U.S. patent application number 17/041459 was filed with the patent office on 2021-01-28 for targeted thrombolysis for treatment of microvascular thrombosis.
This patent application is currently assigned to UMC Utrecht Holding B.V.. The applicant listed for this patent is UMC Utrecht Holding B.V.. Invention is credited to Steven de Maat, Coen Maas.
Application Number | 20210023187 17/041459 |
Document ID | / |
Family ID | 1000005165031 |
Filed Date | 2021-01-28 |
United States Patent
Application |
20210023187 |
Kind Code |
A1 |
Maas; Coen ; et al. |
January 28, 2021 |
Targeted Thrombolysis for Treatment of Microvascular Thrombosis
Abstract
The present invention provides fusion proteins for targeted
delivery of plasminogen activators to platelet-VWF complexes, or
alternatively to the site where these are located, in a
fibrin-independent manner. The fusion protein of the invention are
for use in methods for the prevention or treatment of diseases or
conditions associated with such platelet-VWF complexes, which may
cause microvascular thrombosis in diseases such as e.g. thrombotic
thrombocytopenic purpura. Preferred targeting agents for
incorporation into the fusion proteins are e.g. nanobodies against
VWF or platelets. Preferred plasminogen activators for use in the
fusion proteins comprise the protease domains of uPA or tPA. The
invention further pertains to nucleic acid molecule encoding the
fusion proteins of the invention, e.g. a gene therapy vector, and
to pharmaceutical compositions comprising the fusion proteins of
the invention or such gene therapy vectors.
Inventors: |
Maas; Coen; (Utrecht,
NL) ; de Maat; Steven; (Utrecht, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UMC Utrecht Holding B.V. |
Utrecht |
|
NL |
|
|
Assignee: |
UMC Utrecht Holding B.V.
Utrecht
NL
|
Family ID: |
1000005165031 |
Appl. No.: |
17/041459 |
Filed: |
March 27, 2019 |
PCT Filed: |
March 27, 2019 |
PCT NO: |
PCT/EP2019/057731 |
371 Date: |
September 25, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/49 20130101;
A61P 9/10 20180101; A61K 38/1777 20130101 |
International
Class: |
A61K 38/49 20060101
A61K038/49; A61K 38/17 20060101 A61K038/17; A61P 9/10 20060101
A61P009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 27, 2018 |
EP |
18164232.3 |
Claims
1.-9. (canceled)
10. A method for treating or reducing the risk of microvascular
thrombosis, wherein the method comprises the step of administering
to a subject in need thereof a fusion protein comprising a
plasminogen activator and a targeting agent for targeting the
plasminogen activator to a site of a thrombus comprising al least
one of VWF and platelets, wherein the targeting agent is not a
targeting agent that specifically binds to only the activated form
of the GPIIb/IIIa receptor on platelets, for use in the prevention
or treatment of microvascular thrombosis.
11. The method of claim 10, wherein the microvascular thrombosis is
treated or the risk of its occurrence is reduced in a disease or
condition selected from the group consisting of: acquired or
hereditary thrombotic thrombocytopenic purpura (TTP)
complement-mediated thrombotic microangiopathy, haemolytic uremic
syndrome, antiphospholipid antibody syndrome, non-occlusive
thrombus, the formation of an occlusive thrombus, arterial thrombus
formation, acute coronary occlusion, peripheral arterial occlusive
disease, restenosis and disorders arising from coronary by-pass
graft, coronary artery valve replacement and coronary interventions
such angioplasty, stenting or atherectomy, hyperplasia after
angioplasty, atherectomy or arterial stenting, occlusive syndrome
in a vascular system or lack of patency of diseased arteries,
transient cerebral ischemic attack, unstable or stable angina
pectoris, cerebral infarction, HELLP syndrome, carotid
endarterectomy, carotid artery stenosis, critical limb ischemia,
cardioembolism, peripheral vascular disease, restenosis, sickle
cell disease and myocardial infarct.
12. A fusion protein comprising a plasminogen activator and a
targeting agent for targeting the plasminogen activator to a site
of a thrombus comprising at least one of VWF and platelets, wherein
the targeting agent is one or more of: a) a targeting agent that at
least binds unfolded VWF, wherein preferably the targeting agent
preferentially binds unfolded VWF over globular VWF; b) a targeting
agent that binds the D3 domain of VWF; c) a targeting agent that
binds integrin allb/(3III on platelets; d) a targeting agent that
binds to a receptor that is preferentially expressed by activated
endothelium, wherein preferably the receptor is selected form the
group consisting of E-selectin, P-selectin, uPAR, c1q receptor,
kinin B1 receptor, plasminogen receptor KT (PLGR-KT), endothelial
protein C receptor, thrombomodulin, n-cadherin, ICAM-1 and VCAM-1;
and, e) a targeting agent that binds to a membrane marker for
activated or injured endothelium, wherein the membrane marker is
one or more of anionic phospholipids, phosphatidylserine and
phosphatidylethanolamine.
13. A fusion protein according to claim 12, wherein the fusion
protein comprises more than one targeting agent.
14. A fusion protein according to claim 12, wherein the targeting
agent comprises at least one of: a) an antibody variable domain
that specifically binds to at least one of VWF, platelets, and
activated vascular endothelium; and, b) a binding domain from a
protein that naturally binds VWF, platelets and activated or
injured vascular endothelium, which binding domain specifically
binds to at least one of VWF, platelets, and activated or injured
vascular endothelium.
15. A fusion protein according to claim 14, wherein the antibody
variable domain is a VHH, preferably a humanized VHH, or wherein
the binding domain comprises a binding domain selected from the
group consisting of: i) a VWF-binding domain from one of ADAMTS13,
Factor XII, Factor H (complement regulator), plasminogen and Factor
VIII; and, ii) a membrane binding domain selected from the vitamin
K-dependent carboxylation/gamma-carboxyglutamic (GLA) domain, the
C-domain from factor V and the C-domain from factor VIII.
16. A fusion protein according to claim 12, wherein the plasminogen
activator comprises the protease domain of tissue plasminogen
activator (tPA), urokinase plasminogen activator (uPA),
plasminogen, streptokinase or staphylokinase, wherein preferably
the plasminogen activator further comprises at least the
cysteine-containing part of the connecting peptide that naturally
occurs in the plasminogen activator immediately upstream of its
protease domain, and wherein the fusion protein optionally
comprises a linker amino acid sequence linking the targeting agent
and the plasminogen activator.
17. A fusion protein according to claim 16, wherein the fusion
protein comprises in a N- to C-terminal order: a) one or more
targeting agents wherein the targeting agent is one or more of: a
targeting agent that at least hinds unfolded VWP, wherein
preferably the targeting agent preferentially binds unfolded VWF
over globular VWF; ii) a targeting agent that binds the D3 domain
of VWF; ill) a targeting agent that binds integrin
.alpha.IIb/.beta.III on platelets; iv) a targeting agent that binds
io a receptor that is preferentially expressed by activated
endothelium, wherein preferably the receptor is selected form the
groan consisting of E-selection, P-selection, uPAR, c1q receptor,
kinin B1 receptