ...............L.......................
SEQ ID NO: 54 T0170PMP061A02:
...............L....................... SEQ ID NO: 45
T0170PMP056D11: ...............L....................... SEQ ID NO:
77 T0170PMP069B02: ...............L.................Q..... SEQ ID
NO: 96 T0170PMP070G02: ...........A...L.................Q..... SEQ
ID NO: 2 T0170PMP028B01: ...................G..L...N......Q.....
SEQ ID NO: 4 T0170PMP028G06:
...................G..L...N............ SEQ ID NO: 29
T0170PMP055F06: ...................G..L...N............ SEQ ID NO:
1 T0170PMP027A05: ..............H....G..L...N......Q..... SEQ ID
NO: 6 T0170PMP040C01: ...................G..L...N......Q..... SEQ
ID NO: 3 T0170PMP028F10: ...................G..L...N......Q.....
SEQ ID NO: 5 T0170PMP029F08:
...................G..L...N............ SEQ ID NO: 13
T0170PMP055A08: .......S...........G..L...N............ SEQ ID NO:
32 T0170PMP055G09: .......S...........G..L...N......Q..... SEQ ID
NO: 69 T0170PMP068D05: .......S...........G..L...N......Q..... SEQ
ID NO: 75 T0170PMP068F08: .......S...........G..L...N......Q.....
SEQ ID NO: 95 T0170PMP070F11:
.......S...........G..L...N......Q..... SEQ ID NO: 87
T0170PMP069E11: ...................L..L...N............ SEQ ID NO:
99 T0170PMP084B07: ...................L..L...N............ SEQ ID
NO: 10 T0170PMP055A01: ...................Y......N............ SEQ
ID NO: 11 T0170PMP055A02: ...................Y......N............
SEQ ID NO: 22 T0170PMP055D03:
...................Y......N............ SEQ ID NO: 24
T0170PMP055D10: ...................Y......N............
TABLE-US-00046 TABLE A-3 Sequence alignment of TCR cluster A
binders-part 2 1 10 20 30 40 50 60 70 78 Kabat # | | | | | | | | |
SEQ ID NO: 50 T0170PMP056G05:
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMV SEQ ID NO: 71 T0170PMP068E01:
...............E......A.........I........A....M....T...A.................-
A.... SEQ ID NO: 101 T0170PMP084E03:
......................A.........I........A....M....T...A.................-
A.... SEQ ID NO: 102 T0170PMP084E05:
......................A.........I...H....A....M....T...A.................-
A.... SEQ ID NO: 30 T0170PMP055F08:
......................A.........I........A....M....T...A...........A.....-
A.... SEQ ID NO: 37 T0170PMP056C01:
......................A...G.....I........A....M....T...A.................-
A.... SEQ ID NO: 49 T0170PMP056G02:
...........A..........A.........I........A....M....T...A.................-
A.... SEQ ID NO: 57 T0170PMP067A03:
......................A.........I........A..H.M....T...A.................-
A.... SEQ ID NO: 59 T0170PMP067C09:
......................A.........I........A....M....T...A.................-
AE... SEQ ID NO: 74 T0170PMP068F06:
......................A.........I........A....M....T...A.................-
A..V. SEQ ID NO: 82 T0170PMP069D02:
..........M...........A.........I........A....M....T...A.................-
A.... SEQ ID NO: 93 T0170PMP070D07:
......................A.........I........A....M....T...A.................-
A.... SEQ ID NO: 100 T0170PMP084C02:
......................A.........I........A....M....T...A.................-
A.... SEQ ID NO: 55 T0170PMP061B04:
......................A.........I........A....M....T...A.................-
A.... SEQ ID NO: 83 T0170PMP069D07:
......................A.........I......V.A....M....T...A.................-
A.... SEQ ID NO: 36 T0170PMP056B11:
.............A....T...A.........I........A....M....T...A.................-
A.... SEQ ID NO: 104 T0170PMP084F10:
..........W..A........A.........I........A....M....T...A.................-
A.... SEQ ID NO: 39 T0170PMP056C03:
......................A.........I........A....M....T...T.................-
A.... SEQ ID NO: 80 T0170PMP069C04:
......................A.........I........A.G..M....T...A.................-
A.... SEQ ID NO: 44 T0170PMP056D02:
......................A.........I........A....M....T...A.................-
A.... SEQ ID NO: 56 T0170PMP067A01:
......................A.........I........A...GM....T...A.................-
A.... SEQ ID NO: 58 T0170PMP067B06:
......................A.........I........AR...M....T...A.................-
A.... SEQ ID NO: 88 T0170PMP069F05:
......................A.........I........A....M....T...A.................-
A.... SEQ ID NO: 103 T0170PMP084F04:
......................A.........I...H....A....M....T...A.................-
A.... SEQ ID NO: 21 T0170PMP055C10:
......................A.........V........A....M....T...A.................-
A.... SEQ ID NO: 9 T0170PMP053E10:
......................R.........I........A....M....T...A....E............-
A.... SEQ ID NO: 15 T0170PMP055B01:
......................R.........I........A....M....T...A.V..E............-
A.... SEQ ID NO: 79 T0170PMP069C01:
......................R.........I........A....MI...T...A....E............-
A.... SEQ ID NO: 62 T0170PMP067D09:
......................A.........I........A....M....T...A.S..G............-
A.... SEQ ID NO: 63 T0170PMP067E03:
......................A.........I........A....M....T...A.S...............-
A.... SEQ ID NO: 43 T0170PMP056D01:
.........D............A.......................M....T.A.A....EF...........-
P.... SEQ ID NO: 64 T0170PMP067E06:
.........D............A.......................M....T.A.A....EF...........-
P.... SEQ ID NO: 86 T0170PMP069E09:
......................A.......................M....T.A.A....EF...........-
P.... SEQ ID NO: 81 T0170PMP069C05:
.........D............A.......................M....T.A.A....EF...........-
P.... SEQ ID NO: 90 T0170PMP070B08:
.........D............A.......................M....T.A.A....EF...........-
P.... SEQ ID NO: 12 T0170PMP055A03:
...................................................T....A................-
A....
TABLE-US-00047 TABLE A-4 Sequence alignment of TCR cluster A
binders-part 2 continued 79 90 100 110 Kabat # | ab | |a | SEQ ID
NO: 50 T0170PMP056G05: YLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS SEQ
ID NO: 71 T0170PMP068E01: ...................Y......N............
SEQ ID NO: 101 T0170PMP084E03:
.........G.........Y......N............ SEQ ID NO: 102
T0170PMP084E05: ...................Y......N............ SEQ ID NO:
30 T0170PMP055F08: ...................Y......N............ SEQ ID
NO: 37 T0170PMP056C01: ...................Y......N............ SEQ
ID NO: 49 T0170PMP056G02: ...................Y......N............
SEQ ID NO: 57 T0170PMP067A03:
...................Y......N............ SEQ ID NO: 59
T0170PMP067C09: ...................Y......N............ SEQ ID NO:
74 T0170PMP068F06: ...................Y......N............ SEQ ID
NO: 82 T0170PMP069D02: ...................Y......N............ SEQ
ID NO: 93 T0170PMP070D07: ...........A.......Y......N............
SEQ ID NO: 100 T0170PMP084C02:
....D..............Y......N............ SEQ ID NO: 55
T0170PMP061B04: .............A.....Y......N............ SEQ ID NO:
83 T0170PMP069D07: .............A.....Y......N............ SEQ ID
NO: 36 T0170PMP056B11: ...................Y......N............ SEQ
ID NO: 104 T0170PMP084F10: ...................Y......N............
SEQ ID NO: 39 T0170PMP056C03:
...................Y......N......Q..... SEQ ID NO: 80
T0170PMP069C04: ...................Y......N......Q..... SEQ ID NO:
44 T0170PMP056D02: ...................Y......N...R..Q..... SEQ ID
NO: 56 T0170PMP067A01: ...................Y......N......Q..... SEQ
ID NO: 58 T0170PMP067B06: ...................Y......N......Q.....
SEQ ID NO: 88 T0170PMP069F05:
...............L...Y......N......Q..... SEQ ID NO: 103
T0170PMP084F04: .............A.....Y......N......Q..... SEQ ID NO:
21 T0170PMP055C10: H..................Y......N......Q..... SEQ ID
NO: 9 T0170PMP053E10: ...................Y......N......Q..... SEQ
ID NO: 15 T0170PMP055B01: ...................Y......N......Q.....
SEQ ID NO: 79 T0170PMP069C01:
...................Y......N......Q..... SEQ ID NO: 62
T0170PMP067D09: ...L.N.............Y......N............ SEQ ID NO:
63 T0170PMP067E03: ...L.N.............Y......N............ SEQ ID
NO: 43 T0170PMP056D01: H..............L...G......N......Q..... SEQ
ID NO: 64 T0170PMP067E06: ...............L...G......N......Q.....
SEQ ID NO: 86 T0170PMP069E09:
...............L...G......N......Q..... SEQ ID NO: 81
T0170PMP069C05: ...............L...G......S......Q..... SEQ ID NO:
90 T0170PMP070B08: .........V.....L...G......N............ SEQ ID
NO: 12 T0170PMP055A03: ...................G.............Q.....
TABLE-US-00048 TABLE A-5 Sequence alignment of TCR cluster A
binders-part 3 1 10 20 30 40 50 60 70 78 Kabat # | | | | | | | | |
SEQ ID NO: 50 T0170PMP056G05:
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMV SEQ ID NO: 41 T0170PMP056C07:
...................................................T....A................-
A.... SEQ ID NO: 92 T0170PMP070C09:
.................P.................................T....A................-
A.... SEQ ID NO: 98 T0170PMP082B04:
...................P...............................T.A..A................-
A.... SEQ ID NO: 33 T0170PMP056A02:
.................A.................................T.....................-
A.... SEQ ID NO: 31 T0170PMP055G05:
......................A............H...............T...A.................-
A.... SEQ ID NO: 78 T0170PMP069B08:
......................A.....Y......H...............T...A.................-
A.... SEQ ID NO: 20 T0170PMP055C06:
............H.........A............H..P............T...V.................-
A.... SEQ ID NO: 23 T0170PMP055D06:
......................A............H...............T...V.................-
A.... SEQ ID NO: 25 T0170PMP055E01:
......................A...E.Y......................T.A.VA....F...........-
A.... SEQ ID NO: 27 T0170PMP055F02:
......................A...E.Y......................T.A.VA....F...........-
A.... SEQ ID NO: 38 T0170PMP056C02:
......................A...E.Y......................T.A.VA....F.Q.........-
A.... SEQ ID NO: 97 T0170PMP070G06:
...................P..A...E.Y......................T.A.VA....F...........-
A.... SEQ ID NO: 89 T0170PMP069G08:
......................A...E.Y.......Q..............T.A.VA....F...........-
A.... SEQ ID NO: 94 T0170PMP070E07:
......................A...E.Y......................T.A.VA....F..........G-
A.... SEQ ID NO: 70 T0170PMP068D07:
......................A...E.Y......................T.A.VA....F...........-
V.... SEQ ID NO: 51 T0170PMP056G11:
......................A...E.Y......................T.A.AA....F..........G-
A.... SEQ ID NO: 67 T0170PMP068C07:
......................A...E.Y......................T.A.AA....F...........-
A.... SEQ ID NO: 84 T0170PMP069E02:
......................A...E.Y......................T.A.AA....F...........-
A.... SEQ ID NO: 68 T0170PMP068C11:
......................A...E.Y......................T.A.AA....F...........-
A.... SEQ ID NO: 72 T0170PMP068E08:
......................A...E.Y......................T.A.AA....F...........-
A.... SEQ ID NO: 46 T0170PMP056E02:
..............E.......A...E.Y......................T.A.VA....F....L......-
A.... SEQ ID NO: 35 T0170PMP056A10:
......................AV..S..LL..............GV....T.A.A...SHF...........-
A.... SEQ ID NO: 42 T0170PMP056C10:
......................AV..S..LL...............M.T..T.A.A...SHF...........-
A.... SEQ ID NO: 16 T0170PMP055B02:
......................A...S..LL........C......M....T.A.A...SHF...........-
A.... SEQ ID NO: 34 T0170PMP056A08:
......................A...S..LL...............M....T.A.A...SHF...........-
A.... SEQ ID NO: 40 T0170PMP056C04:
......................A...S..LL...............M....T.A.A...SHF...........-
A.... SEQ ID NO: 53 T0170PMP057D06:
......................A...S..LL...............M....T.A.A...SHF...........-
A.... SEQ ID NO: 7 T0170PMP053A03:
......................AV..S..LL...............M....T.A.A...SHF...........-
A.... SEQ ID NO: 85 T0170PMP069E07:
......................A...S..LL...............M....T.A.A...SHF...........-
A.... SEQ ID NO: 18 T0170PMP055B11:
......................A...S..LL...............M....T.A.A....HF...........-
A.... SEQ ID NO: 48 T0170PMP056F08:
..................G...A...S..LL...............M....T.A.A....HF...........-
A.... SEQ ID NO: 91 T0170PMP070B09:
......................A...S..LL....................T.A.A...SHF...........-
A.... SEQ ID NO: 73 T0170PMP068F04:
......................A...S..LL...............M....T.A.V...SYF...........-
A.... SEQ ID NO: 14 T0170PMP055A10:
......................A...S..LL...............M......A.A....HF...........-
A.... SEQ ID NO: 17 T0170PMP055B03:
...................P..A...S..LL...............M....T.A.A....HF...........-
A.... SEQ ID NO: 52 T0170PMP057B02:
..........W...........A...S.Y.....S.......H...L..T.T...AA................-
AR...
TABLE-US-00049 TABLE A-6 Sequence alignment of TCR cluster A
binders-part 3 continued 79 90 100 110 Kabat # | ab | |a | SEQ ID
NO: 50 T0170PMP056G05: YLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS SEQ
ID NO: 41 T0170PMP056C07: ...................G..........R........
SEQ ID NO: 92 T0170PMP070C09:
...................G................... SEQ ID NO: 98
T0170PMP082B04: ...................G................... SEQ ID NO:
33 T0170PMP056A02: ...................G...W.........Q..... SEQ ID
NO: 31 T0170PMP055G05: ...................G.............Q..... SEQ
ID NO: 78 T0170PMP069B08: ...................G...................
SEQ ID NO: 20 T0170PMP055C06:
.....N.............G.............Q..... SEQ ID NO: 23
T0170PMP055D06: F....N.............G.............Q..... SEQ ID NO:
25 T0170PMP055E01: ...................G...W.........Q..... SEQ ID
NO: 27 T0170PMP055F02: ...................G...W............... SEQ
ID NO: 38 T0170PMP056C02: ...................G...W.........Q.....
SEQ ID NO: 97 T0170PMP070G06:
...................G...W.........Q..... SEQ ID NO: 89
T0170PMP069G08: ...................G...W............... SEQ ID NO:
94 T0170PMP070E07: ...................G...W............... SEQ ID
NO: 70 T0170PMP068D07: ...................G...W............... SEQ
ID NO: 51 T0170PMP056G11: ...................G...W.........Q.....
SEQ ID NO: 67 T0170PMP068C07:
...................G...W.........Q..... SEQ ID NO: 84
T0170PMP069E02: ...................G...W............... SEQ ID NO:
68 T0170PMP068C11: ...................G.............Q..... SEQ ID
NO: 72 T0170PMP068E08: .......R...........G...W.........Q..... SEQ
ID NO: 46 T0170PMP056E02: ...................G...W...............
SEQ ID NO: 35 T0170PMP056A10:
.......R...........G.............Q..... SEQ ID NO: 42
T0170PMP056C10: .......R...........G.............Q..... SEQ ID NO:
16 T0170PMP055B02: .......R...........G................... SEQ ID
NO: 34 T0170PMP056A08: .......R...........G................... SEQ
ID NO: 40 T0170PMP056C04: .......R...........G..........H........
SEQ ID NO: 53 T0170PMP057D06:
.......R...........G.............Q..... SEQ ID NO: 7
T0170PMP053A03: .......R...........G................... SEQ ID NO:
85 T0170PMP069E07: .....G.R...........G................... SEQ ID
NO: 18 T0170PMP055B11: .......R...........G................... SEQ
ID NO: 48 T0170PMP056F08: .......R...........G...................
SEQ ID NO: 91 T0170PMP070B09:
.....N.R...........G................... SEQ ID NO: 73
T0170PMP068F04: .......R...........G.............Q..... SEQ ID NO:
14 T0170PMP055A10: .......R...........G..........R..Q..... SEQ ID
NO: 17 T0170PMP055B03: .......R...........G.............Q..... SEQ
ID NO: 52 T0170PMP057B02:
.............L...H.G..L...N......Q.....
TABLE-US-00050 TABLE A-7 Sequence alignment of TCR cluster B
binders 1 10 20 30 40 50 60 70 77 Kabat # | a | | | | | | | | SEQ
ID NO: 106 T0170PMP055C07:
EVQLVE-SGGGLVQPGGSLRLSCITSGETFKINIWGWYRQAPGKQRELVASLTIGGATNYADSVKGRFTISED-
SAKNT SEQ ID NO: 105 T0170PMP055B06:
......-...................................................D..............-
..... SEQ ID NO: 115 T0170PMP070C01:
......-..................................................................-
...D. SEQ ID NO: 108 T0170PMP056B02:
......-.................................T................................-
..... SEQ ID NO: 109 T0170PMP056D07:
......-....................Q.............................................-
..... SEQ ID NO: 112 T0170PMP069E03:
......-..................................................................-
..... SEQ ID NO: 111 T0170PMP068G05:
......E..................................................................-
..... SEQ ID NO: 114 T0170PMP070A09:
......-..................................................................-
..... SEQ ID NO: 107 T0170PMP055D01:
......-.....................................R............................-
..... SEQ ID NO: 110 T0170PMP068B03:
......-........................V........G................................-
..... SEQ ID NO: 113 T0170PMP069E06:
......-................L.......V........G................................-
..... 78 90 100 110 Kabat # | abc | |a | SEQ ID NO: 106
T0170PMP055C07: VYLQMNSLKPEDTAVYFCNAKSRLYPYDYWGQGTLVTVSS SEQ ID NO:
105 T0170PMP055B06: .........A........................Q..... SEQ ID
NO: 115 T0170PMP070C01: ........................................
SEQ ID NO: 108 T0170PMP056B02:
..................................Q..... SEQ ID NO: 109
T0170PMP056D07: ..................................Q..... SEQ ID NO:
112 T0170PMP069E03: ..............................D...Q..... SEQ ID
NO: 111 T0170PMP068G05: .......................I.......R..Q.....
SEQ ID NO: 114 T0170PMP070A09:
.......................I.......R..Q..... SEQ ID NO: 107
T0170PMP055D01: ..........................I.......Q..... SEQ ID NO:
110 T0170PMP068B03: ...............................R..Q..... SEQ ID
NO: 113 T0170PMP069E06:
.....S............................Q.....
TABLE-US-00051 TABLE A-8 Sequence alignment of TCR cluster C
binders 1 10 20 30 40 50 60 70 78 Kabat # | | | | | | | | | SEQ ID
NO: 116 T0170PMP061G01
EVQLVESGGGLVQPGGSLRLSCAASGEIGRINFYRWYRQAPGNQREVVATITIADKTDYADSAKGRFTISRDE-
SRNMV SEQ ID NO: 118 T0170PMP075D02
.........................................................................-
..... SEQ ID NO: 117 T0170PMP062D02
.............................................G..........I................-
..... 79 90 100 110 Kabat # | abc | |a | SEQ ID NO: 116
T0170PMP061G01 YLQMSSLKPEDTAVYFCHAGSRLYPYDYWGQGTQVTVSS SEQ ID NO:
118 T0170PMP075D02 ..........N............................ SEQ ID
NO: 117 T0170PMP062D02 ....G..................................
TABLE-US-00052 TABLE A-4 Sequences for CDRs and frameworks, plus
preferred combinations as provided in formula I, namely
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. ''SEQ'' refers to the given SEQ ID
NO. The first column refers to the SEQ ID NO of the complete ISV,
i.e. FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4. CDR1, CDR2 and CDR3 were
determined according to Kontermann, 2010. SEQ Nanobody SEQ FR1 SEQ
CDR1 SEQ FR2 SEQ CDR2 SEQ FR3 SEQ CDR3 SEQ FR4 1 T0170PMP 175
EVQLVESGGGLVQ 119 GSVHKIN 203 WYRQAPGKE 134 TITIGD 235
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 027A05 PGGSLRLSCAAS
FLG RELVA TTD YLQMNSLKPEDTAVHFCRA VTVSS 2 T0170PMP 175
EVQLVESGGGLVQ 119 GSVHKIN 204 WYRQAPGKE 134 TITIGD 236
YADYAKGRFTISRDEARNMV 164 GSRLYPYNY 285 WGQGTQ 028B01 PGGSLRLSCAAS
FLG RGLVA TTD YLQMNSLKPEDTAVYFCRA VTVSS 3 T0170PMP 176
EVQLVESGGGLVQ 119 GSVHKIN 205 WYRQAPGKE 135 TITIGD 237
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 028F10 PGRSLRLPCAAS
FLG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 4 T0170PMP 175
EVQLVESGGGLVQ 119 GSVHKIN 203 WYRQAPGKE 134 TITIGD 236
YADYAKGRFTISRDEARNMV 164 GSRLYPYNY 286 WGQGTL 028G06 PGGSLRLSCAAS
FLG RELVA TTD YLQMNSLKPEDTAVYFCRA VTVSS 5 T0170PMP 175
EVQLVESGGGLVQ 119 GSVHKIN 205 WYRQAPGKE 135 TITIGD 237
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 286 WGQGTL 029F08 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 6 T0170PMP 175
EVQLVESGGGLVQ 119 GSVHKIN 203 WYRQAPGKE 134 TITIGD 237
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 040C01 PGGSLRLSCAAS
FLG RELVA TTD YLQMNSLKPEDTAVYFCRA VTVSS 7 T0170PMP 177
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 238
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 053A03 PGGSLRLSCAVS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 8 T0170PMP 178
EVQLVESGGGLVQ 121 GAVHKIN 206 WYRQTPEKE 137 TITIGD 239
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 286 WGQGTL 053D01 PGGSLKLSCAAS
FLG REMVA DVD YLQMTSLKPEDTAVYVCRA VTVSS 9 T0170PMP 179
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 240
YAESAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 053E10 PGGSLRLSCRAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 10 T0170PMP 180
EVQLVESGGGLVR 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 055A01 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 11 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 055A02 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 12 T0170PMP 181
EVQLVESGGGLVQ 123 GDVHKIN 208 WYRQAPGKE 139 HITIGD 237
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 055A03 PGGSLRLSCVAS
FLG REKVA QAD YLQMNSLKPEDTAVYFCRA VTVSS 13 T0170PMP 182
EVQLVESGGGSVQ 119 GSVHKIN 205 WYRQAPGKE 135 TITIGD 241
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 286 WGQGTL 055A08 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLSPEDTAVYFCRA VTVSS 14 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 140 HISIAD 242
YAHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 287 WGRGTQ 055A10 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 15 T0170PMP 179
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 141 HITIGD 240
YAESAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 055B01 PGGSLRLSCRAS
ILG REMVA ATV YLQMNSLKPEDTAVYFCRA VTVSS 16 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 209 WYRQCPGKE 136 HITIAD 238
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 055B02 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 17 T0170PMP 183
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 242
YAHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 055B03 PGGSLRPSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 18 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 242
YAHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 055B11 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 19 T0170PMP 178
EVQLVESGGGLVQ 121 GAVHKIN 206 WYRQTPEKE 143 HITIGD 244
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 055C02 PGGSLKLSCAAS
FLG REMVA EVD YLQMTSLTPEDTAVYVCRA VTVSS 20 T0170PMP 185
EVQLVESGGGLVH 123 GDVHKIN 211 WHRQPPGKE 144 HITIGD 245
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 055C06 PGGSLRLSCAAS
FLG REKVA VTD YLQMNNLKPEDTAVYFCRA VTVSS 21 T0170PMP 175
EVQLVESGGGLVQ 125 GDVHKIN 207 WYRQAPAKE 138 HITIGD 247
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 055C10 PGGSLRLSCAAS
VLG REMVA AID HLQMNSLKPEDTAVYFCRA VTVSS 22 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 146 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 055D03 PGGSLRLSCAAS
ILG REMVA ATN YLQMNSLKPEDTAVYFCRA VTVSS 23 T0170PMP 175
EVQLVESGGGLVQ 123 GDVHKIN 213 WHRQAPGKE 144 HITIGD 248
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 055D06 PGGSLRLSCAAS
FLG REKVA VTD FLQMNNLKPEDTAVYFCRA VTVSS 24 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 249
YAGSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 055D10 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 25 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 147 HITIAD 250
YADFAKGRFTISRDEAKNMV 169 GSRIWPYDY 285 WGQGTQ 055E01 PGGSLRLSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 26 T0170PMP 178
EVQLVESGGGLVQ 121 GAVHKIN 214 WYRQAPEKE 148 TITIGD 251
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 055E05 PGGSLKLSCAAS
FLG REMVA EVD YLQMTSLKPEDTTVYVCRA VTVSS 27 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 147 HITIAD 250
YADFAKGRFTISRDEAKNMV 169 GSRIWPYDY 286 WGQGTL 055F02 PGGSLRLSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 28 T0170PMP 181
EVQLVESGGGLVQ 122 GDVHKIN 208 WYRQAPGKE 149 HISISD 252
YAESAKGRFTISRDESKNMV 170 FSRIYPYDY 286 WGQGTL 055F03 PGGSLRLSCVAS
ILG REKVA QTD YLQMNSLKPEDTAVYLCRA VTVSS 29 T0170PMP 175
EVQLVESGGGLVQ 119 GSVHKIN 203 WYRQAPGKE 135 TITIGD 236
YADYAKGRFTISRDEARNMV 164 GSRLYPYNY 286 WGQGTL 055F06 PGGSLRLSCAAS
FLG RELVA AID YLQMNSLKPEDTAVYFCRA VTVSS 30 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 253
YADSAKGRFAISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 055F08 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 31 T0170PMP 175
EVQLVESGGGLVQ 123 GDVHKIN 213 WHRQAPGKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 055G05 PGGSLRLSCAAS
FLG REKVA AID YLQMNSLKPEDTAVYFCRA VTVSS 32 T0170PMP 182
EVQLVESGGGSVQ 119 GSVHKIN 215 WYRQAPGKE 135 TITIGD 241
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 055G09 PGGSLRLSCAAS
FLG REVVA AID YLQMNSLSPEDTAVYFCRA VTVSS 33 T0170PMP 186
EVQLVESGGGLVQ 123 GDVHKIN 208 WYRQAPGKE 150 HITIGD 237
YADSAKGRFTISRDEAKNMV 169 GSRIWPYDY 285 WGQGTQ 056A02 PGGSARLSCVAS
FLG REKVA QTD YLQMNSLKPEDTAVYFCRA VTVSS 34 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 238
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 056A08 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 35 T0170PMP 177
EVQLVESGGGLVQ 120 GSVHLLN 216 WYRQAPGKE 136 HITIAD 238
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 056A10 PGGSLRLSCAVS
FLG RGVVA AID YLQMNSLRPEDTAVYFCRA VTVSS 36 T0170PMP 187
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 056B11 AGGSLTLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 37 T0170PMP 175
EVQLVESGGGLVQ 127 GGVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 056C01 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 38 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 147 HITIAD 254
YADFAQGRFTISRDEAKNMV 169 GSRIWPYDY 285 WGQGTQ 056C02 PGGSLRLSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 39 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 151 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 056C03 PGGSLRLSCAAS
ILG REMVA TTD YLQMNSLKPEDTAVYFCRA VTVSS 40 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 238
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 288 WGHGTL 056C04 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 41 T0170PMP 181
EVQLVESGGGLVQ 123 GDVHKIN 208 WYRQAPGKE 139 HITIGD 237
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 289 WGRGTL 056C07 PGGSLRLSCVAS
FLG REKVA QAD YLQMNSLKPEDTAVYFCRA VTVSS 42 T0170PMP 177
EVQLVESGGGLVQ 120 GSVHLLN 218 WYRQAPGKE 136 HITIAD 238
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 056C10 PGGSLRLSCAVS
FLG REMVT AID YLQMNSLRPEDTAVYFCRA VTVSS 43 T0170PMP 188
EVQLVESGGDLVQ 123 GDVHKIN 205 WYRQAPGKE 136 HITIAD 255
YAEFAKGRFTISRDEPKNMV 171 GSRIYPYNY 285 WGQGTQ 056D01 PGGSLRLSCAAS
FLG REMVA AID HLQMNSLKPEDTAVYLCRA VTVSS 44 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 287 WGRGTQ 056D02 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 45 T170PMP 181
EVQLVESGGGLVQ 122 GDVHKIN 208 WYRQAPGKE 152 RISISD 252
YAESAKGRFTISRDESKNMV 170 FSRIYPYDY 286 WGQGTL 056D11 PGGSLRLSCVAS
ILG REKVA QTD YLQMNSLKPEDTAVYLCRA VTVSS 46 T0170PMP 189
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 147 HITIAD 256
YADFAKGRLTISRDEAKNMV 169 GSRIWPYDY 286 WGQGTL 056E02 PEGSLRLSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 47 T0170PMP 190
EVQLVESGGGLVQ 121 GAVHKIN 206 WYRQTPEKE 148 TITIGD 239
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 286 WGQGTL 056F01 PGGSLKLPCAAS
FLG REMVA EVD YLQMTSLKPEDTAVYVCRA VTVSS 48 T0170PMP 191
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 242
YAHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 056F08 PGGSLGLSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 49 T0170PMP 192
EVQLVESGGGLAQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 056G02 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 50 T0170PMP 181
EVQLVESGGGLVQ 123 GDVHKIN 208 WYRQAPGKE 153 HISIGD 257
YADSAKGRFTISRDESKNMV 170 FSRIYPYDY 286 WGQGTL 056G05/ PGGSLRLSCVAS
FLG REKVA QTD YLQMNSLKPEDTAVYFCRA VTVSS T0170PMP 069C08 51 T0170PMP
175 EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 154 HITIAD 258
YADFAKGRFTISRDGAKNMV 169 GSRIWPYDY 285 WGQGTQ 056G11 PGGSLRLSCAAS
FLG REKVA AAD YLQMNSLKPEDTAVYFCRA VTVSS 52 T0170PMP 193
EVQLVESGGGWVQ 129 GSVYKIN 219 WYRQAPGHE 155 TITIGD 259
YADSAKGRFTISRDEARNMV 164 GSRLYPYNY 285 WGQGTQ 057B02 PGGSLRLSCAAS
FLS RELVA AAD YLQMNSLKPEDTALYFCHA VTVSS 53 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 238
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 057D06 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLRPEDTAVYFCRA VTVSS 54 T0170PMP 181
EVQLVESGGGLVQ 122 GDVHKIN 208 WYRQAPGKE 156 HIAISD 252
YAESAKGRFTISRDESKNMV 170 FSRIYPYDY 286 WGQGTL 061A02 PGGSLRLSCVAS
ILG REKVA QTD YLQMNSLKPEDTAVYLCRA VTVSS 55 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 260
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 061B04 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAAYFCRA VTVSS 56 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 222 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 067A01 PGGSLRLSCAAS
ILG RGMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 57 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 223 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 067A03 PGGSLRLSCAAS
ILG HEMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 58 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 224 WYRQAPARE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 067B06 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 59 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 263
YADSAKGRFTISRDEAENMV 166 YSRIYPYNY 286 WGQGTL 067C09 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 60 T0170PMP 178
EVQLVESGGGLVQ 121 GAVHKIN 206 WYRQTPEKE 148 TITIGD 239
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 286 WGQGTL
067D01 PGGSLKLSCAAS FLG REMVA EVD YLQMTSLKPEDTAVYVCRA VTVSS 61
T0170PMP 178 EVQLVESGGGLVQ 121 GAVHKIN 214 WYRQAPEKE 148 TITIGD 264
YADSAKGRFTISRDEATNMV 171 GSRIYPYNY 285 WGQGTQ 067D06 PGGSLKLSCAAS
FLG REMVA EVD YLQMTSLKPEDTAVYFCRA VTVSS 62 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 159 HITIGD 265
YAGSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 067D09 PGGSLRLSCAAS
ILG REMVA ATS YLQLNNLKPEDTAVYFCRA VTVSS 63 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 159 HITIGD 266
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 067E03 PGGSLRLSCAAS
ILG REMVA ATS YLQLNNLKPEDTAVYFCRA VTVSS 64 T0170PMP 188
EVQLVESGGDLVQ 123 GDVHKIN 205 WYRQAPGKE 136 HITIAD 267
YAEFAKGRFTISRDEPKNMV 171 GSRIYPYNY 285 WGQGTQ 067E06 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLKPEDTAVYLCRA VTVSS 65 T0170PMP 178
EVQLVESGGGLVQ 121 GAVHKIN 206 WYRQTPEKE 148 TITIGD 268
YAHSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 067F02 PGGSLKLSCAAS
FLG REMVA EVD YLQMTSLKPEDTAVYVCRA VTVSS 66 T0170PMP 178
EVQLVESGGGLVQ 121 GAVHKIN 206 WYRQTPEKE 160 TITIGD 239
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 068C03 PGGSLKLSCAAS
FLG REMVA EVA YLQMTSLKPEDTAVYVCRA VTVSS 67 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 154 HITIAD 250
YADFAKGRFTISRDEAKNMV 169 GSRIWPYDY 285 WGQGTQ 068C07 PGGSLRLSCAAS
FLG REKVA AAD YLQMNSLKPEDTAVYFCRA VTVSS 68 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 154 HITIAD 250
YADFAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 068C11 PGGSLRLSCAAS
FLG REKVA AAD YLQMNSLKPEDTAVYFCRA VTVSS 69 T0170PMP 194
EVQLVESGGGSVQ 119 GSVHKIN 205 WYRQAPGKE 135 TITIGD 241
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 068D05 PGGSLRPSCAAS
FLG REMVA AID YLQMNSLSPEDTAVYFCRA VTVSS 70 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 147 HITIAD 269
YADFAKGRFTISRDEVKNMV 169 GSRIWPYDY 286 WGQGTL 068D07 PGGSLRLSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 71 T0170PMP 195
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 068E01 PGESLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 72 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 154 HITIAD 270
YADFAKGRFTISRDEAKNMV 169 GSRIWPYDY 285 WGQGTQ 068E08 PGGSLRLSCAAS
FLG REKVA AAD YLQMNSLRPEDTAVYFCRA VTVSS 73 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 161 HITIAD 271
YSYFAKGRFTISRDEAKNMV 165 GSRIYPYDY 285 WGQGTQ 068F04 PGGSLRLSCAAS
FLG REMVA VTD YLQMNSLRPEDTAVYFCRA VTVSS 74 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 272
YADSAKGRFTISRDEAKNVV 166 YSRIYPYNY 286 WGQGTL 068F06 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 75 T0170PMP 182
EVQLVESGGGSVQ 119 GSVHKIN 226 WYRQAPGKE 135 TITIGD 241
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 068F08 PGGSLRLSCAAS
FLG RGMVA AID YLQMNSLSPEDTAVYFCRA VTVSS 76 T0170PMP 178
EVQLVESGGGLVQ 121 GAVHKIN 206 WYRQTPEKE 148 TITIGD 273
YEDSAKGRFTISRDEAKNMV 164 GSRLYPYNY 286 WGQGTL 069A06 PGGSLKLSCAAS
FLG REMVA EVD YLQMTGLKPEDTAVYVCRA VTVSS 77 T0170PMP 197
EVQLVESGGGLVR 122 GDVHKIN 208 WYRQAPGKE 149 HISISD 252
YAESAKGRFTISRDESKNMV 170 FSRIYPYDY 285 WGQGTQ 069B02 PGGSLRLSCVAS
ILG REKVA QTD YLQMNSLKPEDTAVYLCRA VTVSS 78 T0170PMP 175
EVQLVESGGGLVQ 132 GDVYKIN 213 WHRQAPGKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 069B08 PGGSLRLSCAAS
FLG REKVA AID YLQMNSLKPEDTAVYFCRA VTVSS 79 T0170PMP 179
EVQLVESGGGLVQ 122 GDVHKIN 227 WYRQAPAKE 138 HITIGD 240
YAESAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 069C01 PGGSLRLSCRAS
ILG REMIA AID YLQMNSLKPEDTAVYFCRA VTVSS 80 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 228 WYRQAPAKG 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 069C04 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 81 T0170PMP 188
EVQLVESGGDLVQ 123 GDVHKIN 205 WYRQAPGKE 136 HITIAD 267
YAEFAKGRFTISRDEPKNMV 173 GSRIYPYSY 285 WGQGTQ 069C05 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLKPEDTAVYLCRA VTVSS 82 T0170PMP 198
EVQLVESGGGMVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 069D02 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 83 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 229 WYRQVPAKE 138 HITIGD 260
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 069D07 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAAYFCRA VTVSS 84 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 154 HITIAD 250
YADFAKGRFTISRDEAKNMV 169 GSRIWPYDY 286 WGQGTL 069E02 PGGSLRLSCAAS
FLG REKVA AAD YLQMNSLKPEDTAVYFCRA VTVSS 85 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 205 WYRQAPGKE 136 HITIAD 275
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 069E07 PGGSLRLSCAAS
FLG REMVA AID YLQMNGLRPEDTAVYFCRA VTVSS 86 T0170PMP 175
EVQLVESGGGLVQ 123 GDVHKIN 205 WYRQAPGKE 136 HITIAD 267
YAEFAKGRFTISRDEPKNMV 171 GSRIYPYNY 285 WGQGTQ 069E09 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLKPEDTAVYLCRA VTVSS 87 T0170PMP 175
EVQLVESGGGLVQ 133 GEVHKIN 230 WYRQCPGKE 135 TITIGD 237
YADSAKGRFTISRDEAKNMV 174 LSRLYPYNY 286 WGQGTL 069E11 PGGSLRLSCAAS
ILG RDMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 88 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 276
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 069F05 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYLCRA VTVSS 89 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 231 WQRQAPGKE 147 HITIAD 250
YADFAKGRFTISRDEAKNMV 169 GSRIWPYDY 286 WGQGTL 069G08 PGGSLRLSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 90 T0170PMP 188
EVQLVESGGDLVQ 123 GDVHKIN 205 WYRQAPGKE 136 HITIAD 277
YAEFAKGRFTISRDEPKNMV 171 GSRIYPYNY 286 WGQGTL 070B08 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLKPVDTAVYLCRA VTVSS 91 T0170PMP 175
EVQLVESGGGLVQ 120 GSVHLLN 208 WYRQAPGKE 136 HITIAD 278
YSHFAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 070B09 PGGSLRLSCAAS
FLG REKVA AID YLQMNNLRPEDTAVYFCRA VTVSS 92 T0170PMP 200
EVQLVESGGGLVQ 123 GDVHKIN 208 WYRQAPGKE 139 HITIGD 237
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 070C09 PGGSPRLSCVAS
FLG REKVA QAD YLQMNSLKPEDTAVYFCRA VTVSS 93 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 280
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 070D07 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDAAVYFCRA VTVSS 94 T0170PMP 175
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 147 HITIAD 258
YADFAKGRFTISRDGAKNMV 169 GSRIWPYDY 286 WGQGTL 070E07 PGGSLRLSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 95 T0170PMP 182
EVQLVESGGGSVQ 119 GSVHKIN 232 WYCQAPGKE 135 TITIGD 241
YADSAKGRFTISRDEAKNMV 164 GSRLYPYNY 285 WGQGTQ 070F11 PGGSLRLSCAAS
FLG REMVA AID YLQMNSLSPEDTAVYFCRA VTVSS 96 T0170PMP 181
EVQLVESGGGLVQ 122 GDVHKIN 208 WYRQAPGKE 149 HISISD 281
YAESAKGRFTISRDESKNMV 170 FSRIYPYDY 285 WGQGTQ 070G02 PGGSLRLSCVAS
ILG REKVA QTD YLQMNSLKPEDAAVYLCRA VTVSS 97 T0170PMP 183
EVQLVESGGGLVQ 126 GEVYKIN 208 WYRQAPGKE 147 HITIAD 250
YADFAKGRFTISRDEAKNMV 169 GSRIWPYDY 285 WGQGTQ 070G06 PGGSLRPSCAAS
FLG REKVA VAD YLQMNSLKPEDTAVYFCRA VTVSS 98 T0170PMP 201
EVQLVESGGGLVQ 123 GDVHKIN 208 WYRQAPGKE 162 HITIAD 237
YADSAKGRFTISRDEAKNMV 165 GSRIYPYDY 286 WGQGTL 082B04 PGGSLRPSCVAS
FLG REKVA QAD YLQMNSLKPEDTAVYFCRA VTVSS 99 T0170PMP 175
EVQLVESGGGLVQ 133 GEVHKIN 233 WYRQAPGKE 163 TITIGD 237
YADSAKGRFTISRDEAKNMV 174 LSRLYPYNY 286 WGQGTL 084B07 PGGSLRLSCAAS
ILG RDMVA ETQ YLQMNSLKPEDTAVYFCRA VTVSS 100 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 283
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 084C02 PGGSLRLSCAAS
ILG REMVA AID YLQMDSLKPEDTAVYFCRA VTVSS 101 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 284
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 084E03 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPGDTAVYFCRA VTVSS 102 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 234 WHRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 084E05 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 103 T0170PMP 175
EVQLVESGGGLVQ 122 GDVHKIN 234 WHRQAPAKE 138 HITIGD 260
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 285 WGQGTQ 084F04 PGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAAYFCRA VTVSS 104 T0170PMP 202
EVQLVESGGGWVQ 122 GDVHKIN 207 WYRQAPAKE 138 HITIGD 237
YADSAKGRFTISRDEAKNMV 166 YSRIYPYNY 286 WGQGTL 084F10 AGGSLRLSCAAS
ILG REMVA AID YLQMNSLKPEDTAVYFCRA VTVSS 105 T0170PMP 184
EVQLVESGGGLVQ 124 GETFKIN 210 WYRQAPGKQ 142 SLTIGG 243
YADSVKGRFTISEDSAKNTV 167 KSRLYPYDY 285 WGQGTQ 055B06 PGGSLRLSCITS
IWG RELVA AID YLQMNSLKAEDTAVYFCNA VTVSS 106 T0170PMP 184
EVQLVESGGGLVQ 124 GETFKIN 210 WYRQAPGKQ 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 167 KSRLYPYDY 286 WGQGTL 055C07 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 107 T0170PMP 184
EVQLVESGGGLVQ 124 GETFKIN 212 WYRQAPGKR 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 168 KSRIYPYDY 285 WGQGTQ 055D01 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 108 T0170PMP 184
EVQLVESGGGLVQ 124 GETFKIN 217 WYRQTPGKQ 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 167 KSRLYPYDY 285 WGQGTQ 056B02 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 109 T0170PMP 184
EVQLVESGGGLVQ 128 GQTFKIN 210 WYRQAPGKQ 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 167 KSRLYPYDY 285 WGQGTQ 056D07 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 110 T0170PMP 184
EVQLVESGGGLVQ 131 GETFKVN 225 WYRQGPGKQ 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 167 KSRLYPYDY 287 WGRGTQ 068B03 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 111 T0170PMP 196
EVQLVEESGGGLV 124 GETFKIN 210 WYRQAPGKQ 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 168 KSRIYPYDY 287 WGRGTQ 068G05 QPGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 112 T0170PMP 184
EVQLVESGGGLVQ 124 GETFKIN 210 WYRQAPGKQ 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 167 KSRLYPYDY 290 WDQGTQ 069E03 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 113 T0170PMP 199
EVQLVESGGGLVQ 131 GETFKVN 225 WYRQGPGKQ 145 SLTIGG 274
YADSVKGRFTISEDSAKNTV 167 KSRLYPYDY 285 WGQGTQ 069E06 PGGSLRLSCLTS
IWG RELVA ATN YLQMSSLKPEDTAVYFCNA VTVSS 114 T0170PMP 184
EVQLVESGGGLVQ 124 GETFKIN 210 WYRQAPGKQ 145 SLTIGG 246
YADSVKGRFTISEDSAKNTV 168 KSRIYPYDY 287 WGRGTQ 070A09 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 115 T0170PMP 184
EVQLVESGGGLVQ 124 GETFKIN 210 WYRQAPGKQ 145 SLTIGG 279
YADSVKGRFTISEDSAKDTV 167 KSRLYPYDY 286 WGQGTL 070C01 PGGSLRLSCITS
IWG RELVA ATN YLQMNSLKPEDTAVYFCNA VTVSS 116 T0170PMP 175
EVQLVESGGGLVQ 130 GEIGRIN 220 WYRQAPGNQ 157 TITIAD 261
YADSAKGRFTISRDESRNMV 172 GSRLYPYDY 285 WGQGTQ 061G01 PGGSLRLSCAAS
FYR REVVA KID YLQMSSLKPEDTAVYFCHA VTVSS 117 T0170PMP 175
EVQLVESGGGLVQ 130 GEIGRIN 221 WYRQAPGNQ 158 TITIAD 262
YADSAKGRFTISRDESRNMV 172 GSRLYPYDY 285 WGQGTQ 062D02 PGGSLRLSCAAS
FYR RGVVA KID YLQMGSLKPEDTAVYFCHA VTVSS 118 T0170PMP 175
EVQLVESGGGLVQ 130 GEIGRIN 220 WYRQAPGNQ 157 TITIAD 282
YADSAKGRFTISRDESRNMV 172 GSRLYPYDY 285 WGQGTQ 075D02 PGGSLRLSCAAS
FYR REVVA KID YLQMSSLKPENTAVYFCHA VTVSS
TABLE-US-00053 TABLE A-5 Sequences of multispecific polypeptides
(with and without tags). ''SEQ'' refers to the given SEQ ID NO;
''ID'' refers to identification name; ''Sequence'' denotes amino
acid sequence SEQ ID Sequence 291 1017000001
EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRD-
NAKNTVYLQMNSLKPEDTAVYYCAA
KIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGLVQGGGSLSLSCAASGRTFSSY
AMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTY-
PDNWYWTKSNNYNYWGQGTLVTVSSG AADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 292
1017000002
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKER-
GLVATITIGDTTDYADYAKGRFTISR
DEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKG-
AAHHHHHH 293 1017000003
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVHKIN-
FLGWYRQAPGKERGLVATITIGDTTD
YADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSSGAADYKDHDGDYK-
DHDIDYKDDDDKGAAHHHHHH 294 1017000006
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKER-
EGVAAINMGGGITYYADSVKGRFTIS
QDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGAADYKDHDG-
DYKDHDIDYKDDDDKGAAHHHHHH 295 1017000007
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFV-
AEVRWGGVTTYSNSLKDRFSISEDSV
KNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKG-
AAHHHHHH 296 1017000008
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAP-
GKERGLVATITIGDTTDYADYAKGRF
TISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDD-
DDKGAAHHHHHH 297 1017000009
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYY.CA
ADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGS-
VHKINFLGWYRQAPGKERGLVATITI
GDTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSSGAADYKDH-
DGDYKDHDIDYKDDDDKGAAHHHHHH 298 1017000012
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAP-
GKEREGVAAINMGGGITYYADSVKGR
FTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGAADYK-
DHDGDYKDHDIDYKDDDDKGAAHHHH HH 299 1017000013
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKE-
REFVAEVRWGGVTTYSNSLKDRFSIS
EDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDD-
DDKGAAHHHHHH 300 1017000014
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHKINFLGWYRQAP
GKERGLVATITIGDTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 301 1017000015
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV-
QLVESGGGLVQPGGSLRLSCAASGSV
HKINFLGWYRQAPGKERGLVATIIIGDTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSR-
LYPYNYWGQGTLVTVSSGAADYKDHD GDYKDHDIDYKDDDDKGAAHHHHHH 302 1017000018
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAG-
GSLRLSCAASGYTIGPYCMGWFRQAP
GKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGL-
STGGYGYDSWGQGTQVTVSSGAADYK DHDGDYKDHDIDYKDDDDKGAAHHHHHH 303
1017000019
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 304 1017000023
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV-
QLVESGGGLVQPGGSLRLSCTFSGGT
FSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQ-
MYMTVVPDYWGQGTLVTVSSGAADYK DHDGDYKDHDIDYKDDDDKGAAHHHHHH 305
1017000025
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAGGSL-
RLSCAASGYTIGPYCMGWFRQAPGKE
REGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTG-
GYGYDSWGQGTQVTVSSGAADYKDHD GDYKDHDIDYKDDDDKGAAHHHHHH 306 1017000029
EVQLVESGGGLVQPGGSLRLSCAASGDVYKINFLGWHRQAPGKEREKVAHITIGDATDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAG
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 307 1017000030
EVQLVESGGGLVQPGGSLKLSCAASGAVHKINFLGWYRQTPEKEREMVATITIGDEVDYADSAKGRFTISRDE-
AKNMVYLQMTSLKPEDTAVYVCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 308 1017000031
EVQLVESGGGLVQPGGSLRLSCITSGETFKINIWGWYRQAPGKQRELVASLTIGGATNYADSVKGRFTISEDS-
AKNTVYLQMNSLKPEDTAVYFCNAK
SRLYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 309 1017000032
EVQLVESGGGLVQPGGSLRLSCAASGSVHLLNFLGWYRQAPGKEREMVAHITIADATDYAHFAKGRFTISRDE-
AKNMVYLQMNSLRPEDTAVYFCRAG
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 310 1017000033
EVQLVESGGGLVQPGGSLRLSCITSGETFKINIWGWYRQAPGKQRELVASLTIGGATDYADSVKGRFTISEDS-
AKNTVYLQMNSLKAEDTAVYFCNAK
SRLYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 311 1017000035
EVQLVESGGGLVQPGGSLRLSCAASGEVYKINFLGWYRQAPGKEREKVAHITIADAADYADFAKGRFTISRDE-
AKNMVYLQMNSLRPEDTAVYFCRAG
SRIWPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 312 1017000037
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMVYLQMNSLKPEDTAVYFCRAF
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 313 1017000038
EVQLVESGGGLVQPGGSLRLSCAASGEIGRINFYRWYRQAPGNQREVVATITIADKTDYADSAKGRFTISRDE-
SRNMVYLQMSSLKPEDTAVYFCHAG
SRLYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 314 1017000041
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHITIGDQADYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAG
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 315 1017000042
EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAY
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 316 1017000044
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERELVATITIGDTTDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 317 1017000046
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 318 1017000049
EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYLCRAY
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD
DDKGAAHHHHHH 319 1017000050
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 320 1017000051
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGEIGRINFYRWYRQAP
GNQREVVATITIADKTDYADSAKGRFTISRDESRNMVYLQMSSLKPEDTAVYFCHAGSRLYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 321 1017000054
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHITIGDQADYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAGSRIYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 322 1017000055
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 323 1017000058
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHKINFLGWYRQAP
GKERELVATITIGDTTDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 324 1017000060
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHKINFLGWYRQAP
GKERGLVATIIIGDTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 325 1017000063
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVYKINFLGWHRQAP
GKEREKVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAGSRIYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 326 1017000064
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGEVYKINFLGWYRQAP
GKEREKVAHITIADAADYADFAKGRFTISRDEAKNMVYLQMNSLRPEDTAVYFCRAGSRIWPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 327 1017000065
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGDLVQPG-
GSLRLSCAASGDVHKINFLGWYRQAP
GKEREMVAHITIADATDYAEFAKGRFTISRDEPKNMVYLQMNSLKPEDTAVYLCRAGSRIYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 328 1017000068
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCITSGETFKINIWGWYRQAP
GKQRELVASLTIGGATNYADSVKGRFTISEDSAKNTVYLQMNSLKPEDTAVYFCNAKSRLYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 329 1017000069
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHLLNFLGWYRQAP
GKEREMVAHITIADATDYAHFAKGRFTISRDEAKNMVYLQMNSLRPEDTAVYFCRAGSRIYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 330 1017000070
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCITSGETFKINIWGWYRQAP
GKQRELVASLTIGGATDYADSVKGRFTISEDSAKNTVYLQMNSLKAEDTAVYFCNAKSRLYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 331 1017000073
EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATSYADSAKGRFTISRDE-
AKNMVYLQLNNLKPEDTAVYFCRAY
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 332 1017000074
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMVYLQMNSLKPEDTAVYFCRAF
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 333 1017000075
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATSYADSAKGRFTISRDEAKNMVYLQLNNLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 334 1017000076
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 335 1017000077
EVQLVESGGDLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREMVAHITIADATDYAEFAKGRFTISRDE-
PKNMVYLQMNSLKPEDTAVYLCRAG
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 336 1017000078
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYLCRAYSRIYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 337 1017000079
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLKLSCAASGAVHKINFLGWYRQTP
EKEREMVATITIGDEVDYADSAKGRFTISRDEAKNMVYLQMTSLKPEDTAVYVCRAGSRLYPYNYWGQGTLV-
TVSSGAADYKDHDGDYKDHDIDYKDD DDKGAAHHHHHH 338 1017000083
DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSSA 339 1017000088
DVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRD-
NAKNTVYLQMNSLKPEDTAVYYCAA
DLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG-
GGLVQPGGSLRLSCAASGDVHKINIL
GWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNY-
WGQGTLVTVSSA 340 1017000093
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSSGGGGSGGGGSGGGGSGGGGSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL-
YADSVKGRFTISRDNAKTTLYLQMNS
LRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH
341 1017000095
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCITSGETFKINIWGWYRQAP
GKQRELVASLTIGGATNYADSVKGRFTISEDSAKNTVYLQMNSLKPEDTAVYFCNAKSRLYPYDYWGQGTLV-
TVSSGGGGSGGGGSGGGGSGGGGSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL-
YADSVKGRFTISRDNAKTTLYLQMNS
LRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH
342 1017000102
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDN-
AKNTVYLQMNSLKPEDTAVYYCKRF
RTAAQGTDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS-
LRLSCVASGDVHKINFLGWYRQAPGK
EREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTV-
SSGAADYKDHDGDYKDHDIDYKDDDD KGAAHHHHHH 343 1017000103
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMVYLQMNSLKPEDTAVYFCRAF
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL-
RLSCAASGITFSINTMGWYRQAPGKQ
RELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTLVTV-
SSGAADYKDHDGDYKDHDIDYKDDDD KGAAHHHHHH 387 1017000104
DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSSGGGGSGGGGSGGGGSGGGGSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL-
YADSVKGRFTISRDNAKTTLYLQMNS LRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA 388
1017000105
DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSSA 389 1017000106
DVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRD-
NAKNTVYLQMNSLKPEDTAVYYCAA
DLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG-
GGLVQPGGSLRLSCAASGDVHKINIL
GWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNY-
WGQGTLVTVSSGGGGSGGGGSGGGGS
GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS-
GSGSDTLYADSVKGRFTISRDNAKTT LYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA
390 1017000107
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRD-
NAKNTVHLQMNSLKPEDTAVYYCAA
SYNVYYNNYYYPISRDEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGSVQAGGSLRLSCAASGDTYGSY
WMGWFRQAPGKEREGVAAINRGGGYTVYADSVKGRFTISRDTAKNTVYLQMNSLRPDDTADYYCAASGVLGG-
LHEDWFNYWGQGTLVTVSSGGGGSGG
GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKE-
REKVAHISIGDQTDYADSAKGRFTIS
RDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKDDDDK-
GAAHHHHHH 391 1017000109
EVQLVESGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV-
QLVESGGGSVQAGGSLRLSCAASGDT
YGSYWMGWFRQAPGKEREGVAAINRGGGYTVYADSVKGRFTISRDTAKNTVYLQMNSLRPDDTADYYCAASG-
VLGGLHEDWFNYWGQGTLVTVSSGGG
GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQA-
PGKEREKVAHISIGDQTDYADSAKGR
FTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGAADYKDHDGDYKDHDIDYKD-
DDDKGAAHHHHHH 392 1017000110
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRD-
NAKNTVHLQMNSLKPEDTAVYYCAA
SYNVYYNNYYYPISRDEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGSVQAGGSLRLSCAASGYTIGPY
CMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYA-
SYYECGHGLSTGGYGYDSWGQGTLVT
VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFL-
GWYRQAPGKEREKVAHISIGDQTDYA
DSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGAADYKDHDGDYKDH-
DIDYKDDDDKGAAHHHHHH 413 1017000001
EVQLVESGGGLVQGGGSLSLSCAASGRTFSSYAMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRD-
NAKNTVYLQMNSLKPEDTAVYYCAA
KIAKTYPDNWYWTKSNNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGLVQGGGSLSLSCAASGRTFSSY
AMAWFRQPPGKEREFVASISWSGENTNYRNSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAKIAKTY-
PDNWYWTKSNNYNYWGQGTLVTVSS 414 1017000002
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKER-
GLVATITIGDTTDYADYAKGRFTISR
DEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSS 415 1017000003
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVHKIN-
FLGWYRQAPGKERGLVATITIGDTTD
YADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSS 416
1017000006
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKER-
EGVAAINMGGGITYYADSVKGRFTIS
QDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 417
1017000007
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFV-
AEVRWGGVTTYSNSLKDRFSISEDSV
KNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 418 1017000008
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAP-
GKERGLVATITIGDTTDYADYAKGRF
IISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSS 419 1017000009
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSV-
HKINFLGWYRQAPGKERGLVATITIG
DTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLVTVSS 420
1017000012
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGSDVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAP-
GKEREGVAAINMGGGITYYADSVKGR
FTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS
421 1017000013
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKE-
REFVAEVRWGGVTTYSNSLKDRFSIS
EDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLVTVSS 422 1017000014
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHKINFLGWYRQAP
GKERGLVATIIIGDTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLV-
TVSS 423 1017000015
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV-
QLVESGGGLVQPGGSLRLSCAASGSV
HKINFLGWYRQAPGKERGLVATIIIGDTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSR-
LYPYNYWGQGTLVTVSS 424 1017000018
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAG-
GSLRLSCAASGYTIGPYCMGWFRQAP
GKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGL-
STGGYGYDSWGQGTQVTVSS 425 1017000019
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 426 1017000023
EVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV-
QLVESGGGLVQPGGSLRLSCTFSGGT
FSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQ-
MYMTVVPDYWGQGTLVTVSS 427 1017000025
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDVQLQASGGGSVQAGGSL-
RLSCAASGYTIGPYCMGWFRQAPGKE
REGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTG-
GYGYDSWGQGTQVTVSS 428 1017000029
EVQLVESGGGLVQPGGSLRLSCAASGDVYKINFLGWHRQAPGKEREKVAHITIGDATDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAG
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 429 1017000030
EVQLVESGGGLVQPGGSLKLSCAASGAVHKINFLGWYRQTPEKEREMVATITIGDEVDYADSAKGRFTISRDE-
AKNMVYLQMTSLKPEDTAVYVCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 430 1017000031
EVQLVESGGGLVQPGGSLRLSCITSGETFKINIWGWYRQAPGKQRELVASLTIGGATNYADSVKGRFTISEDS-
AKNTVYLQMNSLKPEDTAVYFCNAK
SRLYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 431 1017000032
EVQLVESGGGLVQPGGSLRLSCAASGSVHLLNFLGWYRQAPGKEREMVAHITIADATDYAHFAKGRFTISRDE-
AKNMVYLQMNSLRPEDTAVYFCRAG
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 432 1017000033
EVQLVESGGGLVQPGGSLRLSCITSGETFKINIWGWYRQAPGKQRELVASLTIGGATDYADSVKGRFTISEDS-
AKNTVYLQMNSLKAEDTAVYFCNAK
SRLYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 433 1017000035
EVQLVESGGGLVQPGGSLRLSCAASGEVYKINFLGWYRQAPGKEREKVAHITIADAADYADFAKGRFTISRDE-
AKNMVYLQMNSLRPEDTAVYFCRAG
SRIWPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 434 1017000037
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMVYLQMNSLKPEDTAVYFCRAF
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 435 1017000038
EVQLVESGGGLVQPGGSLRLSCAASGEIGRINFYRWYRQAPGNQREVVATITIADKTDYADSAKGRFTISRDE-
SRNMVYLQMSSLKPEDTAVYFCHAG
SRLYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 436 1017000041
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHITIGDQADYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAG
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 437 1017000042
EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAY
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 438 1017000044
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERELVATITIGDTTDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 439 1017000046
EVQLVESGGGLVQPGGSLRLSCAASGSVHKINFLGWYRQAPGKERGLVATITIGDTTDYADYAKGRFTISRDE-
ARNMVYLQMNSLKPEDTAVYFCRAG
SRLYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS
440 1017000049
EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDE-
AKNMVYLQMNSLKPEDTAVYLCRAY
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 441 1017000050
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSS 442 1017000051
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGEIGRINFYRWYRQAP
GNQREVVATIIIADKTDYADSAKGRFTISRDESRNMVYLQMSSLKPEDTAVYFCHAGSRLYPYDYWGQGTLV-
TVSS 443 1017000054
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHITIGDQADYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAGSRIYPYDYWGQGTLV-
TVSS 444 1017000055
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSS 445 1017000058
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHKINFLGWYRQAP
GKERELVATIIIGDTTDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLV-
TVSS 446 1017000060
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHKINFLGWYRQAP
GKERGLVATIIIGDTTDYADYAKGRFTISRDEARNMVYLQMNSLKPEDTAVYFCRAGSRLYPYNYWGQGTLV-
TVSS 447 1017000063
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVYKINFLGWHRQAP
GKEREKVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAGSRIYPYDYWGQGTLV-
TVSS 448 1017000064
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGEVYKINFLGWYRQAP
GKEREKVAHITIADAADYADFAKGRFTISRDEAKNMVYLQMNSLRPEDTAVYFCRAGSRIWPYDYWGQGTLV-
TVSS 449 1017000065
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGDLVQPG-
GSLRLSCAASGDVHKINFLGWYRQAP
GKEREMVAHITIADATDYAEFAKGRFTISRDEPKNMVYLQMNSLKPEDTAVYLCRAGSRIYPYNYWGQGTLV-
TVSS 450 1017000068
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCITSGETFKINIWGWYRQAP
GKQRELVASLTIGGATNYADSVKGRFTISEDSAKNTVYLQMNSLKPEDTAVYFCNAKSRLYPYDYWGQGTLV-
TVSS 451 1017000069
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGSVHLLNFLGWYRQAP
GKEREMVAHITIADATDYAHFAKGRFTISRDEAKNMVYLQMNSLRPEDTAVYFCRAGSRIYPYDYWGQGTLV-
TVSS 452 1017000070
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCITSGETFKINIWGWYRQAP
GKQRELVASLTIGGATDYADSVKGRFTISEDSAKNTVYLQMNSLKAEDTAVYFCNAKSRLYPYDYWGQGTLV-
TVSS 453 1017000073
EVQLVESGGGLVQPGGSLRLSCAASGDVHKINILGWYRQAPAKEREMVAHITIGDATSYADSAKGRFTISRDE-
AKNMVYLQLNNLKPEDTAVYFCRAY
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 454 1017000074
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMVYLQMNSLKPEDTAVYFCRAF
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 455 1017000075
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATSYADSAKGRFTISRDEAKNMVYLQLNNLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSS 456 1017000076
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSS 457 1017000077
EVQLVESGGDLVQPGGSLRLSCAASGDVHKINFLGWYRQAPGKEREMVAHITIADATDYAEFAKGRFTISRDE-
PKNMVYLQMNSLKPEDTAVYLCRAG
SRIYPYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSL-
RLSCTFSGGTFSSYTMGWFRQAPGKE
REFVAEVRWGGVTTYSNSLKDRFSISEDSVKNAVYLQMNSLKPEDTAVYYCAAVRQMYMTVVPDYWGQGTLV-
TVSS 458 1017000078
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYLCRAYSRIYPYNYWGQGTLV-
TVSS 459 1017000079
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLKLSCAASGAVHKINFLGWYRQTP
EKEREMVATIIIGDEVDYADSAKGRFTISRDEAKNMVYLQMTSLKPEDTAVYVCRAGSRLYPYNYWGQGTLV-
TVSS 460 1017000093
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSSGGGGSGGGGSGGGGSGGGGSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL-
YADSVKGRFTISRDNAKTTLYLQMNS LRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 461
1017000095
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCITSGETFKINIWGWYRQAP
GKQRELVASLTIGGATNYADSVKGRFTISEDSAKNTVYLQMNSLKPEDTAVYFCNAKSRLYPYDYWGQGTLV-
TVSSGGGGSGGGGSGGGGSGGGGSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL-
YADSVKGRFTISRDNAKTTLYLQMNS LRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 462
1017000102
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDN-
AKNTVYLQMNSLKPEDTAVYYCKRF
RTAAQGTDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS-
LRLSCVASGDVHKINFLGWYRQAPGK
EREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTV-
SS 463 1017000103
EVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDE-
SKNMVYLQMNSLKPEDTAVYFCRAF
SRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSL-
RLSCAASGITFSINTMGWYRQAPGKQ
RELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTLVTV-
SS 464 1017000107
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRD-
NAKNTVHLQMNSLKPEDTAVYYCAA
SYNVYYNNYYYPISRDEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGSVQAGGSLRLSCAASGDTYGSY
WMGWFRQAPGKEREGVAAINRGGGYTVYADSVKGRFTISRDTAKNTVYLQMNSLRPDDTADYYCAASGVLGG-
LHEDWFNYWGQGTLVTVSSGGGGSGG
GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKE-
REKVAHISIGDQTDYADSAKGRFTIS
RDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS 465 1017000109
EVQLVESGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA
DSTIYASYYECGHGLSTGGYGYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEV-
QLVESGGGSVQAGGSLRLSCAASGDT
YGSYWMGWFRQAPGKEREGVAAINRGGGYTVYADSVKGRFTISRDTAKNTVYLQMNSLRPDDTADYYCAASG-
VLGGLHEDWFNYWGQGTLVTVSSGGG
GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQA-
PGKEREKVAHISIGDQTDYADSAKGR
FTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS 466 1017000110
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRD-
NAKNTVHLQMNSLKPEDTAVYYCAA
SYNVYYNNYYYPISRDEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGSVQAGGSLRLSCAASGYTIGPY
CMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYA-
SYYECGHGLSTGGYGYDSWGQGTLVT
VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFL-
GWYRQAPGKEREKVAHISIGDQTDYA
DSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSS 467
1017000104
DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSSGGGGSGGGGSGGGGSGGGGSGG
GGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL-
YADSVKGRFTISRDNAKTTLYLQMNS LRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 468
1017000105
DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCVASGDVHKINFLGWYRQAP
GKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLV-
TVSS
469 1017000106
DVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRD-
NAKNTVYLQMNSLKPEDTAVYYCAA
DLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG-
GGLVQPGGSLRLSCAASGDVHKINIL
GWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNY-
WGQGTLVTVSSGGGGSGGGGSGGGGS
GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSIS-
GSGSDTLYADSVKGRFTISRDNAKTT LYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS
470 1017000083
DVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV
RQMYMTVVPDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPG-
GSLRLSCAASGDVHKINILGWYRQAP
AKEREMVAHIIIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNYWGQGTLV-
TVSS 471 1017000088
DVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRD-
NAKNTVYLQMNSLKPEDTAVYYCAA
DLTSTNPGSYIYIWAYDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESG-
GGLVQPGGSLRLSCAASGDVHKINIL
GWYRQAPAKEREMVAHITIGDATDYADSAKGRFTISRDEAKNMVYLQMNSLKPEDTAVYFCRAYSRIYPYNY-
WGQGTLVTVSS 486 1017000108
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRD-
NAKNTVHLQMNSLKPEDTAVYYCAA
SYNVYYNNYYYPISRDEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGSVQAGGSLRLSCAASGDTYGSY
WMGWFRQAPGKEREGVAAINRGGGYTVYADSVKGRFTISRDTAKNTVYLQMNSLRPDDTADYYCAASGVLGG-
LHEDWFNYWGQGTLVTVSSGGGGSGG
GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKE-
REKVAHISIGDQTDYADSAKGRFTIS
RDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS-
GGGGSGGGGSEVQLVESGGGLVQPGN
SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRP-
EDTAVYYCTIGGSLSRSSQGTLVTVS SGAADYKDHDGDYKDHDIDYKDDDDKGAAHHHHHH 487
1017000108
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRD-
NAKNTVHLQMNSLKPEDTAVYYCAA
SYNVYYNNYYYPISRDEYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVE-
SGGGSVQAGGSLRLSCAASGDTYGSY
WMGWFRQAPGKEREGVAAINRGGGYTVYADSVKGRFTISRDTAKNTVYLQMNSLRPDDTADYYCAASGVLGG-
LHEDWFNYWGQGTLVTVSSGGGGSGG
GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKE-
REKVAHISIGDQTDYADSAKGRFTIS
RDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGS-
GGGGSGGGGSEVQLVESGGGLVQPGN
SLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRP-
EDTAVYYCTIGGSLSRSSQGTLVTVS S
TABLE-US-00054 TABLE A-6 Sequences of component; of TCR complex.
''SEQ'' refers to the given SEQ ID NO; ID'' refers to
identification name; ''Sequence'' demotes amino add sequence SEQ ID
SEQUENCE 344 Human CD3
MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRC-
NGTDIYKDKESTVQVHYRMCQSCVE delta
LDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGG-
NWARNK (P04234) 345 Human CD3
MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWN-
LGSNAKDPRGMYQCKGSQNKSKPLQ gamma
VYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKD-
REDDQYSHLQGNQLRRN (P09692) 346 Human CD3
MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDK-
NIGSDEDHLSLKEFSELEQSGYYVC epsilon
YPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVT-
RGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQR (P07766) DLYSGLNQRRI 347 Human
CD3
MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYN-
ELNLGRREEYDVLDKRRGRDPEMGG zeta
KPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(P20963) 348 Human TCR
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFA-
CANAFNNSIIPEDTFFPSPESSCDV alpha
KLVEKSFETDTNFRILLLKVAGFNLLMTLRLWSSLNFQNLSVIG constant domain
(P01848) 349 Human TCR
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSR-
YCLSSRLRVSATFWQNPRNHFRCQV beta
QFYGLSENDEWTVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKD-
FQDRAKPVTQI constant domain (P01850) 393 Human TCR
IQVEQSPPDLILQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLYI-
SSSQTTDSGVYFCAALIQGAQKLVF alpha GQGTRLTIN variable domain derived
from 2IAN 476 Human TCR
NAGVTQTPKFRILKIGQSMTLQCTQDMNHNYMYWYRQDPGMGLKLIYYSVGAGITDKGEVPNGYNVSRSTTED-
FPLRLELAAPSQTSVYFCASTYHGT beta GYFGEGSWLTVV variable domain derived
from 2IAN 394 Human TCR
QLLEQSPQFLSIQEGENLTVYCNSSSVFSSLQWYRQEPGEGPVLLVTVVTGGEVKKLKRLTFQFGDARKDSSL-
HITAAQPGDTGLYLCAGAGSQGNLI alpha FGKGTKLSVK variable domain derived
from 2XN9 477 Human TCR
DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKES-
FPLTVTSAQKNPTAFYLCASSSRSS beta YEQYFGPGTRLTVT variable domain
derived from 2XN9 395 Human TCR
GDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSST-
LILHRATLRDAAVYYCTVYGGATNK alpha LIFGTGTLLAVQ variable domain
derived from 310E 478 Human TCR
VVSQHPSWVIAKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLT-
LSTLTVTSAHPEDSSFYICSARGGS beta YNSPLHFGNGTRLTVT variable domain
derived from 310E 396 Cyno TCR
PYIQNPDPAVYQLRGSKSNDTSVCLFTDFDSVMNVSQSKDSDVHITDKTVLDMRSMDFKSNGAVAWSNKSDFA-
CTSAFKDSVIPADTFFPSPESSC alpha constant domain 397 Rhesus TCR
EDLKKVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALEDSR-
YSLSSRLRVSATFWHNPRNHFRCQV beta QFYGLSEDDEWTEDRDKPITQKISAEAWGRADC
constant domain 398 Rhesus TCR
QQIMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIMLVKSGEVKKQKRLIFQFGEAKKNSSL-
HITATQTTDVGTYFCATTGVNNLFF alpha GTGTRLTVL variable domain 399
Rhesus TCR
AGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHDYMYWYRQDPGMGLRLIHYSVGEGSTEKGEVPDGYNVTRS-
NTEDFPLRLESAAPSQTSVYFCASS beta YWTGRSYEQYFGPGTRLTVI variable domain
479 Human TCR
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKSVLDMRSMDFKSNSAVAWSNKSDFA-
CANAFNNSIIPEDTFFPSPESSC alpha constant domain 480 Human TCR
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSR-
YSLSSRLRVSATFWQNPRNHFRCQV beta QFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC
constant domain 484 Human TCR
PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFA-
CANAFNNSIIPEDTFFPSPESSCDV alpha
KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS constant domain
(P01848) 485 Human TCR
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSR-
YCLSSRLRVSATFWQNPRNHFRCQV beta
QFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSAL-
VLMAMVKRKDF constant domain
TABLE-US-00055 TABLE A-7 Sequences of TAA binding building blocks
and control Nanobodies. ''SEQ'' refers to a given SEQ ID NO; ''ID''
refers to identification name; ''Sequence'' denotes amino acid
sequence SEQ ID SEQUENCE 350 HER2005F07
EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDN-
AKNTVYLQMNSLKPEDTAVYYCKRF (Q108L) RTAAQGTDYWGQGTLVTVSS 351
HER2047D05
EVQLVESGGGLVQPGGSLRLSCAASGSIFGFNDMAWYRQAPGKQRELVALISRVGVTSSADSVKGRFTISRVN-
AKDTVYLQMNSLKPEDTAVYYCYMD (L108Q) QRLDGSTLAYWGQGTQVTVSS 352
EGFR009G08
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVVAINWSSGSTYYADSVKGRFTISRD-
NAKNTMYLQMNSLKPEDTAVYYCAA GYQINSGNYNFKDYEYDYWGQGTQVTVSS 353 NbCEA5
EVQLVESGGGSVQAGGSLRLSCAASGDTYGSYWMGWFRQAPGKEREGVAAINRGGGYTVYADS-
VKGRFTISRDTAKNTVYLQMNSLRPDDTADYYCAA (CEA#1)
SGVLGGLHEDWFNYWGQGTLVTVSS 354 T023200005
EVQLVESGGGSVQAGGSLRLSCAASGDTYGSYWMGWFRQAPGQEREAVAAINRGGGYTVYADSVKGRFTISRD-
NAKNTLYLQMNSLRPDDTADYYCAA (CEA#5) SGVLGGLHEDWFNYWGQGTLVTVSS 355
7D12
EVQLVESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVK-
GRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAA (EGFR#1)
AAGSAWYGTLYEYDYWGQGTLVTVSS 356 T023200033
EVQLVESGGGSVQAGGSLRLTCAASGSTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRD-
NAKNTVDLQMNSLKPEDTAIYYCAA (EGFR#33) AAGSTWYGTLYEYDYWGQGTLVTVSS 357
20CD019C07
EVQLVESGGGLVQPGGSLRLSCTFSGGTFSSYTMGWFRQAPGKEREFVAEVRWGGVTTYSNSLKDRFSISEDS-
VKNAVYLQMNSLKPEDTAVYYCAAV RQMYMTVVPDYWGQGTLVTVSS 358 EGFR038G07
EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQATGKEREFVATIAWDSGSTYYADSVKGRFTISRD-
NAKNTVHLQMNSLKPEDTAVYYCAA SYNVYYNNYYYPISRDEYDYWGQGTLVTVSS 359
cAblys3
DVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQD-
NAKNTVYLLMNSLEPEDTAIYYCAA DSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS 360
RSV007B02
EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAISWSDGSTYYADSVKGRFTISRD-
NAKNTVYLQMNSLKPEDTAVYYCAA DLTSTNPGSYTYIWAYDYWGQGTQVTVSS
TABLE-US-00056 TABLE A-8 Sequences for CDRs and frameworks of TAA
binding building blocks, plus preferred combinations as provided in
formula I, namely FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4. ''SEQ'' refers
to the given SEQ ID NO. The first column refers to the SEQ ID NO of
the complete ISV, i.e. FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4. CDR1, CDR2
and CDR3 were determined according to Kontermann, 2010. SEQ
Nanobody SEQ FR1 SEQ CDR1 SEQ FR2 SEQ CDR2 SEQ FR3 SEQ CDR3 SEQ FR4
353 NbCEA5 367 EVQLVESGGGSVQ 361 GDTYGSY 369 WFRQAPGKE 363 AINRGG
372 YADSVKGRFTISRDTAKNTV 365 SGVLGGLHED 375 WGQGTL (CEA#1)
AGGSLRLSCAAS WMG REGVA GYTV YLQMNSLRPDDTADYYCAA WFNY VTVSS 354
T023200005 367 EVQLVESGGGSVQ 361 GDTYGSY 370 WFRQAPGQE 363 AINRGG
373 YADSVKGRFTISRDNAKNTL 365 SGVLGGLHED 375 WGQGTL (CEA#5)
AGGSLRLSCAAS WMG REAVA GYTV YLQMNSLRPDDTADYYCAA WFNY VTVSS 357
20CD019C07 368 EVQLVESGGGLVQ 362 GGTFSSY 371 WFRQAPGKE 364 EVRWGG
374 YSNSLKDRFSISEDSVKNAV 366 VRQMYMTVVP 375 WGQGTL PGGSLRLSCTFS TMG
REFVA VTT YLQMNSLKPEDTAVYYCAA DY VTVSS
TABLE-US-00057 TABLE C-6 EC50 (M) of multispecific TCR binding
polypeptides for binding to CHO-K1 human TCR(2XN9)/CD3, purified
primary human T cells and Ramos cells as determined in flow
cytometry. CHO-K1 huTCR(2XN9)/CD3 human T cells Ramos cells Cluster
sample ID EC50 (M) 95% LCI 95% UCI EC50 (M) 95% LCI 95% UCI EC50
(M) 95% LCI 95% UCI A T017000055 1.1E-07 9.8E-08 1.3E-07 1.3E-07
3.0E-08 5.6E-07 1.8E-09 1.6E-09 1.9E-09 A T017000042 1.7E-08
1.4E-08 1.9E-08 2.3E-07 2.0E-07 2.6E-07 2.0E-08 1.8E-08 2.2E-08 A
T017000076 7.8E-08 7.1E-08 8.6E-08 1.3E-07 1.3E-08 1.3E-06 1.8E-09
1.7E-09 2.0E-09 A T017000074 1.6E-08 1.5E-08 1.7E-08 1.9E-07
1.7E-07 2.1E-07 1.6E-08 1.4E-08 1.7E-08 B T017000068 1.9E-07
1.5E-07 2.3E-07 6.2E-09 4.2E-09 8.9E-09 1.8E-09 1.7E-09 2.0E-09 B
T017000031 2.0E-08 1.8E-08 2.3E-08 3.5E-07 3.1E-07 4.0E-07 2.3E-08
2.0E-08 2.5E-08 C T017000051 4.6E-07 4.1E-07 5.1E-07 >1E-07 / /
2.7E-09 2.4E-09 2.9E-09 C T017000038 1.7E-08 1.6E-08 1.9E-08
>1E-07 / / 2.1E-08 1.9E-08 2.3E-08
TABLE-US-00058 TABLE C-7 IC50 (M) of the multispecific polypeptides
in the flow cytometry based T cell mediated Ramos killing assay
using an effector to target ratio of 10 to 1. ID monovalent ID
construct IC50 % % lysis ID construct IC50 % % lysis Cluster
Nanobody (CD20 .times. TCR) n (M) lysis (stdev) (TCR .times. CD20)
n (M) lysis (stdev) A T0170056G05 T017000076 8 3.0E-10 25 14
T017000074 6 9.2E-10 18 11 A T0170055A02 T017000055 19 5.3E-10 29
13 T017000042 5 5.7E-09 17 11 B T0170055C07 T017000068 7 1.0E-09 22
7 T017000031 3 1.1E-09 29 9 C T0170061G01 T017000051 2 5.7E-10 16 2
T017000038 2 2.7E-09 27 4
TABLE-US-00059 TABLE C-12 EC50 values of the half-life extended
polypeptides in cell based binding to CHO-K1 human TCR(2XN9)/CD3,
primary human T cells and Ramos cells as determined in flow
cytometry. CHO-K1-K1 huTCR (2XN9)/CD3 human T cells Ramos cells
Cluster sample ID EC50 (M) 95% LCI 95% UCI EC50 (M) 95% LCI 95% UCI
EC50 (M) 95% LCI 95% UCI A T017000093 7.7E-08 7.0E-08 8.4E-08
4.8E-07 4.8E-08 4.8E-06 1.5E-09 1.3E-09 1.6E-09 B T017000095
2.7E-07 2.2E-07 3.4E-07 2.2E-07 5.1E-08 9.8E-07 3.7E-09 3.3E-09
4.1E-09
TABLE-US-00060 TABLE C-15 IC50 values of the HER2/TCR binding
polypeptides in the T cell mediated HER2-positive tumour killing
assay. xCELLigence based cytotoxicity (readout 18 h) - SKBR3 (E/T =
15:1) ID monovalent ID construct IC50 95% 95% ID construct IC50 95%
95% Cluster Nb (HER2 .times. TCR) n (M) LCI UCI (TCR .times. HER2)
n (M) LCI UCI A T0170056G05 T017000102 1 1.4E-11 1.1E-11 1.7E-11
T017000103 1 3.8E-12 3.2E-12 4.7E-12 ID monovalent ID construct
EC50 95% 95% ID construct IC50 95% 95% Cluster Nb (HER2 .times.
TCR) n (M) LCI2 UCI2 (TCR .times. HER2) n (M) LCI UCI xCELLigence
based cytotoxicity (readout 18 h) - MCF-7 (E/T = 15:1) A
T0170056G05 T017000102 1 1.2E-10 9.0E-11 1.0E-10 T017000103 1
5.9E-11 5.0E-11 7.0E-11 xCELLigence based cytotoxicity (readout 18
h) - MDA-MB-468(E/T = 15:1) A T0170056G05 T017000102 1 No fit
T017000103 1 No fit
TABLE-US-00061 TABLE C-16 IC50 (M) of the HER2/TCR bindins
polypeptides for IFN-13 secretion bv human T cells in the human T
cell mediated xCELLigence based HER2 dependent killing assay. ID
monovalent ID construct IC50 95% 95% ID construct IC50 95% 95% Nb
(HER2 .times. TCR) n (M) LCI UCI (TCR .times. HER2) n (M) LCI UCI
T0170056G05 T017000102 1 2.8E-11 1.4E-11 5.6E-11 T017000103 1
1.1E-10 5.6E-11 2.2E-10
TABLE-US-00062 TABLE C-17 IC50 (M) of and % lysis by the TCR/CD20
binding multispecific constructs in the cynomolgus T cell mediated
B cell (Ramos) killing assay to evaluate the cynomolgus
functionality of the TCR building block. ID monovalent ID construct
IC50 % % lysis ID construct IC50 % % lysis Cluster Nb (CD20 .times.
TCR) n (M) lysis (stdev) (TCR .times. CD20) n (M) lysis (stdev) A
T0170055A02 T017000055 2 5.69E-10 17 6 T017000042 1 1.7E-10 18 A
T0170056G05 T017000076 2 3.09E-10 20 1 T017000074 2 2.4E-10 31 2.21
B T0170055C07 T017000068 1 T017000031 1 7.9E-10 10
TABLE-US-00063 TABLE C-18 IC50 (M) of the TCR/CD20 binding
multispecific polypeptides in the cynomolgus T cell mediated
xCELLigence based CHO-K1 human CD20 killing assay. ID monovalent ID
construct IC50 95% 95% ID construct IC50 95% 95% Cluster Nb (CD20
.times. TCR) n (M) LCI UCI (TCR .times. CD20) n (M) LCI UCI A
T0170055A02 T017000055 1 9.9E-10 4.2E-10 2.3E-09 T017000042 1
8.9E-10 6.0E-10 1.3E-09 A T0170056G05 T017000076 1 6.7E-10 2.2E-10
2.0E-09 T017000074 1 1.2E-10 6.4E-11 2.3E-10 B T0170055C07
T017000068 ND ND ND T017000031 1 1.2E-09 7.2E-10 1.9E-09
TABLE-US-00064 TABLE C-23 IC50 (M) of the TCR/Her2 binding
multispecific polypeptides in the cynomolgus T cell mediated
xCELLigence based SKBR3 killing assay. ID monovalent ID construct
IC50 95% 95% ID construct IC50 95% 95% Nb (HER2 .times. TCR) n (M)
LCI UCI (TCR .times. HER2) n (M) LCI UCI T0170056G05 T017000102 1
4.6E-11 1.3E-11 1.7E-10 T017000103 1 1.2E-11 7.3E-12 2.1E-11
TABLE-US-00065 TABLE 31 IC50 (M) of the multispecific polypeptides
in the human T cell mediated xCELLigence based killing assay using
an effector to target ratio of 15. Data were analysed after 50-60
h. LoVo LS174T HER14 ID construct n IC50 (M) 95% LCI 95% UCI n IC50
(M) 95% LCI 95% UCI n IC50 (M) 95% LCI 95% UCI T017000107 1 6.1E-11
5.4E-11 7.0E-11 1 5.3E-09 4.2E-09 6.6E-09 1 1.4E-10 1.2E-10 1.6E-10
T017000109 1 2.0E-09 1.8E-09 2.2E-09 1 3.6E-08 2.7E-08 4.9E-08 1 /
/ / T017000110 1 6.0E-10 5.3E-10 6.8E-10 1 8.9E-09 5.8E-09 1.4E-08
1 4.2E-10 3.7E-10 4.7E-10
Sequence CWU 1
1
4871117PRTArtificial SequenceNanobody sequence 1Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu Gly
Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45Ala Thr
Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75
80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val His Phe Cys Arg
85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr
Gln 100 105 110Val Thr Val Ser Ser 1152117PRTArtificial
SequenceNanobody sequence 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Gly Leu Val 35 40 45Ala Thr Ile Thr Ile Gly Asp
Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 1153117PRTArtificial SequenceNanobody sequence 3Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Pro Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn
20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met
Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr
Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
1154117PRTArtificial SequenceNanobody sequence 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu Gly
Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45Ala Thr
Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55 60Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65 70 75
80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 1155117PRTArtificial
SequenceNanobody sequence 5Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 1156117PRTArtificial SequenceNanobody sequence 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 Ser Val His Lys Ile Asn
20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu
Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr
Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
1157117PRTArtificial SequenceNanobody sequence 7Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Val Ser Gly Ser Val His Leu Leu Asn 20 25 30Phe Leu Gly
Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His
Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys 50 55 60Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75
80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 1158117PRTArtificial
SequenceNanobody sequence 8Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Ala Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Thr
Pro Glu Lys Glu Arg Glu Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp
Asp Val Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Thr Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg 85 90 95Ala Gly Ser
Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 1159117PRTArtificial SequenceNanobody sequence 9Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Asp Val His Lys Ile Asn
20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met
Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Glu Ser
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr
Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11510117PRTArtificial SequenceNanobody sequence 10Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11511117PRTArtificial
SequenceNanobody sequence 11Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11512117PRTArtificial SequenceNanobody sequence
12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Thr Ile Gly Asp Gln Ala Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11513117PRTArtificial SequenceNanobody sequence 13Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11514117PRTArtificial
SequenceNanobody sequence 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Ser Ile Ala Asp
Ala Thr Asp Tyr Ala His Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Arg Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11515117PRTArtificial SequenceNanobody sequence
15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Arg Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Val Tyr Ala Glu
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11516117PRTArtificial SequenceNanobody sequence 16Glu 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Cys Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11517117PRTArtificial
SequenceNanobody sequence 17Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Pro Ser Cys Ala Ala Ser
Gly Ser Val His Leu Leu Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Ala Asp
Ala Thr Asp Tyr Ala His Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11518117PRTArtificial SequenceNanobody sequence
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 Ser Val His Leu Leu
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala His
Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11519117PRTArtificial SequenceNanobody sequence 19Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Thr Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Val Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11520117PRTArtificial
SequenceNanobody sequence 20Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val His Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp His Arg Gln Pro
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Gly Asp
Val Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Asn
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11521117PRTArtificial SequenceNanobody sequence
21Glu 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 Asp Val His Lys Ile Asn 20 25 30Val Leu Gly Trp
Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile
Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val His Leu65 70 75
80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr
Gln 100 105 110Val Thr Val Ser Ser 11522117PRTArtificial
SequenceNanobody sequence 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asn Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11523117PRTArtificial SequenceNanobody sequence
23Glu 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 Asp Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Thr Ile Gly Asp Val Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Phe Leu65 70 75 80Gln Met Asn Asn Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11524117PRTArtificial SequenceNanobody sequence 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Gly Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11525117PRTArtificial
SequenceNanobody sequence 25Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp
Val Ala Asp Tyr Ala Asp Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11526117PRTArtificial SequenceNanobody sequence
26Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Glu Lys Glu Arg Glu
Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Thr Ser Leu Lys Pro Glu Asp Thr
Thr Val Tyr Val Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11527117PRTArtificial SequenceNanobody sequence 27Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11528117PRTArtificial
SequenceNanobody sequence 28Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Ser Ile Ser Asp
Gln Thr Asp Tyr Ala Glu Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ser Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg 85 90 95Ala Phe Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11529117PRTArtificial SequenceNanobody sequence
29Glu 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 Ser Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Leu Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Tyr Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11530117PRTArtificial SequenceNanobody sequence 30Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Ala Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11531117PRTArtificial
SequenceNanobody sequence 31Glu 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 Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp His Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11532117PRTArtificial SequenceNanobody sequence
32Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Val Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Ser Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11533117PRTArtificial SequenceNanobody sequence 33Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Ala Arg
Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11534117PRTArtificial
SequenceNanobody sequence 34Glu 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Ala Asp
Ala Thr Asp Tyr Ser His Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11535117PRTArtificial SequenceNanobody sequence
35Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Val His Leu Leu
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly
Val Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His
Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11536117PRTArtificial SequenceNanobody sequence 36Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Thr
Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11537117PRTArtificial
SequenceNanobody sequence 37Glu 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 Gly Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11538117PRTArtificial SequenceNanobody sequence
38Glu 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 Glu Val Tyr Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp
Phe Ala Gln 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11539117PRTArtificial SequenceNanobody sequence 39Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11540117PRTArtificial
SequenceNanobody sequence 40Glu 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Ala Asp
Ala Thr Asp Tyr Ser His Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly His Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11541117PRTArtificial SequenceNanobody sequence
41Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5
10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Thr Ile Gly Asp Gln Ala Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Arg Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11542117PRTArtificial SequenceNanobody sequence 42Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Val Ser Gly Ser Val His Leu Leu Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Thr
His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11543117PRTArtificial
SequenceNanobody sequence 43Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Ala Asp
Ala Thr Asp Tyr Ala Glu Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Pro Lys Asn Met Val His Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11544117PRTArtificial SequenceNanobody sequence
44Glu 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 Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Arg Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11545117PRTArtificial SequenceNanobody sequence 45Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
Arg Ile Ser Ile Ser Asp Gln Thr Asp Tyr Ala Glu Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys
Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11546117PRTArtificial
SequenceNanobody sequence 46Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Glu Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp
Val Ala Asp Tyr Ala Asp Phe Ala Lys 50 55 60Gly Arg Leu Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11547117PRTArtificial SequenceNanobody sequence
47Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Lys Leu Pro Cys Ala Ala Ser Gly Ala Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu
Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Thr Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Val Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11548117PRTArtificial SequenceNanobody sequence 48Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Gly
Leu Ser Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala His Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11549117PRTArtificial
SequenceNanobody sequence 49Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Ala Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11550117PRTArtificial SequenceNanobody sequence
50Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11551117PRTArtificial SequenceNanobody sequence 51Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11552117PRTArtificial
SequenceNanobody sequence 52Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Trp Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ser Val Tyr Lys Ile Asn 20 25 30Phe Leu Ser Trp Tyr Arg Gln Ala
Pro Gly His Glu Arg Glu Leu Val 35 40 45Ala Thr Ile Thr Ile Gly Asp
Ala Ala Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Phe Cys His 85 90 95Ala Gly Ser
Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11553117PRTArtificial SequenceNanobody sequence
53Glu 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 Ser Val His Leu Leu
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His
Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11554117PRTArtificial SequenceNanobody sequence 54Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Ala Ile Ser Asp Gln Thr Asp Tyr Ala Glu Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys
Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11555117PRTArtificial
SequenceNanobody sequence 55Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Ala Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11556117PRTArtificial SequenceNanobody sequence
56Glu 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 Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Gly
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11557117PRTArtificial SequenceNanobody sequence 57Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu His Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11558117PRTArtificial
SequenceNanobody sequence 58Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Ala Arg Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11559117PRTArtificial SequenceNanobody sequence
59Glu 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 Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Glu Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11560117PRTArtificial SequenceNanobody sequence 60Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11561117PRTArtificial
SequenceNanobody sequence 61Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Ala Val
His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Glu Lys
Glu Arg Glu Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Glu Val Asp
Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu
Ala Thr Asn Met Val Tyr Leu65 70 75 80Gln Met Thr Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr
Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser
Ser 11562117PRTArtificial SequenceNanobody sequence 62Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile
Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40
45Ala His Ile Thr Ile Gly Asp Ala Thr Ser Tyr Ala Gly Ser Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu65 70 75 80Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11563117PRTArtificial SequenceNanobody sequence 63Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Ser Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11564117PRTArtificial
SequenceNanobody sequence 64Glu Val Gln Leu Val Glu Ser Gly Gly Asp
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Ala Asp
Ala Thr Asp Tyr Ala Glu Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Pro Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11565117PRTArtificial SequenceNanobody sequence
65Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu
Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala His
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Thr Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Val Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11566117PRTArtificial SequenceNanobody sequence 66Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys
Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Glu Val Ala Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11567117PRTArtificial
SequenceNanobody sequence 67Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp
Ala Ala Asp Tyr Ala Asp Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11568117PRTArtificial SequenceNanobody sequence
68Glu 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 Glu Val Tyr Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp
Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11569117PRTArtificial SequenceNanobody sequence 69Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Pro Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11570117PRTArtificial
SequenceNanobody sequence 70Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp
Val Ala Asp Tyr Ala Asp Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Val Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11571117PRTArtificial SequenceNanobody sequence
71Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11572117PRTArtificial SequenceNanobody sequence 72Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11573117PRTArtificial
SequenceNanobody sequence 73Glu 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Ala Asp
Val Thr Asp Tyr Ser Tyr Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11574117PRTArtificial SequenceNanobody sequence
74Glu 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 Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Val Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11575117PRTArtificial SequenceNanobody sequence 75Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Met Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11576117PRTArtificial
SequenceNanobody sequence 76Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Ala Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Thr
Pro Glu Lys Glu Arg Glu Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp
Glu Val Asp Tyr Glu Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Thr Gly
Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg 85 90 95Ala Gly Ser
Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11577117PRTArtificial SequenceNanobody sequence
77Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Ser Ile Ser Asp Gln Thr Asp Tyr Ala Glu
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Leu Cys Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11578117PRTArtificial SequenceNanobody sequence 78Glu 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 Asp Val Tyr Lys Ile Asn 20 25 30Phe Leu
Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11579117PRTArtificial
SequenceNanobody sequence 79Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Arg Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala
Pro Ala Lys Glu Arg Glu Met Ile 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Glu Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11580117PRTArtificial SequenceNanobody sequence
80Glu 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 Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Gly Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11581117PRTArtificial SequenceNanobody sequence 81Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe
Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40
45Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Leu Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Ser Tyr Trp Gly
Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11582117PRTArtificial SequenceNanobody sequence 82Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11583117PRTArtificial
SequenceNanobody sequence 83Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Val
Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Ala Tyr Phe Cys Arg 85 90 95Ala Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11584117PRTArtificial SequenceNanobody sequence
84Glu 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 Glu Val Tyr Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp
Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11585117PRTArtificial SequenceNanobody sequence 85Glu 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Gly Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11586117PRTArtificial
SequenceNanobody sequence 86Glu 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 Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Ala Asp
Ala Thr Asp Tyr Ala Glu Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Pro Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11587117PRTArtificial SequenceNanobody sequence
87Glu 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 Glu Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Cys Pro Gly Lys Glu Arg Asp
Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Leu Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11588117PRTArtificial SequenceNanobody sequence 88Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11589117PRTArtificial
SequenceNanobody sequence 89Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu Gly Trp Gln Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp
Val Ala Asp Tyr Ala Asp Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11590117PRTArtificial SequenceNanobody sequence
90Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu
Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Val Asp Thr
Ala Val Tyr Leu Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11591117PRTArtificial SequenceNanobody sequence 91Glu 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ser His Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Asn Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11592117PRTArtificial
SequenceNanobody sequence 92Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Pro Arg Leu Ser Cys Val Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Gly Asp
Gln Ala Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11593117PRTArtificial SequenceNanobody sequence
93Glu 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 Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Ala
Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
11594117PRTArtificial SequenceNanobody sequence 94Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 11595117PRTArtificial
SequenceNanobody sequence 95Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Ser Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ser Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Cys Gln Ala
Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp
Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Ser Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val
Thr Val Ser Ser 11596117PRTArtificial SequenceNanobody sequence
96Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Ser Ile Ser Asp Gln Thr Asp Tyr Ala Glu
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Ala
Ala Val Tyr Leu Cys Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
11597117PRTArtificial SequenceNanobody sequence 97Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Pro Ser Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Ala Asp Val Ala Asp Tyr Ala Asp Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 11598117PRTArtificial
SequenceNanobody sequence 98Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Pro Ser Cys Val Ala Ser
Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp
Gln Ala Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser
Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val
Thr Val Ser Ser 11599117PRTArtificial SequenceNanobody sequence
99Glu 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 Glu Val His Lys Ile
Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Asp
Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Glu Thr Gln Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Leu Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115100117PRTArtificial SequenceNanobody sequence 100Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 115101117PRTArtificial
SequenceNanobody sequence 101Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln
Ala Pro Ala Lys Glu Arg Glu Met Val
35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Gly Asp Thr Ala Val
Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115102117PRTArtificial SequenceNanobody sequence 102Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu
Gly Trp His Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser 115103117PRTArtificial
SequenceNanobody sequence 103Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp His Arg Gln
Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly
Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Ala Tyr Phe Cys Arg 85 90 95Ala Tyr
Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 100 105
110Val Thr Val Ser Ser 115104117PRTArtificial SequenceNanobody
sequence 104Glu Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His
Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu
Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr
Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115105117PRTArtificial SequenceNanobody sequence 105Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile Trp
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala
Ser Leu Thr Ile Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys
Asn 85 90 95Ala Lys Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 115106117PRTArtificial
SequenceNanobody sequence 106Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr
Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly
Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys
Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser 115107117PRTArtificial SequenceNanobody
sequence 107Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe
Lys Ile Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln Ala Pro Gly Lys Arg
Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala
Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys Ser Arg Ile Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
115108117PRTArtificial SequenceNanobody sequence 108Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile Trp
Gly Trp Tyr Arg Gln Thr Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala
Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Asn 85 90 95Ala Lys Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 115109117PRTArtificial
SequenceNanobody sequence 109Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr
Ser Gly Gln Thr Phe Lys Ile Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly
Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys
Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105
110Val Thr Val Ser Ser 115110117PRTArtificial SequenceNanobody
sequence 110Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe
Lys Val Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln Gly Pro Gly Lys Gln
Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala
Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys Ser Arg Leu Tyr Pro
Tyr Asp Tyr Trp Gly Arg Gly Thr Gln 100 105 110Val Thr Val Ser Ser
115111118PRTArtificial SequenceNanobody sequence 111Glu Val Gln Leu
Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile 20 25 30Asn Ile
Trp Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu 35 40 45Val
Ala Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys 85 90 95Asn Ala Lys Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Arg
Gly Thr 100 105 110Gln Val Thr Val Ser Ser 115112117PRTArtificial
SequenceNanobody sequence 112Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr
Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly
Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys
Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Asp Gln Gly Thr Gln 100 105
110Val Thr Val Ser Ser 115113117PRTArtificial SequenceNanobody
sequence 113Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Leu Thr Ser Gly Glu Thr Phe
Lys Val Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln Gly Pro Gly Lys Gln
Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala
Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Ser Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys Ser Arg Leu Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
115114117PRTArtificial SequenceNanobody sequence 114Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile Trp
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala
Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Asn 85 90 95Ala Lys Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Arg Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 115115117PRTArtificial
SequenceNanobody sequence 115Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr
Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly
Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Glu Asp Ser Ala Lys Asp Thr Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys
Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser 115116117PRTArtificial SequenceNanobody
sequence 116Glu 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 Glu Ile Gly
Arg Ile Asn 20 25 30Phe Tyr Arg Trp Tyr Arg Gln Ala Pro Gly Asn Gln
Arg Glu Val Val 35 40 45Ala Thr Ile Thr Ile Ala Asp Lys Thr Asp Tyr
Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser
Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Ser Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys His 85 90 95Ala Gly Ser Arg Leu Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105 110Val Thr Val Ser Ser
115117117PRTArtificial SequenceNanobody sequence 117Glu 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 Glu Ile Gly Arg Ile Asn 20 25 30Phe Tyr
Arg Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Gly Val Val 35 40 45Ala
Thr Ile Thr Ile Ala Asp Lys Ile Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Arg Asn Met Val Tyr Leu65
70 75 80Gln Met Gly Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
His 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln 100 105 110Val Thr Val Ser Ser 115118117PRTArtificial
SequenceNanobody sequence 118Glu 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 Glu Ile Gly Arg Ile Asn 20 25 30Phe Tyr Arg Trp Tyr Arg Gln
Ala Pro Gly Asn Gln Arg Glu Val Val 35 40 45Ala Thr Ile Thr Ile Ala
Asp Lys Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Glu Ser Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Ser
Ser Leu Lys Pro Glu Asn Thr Ala Val Tyr Phe Cys His 85 90 95Ala Gly
Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 100 105
110Val Thr Val Ser Ser 11511910PRTArtificial SequenceCDR1 119Gly
Ser Val His Lys Ile Asn Phe Leu Gly1 5 1012010PRTArtificial
SequenceCDR1 120Gly Ser Val His Leu Leu Asn Phe Leu Gly1 5
1012110PRTArtificial SequenceCDR1 121Gly Ala Val His Lys Ile Asn
Phe Leu Gly1 5 1012210PRTArtificial SequenceCDR1 122Gly Asp Val His
Lys Ile Asn Ile Leu Gly1 5 1012310PRTArtificial SequenceCDR1 123Gly
Asp Val His Lys Ile Asn Phe Leu Gly1 5 1012410PRTArtificial
SequenceCDR1 124Gly Glu Thr Phe Lys Ile Asn Ile Trp Gly1 5
1012510PRTArtificial SequenceCDR1 125Gly Asp Val His Lys Ile Asn
Val Leu Gly1 5 1012610PRTArtificial SequenceCDR1 126Gly Glu Val Tyr
Lys Ile Asn Phe Leu Gly1 5 1012710PRTArtificial SequenceCDR1 127Gly
Gly Val His Lys Ile Asn Ile Leu Gly1 5 1012810PRTArtificial
SequenceCDR1 128Gly Gln Thr Phe Lys Ile Asn Ile Trp Gly1 5
1012910PRTArtificial SequenceCDR1 129Gly Ser Val Tyr Lys Ile Asn
Phe Leu Ser1 5 1013010PRTArtificial SequenceCDR1 130Gly Glu Ile Gly
Arg Ile Asn Phe Tyr Arg1 5 1013110PRTArtificial SequenceCDR1 131Gly
Glu Thr Phe Lys Val Asn Ile Trp Gly1 5 1013210PRTArtificial
SequenceCDR1 132Gly Asp Val Tyr Lys Ile Asn Phe Leu Gly1 5
1013310PRTArtificial SequenceCDR1 133Gly Glu Val His Lys Ile Asn
Ile Leu Gly1 5 101349PRTArtificial SequenceCDR2 134Thr Ile Thr Ile
Gly Asp Thr Thr Asp1 51359PRTArtificial SequenceCDR2 135Thr Ile Thr
Ile Gly Asp Ala Thr Asp1 51369PRTArtificial SequenceCDR2 136His Ile
Thr Ile Ala Asp Ala Thr Asp1 51379PRTArtificial SequenceCDR2 137Thr
Ile Thr Ile Gly Asp Asp Val Asp1 51389PRTArtificial SequenceCDR2
138His Ile Thr Ile Gly Asp Ala Thr Asp1 51399PRTArtificial
SequenceCDR2 139His Ile Thr Ile Gly Asp Gln Ala Asp1
51409PRTArtificial SequenceCDR2 140His Ile Ser Ile Ala Asp Ala Thr
Asp1 51419PRTArtificial SequenceCDR2 141His Ile Thr Ile Gly Asp Ala
Thr Val1 51429PRTArtificial SequenceCDR2 142Ser Leu Thr Ile Gly Gly
Ala Thr Asp1 51439PRTArtificial SequenceCDR2 143His Ile Thr Ile Gly
Asp Glu Val Asp1 51449PRTArtificial SequenceCDR2 144His Ile Thr Ile
Gly Asp Val Thr Asp1 51459PRTArtificial SequenceCDR2 145Ser Leu Thr
Ile Gly Gly Ala Thr Asn1 51469PRTArtificial SequenceCDR2 146His Ile
Thr Ile Gly Asp Ala Thr Asn1 51479PRTArtificial SequenceCDR2 147His
Ile Thr Ile Ala Asp Val Ala Asp1 51489PRTArtificial SequenceCDR2
148Thr Ile Thr Ile Gly Asp Glu Val Asp1 51499PRTArtificial
SequenceCDR2 149His Ile Ser Ile Ser Asp Gln Thr Asp1
51509PRTArtificial SequenceCDR2 150His Ile Thr Ile Gly Asp Gln Thr
Asp1 51519PRTArtificial SequenceCDR2 151His Ile Thr Ile Gly Asp Thr
Thr Asp1 51529PRTArtificial SequenceCDR2 152Arg Ile Ser Ile Ser Asp
Gln Thr Asp1 51539PRTArtificial SequenceCDR2 153His Ile Ser Ile Gly
Asp Gln Thr Asp1 51549PRTArtificial SequenceCDR2 154His Ile Thr Ile
Ala Asp Ala Ala Asp1 51559PRTArtificial SequenceCDR2 155Thr Ile Thr
Ile Gly Asp Ala Ala Asp1 51569PRTArtificial SequenceCDR2 156His Ile
Ala Ile Ser Asp Gln Thr Asp1 51579PRTArtificial SequenceCDR2 157Thr
Ile Thr Ile Ala Asp Lys Thr Asp1 51589PRTArtificial SequenceCDR2
158Thr Ile Thr Ile Ala Asp Lys Ile Asp1 51599PRTArtificial
SequenceCDR2 159His Ile Thr Ile Gly Asp Ala Thr Ser1
51609PRTArtificial SequenceCDR2 160Thr Ile Thr Ile Gly Asp Glu Val
Ala1 51619PRTArtificial SequenceCDR2 161His Ile Thr Ile Ala Asp Val
Thr Asp1 51629PRTArtificial SequenceCDR2 162His Ile Thr Ile Ala Asp
Gln Ala Asp1 51639PRTArtificial SequenceCDR2 163Thr Ile Thr Ile Gly
Asp Glu Thr Gln1 51649PRTArtificial SequenceCDR3 164Gly Ser Arg Leu
Tyr Pro Tyr Asn Tyr1 51659PRTArtificial SequenceCDR3 165Gly Ser Arg
Ile Tyr Pro Tyr Asp Tyr1 51669PRTArtificial SequenceCDR3 166Tyr Ser
Arg Ile Tyr Pro Tyr Asn Tyr1 51679PRTArtificial SequenceCDR3 167Lys
Ser Arg Leu Tyr Pro Tyr Asp Tyr1 51689PRTArtificial SequenceCDR3
168Lys Ser Arg Ile Tyr Pro Tyr Asp Tyr1 51699PRTArtificial
SequenceCDR3 169Gly Ser Arg Ile Trp Pro Tyr Asp Tyr1
51709PRTArtificial SequenceCDR3 170Phe Ser Arg Ile Tyr Pro Tyr Asp
Tyr1 51719PRTArtificial SequenceCDR3 171Gly Ser Arg Ile Tyr Pro Tyr
Asn Tyr1 51729PRTArtificial SequenceCDR3 172Gly Ser Arg Leu Tyr Pro
Tyr Asp Tyr1 51739PRTArtificial SequenceCDR3 173Gly Ser Arg Ile Tyr
Pro Tyr Ser Tyr1 51749PRTArtificial SequenceCDR3 174Leu Ser Arg Leu
Tyr Pro Tyr Asn Tyr1 517525PRTArtificial SequenceFR1 175Glu 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 20 2517625PRTArtificial SequenceFR1
176Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu Pro Cys Ala Ala Ser 20 2517725PRTArtificial
SequenceFR1 177Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Val Ser 20
2517825PRTArtificial SequenceFR1 178Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala
Ala Ser 20 2517925PRTArtificial SequenceFR1 179Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Arg Ala Ser 20 2518025PRTArtificial SequenceFR1 180Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser 20 2518125PRTArtificial SequenceFR1
181Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Val Ala Ser 20 2518225PRTArtificial
SequenceFR1 182Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20
2518325PRTArtificial SequenceFR1 183Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Pro Ser Cys Ala
Ala Ser 20 2518425PRTArtificial SequenceFR1 184Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ile Thr Ser 20 2518525PRTArtificial SequenceFR1 185Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser 20 2518625PRTArtificial SequenceFR1
186Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Ala Arg Leu Ser Cys Val Ala Ser 20 2518725PRTArtificial
SequenceFR1 187Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Ala Gly Gly1 5 10 15Ser Leu Thr Leu Ser Cys Ala Ala Ser 20
2518825PRTArtificial SequenceFR1 188Glu Val Gln Leu Val Glu Ser Gly
Gly Asp Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser 20 2518925PRTArtificial SequenceFR1 189Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Glu Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser 20 2519025PRTArtificial SequenceFR1 190Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Lys Leu Pro Cys Ala Ala Ser 20 2519125PRTArtificial SequenceFR1
191Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Gly Leu Ser Cys Ala Ala Ser 20 2519225PRTArtificial
SequenceFR1 192Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ala Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20
2519325PRTArtificial SequenceFR1 193Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Trp Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser 20 2519425PRTArtificial SequenceFR1 194Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Pro
Ser Cys Ala Ala Ser 20 2519525PRTArtificial SequenceFR1 195Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser 20 2519626PRTArtificial SequenceFR1
196Glu Val Gln Leu Val Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1
5 10 15Gly Ser Leu Arg Leu Ser Cys Ile Thr Ser 20
2519725PRTArtificial SequenceFR1 197Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Arg Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val
Ala Ser 20 2519825PRTArtificial SequenceFR1 198Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Met Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser 20 2519925PRTArtificial SequenceFR1 199Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Leu Thr Ser 20 2520025PRTArtificial SequenceFR1
200Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Pro Arg Leu Ser Cys Val Ala Ser 20 2520125PRTArtificial
SequenceFR1 201Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Pro Ser Cys Val Ala Ser 20
2520225PRTArtificial SequenceFR1 202Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Trp Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser 20 2520314PRTArtificial SequenceFR2 203Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Glu Leu Val Ala1 5 1020414PRTArtificial
SequenceFR2 204Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val
Ala1 5 1020514PRTArtificial SequenceFR2 205Trp Tyr Arg Gln Ala Pro
Gly Lys Glu Arg Glu Met Val Ala1 5 1020614PRTArtificial SequenceFR2
206Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val Ala1 5
1020714PRTArtificial SequenceFR2 207Trp Tyr Arg Gln Ala Pro Ala Lys
Glu Arg Glu Met Val Ala1 5 1020814PRTArtificial SequenceFR2 208Trp
Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala1 5
1020914PRTArtificial SequenceFR2 209Trp Tyr Arg Gln Cys Pro Gly Lys
Glu Arg Glu Met Val Ala1 5 1021014PRTArtificial SequenceFR2 210Trp
Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala1 5
1021114PRTArtificial SequenceFR2 211Trp His Arg Gln Pro Pro Gly Lys
Glu Arg Glu Lys Val Ala1 5 1021214PRTArtificial SequenceFR2 212Trp
Tyr Arg Gln Ala Pro Gly Lys Arg Arg Glu Leu Val Ala1 5
1021314PRTArtificial SequenceFR2 213Trp His Arg Gln Ala Pro Gly Lys
Glu Arg Glu Lys Val Ala1 5 1021414PRTArtificial SequenceFR2 214Trp
Tyr Arg Gln Ala Pro Glu Lys Glu Arg Glu Met Val Ala1 5
1021514PRTArtificial SequenceFR2 215Trp Tyr Arg Gln Ala Pro Gly Lys
Glu Arg Glu Val Val Ala1 5 1021614PRTArtificial SequenceFR2 216Trp
Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Val Val Ala1 5
1021714PRTArtificial SequenceFR2 217Trp Tyr Arg Gln Thr Pro Gly Lys
Gln Arg Glu Leu Val Ala1 5 1021814PRTArtificial SequenceFR2 218Trp
Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val Thr1 5
1021914PRTArtificial SequenceFR2 219Trp Tyr Arg Gln Ala Pro Gly His
Glu Arg Glu Leu Val Ala1 5 1022014PRTArtificial SequenceFR2 220Trp
Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Val Val Ala1 5
1022114PRTArtificial SequenceFR2 221Trp Tyr Arg Gln Ala Pro Gly Asn
Gln Arg Gly Val Val Ala1 5 1022214PRTArtificial SequenceFR2 222Trp
Tyr Arg Gln Ala Pro Ala Lys Glu Arg Gly Met Val Ala1 5
1022314PRTArtificial SequenceFR2 223Trp Tyr Arg Gln Ala Pro Ala Lys
Glu His Glu Met Val Ala1 5 1022414PRTArtificial SequenceFR2 224Trp
Tyr Arg Gln Ala Pro Ala Arg Glu Arg Glu Met Val Ala1 5
1022514PRTArtificial SequenceFR2 225Trp Tyr Arg Gln Gly Pro Gly Lys
Gln Arg Glu Leu Val Ala1 5 1022614PRTArtificial SequenceFR2 226Trp
Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Met Val Ala1 5
1022714PRTArtificial SequenceFR2 227Trp Tyr Arg Gln Ala Pro Ala Lys
Glu Arg Glu Met Ile Ala1 5 1022814PRTArtificial SequenceFR2 228Trp
Tyr Arg Gln Ala Pro Ala Lys Gly Arg Glu Met Val Ala1 5
1022914PRTArtificial SequenceFR2 229Trp Tyr Arg Gln Val Pro Ala Lys
Glu Arg Glu Met Val Ala1 5 1023014PRTArtificial SequenceFR2 230Trp
Tyr Arg Gln Cys Pro Gly Lys Glu Arg Asp Met Val Ala1 5
1023114PRTArtificial SequenceFR2 231Trp Gln Arg Gln Ala Pro Gly Lys
Glu Arg Glu Lys Val Ala1 5 1023214PRTArtificial SequenceFR2 232Trp
Tyr Cys Gln Ala Pro Gly Lys Glu Arg Glu Met Val Ala1 5
1023314PRTArtificial SequenceFR2 233Trp Tyr Arg Gln Ala Pro Gly Lys
Glu Arg Asp Met Val Ala1 5 1023414PRTArtificial SequenceFR2 234Trp
His Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala1 5
1023539PRTArtificial SequenceFR3 235Tyr Ala Asp Ser Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val His Phe Cys Arg
Ala 3523639PRTArtificial SequenceFR3 236Tyr Ala Asp Tyr Ala Lys Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Arg Asn Met Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys
Arg Ala 3523739PRTArtificial SequenceFR3 237Tyr Ala Asp Ser Ala Lys
Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe
Cys Arg Ala 3523839PRTArtificial SequenceFR3 238Tyr Ser His Phe Ala
Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val
Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr
Phe Cys Arg Ala 3523939PRTArtificial SequenceFR3 239Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met
Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val
Tyr Val Cys Arg Ala 3524039PRTArtificial SequenceFR3 240Tyr Ala Glu
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn
Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala
Val Tyr Phe Cys Arg Ala 3524139PRTArtificial SequenceFR3 241Tyr Ala
Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys
Asn Met Val Tyr Leu Gln Met Asn Ser Leu Ser Pro Glu Asp Thr 20 25
30Ala Val Tyr Phe Cys Arg Ala 3524239PRTArtificial SequenceFR3
242Tyr Ala His Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1
5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala 3524339PRTArtificial
SequenceFR3 243Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Glu
Asp Ser Ala1 5 10 15Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys
Ala Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys Asn Ala
3524439PRTArtificial SequenceFR3 244Tyr Ala Asp Ser Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu Gln
Met Thr Ser Leu Thr Pro Glu Asp Thr 20 25 30Ala Val Tyr Val Cys Arg
Ala 3524539PRTArtificial SequenceFR3 245Tyr Ala Asp Ser Ala Lys Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys
Arg Ala 3524639PRTArtificial SequenceFR3 246Tyr Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala1 5 10 15Lys Asn Thr Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe
Cys Asn Ala 3524739PRTArtificial SequenceFR3 247Tyr Ala Asp Ser Ala
Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val
His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr
Phe Cys Arg Ala 3524839PRTArtificial SequenceFR3 248Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5
10 15Lys Asn Met Val Phe Leu Gln Met Asn Asn Leu Lys Pro Glu Asp
Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala 3524939PRTArtificial
SequenceFR3 249Tyr Ala Gly Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg
Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala
3525039PRTArtificial SequenceFR3 250Tyr Ala Asp Phe Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys Arg
Ala 3525139PRTArtificial SequenceFR3 251Tyr Ala Asp Ser Ala Lys Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu
Gln Met Thr Ser Leu Lys Pro Glu Asp Thr 20 25 30Thr Val Tyr Val Cys
Arg Ala 3525239PRTArtificial SequenceFR3 252Tyr Ala Glu Ser Ala Lys
Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser1 5 10 15Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Leu
Cys Arg Ala 3525339PRTArtificial SequenceFR3 253Tyr Ala Asp Ser Ala
Lys Gly Arg Phe Ala Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr
Phe Cys Arg Ala 3525439PRTArtificial SequenceFR3 254Tyr Ala Asp Phe
Ala Gln Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val
Tyr Phe Cys Arg Ala 3525539PRTArtificial SequenceFR3 255Tyr Ala Glu
Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro1 5 10 15Lys Asn
Met Val His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala
Val Tyr Leu Cys Arg Ala 3525639PRTArtificial SequenceFR3 256Tyr Ala
Asp Phe Ala Lys Gly Arg Leu Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys
Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25
30Ala Val Tyr Phe Cys Arg Ala 3525739PRTArtificial SequenceFR3
257Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser1
5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala 3525839PRTArtificial
SequenceFR3 258Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg
Asp Gly Ala1 5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala
3525939PRTArtificial SequenceFR3 259Tyr Ala Asp Ser Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Arg Asn Met Val Tyr Leu Gln
Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Leu Tyr Phe Cys His
Ala 3526039PRTArtificial SequenceFR3 260Tyr Ala Asp Ser Ala Lys Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Ala Tyr Phe Cys
Arg Ala 3526139PRTArtificial SequenceFR3 261Tyr Ala Asp Ser Ala Lys
Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser1 5 10 15Arg Asn Met Val Tyr
Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe
Cys His Ala 3526239PRTArtificial SequenceFR3 262Tyr Ala Asp Ser Ala
Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser1 5 10 15Arg Asn Met Val
Tyr Leu Gln Met Gly Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr
Phe Cys His Ala 3526339PRTArtificial SequenceFR3 263Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Glu Asn Met
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val
Tyr Phe Cys Arg Ala 3526439PRTArtificial SequenceFR3 264Tyr Ala Asp
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Thr Asn
Met Val Tyr Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala
Val Tyr Phe Cys Arg Ala 3526539PRTArtificial SequenceFR3 265Tyr Ala
Gly Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys
Asn Met Val Tyr Leu Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr 20 25
30Ala Val Tyr Phe Cys Arg Ala 3526639PRTArtificial SequenceFR3
266Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1
5 10 15Lys Asn Met Val Tyr Leu Gln Leu Asn Asn Leu Lys Pro Glu Asp
Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala 3526739PRTArtificial
SequenceFR3 267Tyr Ala Glu Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg
Asp Glu Pro1 5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu Asp Thr 20 25 30Ala Val Tyr Leu Cys Arg Ala
3526839PRTArtificial SequenceFR3 268Tyr Ala His Ser Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu Gln
Met Thr Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Val Cys Arg
Ala 3526939PRTArtificial SequenceFR3 269Tyr Ala Asp Phe Ala Lys Gly
Arg Phe Thr Ile Ser Arg Asp Glu Val1 5 10 15Lys Asn Met Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys
Arg Ala 3527039PRTArtificial SequenceFR3 270Tyr Ala Asp Phe Ala Lys
Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe
Cys Arg Ala 3527139PRTArtificial SequenceFR3 271Tyr Ser Tyr Phe Ala
Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val
Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr
Phe Cys Arg Ala 3527239PRTArtificial SequenceFR3 272Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Val
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val
Tyr Phe Cys Arg Ala 3527339PRTArtificial SequenceFR3 273Tyr Glu Asp
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn
Met Val Tyr Leu Gln Met Thr Gly Leu Lys Pro Glu Asp Thr 20 25 30Ala
Val Tyr Val Cys Arg Ala 3527439PRTArtificial SequenceFR3 274Tyr Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala1 5 10 15Lys
Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr 20 25
30Ala Val Tyr Phe Cys Asn Ala 3527539PRTArtificial SequenceFR3
275Tyr Ser His Phe Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1
5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Gly Leu Arg Pro Glu Asp
Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala 3527639PRTArtificial
SequenceFR3 276Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg
Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu Asp Thr 20 25 30Ala Val Tyr Leu Cys Arg Ala
3527739PRTArtificial SequenceFR3 277Tyr Ala Glu Phe Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Pro1 5 10 15Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu Lys Pro Val Asp Thr 20 25 30Ala Val Tyr Leu Cys Arg
Ala 3527839PRTArtificial SequenceFR3 278Tyr Ser His Phe Ala Lys Gly
Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val Tyr Leu
Gln Met Asn Asn Leu Arg Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe Cys
Arg Ala 3527939PRTArtificial SequenceFR3 279Tyr Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala1 5 10 15Lys Asp Thr Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Phe
Cys Asn Ala 3528039PRTArtificial SequenceFR3 280Tyr Ala Asp Ser Ala
Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys Asn Met Val
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ala 20 25 30Ala Val Tyr
Phe Cys Arg Ala 3528139PRTArtificial SequenceFR3 281Tyr Ala Glu Ser
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser1 5 10 15Lys Asn Met
Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ala 20 25 30Ala Val
Tyr Leu Cys Arg Ala 3528239PRTArtificial SequenceFR3 282Tyr Ala Asp
Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser1 5 10 15Arg Asn
Met Val Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asn Thr 20 25 30Ala
Val Tyr Phe Cys His Ala 3528339PRTArtificial SequenceFR3 283Tyr Ala
Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1 5 10 15Lys
Asn Met Val Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr 20 25
30Ala Val Tyr Phe Cys Arg Ala 3528439PRTArtificial SequenceFR3
284Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala1
5 10 15Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Gly Asp
Thr 20 25 30Ala Val Tyr Phe Cys Arg Ala 3528511PRTArtificial
SequenceFR4 285Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser1 5
1028611PRTArtificial SequenceFR4 286Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser1 5 1028711PRTArtificial SequenceFR4 287Trp Gly Arg Gly
Thr Gln Val Thr Val Ser Ser1 5 1028811PRTArtificial SequenceFR4
288Trp Gly His Gly Thr Leu Val Thr Val Ser Ser1 5
1028911PRTArtificial SequenceFR4 289Trp Gly Arg Gly Thr Leu Val Thr
Val Ser Ser1 5 1029011PRTArtificial SequenceFR4 290Trp Asp Gln Gly
Thr Gln Val Thr Val Ser Ser1 5 10291327PRTArtificial
SequenceNanobody sequence 291Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Gly Gly Gly1 5 10 15Ser Leu Ser Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met Ala Trp Phe Arg Gln
Pro Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ser Ile Ser Trp Ser
Gly Glu Asn Thr Asn Tyr Arg Asn Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Lys Ile Ala Lys Thr Tyr Pro Asp Asn Trp Tyr Trp Thr Lys 100 105
110Ser Asn Asn Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly145 150 155 160Gly Gly Gly Ser Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val 165 170 175Gln Gly Gly Gly Ser Leu Ser
Leu Ser Cys Ala Ala Ser Gly Arg Thr 180 185 190Phe Ser Ser Tyr Ala
Met Ala Trp Phe Arg Gln Pro Pro Gly Lys Glu 195 200 205Arg Glu Phe
Val Ala Ser Ile Ser Trp Ser Gly Glu Asn Thr Asn Tyr 210 215 220Arg
Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys225 230
235 240Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala 245 250 255Val Tyr Tyr Cys Ala Ala Lys Ile Ala Lys Thr Tyr Pro
Asp Asn Trp 260 265 270Tyr Trp Thr Lys Ser Asn Asn Tyr Asn Tyr Trp
Gly Gln Gly Thr Leu 275 280 285Val Thr Val Ser Ser Gly Ala Ala Asp
Tyr Lys Asp His Asp Gly Asp 290 295 300Tyr Lys Asp His Asp Ile Asp
Tyr Lys Asp Asp Asp Asp Lys Gly Ala305 310 315 320Ala His His His
His His His 325292276PRTArtificial SequenceNanobody sequence 292Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser
Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val
Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Glu Val Gln 115 120 125Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 130 135 140Leu Ser Cys Ala
Ala Ser Gly Ser Val His Lys Ile Asn Phe Leu Gly145 150 155 160Trp
Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile 165 170
175Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys Gly Arg Phe
180 185 190Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu Gln
Met Asn 195 200 205Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg Ala Gly Ser 210 215 220Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val225 230 235 240Ser Ser Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp 245 250 255His Asp Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His 260 265 270His His His
His 275293289PRTArtificial SequenceNanobody sequence 293Glu Val Gln
Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25 30Cys
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40
45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val
Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile
Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu
Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser
Trp Gly Gln Gly Thr Gln 115 120 125Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu 130 135 140Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys145 150 155 160Ala Ala Ser
Gly Ser Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg 165 170 175Gln
Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile Thr Ile Gly 180 185
190Asp Thr Thr Asp Tyr
Ala Asp Tyr Ala Lys Gly Arg Phe Thr Ile Ser 195 200 205Arg Asp Glu
Ala Arg Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys 210 215 220Pro
Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Gly Ser Arg Leu Tyr225 230
235 240Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Gly 245 250 255Ala Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp
His Asp Ile 260 265 270Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala
His His His His His 275 280 285His294292PRTArtificial
SequenceNanobody sequence 294Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Val Gln
115 120 125Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser
Leu Arg 130 135 140Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro
Tyr Cys Met Gly145 150 155 160Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Gly Val Ala Ala Ile 165 170 175Asn Met Gly Gly Gly Ile Thr
Tyr Tyr Ala Asp Ser Val Lys Gly Arg 180 185 190Phe Thr Ile Ser Gln
Asp Asn Ala Lys Asn Thr Val Tyr Leu Leu Met 195 200 205Asn Ser Leu
Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala Asp 210 215 220Ser
Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly Leu Ser Thr225 230
235 240Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Gln Val Thr
Val 245 250 255Ser Ser Gly Ala Ala Asp Tyr Lys Asp His Asp Gly Asp
Tyr Lys Asp 260 265 270His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys
Gly Ala Ala His His 275 280 285His His His His
290295276PRTArtificial SequenceNanobody sequence 295Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val
Gln Leu Val Glu 115 120 125Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys 130 135 140Thr Phe Ser Gly Gly Thr Phe Ser
Ser Tyr Thr Met Gly Trp Phe Arg145 150 155 160Gln Ala Pro Gly Lys
Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly 165 170 175Gly Val Thr
Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser 180 185 190Glu
Asp Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys 195 200
205Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr
210 215 220Met Thr Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val225 230 235 240Ser Ser Gly Ala Ala Asp Tyr Lys Asp His Asp
Gly Asp Tyr Lys Asp 245 250 255His Asp Ile Asp Tyr Lys Asp Asp Asp
Asp Lys Gly Ala Ala His His 260 265 270His His His His
275296280PRTArtificial SequenceNanobody sequence 296Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Ser Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly 130 135 140Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Ser Val His Lys Ile145 150 155 160Asn Phe Leu Gly Trp
Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu 165 170 175Val Ala Thr
Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala 180 185 190Lys
Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr 195 200
205Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
210 215 220Arg Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln
Gly Thr225 230 235 240Leu Val Thr Val Ser Ser Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly 245 250 255Asp Tyr Lys Asp His Asp Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly 260 265 270Ala Ala His His His His His
His 275 280297293PRTArtificial SequenceNanobody sequence 297Glu Val
Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25
30Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr
Val Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala
Ile Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr
Glu Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp
Ser Trp Gly Gln Gly Thr Gln 115 120 125Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Ser Glu Val 130 135 140Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu145 150 155 160Arg Leu
Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn Phe Leu 165 170
175Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr
180 185 190Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys
Gly Arg 195 200 205Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val
Tyr Leu Gln Met 210 215 220Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Arg Ala Gly225 230 235 240Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255Val Ser Ser Gly Ala
Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys 260 265 270Asp His Asp
Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His 275 280 285His
His His His His 290298296PRTArtificial SequenceNanobody sequence
298Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn
Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn
Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val
Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Ser Asp Val Gln Leu
Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly 130 135 140Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro145 150 155
160Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
165 170 175Val Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala
Asp Ser 180 185 190Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala
Lys Asn Thr Val 195 200 205Tyr Leu Leu Met Asn Ser Leu Glu Pro Glu
Asp Thr Ala Ile Tyr Tyr 210 215 220Cys Ala Ala Asp Ser Thr Ile Tyr
Ala Ser Tyr Tyr Glu Cys Gly His225 230 235 240Gly Leu Ser Thr Gly
Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr 245 250 255Gln Val Thr
Val Ser Ser Gly Ala Ala Asp Tyr Lys Asp His Asp Gly 260 265 270Asp
Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly 275 280
285Ala Ala His His His His His His 290 295299280PRTArtificial
SequenceNanobody sequence 299Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Glu Arg Gly Leu Val 35 40 45Ala Thr Ile Thr Ile Gly
Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly
Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val
115 120 125Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu 130 135 140Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser
Ser Tyr Thr Met145 150 155 160Gly Trp Phe Arg Gln Ala Pro Gly Lys
Glu Arg Glu Phe Val Ala Glu 165 170 175Val Arg Trp Gly Gly Val Thr
Thr Tyr Ser Asn Ser Leu Lys Asp Arg 180 185 190Phe Ser Ile Ser Glu
Asp Ser Val Lys Asn Ala Val Tyr Leu Gln Met 195 200 205Asn Ser Leu
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val 210 215 220Arg
Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln Gly Thr225 230
235 240Leu Val Thr Val Ser Ser Gly Ala Ala Asp Tyr Lys Asp His Asp
Gly 245 250 255Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp
Asp Lys Gly 260 265 270Ala Ala His His His His His His 275
280300306PRTArtificial SequenceNanobody sequence 300Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala
Ser Gly Ser Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185 190Arg
Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile Thr Ile 195 200
205Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys Gly Arg Phe Thr Ile
210 215 220Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu Gln Met Asn
Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
Ala Gly Ser Arg Leu 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr Lys Asp
His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr Lys Asp
Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305301319PRTArtificial SequenceNanobody sequence 301Glu Val Gln
Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25 30Cys
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40
45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val
Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile
Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu
Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser
Trp Gly Gln Gly Thr Gln 115 120 125Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly145 150 155 160Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 165 170 175Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 180 185
190Ser Gly Ser Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala
195 200 205Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile Thr Ile Gly
Asp Thr 210 215 220Thr Asp Tyr Ala Asp Tyr Ala Lys Gly Arg Phe Thr
Ile Ser Arg Asp225 230 235 240Glu Ala Arg Asn Met Val Tyr Leu Gln
Met Asn Ser Leu Lys Pro Glu 245 250 255Asp Thr Ala Val Tyr Phe Cys
Arg Ala Gly Ser Arg Leu Tyr Pro Tyr 260 265 270Asn Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Gly Ala Ala 275 280 285Asp Tyr Lys
Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr 290 295
300Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His His His305
310 315302322PRTArtificial SequenceNanobody sequence 302Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Asp Val Gln Leu Gln145 150 155 160Ala Ser Gly
Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr Cys Met Gly Trp Phe 180 185
190Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asn Met
195 200 205Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr 210 215 220Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu
Leu Met Asn Ser225 230 235 240Leu Glu Pro Glu Asp Thr Ala Ile Tyr
Tyr Cys Ala Ala Asp Ser Thr 245 250 255Ile Tyr Ala Ser Tyr Tyr Glu
Cys Gly His Gly Leu Ser Thr Gly Gly 260 265 270Tyr Gly Tyr Asp Ser
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 275 280 285Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 290 295 300Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His305 310
315 320His His303306PRTArtificial SequenceNanobody sequence 303Glu
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 Ser Val His Lys Ile Asn
20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu
Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170
175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala
180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly
Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser
Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met
Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305304322PRTArtificial SequenceNanobody sequence 304Glu
Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr
20 25 30Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
Val 35 40 45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn
Thr Val Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr
Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr
Tyr Glu Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr
Asp Ser Trp Gly Gln Gly Thr Gln 115 120 125Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly145 150 155 160Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 165 170
175Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe
180 185 190Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg
Gln Ala 195 200 205Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg
Trp Gly Gly Val 210 215 220Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg
Phe Ser Ile Ser Glu Asp225 230 235 240Ser Val Lys Asn Ala Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu 245 250 255Asp Thr Ala Val Tyr
Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 260 265 270Val Val Pro
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 275 280 285Gly
Ala Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 290 295
300Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His
His305 310 315 320His His305319PRTArtificial SequenceNanobody
sequence 305Glu 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 Ser Val His
Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
Arg Gly Leu Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr
Ala Asp Tyr Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Gln Ala Ser Gly145 150
155 160Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala 165 170 175Ser Gly Tyr Thr Ile Gly Pro Tyr Cys Met Gly Trp Phe
Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile
Asn Met Gly Gly Gly 195 200 205Ile Thr Tyr Tyr Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Gln 210 215 220Asp Asn Ala Lys Asn Thr Val
Tyr Leu Leu Met Asn Ser Leu Glu Pro225 230 235 240Glu Asp Thr Ala
Ile Tyr Tyr Cys Ala Ala Asp Ser Thr Ile Tyr Ala 245 250 255Ser Tyr
Tyr Glu Cys Gly His Gly Leu Ser Thr Gly Gly Tyr Gly Tyr 260 265
270Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Ala Ala
275 280 285Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile
Asp Tyr 290 295 300Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His
His His His305 310 315306306PRTArtificial SequenceNanobody sequence
306Glu 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 Asp Val Tyr Lys Ile
Asn 20 25 30Phe Leu Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155
160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe
165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg
Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg
Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg
Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr
Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly
Ala Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280
285Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His
290 295 300His His305307306PRTArtificial SequenceNanobody sequence
307Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ala Val His Lys Ile
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Thr Pro Glu Lys Glu Arg Glu
Met Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Glu Val Asp Tyr Ala Asp
Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Thr Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Val Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155
160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe
165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg
Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg
Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg
Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr
Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly
Ala Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280
285Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His
290 295 300His His305308306PRTArtificial SequenceNanobody sequence
308Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile
Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu
Leu Val 35 40 45Ala Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr Ala Asp
Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala Lys Asn
Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys Ser Arg Leu Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155
160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe
165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg
Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg
Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg
Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr
Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly
Ala Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280
285Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His
290 295 300His His305309306PRTArtificial SequenceNanobody sequence
309Glu 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 Ser Val His Leu Leu
Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Met Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala His
Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp
Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155
160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe
165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg
Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg
Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg
Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu225
230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr
Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr Lys Asp His Asp Gly
Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr Lys Asp Asp Asp Asp
Lys Gly Ala Ala His His His His 290 295 300His
His305310306PRTArtificial SequenceNanobody sequence 310Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile
Trp Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45Ala Ser Leu Thr Ile Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr
Phe Cys Asn 85 90 95Ala Lys Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305311306PRTArtificial SequenceNanobody sequence 311Glu 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 Glu Val Tyr Lys Ile Asn 20 25 30Phe
Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40
45Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305312306PRTArtificial SequenceNanobody sequence 312Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe
Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40
45Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305313306PRTArtificial SequenceNanobody sequence 313Glu 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 Glu Ile Gly Arg Ile Asn 20 25 30Phe
Tyr Arg Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Val Val 35 40
45Ala Thr Ile Thr Ile Ala Asp Lys Thr Asp Tyr Ala Asp Ser Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Arg Asn Met Val Tyr
Leu65 70 75 80Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys His 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305314306PRTArtificial SequenceNanobody sequence 314Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe
Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40
45Ala His Ile Thr Ile Gly Asp Gln Ala Asp Tyr Ala Asp Ser Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305315306PRTArtificial SequenceNanobody sequence 315Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile
Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val 35 40
45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305316306PRTArtificial SequenceNanobody sequence 316Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe
Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40
45Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305317306PRTArtificial SequenceNanobody sequence 317Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe
Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val 35 40
45Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys
Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu225 230 235
240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr
245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 260 265 270Gly Ala Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr
Lys Asp His Asp 275 280 285Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly
Ala Ala His His His His 290 295 300His His305318306PRTArtificial
SequenceNanobody sequence 318Glu 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 Asp Val His Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln
Ala Pro Ala Lys Glu Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly
Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg 85 90 95Ala Tyr
Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser
Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg
Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr
Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser
Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu225 230
235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met
Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr Lys Asp His Asp Gly Asp
Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr Lys Asp Asp Asp Asp Lys
Gly Ala Ala His His His His 290 295 300His
His305319306PRTArtificial SequenceNanobody sequence 319Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile
195 200 205Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Phe Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305320306PRTArtificial SequenceNanobody sequence 320Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Glu Ile Gly Arg Ile Asn Phe Tyr Arg Trp Tyr 180 185
190Arg Gln Ala Pro Gly Asn Gln Arg Glu Val Val Ala Thr Ile Thr Ile
195 200 205Ala Asp Lys Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ser Arg Asn Met Val Tyr Leu Gln
Met Ser Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys His Ala Gly Ser Arg Leu 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305321306PRTArtificial SequenceNanobody sequence 321Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Thr Ile
195 200 205Gly Asp Gln Ala Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Gly Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305322306PRTArtificial SequenceNanobody sequence 322Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Asp Val His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala His Ile Thr Ile
195 200 205Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Tyr Ser Arg Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305323306PRTArtificial SequenceNanobody sequence 323Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Ser Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala Thr Ile Thr Ile
195 200 205Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Gly Ser Arg Leu 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305324306PRTArtificial SequenceNanobody sequence 324Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Ser Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile Thr Ile
195 200 205Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Gly Ser Arg Leu 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305325306PRTArtificial SequenceNanobody sequence 325Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Asp Val Tyr Lys Ile Asn Phe Leu Gly Trp His 180 185
190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Thr Ile
195 200 205Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys Gly
Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Arg Ala Gly Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305326306PRTArtificial SequenceNanobody sequence 326Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser
Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val
Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170
175Cys Ala Ala Ser Gly Glu Val Tyr Lys Ile Asn Phe Leu Gly Trp Tyr
180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile
Thr Ile 195 200 205Ala Asp Ala Ala Asp Tyr Ala Asp Phe Ala Lys Gly
Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Arg Pro Glu Asp Thr Ala Val
Tyr Phe Cys Arg Ala Gly Ser Arg Ile 245 250 255Trp Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305327306PRTArtificial SequenceNanobody sequence 327Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser
Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val
Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu
Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170
175Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr
180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val Ala His Ile
Thr Ile 195 200 205Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys Gly
Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Pro Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val
Tyr Leu Cys Arg Ala Gly Ser Arg Ile 245 250 255Tyr Pro Tyr Asn Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305328306PRTArtificial SequenceNanobody sequence 328Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser
Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val
Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170
175Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn Ile Trp Gly Trp Tyr
180 185 190Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ser Leu
Thr Ile 195 200 205Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile 210 215 220Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Asn Ala Lys Ser Arg Leu 245 250 255Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305329306PRTArtificial SequenceNanobody sequence 329Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser
Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val
Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170
175Cys Ala Ala Ser Gly Ser Val His Leu Leu Asn Phe Leu Gly Trp Tyr
180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val Ala His Ile
Thr Ile 195 200 205Ala Asp Ala Thr Asp Tyr Ala His Phe Ala Lys Gly
Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Arg Pro Glu Asp Thr Ala Val
Tyr Phe Cys Arg Ala Gly Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305330306PRTArtificial SequenceNanobody sequence 330Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser
Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val
Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170
175Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn Ile Trp Gly Trp Tyr
180 185 190Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ser Leu
Thr Ile 195 200 205Gly Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile 210 215 220Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Lys Ala Glu Asp Thr Ala Val
Tyr Phe Cys Asn Ala Lys Ser Arg Leu 245 250 255Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305331306PRTArtificial SequenceNanobody sequence 331Glu
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 Asp Val His Lys Ile Asn
20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu Met
Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Ser Tyr Ala Asp Ser
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met
Val Tyr Leu65 70 75 80Gln Leu Asn Asn Leu Lys Pro Glu Asp Thr Ala
Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170
175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala
180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly
Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser
Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met
Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305332306PRTArtificial SequenceNanobody sequence 332Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn
20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys
Val 35 40 45Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Phe Cys Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170
175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala
180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly
Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser
Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met
Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala
Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile
Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295
300His His305333306PRTArtificial SequenceNanobody sequence 333Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser
Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val
Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170
175Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn Ile Leu Gly Trp Tyr
180 185 190Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala His Ile
Thr Ile 195
200 205Gly Asp Ala Thr Ser Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr
Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Leu
Asn Asn Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg Ala Tyr Ser Arg Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr Lys
Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr Lys
Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305334306PRTArtificial SequenceNanobody sequence 334Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile
195 200 205Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Phe Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305335306PRTArtificial SequenceNanobody sequence 335Glu Val Gln
Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe
Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40
45Ala His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Leu Cys Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser
Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185
190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val
195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser
Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala
Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305336306PRTArtificial SequenceNanobody sequence 336Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Asp Val His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala His Ile Thr Ile
195 200 205Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Leu
Cys Arg Ala Tyr Ser Arg Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305337306PRTArtificial SequenceNanobody sequence 337Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser 165 170 175Cys
Ala Ala Ser Gly Ala Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185
190Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val Ala Thr Ile Thr Ile
195 200 205Gly Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln
Met Thr Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Val
Cys Arg Ala Gly Ser Arg Leu 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Ala Ala Asp Tyr
Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp 275 280 285Ile Asp Tyr
Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His 290 295 300His
His305338273PRTArtificial SequenceNanobody sequence 338Asp Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Asp Val His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala His Ile Thr Ile
195 200 205Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Tyr Ser Arg Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270Ala339280PRTArtificial SequenceNanobody sequence 339Asp Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Ala Ile Ser Trp Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr
Ile Tyr Ile Trp 100 105 110Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly145 150 155 160Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 165 170 175Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys 180 185
190Ile Asn Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu
195 200 205Met Val Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala
Asp Ser 210 215 220Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Lys Asn Met Val225 230 235 240Tyr Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Phe 245 250 255Cys Arg Ala Tyr Ser Arg Ile
Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly 260 265 270Thr Leu Val Thr Val
Ser Ser Ala 275 280340456PRTArtificial SequenceNanobody sequence
340Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn
Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn
Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val
Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155
160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
165 170 175Cys Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly
Trp Tyr 180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala
His Ile Ser Ile 195 200 205Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala
Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ser Lys Asn Met
Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr
Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile 245 250 255Tyr Pro Tyr
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 275 280
285Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
290 295 300Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln305 310 315 320Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe 325 330 335Ser Ser Phe Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu 340 345 350Glu Trp Val Ser Ser Ile Ser
Gly Ser Gly Ser Asp Thr Leu Tyr Ala 355 360 365Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr 370 375 380Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val385 390 395
400Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr
405 410 415Leu Val Thr Val Ser Ser Gly Ala Ala Asp Tyr Lys Asp His
Asp Gly 420 425 430Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp
Asp Asp Lys Gly 435 440 445Ala Ala His His His His His His 450
455341456PRTArtificial SequenceNanobody sequence 341Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170
175Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn Ile Trp Gly Trp Tyr
180 185 190Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ser Leu
Thr Ile 195 200 205Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile 210 215 220Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Asn Ala Lys Ser Arg Leu 245 250 255Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 275 280 285Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 290 295
300Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln305 310 315 320Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe 325 330 335Ser Ser Phe Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 340 345 350Glu Trp Val Ser Ser Ile Ser Gly
Ser Gly Ser Asp Thr Leu Tyr Ala 355 360 365Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr 370 375 380Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val385 390 395 400Tyr
Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr 405 410
415Leu Val Thr Val Ser Ser Gly Ala Ala Asp Tyr Lys Asp His Asp Gly
420 425 430Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp Asp
Lys Gly 435 440 445Ala Ala His His His His His His 450
455342304PRTArtificial SequenceNanobody sequence 342Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn 20 25 30Thr Met
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala
Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Lys 85 90 95Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln
Gly Thr 100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu Val Glu Ser145 150 155 160Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val 165 170 175Ala Ser Gly
Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln 180 185 190Ala
Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp 195 200
205Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg
210 215 220Asp Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu
Lys Pro225 230 235 240Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe
Ser Arg Ile Tyr Pro 245 250 255Tyr Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Gly Ala 260 265 270Ala Asp Tyr Lys Asp His Asp
Gly Asp Tyr Lys Asp His Asp Ile Asp 275 280 285Tyr Lys Asp Asp Asp
Asp Lys Gly Ala Ala His His His His His His 290 295
300343304PRTArtificial SequenceNanobody sequence 343Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln
Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 165 170 175Ser Gly Ile
Thr Phe Ser Ile Asn Thr Met Gly Trp Tyr Arg Gln Ala 180 185 190Pro
Gly Lys Gln Arg Glu Leu Val Ala Leu Ile Ser Ser Ile Gly Asp 195 200
205Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
210 215 220Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Lys Arg Phe Arg
Thr Ala Ala Gln Gly 245 250 255Thr Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Gly Ala 260 265 270Ala Asp Tyr Lys Asp His Asp
Gly Asp Tyr Lys Asp His Asp Ile Asp 275 280 285Tyr Lys Asp Asp Asp
Asp Lys Gly Ala Ala His His His His His His 290 295
300344171PRTHomo sapiens 344Met Glu His Ser Thr Phe Leu Ser Gly Leu
Val Leu Ala Thr Leu Leu1 5 10 15Ser Gln Val Ser Pro Phe Lys Ile Pro
Ile Glu Glu Leu Glu Asp Arg 20 25 30Val Phe Val Asn Cys Asn Thr Ser
Ile Thr Trp Val Glu Gly Thr Val 35 40 45Gly Thr Leu Leu Ser Asp Ile
Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60Leu Asp Pro Arg Gly Ile
Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys65 70 75 80Asp Lys Glu Ser
Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys 85 90 95Val Glu Leu
Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val 100 105 110Ile
Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His 115 120
125Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg
130 135 140Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala
Gln Tyr145 150 155 160Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys
165 170345182PRTHomo sapiens 345Met Glu Gln Gly Lys Gly Leu Ala Val
Leu Ile Leu Ala Ile Ile Leu1 5 10 15Leu Gln Gly Thr Leu Ala Gln Ser
Ile Lys Gly Asn His Leu Val Lys 20 25 30Val Tyr Asp Tyr Gln Glu Asp
Gly Ser Val Leu Leu Thr Cys Asp Ala 35 40 45Glu Ala Lys Asn Ile Thr
Trp Phe Lys Asp Gly Lys Met Ile Gly Phe 50 55 60Leu Thr Glu Asp Lys
Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp65 70 75 80Pro Arg Gly
Met Tyr Gln Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro 85 90 95Leu Gln
Val Tyr Tyr Arg Met Cys Gln Asn Cys Ile Glu Leu Asn Ala 100 105
110Ala Thr Ile Ser Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val
115 120 125Leu Ala Val Gly Val Tyr Phe Ile Ala Gly Gln Asp Gly Val
Arg Gln 130 135 140Ser Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn
Asp Gln Leu Tyr145 150 155 160Gln Pro Leu Lys Asp Arg Glu Asp Asp
Gln Tyr Ser His Leu Gln Gly 165 170 175Asn Gln Leu Arg Arg Asn
180346207PRTHomo sapiens 346Met Gln Ser Gly Thr His Trp Arg Val Leu
Gly Leu Cys Leu Leu Ser1 5 10 15Val Gly Val Trp Gly Gln Asp Gly Asn
Glu Glu Met Gly Gly Ile Thr 20 25 30Gln Thr Pro Tyr Lys Val Ser Ile
Ser Gly Thr Thr Val Ile Leu Thr 35 40 45Cys Pro Gln Tyr Pro Gly Ser
Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60Asn Ile Gly Gly Asp Glu
Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp65 70 75 80His Leu Ser Leu
Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95Val Cys Tyr
Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110Tyr
Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met 115 120
125Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu
130 135 140Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys
Ala Lys145 150 155 160Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg
Gln Arg Gly Gln Asn 165 170 175Lys Glu Arg Pro Pro Pro Val Pro Asn
Pro Asp Tyr Glu Pro Ile Arg 180 185 190Lys Gly Gln Arg Asp Leu Tyr
Ser Gly Leu Asn Gln Arg Arg Ile 195 200 205347164PRTHomo sapiens
347Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu1
5 10 15Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu
Cys 20 25 30Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu
Thr Ala 35 40 45Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
Pro Ala Tyr 50 55 60Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
Leu Gly Arg Arg65 70 75 80Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
Gly Arg Asp Pro Glu Met 85 90 95Gly Gly Lys Pro Gln Arg Arg Lys Asn
Pro Gln Glu Gly Leu Tyr Asn 100 105 110Glu Leu Gln Lys Asp Lys Met
Ala Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125Lys Gly Glu Arg Arg
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 130 135 140Leu Ser Thr
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala145 150 155
160Leu Pro Pro Arg348142PRTHomo sapiens 348Pro Asn Ile Gln Asn Pro
Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser1 5 10 15Lys Ser Ser Asp Lys
Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln 20 25 30Thr Asn Val Ser
Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 35 40 45Thr Val Leu
Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val 50 55 60Ala Trp
Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn65 70 75
80Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys
85 90 95Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Phe
Arg 100 105 110Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met
Thr Leu Arg 115 120 125Leu Trp Ser Ser Leu Asn Phe Gln Asn Leu Ser
Val Ile Gly 130 135 140349177PRTHomo sapiens 349Glu Asp Leu Asn Lys
Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro1 5 10 15Ser Glu Ala Glu
Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu 20 25 30Ala Thr Gly
Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45Gly Lys
Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60Glu
Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu65 70 75
80Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys
85 90 95Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Val
Ser 100 105 110Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser
Val Ser Tyr 115 120 125Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr
Glu Ile Leu Leu Gly 130 135 140Lys Ala Thr Leu Tyr Ala Val Leu Val
Ser Ala Leu Val Leu Met Ala145 150 155 160Met Val Lys Arg Lys Asp
Phe Gln Asp Arg Ala Lys Pro Val Thr Gln 165 170
175Ile350118PRTArtificial SequenceNanobody sequence 350Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Ser Ile Asn 20 25 30Thr
Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40
45Ala Leu Ile Ser Ser Ile Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Lys 85 90 95Arg Phe Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser
115351119PRTArtificial SequenceNanobody sequence 351Glu 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 Ser Ile Phe Gly Phe Asn 20 25 30Asp Met
Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala
Leu Ile Ser Arg Val Gly Val Thr Ser Ser Ala Asp Ser Val Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Val Asn Ala Lys Asp Thr Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Tyr 85 90 95Met Asp Gln Arg Leu Asp Gly Ser Thr Leu Ala Tyr Trp Gly
Gln Gly 100 105 110Thr Gln Val Thr Val Ser Ser
115352127PRTArtificial SequenceNanobody sequence 352Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Val
Ala Ile Asn Trp Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Gly Tyr Gln Ile Asn Ser Gly Asn Tyr Asn Phe Lys
Asp Tyr 100 105 110Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 125353123PRTArtificial SequenceNanobody
sequence 353Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Tyr
Gly Ser Tyr 20 25 30Trp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Gly Val 35 40 45Ala Ala Ile Asn Arg Gly Gly Gly Tyr Thr Val
Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr
Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr Ala
Asp Tyr Tyr Cys 85 90 95Ala Ala Ser Gly Val Leu Gly Gly Leu His Glu
Asp Trp Phe Asn Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120354123PRTArtificial SequenceNanobody sequence 354Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Tyr Gly Ser Tyr
20 25 30Trp Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Ala
Val 35 40 45Ala Ala Ile Asn Arg Gly Gly Gly Tyr Thr Val Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr
Ala Asp Tyr Tyr Cys 85 90 95Ala Ala Ser Gly Val Leu Gly Gly Leu His
Glu Asp Trp Phe Asn Tyr 100 105 110Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120355124PRTArtificial SequenceNanobody sequence
355Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Thr Gly Gly1
5 10 15Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser
Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Thr Val Asp65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Ala Ala Gly Ser Ala Trp Tyr
Gly Thr Leu Tyr Glu Tyr Asp 100 105 110Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120356124PRTArtificial SequenceNanobody
sequence 356Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Thr Cys Ala Ala Ser Gly Ser Thr Ser
Arg Ser Tyr 20 25 30Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ser Gly Ile Ser Trp Arg Gly Asp Ser Thr Gly
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Thr Val Asp65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Ala Ala Gly Ser Thr
Trp Tyr Gly Thr Leu Tyr Glu Tyr Asp 100 105 110Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120357120PRTArtificial
SequenceNanobody sequence 357Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser 115 120358129PRTArtificial
SequenceNanobody sequence 358Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Val Met Gly Trp Phe Arg Gln
Ala Thr Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Thr Ile Ala Trp Asp
Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Ser Tyr Asn Val Tyr Tyr Asn Asn Tyr Tyr Tyr Pro Ile Ser 100 105
110Arg Asp Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125Ser359133PRTArtificial SequenceNanobody sequence 359Asp
Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr
20 25 30Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
Val 35 40 45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn
Thr Val Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr
Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr
Tyr Glu Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr
Asp Ser Trp Gly Gln Gly Thr Gln 115 120 125Val Thr Val Ser Ser
130360127PRTArtificial SequenceNanobody sequence 360Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Ala Ile Ser Trp Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr Ile Tyr
Ile Trp 100 105 110Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 120 12536110PRTArtificial SequenceCDR1 361Gly Asp
Thr Tyr Gly Ser Tyr Trp Met Gly1 5 1036210PRTArtificial
SequenceCDR1 362Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly1 5
1036310PRTArtificial SequenceCDR2 363Ala Ile Asn Arg Gly Gly Gly
Tyr Thr Val1 5 103649PRTArtificial SequenceCDR2 364Glu Val Arg Trp
Gly Gly Val Thr Thr1 536514PRTArtificial SequenceCDR3 365Ser Gly
Val Leu Gly Gly Leu His Glu Asp Trp Phe Asn Tyr1 5
1036612PRTArtificial SequenceCDR3 366Val Arg Gln Met Tyr Met Thr
Val Val Pro Asp Tyr1 5 1036725PRTArtificial SequenceFR1 367Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser 20 2536825PRTArtificial SequenceFR1
368Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser 20 2536914PRTArtificial
SequenceFR2 369Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
Ala1 5 1037014PRTArtificial SequenceFR2 370Trp Phe Arg Gln Ala Pro
Gly Gln Glu Arg Glu Ala Val Ala1 5 1037114PRTArtificial SequenceFR2
371Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala1 5
1037239PRTArtificial SequenceFR3 372Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Thr Ala1 5 10 15Lys Asn Thr Val Tyr Leu Gln
Met Asn Ser Leu Arg Pro Asp Asp Thr 20 25 30Ala Asp Tyr Tyr Cys Ala
Ala 3537339PRTArtificial SequenceFR3 373Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala1 5 10 15Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Pro Asp Asp Thr 20 25 30Ala Asp Tyr Tyr Cys
Ala Ala 3537439PRTArtificial SequenceFR3 374Tyr Ser Asn Ser Leu Lys
Asp Arg Phe Ser Ile Ser Glu Asp Ser Val1 5 10 15Lys Asn Ala Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 20 25 30Ala Val Tyr Tyr
Cys Ala Ala 3537511PRTArtificial SequenceFR4 375Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser1 5 103765PRTArtificial SequenceLinker
376Gly Gly Gly Gly Ser1 53777PRTArtificial SequenceLinker 377Ser
Gly Gly Ser Gly Gly Ser1 53789PRTArtificial SequenceLinker 378Gly
Gly Gly Gly Ser Gly Gly Gly Ser1 537910PRTArtificial SequenceLinker
379Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 1038015PRTArtificial
SequenceLinker 380Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser1 5 10 1538118PRTArtificial SequenceLinker 381Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly1 5 10 15Gly
Ser38220PRTArtificial SequenceLinker 382Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser
2038325PRTArtificial SequenceLinker 383Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly
Gly Gly Ser 20 2538430PRTArtificial SequenceLinker 384Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25
3038535PRTArtificial SequenceLinker 385Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30Gly Gly Ser
353863PRTArtificial SequenceLinker 386Ala Ala
Ala1387423PRTArtificial SequenceNanobody sequence 387Asp Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys
50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr
Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys
Ala Ala Ser Gly Asp Val His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185
190Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala His Ile Thr Ile
195 200 205Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe
Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln
Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Tyr Ser Arg Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 275 280 285Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 290 295 300Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln305 310
315 320Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe 325 330 335Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 340 345 350Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser
Asp Thr Leu Tyr Ala 355 360 365Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Thr 370 375 380Thr Leu Tyr Leu Gln Met Asn
Ser Leu Arg Pro Glu Asp Thr Ala Val385 390 395 400Tyr Tyr Cys Thr
Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr 405 410 415Leu Val
Thr Val Ser Ser Ala 420388273PRTArtificial SequenceNanobody
sequence 388Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser 165 170 175Cys Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu
Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val
Ala His Ile Ser Ile 195 200 205Gly Asp Gln Thr Asp Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ser Lys Asn
Met Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile 245 250 255Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270Ala389430PRTArtificial SequenceNanobody sequence 389Asp Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Ala Ile Ser Trp Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr
Ile Tyr Ile Trp 100 105 110Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly145 150 155 160Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
165 170 175Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val
His Lys 180 185 190Ile Asn Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala
Lys Glu Arg Glu 195 200 205Met Val Ala His Ile Thr Ile Gly Asp Ala
Thr Asp Tyr Ala Asp Ser 210 215 220Ala Lys Gly Arg Phe Thr Ile Ser
Arg Asp Glu Ala Lys Asn Met Val225 230 235 240Tyr Leu Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe 245 250 255Cys Arg Ala
Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly 260 265 270Thr
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 275 280
285Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
290 295 300Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
Val Glu305 310 315 320Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser
Leu Arg Leu Ser Cys 325 330 335Ala Ala Ser Gly Phe Thr Phe Ser Ser
Phe Gly Met Ser Trp Val Arg 340 345 350Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser Ser Ile Ser Gly Ser 355 360 365Gly Ser Asp Thr Leu
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 370 375 380Ser Arg Asp
Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu385 390 395
400Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu
405 410 415Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Ala
420 425 430390473PRTArtificial SequenceNanobody sequence 390Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25
30Val Met Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe Val
35 40 45Ala Thr Ile Ala Trp Asp Ser Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
Val His65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Ala Ser Tyr Asn Val Tyr Tyr Asn Asn Tyr
Tyr Tyr Pro Ile Ser 100 105 110Arg Asp Glu Tyr Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 115 120 125Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly145 150 155 160Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val 165 170
175Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr
180 185 190Tyr Gly Ser Tyr Trp Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Glu 195 200 205Arg Glu Gly Val Ala Ala Ile Asn Arg Gly Gly Gly
Tyr Thr Val Tyr 210 215 220Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Thr Ala Lys225 230 235 240Asn Thr Val Tyr Leu Gln Met
Asn Ser Leu Arg Pro Asp Asp Thr Ala 245 250 255Asp Tyr Tyr Cys Ala
Ala Ser Gly Val Leu Gly Gly Leu His Glu Asp 260 265 270Trp Phe Asn
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 275 280 285Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 290 295
300Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly305 310 315 320Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro 325 330 335Gly Gly Ser Leu Arg Leu Ser Cys Val Ala
Ser Gly Asp Val His Lys 340 345 350Ile Asn Phe Leu Gly Trp Tyr Arg
Gln Ala Pro Gly Lys Glu Arg Glu 355 360 365Lys Val Ala His Ile Ser
Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser 370 375 380Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val385 390 395 400Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe 405 410
415Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
420 425 430Thr Leu Val Thr Val Ser Ser Gly Ala Ala Asp Tyr Lys Asp
His Asp 435 440 445Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp
Asp Asp Asp Lys 450 455 460Gly Ala Ala His His His His His His465
470391477PRTArtificial SequenceNanobody sequence 391Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25 30Cys Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ala
Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr
Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly
His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly
Gln Gly Thr Leu 115 120 125Val Thr Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly145 150 155 160Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 165 170 175Gly Gly Ser
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 180 185 190Ser
Gly Asp Thr Tyr Gly Ser Tyr Trp Met Gly Trp Phe Arg Gln Ala 195 200
205Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asn Arg Gly Gly Gly
210 215 220Tyr Thr Val Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg225 230 235 240Asp Thr Ala Lys Asn Thr Val Tyr Leu Gln Met
Asn Ser Leu Arg Pro 245 250 255Asp Asp Thr Ala Asp Tyr Tyr Cys Ala
Ala Ser Gly Val Leu Gly Gly 260 265 270Leu His Glu Asp Trp Phe Asn
Tyr Trp Gly Gln Gly Thr Leu Val Thr 275 280 285Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 290 295 300Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly305 310 315
320Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
325 330 335Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala
Ser Gly 340 345 350Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr Arg
Gln Ala Pro Gly 355 360 365Lys Glu Arg Glu Lys Val Ala His Ile Ser
Ile Gly Asp Gln Thr Asp 370 375 380Tyr Ala Asp Ser Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ser385 390 395 400Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 405 410 415Ala Val Tyr
Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr 420 425 430Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ala Ala Asp Tyr 435 440
445Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp
450 455 460Asp Asp Asp Lys Gly Ala Ala His His His His His His465
470 475392483PRTArtificial SequenceNanobody sequence 392Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Val
Met Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe Val 35 40
45Ala Thr Ile Ala Trp Asp Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
His65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Ala Ser Tyr Asn Val Tyr Tyr Asn Asn Tyr Tyr
Tyr Pro Ile Ser 100 105 110Arg Asp Glu Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly145 150 155 160Gly Gly Gly
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val 165 170 175Gln
Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr 180 185
190Ile Gly Pro Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
195 200 205Arg Glu Gly Val Ala Ala Ile Asn Met Gly Gly Gly Ile Thr
Tyr Tyr 210 215 220Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln
Asp Asn Ala Lys225 230 235 240Asn Thr Val Tyr Leu Leu Met Asn Ser
Leu Glu Pro Glu Asp Thr Ala 245 250 255Ile Tyr Tyr Cys Ala Ala Asp
Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu 260 265 270Cys Gly His Gly Leu
Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly 275 280 285Gln Gly Thr
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 290 295 300Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly305 310
315 320Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
Leu 325 330 335Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu 340 345 350Ser Cys Val Ala Ser Gly Asp Val His Lys Ile
Asn Phe Leu Gly Trp 355 360 365Tyr Arg Gln Ala Pro Gly Lys Glu Arg
Glu Lys Val Ala His Ile Ser 370 375 380Ile Gly Asp Gln Thr Asp Tyr
Ala Asp Ser Ala Lys Gly Arg Phe Thr385 390 395 400Ile Ser Arg Asp
Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn Ser 405 410 415Leu Lys
Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg 420 425
430Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
435 440 445Ser Gly Ala Ala Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys
Asp His 450 455 460Asp Ile Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ala
Ala His His His465 470 475 480His His His393107PRTHomo sapiens
393Ile Gln Val Glu Gln Ser Pro Pro Asp Leu Ile Leu Gln Glu Gly Ala1
5 10 15Asn Ser Thr Leu Arg Cys Asn Phe Ser Asp Ser Val Asn Asn Leu
Gln 20 25 30Trp Phe His Gln Asn Pro Trp Gly Gln Leu Ile Asn Leu Phe
Tyr Ile 35 40 45Pro Ser Gly Thr Lys Gln Asn Gly Arg Leu Ser Ala Thr
Thr Val Ala 50 55 60Thr Glu Arg Tyr Ser Leu Leu Tyr Ile Ser Ser Ser
Gln Thr Thr Asp65 70 75 80Ser Gly Val Tyr Phe Cys Ala Ala Leu Ile
Gln Gly Ala Gln Lys Leu 85 90 95Val Phe Gly Gln Gly Thr Arg Leu Thr
Ile Asn 100 105394108PRTHomo sapiens 394Gln Leu Leu Glu Gln Ser Pro
Gln Phe Leu Ser Ile Gln Glu Gly Glu1 5 10 15Asn Leu Thr Val Tyr Cys
Asn Ser Ser Ser Val Phe Ser Ser Leu Gln 20 25 30Trp Tyr Arg Gln Glu
Pro Gly Glu Gly Pro Val Leu Leu Val Thr Val 35 40 45Val Thr Gly Gly
Glu Val Lys Lys Leu Lys Arg Leu Thr Phe Gln Phe 50 55 60Gly Asp Ala
Arg Lys Asp Ser Ser Leu His Ile Thr Ala Ala Gln Pro65 70 75 80Gly
Asp Thr Gly Leu Tyr Leu Cys Ala Gly Ala Gly Ser Gln Gly Asn 85 90
95Leu Ile Phe Gly Lys Gly Thr Lys Leu Ser Val Lys 100
105395110PRTHomo sapiens 395Gly Asp Ala Lys Thr Thr Gln Pro Asn Ser
Met Glu Ser Asn Glu Glu1 5 10 15Glu Pro Val His Leu Pro Cys Asn His
Ser Thr Ile Ser Gly Thr Asp 20 25 30Tyr Ile His Trp Tyr Arg Gln Leu
Pro Ser Gln Gly Pro Glu Tyr Val 35 40 45Ile His Gly Leu Thr Ser Asn
Val Asn Asn Arg Met Ala Ser Leu Ala 50 55 60Ile Ala Glu Asp Arg Lys
Ser Ser Thr Leu Ile Leu His Arg Ala Thr65 70 75 80Leu Arg Asp Ala
Ala Val Tyr Tyr Cys Thr Val Tyr Gly Gly Ala Thr 85 90 95Asn Lys Leu
Ile Phe Gly Thr Gly Thr Leu Leu Ala Val Gln 100 105
11039696PRTMacaca fascicularis 396Pro Tyr Ile Gln Asn Pro Asp Pro
Ala Val Tyr Gln Leu Arg Gly Ser1 5 10 15Lys Ser Asn Asp Thr Ser Val
Cys Leu Phe Thr Asp Phe Asp Ser Val 20 25 30Met Asn Val Ser Gln Ser
Lys Asp Ser Asp Val His Ile Thr Asp Lys 35 40 45Thr Val Leu Asp Met
Arg Ser Met Asp Phe Lys Ser Asn Gly Ala Val 50 55 60Ala Trp Ser Asn
Lys Ser Asp Phe Ala Cys Thr Ser Ala Phe Lys Asp65 70 75 80Ser Val
Ile Pro Ala Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys 85 90
95397131PRTMacaca mulatta 397Glu Asp Leu Lys Lys Val Phe Pro Pro
Lys Val Ala Val Phe Glu Pro1 5 10 15Ser Glu Ala Glu Ile Ser His Thr
Gln Lys Ala Thr Leu Val Cys Leu 20 25 30Ala Thr Gly Phe Tyr Pro Asp
His Val Glu Leu Ser Trp Trp Val Asn 35 40 45Gly Lys Glu Val His Ser
Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60Glu Gln Pro Ala Leu
Glu Asp Ser Arg Tyr Ser Leu Ser Ser Arg Leu65 70 75 80Arg Val Ser
Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 85 90 95Gln Val
Gln Phe Tyr Gly Leu Ser Glu Asp Asp Glu Trp Thr Glu Asp 100 105
110Arg Asp Lys Pro Ile Thr Gln Lys Ile Ser Ala Glu Ala Trp Gly Arg
115 120 125Ala Asp Cys 130398107PRTMacaca mulatta 398Gln Gln Ile
Met Gln Ile Pro Gln Tyr Gln His Val Gln Glu Gly Glu1 5 10 15Asp Phe
Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser Asn Ile Gln 20 25 30Trp
Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu Ile Met Leu 35 40
45Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Ile Phe Gln Phe
50 55 60Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala Thr Gln
Thr65 70 75 80Thr Asp Val Gly Thr Tyr Phe Cys Ala Thr Thr Gly Val
Asn Asn Leu 85 90 95Phe Phe Gly Thr Gly Thr Arg Leu Thr Val Leu 100
105399118PRTMacaca mulatta 399Ala Gly Pro Val Asn Ala Gly Val Thr
Gln Thr Pro Lys Phe Gln Val1 5 10 15Leu Lys Thr Gly Gln Ser Met Thr
Leu Gln Cys Ala Gln Asp Met Asn 20 25 30His Asp Tyr Met Tyr Trp Tyr
Arg Gln Asp Pro Gly Met Gly Leu Arg 35 40 45Leu Ile His Tyr Ser Val
Gly Glu Gly Ser Thr Glu Lys Gly Glu Val 50 55 60Pro Asp Gly Tyr Asn
Val Thr Arg Ser Asn Thr Glu Asp Phe Pro Leu65 70 75 80Arg Leu Glu
Ser Ala Ala Pro Ser Gln Thr Ser
Val Tyr Phe Cys Ala 85 90 95Ser Ser Tyr Trp Thr Gly Arg Ser Tyr Glu
Gln Tyr Phe Gly Pro Gly 100 105 110Thr Arg Leu Thr Val Ile
115400115PRTArtificial SequenceNanobody sequence 400Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu
Val Thr 100 105 110Val Ser Ser 115401115PRTArtificial
SequenceNanobody sequence 401Glu 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 Arg Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser
Gly Ser Asp Thr Leu 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 Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile
Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 115402116PRTArtificial SequenceNanobody sequence
402Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala
115403116PRTArtificial SequenceNanobody sequence 403Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val 35 40 45Ser
Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu 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 Pro Glu Asp Thr Ala Thr Tyr Tyr
Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu
Val Thr 100 105 110Val Ser Ser Ala 115404115PRTArtificial
SequenceNanobody sequence 404Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser
Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile
Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser 115405116PRTArtificial SequenceNanobody sequence
405Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Lys 100 105 110Val Ser Ser Ala
115406115PRTArtificial SequenceNanobody sequence 406Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu
Val Thr 100 105 110Val Ser Ser 115407116PRTArtificial
SequenceNanobody sequence 407Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser
Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile
Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser Ala 115408117PRTArtificial SequenceNanobody sequence
408Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Ala Ala
115409118PRTArtificial SequenceNanobody sequence 409Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu
Val Thr 100 105 110Val Ser Ser Ala Ala Ala 115410116PRTArtificial
SequenceNanobody sequence 410Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ser Ile Ser Gly Ser
Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile
Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105
110Val Ser Ser Gly 115411117PRTArtificial SequenceNanobody sequence
411Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Phe 20 25 30Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Thr Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
Thr Ala Leu Tyr Tyr Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser
Ser Gln Gly Thr Leu Val Thr 100 105 110Val Ser Ser Gly Gly
115412118PRTArtificial SequenceNanobody sequence 412Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Asn1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30Gly Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu
Val Thr 100 105 110Val Ser Ser Gly Gly Gly 115413293PRTArtificial
SequenceNanobody sequence 413Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Gly Gly Gly1 5 10 15Ser Leu Ser Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met Ala Trp Phe Arg Gln
Pro Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ser Ile Ser Trp Ser
Gly Glu Asn Thr Asn Tyr Arg Asn Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Lys Ile Ala Lys Thr Tyr Pro Asp Asn Trp Tyr Trp Thr Lys 100 105
110Ser Asn Asn Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly145 150 155 160Gly Gly Gly Ser Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val 165 170 175Gln Gly Gly Gly Ser Leu Ser
Leu Ser Cys Ala Ala Ser Gly Arg Thr 180 185 190Phe Ser Ser Tyr Ala
Met Ala Trp Phe Arg Gln Pro Pro Gly Lys Glu 195 200 205Arg Glu Phe
Val Ala Ser Ile Ser Trp Ser Gly Glu Asn Thr Asn Tyr 210 215 220Arg
Asn Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys225 230
235 240Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala 245 250 255Val Tyr Tyr Cys Ala Ala Lys Ile Ala Lys Thr Tyr Pro
Asp Asn Trp 260 265 270Tyr Trp Thr Lys Ser Asn Asn Tyr Asn Tyr Trp
Gly Gln Gly Thr Leu 275 280 285Val Thr Val Ser Ser
290414242PRTArtificial SequenceNanobody sequence 414Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Glu Val Gln 115 120 125Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg 130 135 140Leu Ser Cys Ala Ala Ser Gly Ser
Val His Lys Ile Asn Phe Leu Gly145 150 155 160Trp Tyr Arg Gln Ala
Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile 165 170 175Thr Ile Gly
Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys Gly Arg Phe 180 185 190Thr
Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu Gln Met Asn 195 200
205Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Gly Ser
210 215 220Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val225 230 235 240Ser Ser415255PRTArtificial SequenceNanobody
sequence 415Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile
Gly Pro Tyr 20 25 30Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Gly Val 35 40 45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
Ala Lys Asn Thr Val Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro
Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr
Ala Ser Tyr Tyr Glu Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly
Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Gln 115 120 125Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 130 135 140Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys145 150
155 160Ala Ala Ser Gly Ser Val His Lys Ile Asn Phe Leu Gly Trp Tyr
Arg 165 170 175Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile
Thr Ile Gly 180 185 190Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys Gly
Arg Phe Thr Ile Ser 195 200 205Arg Asp Glu Ala Arg Asn Met Val Tyr
Leu Gln Met Asn Ser Leu Lys 210 215 220Pro Glu Asp Thr Ala Val Tyr
Phe Cys Arg Ala Gly Ser Arg Leu Tyr225 230 235 240Pro Tyr Asn Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250
255416258PRTArtificial SequenceNanobody sequence 416Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25
30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu
Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Asp Val Gln 115 120 125Leu Gln Ala Ser Gly Gly Gly
Ser Val Gln Ala Gly Gly Ser Leu Arg 130 135 140Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Ile Gly Pro Tyr Cys Met Gly145 150 155 160Trp Phe
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile 165 170
175Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
180 185 190Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu
Leu Met 195 200 205Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr
Cys Ala Ala Asp 210 215 220Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys
Gly His Gly Leu Ser Thr225 230 235 240Gly Gly Tyr Gly Tyr Asp Ser
Trp Gly Gln Gly Thr Gln Val Thr Val 245 250 255Ser
Ser417242PRTArtificial SequenceNanobody sequence 417Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Val
Gln Leu Val Glu 115 120 125Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys 130 135 140Thr Phe Ser Gly Gly Thr Phe Ser
Ser Tyr Thr Met Gly Trp Phe Arg145 150 155 160Gln Ala Pro Gly Lys
Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly 165 170 175Gly Val Thr
Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser 180 185 190Glu
Asp Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys 195 200
205Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr
210 215 220Met Thr Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val225 230 235 240Ser Ser418246PRTArtificial SequenceNanobody
sequence 418Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 130 135 140Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile145 150
155 160Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly
Leu 165 170 175Val Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala
Asp Tyr Ala 180 185 190Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Arg Asn Met Val Tyr 195 200 205Leu Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys 210 215 220Arg Ala Gly Ser Arg Leu Tyr
Pro Tyr Asn Tyr Trp Gly Gln Gly Thr225 230 235 240Leu Val Thr Val
Ser Ser 245419259PRTArtificial SequenceNanobody sequence 419Glu Val
Gln Leu Gln Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25
30Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr
Val Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala
Ile Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr
Glu Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp
Ser Trp Gly Gln Gly Thr Gln 115 120 125Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Ser Glu Val 130 135 140Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu145 150 155 160Arg Leu
Ser Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn Phe Leu 165 170
175Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr
180 185 190Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys
Gly Arg 195 200 205Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val
Tyr Leu Gln Met 210 215 220Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Arg Ala Gly225 230 235 240Ser Arg Leu Tyr Pro Tyr Asn
Tyr Trp Gly Gln Gly Thr Leu Val Thr 245 250 255Val Ser
Ser420262PRTArtificial SequenceNanobody sequence 420Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Ser Asp Val Gln Leu Gln Ala Ser Gly Gly
Gly Ser Val Gln Ala Gly 130 135 140Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Tyr Thr Ile Gly Pro145 150 155 160Tyr Cys Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 165 170 175Val Ala Ala
Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser 180 185 190Val
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val 195 200
205Tyr Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr
210 215 220Cys Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys
Gly His225 230 235 240Gly Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser
Trp Gly Gln Gly Thr 245 250 255Gln Val Thr Val Ser Ser
260421246PRTArtificial SequenceNanobody sequence 421Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Ser Glu Val 115 120 125Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu 130 135 140Arg Leu Ser Cys Thr Phe Ser Gly
Gly Thr Phe Ser Ser Tyr Thr Met145 150 155 160Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Glu 165 170 175Val Arg Trp
Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg 180 185 190Phe
Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu Gln Met 195 200
205Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val
210 215 220Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln
Gly Thr225 230 235 240Leu Val Thr Val Ser Ser
245422272PRTArtificial SequenceNanobody sequence 422Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala
Ser Gly Ser Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185 190Arg
Gln Ala Pro Gly Lys Glu Arg Gly Leu Val Ala Thr Ile Thr Ile 195 200
205Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys Gly Arg Phe Thr Ile
210 215 220Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu Gln Met Asn
Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
Ala Gly Ser Arg Leu 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270423285PRTArtificial
SequenceNanobody sequence 423Glu Val Gln Leu Gln Ala Ser Gly Gly
Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25 30Cys Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ala Ala Ile Asn Met Gly
Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Leu Met
Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Ala
Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly 100 105
110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Gln
115 120 125Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly145 150 155 160Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly 165 170 175Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala 180 185 190Ser Gly Ser Val His
Lys Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala 195 200 205Pro Gly Lys
Glu Arg Gly Leu Val Ala Thr Ile Thr Ile Gly Asp Thr 210 215 220Thr
Asp Tyr Ala Asp Tyr Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp225 230
235 240Glu Ala Arg Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
Glu 245 250 255Asp Thr Ala Val Tyr Phe Cys Arg Ala Gly Ser Arg Leu
Tyr Pro Tyr 260 265 270Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 275 280 285424288PRTArtificial SequenceNanobody sequence
424Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn
Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn
Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val
Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Gln145 150 155
160Ala Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
165 170 175Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr Cys Met Gly
Trp Phe 180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
Ala Ile Asn Met 195 200 205Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser
Val Lys Gly Arg Phe Thr 210 215 220Ile Ser Gln Asp Asn Ala Lys Asn
Thr Val Tyr Leu Leu Met Asn Ser225 230 235 240Leu Glu Pro Glu Asp
Thr Ala Ile Tyr Tyr Cys Ala Ala Asp Ser Thr 245 250 255Ile Tyr Ala
Ser Tyr Tyr Glu Cys Gly His Gly Leu Ser Thr Gly Gly 260 265 270Tyr
Gly Tyr Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 275 280
285425272PRTArtificial SequenceNanobody sequence 425Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85
90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly Thr
Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro Gly
Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200
205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp
210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg
Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270426288PRTArtificial
SequenceNanobody sequence 426Glu Val Gln Leu Gln Ala Ser Gly Gly
Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25 30Cys Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ala Ala Ile Asn Met Gly
Gly Gly Ile Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Leu Met
Asn Ser Leu Glu Pro Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Ala
Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr Glu Cys Gly His Gly 100 105
110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp Ser Trp Gly Gln Gly Thr Gln
115 120 125Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly145 150 155 160Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly 165 170 175Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys Thr Phe 180 185 190Ser Gly Gly Thr Phe
Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 195 200 205Pro Gly Lys
Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 210 215 220Thr
Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp225 230
235 240Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
Glu 245 250 255Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met
Tyr Met Thr 260 265 270Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser 275 280 285427285PRTArtificial SequenceNanobody
sequence 427Glu 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 Ser Val His
Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
Arg Gly Leu Val 35 40 45Ala Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr
Ala Asp Tyr Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Gln Ala Ser Gly145 150
155 160Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala 165 170 175Ser Gly Tyr Thr Ile Gly Pro Tyr Cys Met Gly Trp Phe
Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile
Asn Met Gly Gly Gly 195 200 205Ile Thr Tyr Tyr Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Gln 210 215 220Asp Asn Ala Lys Asn Thr Val
Tyr Leu Leu Met Asn Ser Leu Glu Pro225 230 235 240Glu Asp Thr Ala
Ile Tyr Tyr Cys Ala Ala Asp Ser Thr Ile Tyr Ala 245 250 255Ser Tyr
Tyr Glu Cys Gly His Gly Leu Ser Thr Gly Gly Tyr Gly Tyr 260 265
270Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 275 280
285428272PRTArtificial SequenceNanobody sequence 428Glu 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 Asp Val Tyr Lys Ile Asn 20 25 30Phe Leu
Gly Trp His Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly
Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro
Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200
205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp
210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg
Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270429272PRTArtificial
SequenceNanobody sequence 429Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Lys Leu Ser Cys Ala Ala
Ser Gly Ala Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln
Thr Pro Glu Lys Glu Arg Glu Met Val 35 40 45Ala Thr Ile Thr Ile Gly
Asp Glu Val Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Thr
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Arg 85 90 95Ala Gly
Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser
Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg
Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr
Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser
Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu225 230
235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met
Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270430272PRTArtificial SequenceNanobody
sequence 430Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr Ser Gly Glu Thr Phe
Lys Ile Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln
Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly Gly Ala Thr Asn Tyr
Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Glu Asp Ser Ala
Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys Ser Arg Leu Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150
155 160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr
Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe
Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val
Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp
Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val
Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val
Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270431272PRTArtificial SequenceNanobody sequence 431Glu 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 Ser Val His Leu Leu Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala His Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly
Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro
Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200
205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp
210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg
Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270432272PRTArtificial
SequenceNanobody sequence 432Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ile Thr
Ser Gly Glu Thr Phe Lys Ile Asn 20 25 30Ile Trp Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Ser Leu Thr Ile Gly
Gly Ala Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95Ala Lys
Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser
Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg
Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr
Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser
Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu225 230
235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met
Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270433272PRTArtificial SequenceNanobody
sequence 433Glu 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 Glu Val Tyr
Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu
Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Ala Asp Ala Ala Asp Tyr
Ala Asp Phe Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Pro Glu
Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly Ser Arg Ile Trp Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150
155 160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr
Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe
Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val
Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp
Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val
Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val
Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270434272PRTArtificial SequenceNanobody sequence 434Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25
30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val
35 40 45Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala
Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val
Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170
175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala
180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly
Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser
Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met
Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys
Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270435272PRTArtificial SequenceNanobody sequence 435Glu 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 Glu Ile Gly Arg Ile Asn 20 25 30Phe Tyr
Arg Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Val Val 35 40 45Ala
Thr Ile Thr Ile Ala Asp Lys Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Arg Asn Met Val Tyr Leu65
70 75 80Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
His 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly
Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro
Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200
205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp
210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg
Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270436272PRTArtificial
SequenceNanobody sequence 436Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Val Ala
Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala His Ile Thr Ile Gly
Asp Gln Ala Asp Tyr Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly
Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser
Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg
Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr
Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser
Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu225 230
235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met
Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270437272PRTArtificial SequenceNanobody
sequence 437Glu 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 Asp Val His
Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu
Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr
Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150
155 160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr
Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe
Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val
Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp
Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val
Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val
Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270438272PRTArtificial SequenceNanobody sequence 438Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val 35 40 45Ala
Thr Ile Thr Ile Gly Asp Thr Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Gly Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly
Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro
Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200
205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp
210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg
Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270439272PRTArtificial
SequenceNanobody sequence 439Glu 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 Ser Val His Lys Ile Asn 20 25 30Phe Leu Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Glu Arg Gly Leu Val 35 40 45Ala Thr Ile Thr Ile Gly
Asp Thr Thr Asp Tyr Ala Asp Tyr Ala Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Glu Ala Arg Asn Met Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Gly
Ser Arg Leu Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105
110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser
Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg
Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr
Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser
Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu225 230
235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met
Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270440272PRTArtificial SequenceNanobody
sequence 440Glu 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 Asp Val His
Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu
Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr
Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Lys Asn Met Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Leu Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150
155 160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr
Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe
Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val
Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp
Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val
Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val
Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270441272PRTArtificial SequenceNanobody sequence 441Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Val Ala
Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185 190Arg
Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile 195 200
205Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile
210 215 220Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn
Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
Ala Phe Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270442272PRTArtificial
SequenceNanobody sequence 442Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala Ser Gly Glu Ile
Gly Arg Ile Asn Phe Tyr Arg Trp Tyr 180 185 190Arg Gln Ala Pro Gly
Asn Gln Arg Glu Val Val Ala Thr Ile Thr Ile 195 200 205Ala Asp Lys
Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser
Arg Asp Glu Ser Arg Asn Met Val Tyr Leu Gln Met Ser Ser Leu225 230
235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys His Ala Gly Ser Arg
Leu 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270443272PRTArtificial SequenceNanobody
sequence 443Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser 165 170 175Cys Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu
Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val
Ala His Ile Thr Ile 195 200 205Gly Asp Gln Ala Asp Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Phe Cys Arg Ala Gly Ser Arg Ile 245 250 255Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270444272PRTArtificial SequenceNanobody sequence 444Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala
Ser Gly Asp Val His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185 190Arg
Gln Ala Pro Ala Lys Glu Arg Glu Met Val Ala His Ile Thr Ile 195 200
205Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile
210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Met Asn
Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
Ala Tyr Ser Arg Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270445272PRTArtificial
SequenceNanobody sequence 445Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala Ser Gly Ser Val
His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly
Lys Glu Arg Glu Leu Val Ala Thr Ile Thr Ile 195 200 205Gly Asp Thr
Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu225 230
235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Gly Ser Arg
Leu 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270446272PRTArtificial SequenceNanobody
sequence 446Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser 165 170 175Cys Ala Ala Ser Gly Ser Val His Lys Ile Asn Phe Leu
Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Gly Leu Val
Ala Thr Ile Thr Ile 195 200 205Gly Asp Thr Thr Asp Tyr Ala Asp Tyr
Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ala Arg Asn
Met Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Phe Cys Arg Ala Gly Ser Arg Leu 245 250 255Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270447272PRTArtificial SequenceNanobody sequence 447Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala
Ser Gly Asp Val Tyr Lys Ile Asn Phe Leu Gly Trp His 180 185 190Arg
Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Thr Ile 195 200
205Gly Asp Ala Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile
210 215 220Ser Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Met Asn
Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
Ala Gly Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270448272PRTArtificial
SequenceNanobody sequence 448Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala Ser Gly Glu Val
Tyr Lys Ile Asn Phe Leu Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly
Lys Glu Arg Glu Lys Val Ala His Ile Thr Ile 195 200 205Ala Asp Ala
Ala Asp Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu225 230
235 240Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Gly Ser Arg
Ile 245 250 255Trp Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270449272PRTArtificial SequenceNanobody
sequence 449Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser 165 170 175Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu
Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val
Ala His Ile Thr Ile 195 200 205Ala Asp Ala Thr Asp Tyr Ala Glu Phe
Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Pro Lys Asn
Met Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Leu Cys Arg Ala Gly Ser Arg Ile 245 250 255Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270450272PRTArtificial SequenceNanobody sequence 450Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ile Thr
Ser Gly Glu Thr Phe Lys Ile Asn Ile Trp Gly Trp Tyr 180 185 190Arg
Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ser Leu Thr Ile 195 200
205Gly Gly Ala Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
210 215 220Ser Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu Gln Met Asn
Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Asn
Ala Lys Ser Arg Leu 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270451272PRTArtificial
SequenceNanobody sequence 451Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala Ser Gly Ser Val
His Leu Leu Asn Phe Leu Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly
Lys Glu Arg Glu Met Val Ala His Ile Thr Ile 195 200 205Ala Asp Ala
Thr Asp Tyr Ala His Phe Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu225 230
235 240Arg Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg Ala Gly Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 260 265 270452272PRTArtificial
SequenceNanobody sequence 452Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ile Thr Ser Gly Glu Thr
Phe Lys Ile Asn Ile Trp Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly
Lys Gln Arg Glu Leu Val Ala Ser Leu Thr Ile 195 200 205Gly Gly Ala
Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 210 215 220Ser
Glu Asp Ser Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu225 230
235 240Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Asn Ala Lys Ser Arg
Leu 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270453272PRTArtificial SequenceNanobody
sequence 453Glu 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 Asp Val His
Lys Ile Asn 20 25 30Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu
Arg Glu Met Val 35 40 45Ala His Ile Thr Ile Gly Asp Ala Thr Ser Tyr
Ala Asp Ser Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala
Lys Asn Met Val Tyr Leu65 70 75 80Gln Leu Asn Asn Leu Lys Pro Glu
Asp Thr Ala Val Tyr Phe Cys Arg 85 90 95Ala Tyr Ser Arg Ile Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150
155 160Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr
Phe 165 170 175Ser Gly Gly Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe
Arg Gln Ala 180 185 190Pro Gly Lys Glu Arg Glu Phe Val Ala Glu Val
Arg Trp Gly Gly Val 195 200 205Thr Thr Tyr Ser Asn Ser Leu Lys Asp
Arg Phe Ser Ile Ser Glu Asp 210 215 220Ser Val Lys Asn Ala Val Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val
Tyr Tyr Cys Ala Ala Val Arg Gln Met Tyr Met Thr 245 250 255Val Val
Pro Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270454272PRTArtificial SequenceNanobody sequence 454Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val 35 40 45Ala
His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys
Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly
Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro
Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200
205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp
210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg
Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270455272PRTArtificial
SequenceNanobody sequence 455Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala Ser Gly Asp Val
His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185 190Arg Gln Ala Pro Ala
Lys Glu Arg Glu Met Val Ala His Ile Thr Ile 195 200 205Gly Asp Ala
Thr Ser Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Leu Asn Asn Leu225 230
235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Tyr Ser Arg
Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270456272PRTArtificial SequenceNanobody
sequence 456Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser 165 170 175Cys Val Ala Ser Gly Asp Val His Lys Ile Asn Phe Leu
Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys Val
Ala His Ile Ser Ile 195 200 205Gly Asp Gln Thr Asp Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ser Lys Asn
Met Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile 245 250 255Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270457272PRTArtificial SequenceNanobody sequence 457Glu Val Gln Leu
Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn 20 25 30Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Met Val 35 40 45Ala
His Ile Thr Ile Ala Asp Ala Thr Asp Tyr Ala Glu Phe Ala Lys 50 55
60Gly Arg Phe Thr Ile Ser Arg Asp Glu Pro Lys Asn Met Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys
Arg 85 90 95Ala Gly Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly
Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly Gly Leu Val Gln
Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Phe 165 170 175Ser Gly Gly
Thr Phe Ser Ser Tyr Thr Met Gly Trp Phe Arg Gln Ala 180 185 190Pro
Gly Lys Glu Arg Glu Phe Val Ala Glu Val Arg Trp Gly Gly Val 195 200
205Thr Thr Tyr Ser Asn Ser Leu Lys Asp Arg Phe Ser Ile Ser Glu Asp
210 215 220Ser Val Lys Asn Ala Val Tyr Leu Gln Met Asn Ser Leu Lys
Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys Ala Ala Val Arg
Gln Met Tyr Met Thr 245 250 255Val Val Pro Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270458272PRTArtificial
SequenceNanobody sequence 458Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala Ser Gly Asp Val
His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185 190Arg Gln Ala Pro Ala
Lys Glu Arg Glu Met Val Ala His Ile Thr Ile 195 200 205Gly Asp Ala
Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu225 230
235 240Lys Pro Glu Asp Thr Ala Val Tyr Leu Cys Arg Ala Tyr Ser Arg
Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270459272PRTArtificial SequenceNanobody
sequence 459Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu
Ser 165 170 175Cys Ala Ala Ser Gly Ala Val His Lys Ile Asn Phe Leu
Gly Trp Tyr 180 185 190Arg Gln Thr Pro Glu Lys Glu Arg Glu Met Val
Ala Thr Ile Thr Ile 195 200 205Gly Asp Glu Val Asp Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu Gln Met Thr Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Val Cys Arg Ala Gly Ser Arg Leu 245 250 255Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270460422PRTArtificial SequenceNanobody sequence 460Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Val Ala
Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185 190Arg
Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile 195 200
205Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly
Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ser Lys Asn Met Val Tyr
Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val
Tyr Phe Cys Arg Ala Phe Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 275 280 285Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 290 295
300Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln305 310 315 320Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe 325 330 335Ser Ser Phe Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu 340 345 350Glu Trp Val Ser Ser Ile Ser Gly
Ser Gly Ser Asp Thr Leu Tyr Ala 355 360 365Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr 370 375 380Thr Leu Tyr Leu
Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val385 390 395 400Tyr
Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr 405 410
415Leu Val Thr Val Ser Ser 420461422PRTArtificial SequenceNanobody
sequence 461Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser 165 170 175Cys Ile Thr Ser Gly Glu Thr Phe Lys Ile Asn Ile Trp
Gly Trp Tyr 180 185 190Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
Ala Ser Leu Thr Ile 195 200 205Gly Gly Ala Thr Asn Tyr Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile 210 215 220Ser Glu Asp Ser Ala Lys Asn
Thr Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Phe Cys Asn Ala Lys Ser Arg Leu 245 250 255Tyr Pro
Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
275 280 285Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 290 295 300Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln305 310 315 320Pro Gly Asn Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe 325 330 335Ser Ser Phe Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345 350Glu Trp Val Ser Ser
Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala 355 360 365Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr 370 375 380Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val385 390
395 400Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly
Thr 405 410 415Leu Val Thr Val Ser Ser 420462270PRTArtificial
SequenceNanobody sequence 462Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Ile Thr Phe Ser Ile Asn 20 25 30Thr Met Gly Trp Tyr Arg Gln
Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45Ala Leu Ile Ser Ser Ile
Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Lys 85 90 95Arg Phe
Arg Thr Ala Ala Gln Gly Thr Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln
Leu Val Glu Ser145 150 155 160Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys Val 165 170 175Ala Ser Gly Asp Val His Lys
Ile Asn Phe Leu Gly Trp Tyr Arg Gln 180 185 190Ala Pro Gly Lys Glu
Arg Glu Lys Val Ala His Ile Ser Ile Gly Asp 195 200 205Gln Thr Asp
Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg 210 215 220Asp
Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro225 230
235 240Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr
Pro 245 250 255Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 260 265 270463270PRTArtificial SequenceNanobody sequence 463Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn
20 25 30Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Lys
Val 35 40 45Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser
Ala Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met
Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
Val Tyr Phe Cys Arg 85 90 95Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr
Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly145 150 155 160Gly
Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 165 170
175Ser Gly Ile Thr Phe Ser Ile Asn Thr Met Gly Trp Tyr Arg Gln Ala
180 185 190Pro Gly Lys Gln Arg Glu Leu Val Ala Leu Ile Ser Ser Ile
Gly Asp 195 200 205Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp 210 215 220Asn Ala Lys Asn Thr Val Tyr Leu Gln Met
Asn Ser Leu Lys Pro Glu225 230 235 240Asp Thr Ala Val Tyr Tyr Cys
Lys Arg Phe Arg Thr Ala Ala Gln Gly 245 250 255Thr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 260 265 270464439PRTArtificial
SequenceNanobody sequence 464Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Val Met Gly Trp Phe Arg Gln
Ala Thr Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Thr Ile Ala Trp Asp
Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Ser Tyr Asn Val Tyr Tyr Asn Asn Tyr Tyr Tyr Pro Ile Ser 100 105
110Arg Asp Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly145 150 155 160Gly Gly Gly Ser Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Ser Val 165 170 175Gln Ala Gly Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Asp Thr 180 185 190Tyr Gly Ser Tyr Trp
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 195 200 205Arg Glu Gly
Val Ala Ala Ile Asn Arg Gly Gly Gly Tyr Thr Val Tyr 210 215 220Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys225 230
235 240Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr
Ala 245 250 255Asp Tyr Tyr Cys Ala Ala Ser Gly Val Leu Gly Gly Leu
His Glu Asp 260 265 270Trp Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Gly 275 280 285Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly 290 295 300Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly305 310 315 320Gly Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 325 330 335Gly Gly
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys 340 345
350Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
355 360 365Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala
Asp Ser 370 375 380Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser
Lys Asn Met Val385 390 395 400Tyr Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Phe 405 410 415Cys Arg Ala Phe Ser Arg Ile
Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly 420 425 430Thr Leu Val Thr Val
Ser Ser 435465443PRTArtificial SequenceNanobody sequence 465Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ile Gly Pro Tyr 20 25
30Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr
Val Tyr65 70 75 80Leu Leu Met Asn Ser Leu Glu Pro Glu Asp Thr Ala
Ile Tyr Tyr Cys 85 90 95Ala Ala Asp Ser Thr Ile Tyr Ala Ser Tyr Tyr
Glu Cys Gly His Gly 100 105 110Leu Ser Thr Gly Gly Tyr Gly Tyr Asp
Ser Trp Gly Gln Gly Thr Leu 115 120 125Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly145 150 155 160Gly Gly
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 165 170
175Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
180 185 190Ser Gly Asp Thr Tyr Gly Ser Tyr Trp Met Gly Trp Phe Arg
Gln Ala 195 200 205Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asn
Arg Gly Gly Gly 210 215 220Tyr Thr Val Tyr Ala Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg225 230 235 240Asp Thr Ala Lys Asn Thr Val
Tyr Leu Gln Met Asn Ser Leu Arg Pro 245 250 255Asp Asp Thr Ala Asp
Tyr Tyr Cys Ala Ala Ser Gly Val Leu Gly Gly 260 265 270Leu His Glu
Asp Trp Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr 275 280 285Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 290 295
300Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly305 310 315 320Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu
Ser Gly Gly Gly 325 330 335Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys Val Ala Ser Gly 340 345 350Asp Val His Lys Ile Asn Phe Leu
Gly Trp Tyr Arg Gln Ala Pro Gly 355 360 365Lys Glu Arg Glu Lys Val
Ala His Ile Ser Ile Gly Asp Gln Thr Asp 370 375 380Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser385 390 395 400Lys
Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 405 410
415Ala Val Tyr Phe Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr
420 425 430Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 435
440466449PRTArtificial SequenceNanobody sequence 466Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Val Met
Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Thr Ile Ala Trp Asp Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Ser Tyr Asn Val Tyr Tyr Asn Asn Tyr Tyr Tyr Pro
Ile Ser 100 105 110Arg Asp Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser 115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly145 150 155 160Gly Gly Gly Ser Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val 165 170 175Gln Ala Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr 180 185 190Ile
Gly Pro Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 195 200
205Arg Glu Gly Val Ala Ala Ile Asn Met Gly Gly Gly Ile Thr Tyr Tyr
210 215 220Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
Ala Lys225 230 235 240Asn Thr Val Tyr Leu Leu Met Asn Ser Leu Glu
Pro Glu Asp Thr Ala 245 250 255Ile Tyr Tyr Cys Ala Ala Asp Ser Thr
Ile Tyr Ala Ser Tyr Tyr Glu 260 265 270Cys Gly His Gly Leu Ser Thr
Gly Gly Tyr Gly Tyr Asp Ser Trp Gly 275 280 285Gln Gly Thr Leu Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 290 295 300Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly305 310 315
320Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
325 330 335Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu
Arg Leu 340 345 350Ser Cys Val Ala Ser Gly Asp Val His Lys Ile Asn
Phe Leu Gly Trp 355 360 365Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
Lys Val Ala His Ile Ser 370 375 380Ile Gly Asp Gln Thr Asp Tyr Ala
Asp Ser Ala Lys Gly Arg Phe
Thr385 390 395 400Ile Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu
Gln Met Asn Ser 405 410 415Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe
Cys Arg Ala Phe Ser Arg 420 425 430Ile Tyr Pro Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 435 440 445Ser467422PRTArtificial
SequenceNanobody sequence 467Asp Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe
Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly
Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile
Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn
Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val
Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Ala Ala Ser Gly Asp Val
His Lys Ile Asn Ile Leu Gly Trp Tyr 180 185 190Arg Gln Ala Pro Ala
Lys Glu Arg Glu Met Val Ala His Ile Thr Ile 195 200 205Gly Asp Ala
Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser
Arg Asp Glu Ala Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu225 230
235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg Ala Tyr Ser Arg
Ile 245 250 255Tyr Pro Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 260 265 270Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly 275 280 285Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly 290 295 300Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln305 310 315 320Pro Gly Asn Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 325 330 335Ser Ser
Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 340 345
350Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala
355 360 365Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Thr 370 375 380Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu
Asp Thr Ala Val385 390 395 400Tyr Tyr Cys Thr Ile Gly Gly Ser Leu
Ser Arg Ser Ser Gln Gly Thr 405 410 415Leu Val Thr Val Ser Ser
420468272PRTArtificial SequenceNanobody sequence 468Asp Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Glu Val Arg Trp Gly Gly Val Thr Thr Tyr Ser Asn Ser Leu Lys 50 55
60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val Lys Asn Ala Val Tyr Leu65
70 75 80Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Ala Val Arg Gln Met Tyr Met Thr Val Val Pro Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 130 135 140Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val145 150 155 160Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 165 170 175Cys Val Ala
Ser Gly Asp Val His Lys Ile Asn Phe Leu Gly Trp Tyr 180 185 190Arg
Gln Ala Pro Gly Lys Glu Arg Glu Lys Val Ala His Ile Ser Ile 195 200
205Gly Asp Gln Thr Asp Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile
210 215 220Ser Arg Asp Glu Ser Lys Asn Met Val Tyr Leu Gln Met Asn
Ser Leu225 230 235 240Lys Pro Glu Asp Thr Ala Val Tyr Phe Cys Arg
Ala Phe Ser Arg Ile 245 250 255Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 260 265 270469429PRTArtificial
SequenceNanobody sequence 469Asp Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Ala Ile Ser Trp Ser
Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr Ile Tyr Ile Trp 100 105
110Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly
115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly145 150 155 160Gly Ser Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro 165 170 175Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Asp Val His Lys 180 185 190Ile Asn Ile Leu Gly
Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu 195 200 205Met Val Ala
His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser 210 215 220Ala
Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn Met Val225 230
235 240Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
Phe 245 250 255Cys Arg Ala Tyr Ser Arg Ile Tyr Pro Tyr Asn Tyr Trp
Gly Gln Gly 260 265 270Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly 275 280 285Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser 290 295 300Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Val Glu305 310 315 320Ser Gly Gly Gly
Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys 325 330 335Ala Ala
Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg 340 345
350Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser
355 360 365Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe
Thr Ile 370 375 380Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln
Met Asn Ser Leu385 390 395 400Arg Pro Glu Asp Thr Ala Val Tyr Tyr
Cys Thr Ile Gly Gly Ser Leu 405 410 415Ser Arg Ser Ser Gln Gly Thr
Leu Val Thr Val Ser Ser 420 425470272PRTArtificial SequenceNanobody
sequence 470Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Thr Phe Ser Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
Arg Glu Phe Val 35 40 45Ala Glu Val Arg Trp Gly Gly Val Thr Thr Tyr
Ser Asn Ser Leu Lys 50 55 60Asp Arg Phe Ser Ile Ser Glu Asp Ser Val
Lys Asn Ala Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Ala Val Arg Gln Met Tyr Met
Thr Val Val Pro Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val145 150
155 160Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser 165 170 175Cys Ala Ala Ser Gly Asp Val His Lys Ile Asn Ile Leu
Gly Trp Tyr 180 185 190Arg Gln Ala Pro Ala Lys Glu Arg Glu Met Val
Ala His Ile Thr Ile 195 200 205Gly Asp Ala Thr Asp Tyr Ala Asp Ser
Ala Lys Gly Arg Phe Thr Ile 210 215 220Ser Arg Asp Glu Ala Lys Asn
Met Val Tyr Leu Gln Met Asn Ser Leu225 230 235 240Lys Pro Glu Asp
Thr Ala Val Tyr Phe Cys Arg Ala Tyr Ser Arg Ile 245 250 255Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 260 265
270471279PRTArtificial SequenceNanobody sequence 471Asp Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Ala Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45Ala
Ala Ile Ser Trp Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65
70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Ala Asp Leu Thr Ser Thr Asn Pro Gly Ser Tyr Ile Tyr
Ile Trp 100 105 110Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
Val Ser Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly145 150 155 160Gly Ser Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 165 170 175Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Val His Lys 180 185 190Ile
Asn Ile Leu Gly Trp Tyr Arg Gln Ala Pro Ala Lys Glu Arg Glu 195 200
205Met Val Ala His Ile Thr Ile Gly Asp Ala Thr Asp Tyr Ala Asp Ser
210 215 220Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ala Lys Asn
Met Val225 230 235 240Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
Thr Ala Val Tyr Phe 245 250 255Cys Arg Ala Tyr Ser Arg Ile Tyr Pro
Tyr Asn Tyr Trp Gly Gln Gly 260 265 270Thr Leu Val Thr Val Ser Ser
27547210PRTArtificial SequenceCDR1 472Gly Phe Thr Phe Ser Ser Phe
Gly Met Ser1 5 1047310PRTArtificial SequenceCDR1 473Gly Phe Thr Phe
Arg Ser Phe Gly Met Ser1 5 1047410PRTArtificial SequenceCDR2 474Ser
Ile Ser Gly Ser Gly Ser Asp Thr Leu1 5 104756PRTArtificial
SequenceCDR3 475Gly Gly Ser Leu Ser Arg1 5476110PRTHomo sapiens
476Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Arg Ile Leu Lys Ile Gly1
5 10 15Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His Asn Tyr
Met 20 25 30Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu Ile
Tyr Tyr 35 40 45Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro
Asn Gly Tyr 50 55 60Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu
Arg Leu Glu Leu65 70 75 80Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe
Cys Ala Ser Thr Tyr His 85 90 95Gly Thr Gly Tyr Phe Gly Glu Gly Ser
Trp Leu Thr Val Val 100 105 110477112PRTHomo sapiens 477Asp Gly Gly
Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg Lys Glu Gly1 5 10 15Gln Asn
Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His Asp Ala Met 20 25 30Tyr
Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu Ile Tyr Tyr 35 40
45Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala Glu Gly Tyr
50 55 60Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr Val Thr
Ser65 70 75 80Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser
Ser Ser Arg 85 90 95Ser Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg
Leu Thr Val Thr 100 105 110478114PRTHomo sapiens 478Val Val Ser Gln
His Pro Ser Trp Val Ile Ala Lys Ser Gly Thr Ser1 5 10 15Val Lys Ile
Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met Phe 20 25 30Trp Tyr
Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr Ser 35 40 45Asn
Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp Lys 50 55
60Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val Thr65
70 75 80Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg
Gly 85 90 95Gly Ser Tyr Asn Ser Pro Leu His Phe Gly Asn Gly Thr Arg
Leu Thr 100 105 110Val Thr47996PRTHomo sapiens 479Pro Asn Ile Gln
Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser1 5 10 15Lys Ser Ser
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln 20 25 30Thr Asn
Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 35 40 45Thr
Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val 50 55
60Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn65
70 75 80Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser
Cys 85 90 95480131PRTHomo sapiens 480Glu Asp Leu Asn Lys Val Phe
Pro Pro Glu Val Ala Val Phe Glu Pro1 5 10 15Ser Glu Ala Glu Ile Ser
His Thr Gln Lys Ala Thr Leu Val Cys Leu 20 25 30Ala Thr Gly Phe Phe
Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45Gly Lys Glu Val
His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60Glu Gln Pro
Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu65 70 75 80Arg
Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 85 90
95Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp
100 105 110Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp
Gly Arg 115 120 125Ala Asp Cys 1304815PRTArtificial SequenceCDR1
481Ser Phe Gly Met Ser1 548217PRTArtificial SequenceCDR2 482Ser Ile
Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys1 5 10
15Gly48398PRTArtificial SequenceNanobody sequence 483Gln 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 Leu Asp Tyr Tyr 20 25 30Ala
Ile Gly Trp Phe Arg Gln
Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45Ser Cys Ile Ser Ser Ser
Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Ala484142PRTHomo sapiens 484Pro Asn Ile Gln Asn Pro Asp Pro Ala Val
Tyr Gln Leu Arg Asp Ser1 5 10 15Lys Ser Ser Asp Lys Ser Val Cys Leu
Phe Thr Asp Phe Asp Ser Gln 20 25 30Thr Asn Val Ser Gln Ser Lys Asp
Ser Asp Val Tyr Ile Thr Asp Lys 35 40 45Thr Val Leu Asp Met Arg Ser
Met Asp Phe Lys Ser Asn Ser Ala Val 50 55 60Ala Trp Ser Asn Lys Ser
Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn65 70 75 80Ser Ile Ile Pro
Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys 85 90 95Asp Val Lys
Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn 100 105 110Phe
Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val 115 120
125Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135
140485177PRTHomo sapiens 485Glu Asp Leu Asn Lys Val Phe Pro Pro Glu
Val Ala Val Phe Glu Pro1 5 10 15Ser Glu Ala Glu Ile Ser His Thr Gln
Lys Ala Thr Leu Val Cys Leu 20 25 30Ala Thr Gly Phe Phe Pro Asp His
Val Glu Leu Ser Trp Trp Val Asn 35 40 45Gly Lys Glu Val His Ser Gly
Val Ser Thr Asp Pro Gln Pro Leu Lys 50 55 60Glu Gln Pro Ala Leu Asn
Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu65 70 75 80Arg Val Ser Ala
Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys 85 90 95Gln Val Gln
Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp 100 105 110Arg
Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg 115 120
125Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser
130 135 140Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu
Tyr Ala145 150 155 160Val Leu Val Ser Ala Leu Val Leu Met Ala Met
Val Lys Arg Lys Asp 165 170 175Phe486623PRTArtificial
SequenceNanobody sequence 486Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Ala Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30Val Met Gly Trp Phe Arg Gln
Ala Thr Gly Lys Glu Arg Glu Phe Val 35 40 45Ala Thr Ile Ala Trp Asp
Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val His65 70 75 80Leu Gln Met
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala
Ser Tyr Asn Val Tyr Tyr Asn Asn Tyr Tyr Tyr Pro Ile Ser 100 105
110Arg Asp Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 130 135 140Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly145 150 155 160Gly Gly Gly Ser Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Ser Val 165 170 175Gln Ala Gly Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Asp Thr 180 185 190Tyr Gly Ser Tyr Trp
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 195 200 205Arg Glu Gly
Val Ala Ala Ile Asn Arg Gly Gly Gly Tyr Thr Val Tyr 210 215 220Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys225 230
235 240Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr
Ala 245 250 255Asp Tyr Tyr Cys Ala Ala Ser Gly Val Leu Gly Gly Leu
His Glu Asp 260 265 270Trp Phe Asn Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Gly 275 280 285Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly 290 295 300Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly305 310 315 320Gly Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 325 330 335Gly Gly
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Asp Val His Lys 340 345
350Ile Asn Phe Leu Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu
355 360 365Lys Val Ala His Ile Ser Ile Gly Asp Gln Thr Asp Tyr Ala
Asp Ser 370 375 380Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser
Lys Asn Met Val385 390 395 400Tyr Leu Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Phe 405 410 415Cys Arg Ala Phe Ser Arg Ile
Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly 420 425 430Thr Leu Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 435 440 445Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 450 455 460Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu465 470
475 480Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser
Cys 485 490 495Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser
Trp Val Arg 500 505 510Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
Ser Ile Ser Gly Ser 515 520 525Gly Ser Asp Thr Leu Tyr Ala Asp Ser
Val Lys Gly Arg Phe Thr Ile 530 535 540Ser Arg Asp Asn Ala Lys Thr
Thr Leu Tyr Leu Gln Met Asn Ser Leu545 550 555 560Arg Pro Glu Asp
Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu 565 570 575Ser Arg
Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ala Ala 580 585
590Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr
595 600 605Lys Asp Asp Asp Asp Lys Gly Ala Ala His His His His His
His 610 615 620487589PRTArtificial SequenceNanobody sequence 487Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30Val Met Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe
Val 35 40 45Ala Thr Ile Ala Trp Asp Ser Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Thr Val His65 70 75 80Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ser Tyr Asn Val Tyr Tyr Asn Asn
Tyr Tyr Tyr Pro Ile Ser 100 105 110Arg Asp Glu Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 115 120 125Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly145 150 155 160Gly
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val 165 170
175Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr
180 185 190Tyr Gly Ser Tyr Trp Met Gly Trp Phe Arg Gln Ala Pro Gly
Lys Glu 195 200 205Arg Glu Gly Val Ala Ala Ile Asn Arg Gly Gly Gly
Tyr Thr Val Tyr 210 215 220Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Thr Ala Lys225 230 235 240Asn Thr Val Tyr Leu Gln Met
Asn Ser Leu Arg Pro Asp Asp Thr Ala 245 250 255Asp Tyr Tyr Cys Ala
Ala Ser Gly Val Leu Gly Gly Leu His Glu Asp 260 265 270Trp Phe Asn
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 275 280 285Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 290 295
300Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly305 310 315 320Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro 325 330 335Gly Gly Ser Leu Arg Leu Ser Cys Val Ala
Ser Gly Asp Val His Lys 340 345 350Ile Asn Phe Leu Gly Trp Tyr Arg
Gln Ala Pro Gly Lys Glu Arg Glu 355 360 365Lys Val Ala His Ile Ser
Ile Gly Asp Gln Thr Asp Tyr Ala Asp Ser 370 375 380Ala Lys Gly Arg
Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Met Val385 390 395 400Tyr
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe 405 410
415Cys Arg Ala Phe Ser Arg Ile Tyr Pro Tyr Asp Tyr Trp Gly Gln Gly
420 425 430Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 435 440 445Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 450 455 460Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Glu Val Gln Leu Val Glu465 470 475 480Ser Gly Gly Gly Leu Val Gln
Pro Gly Asn Ser Leu Arg Leu Ser Cys 485 490 495Ala Ala Ser Gly Phe
Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg 500 505 510Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser 515 520 525Gly
Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 530 535
540Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser
Leu545 550 555 560Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile
Gly Gly Ser Leu 565 570 575Ser Arg Ser Ser Gln Gly Thr Leu Val Thr
Val Ser Ser 580 585
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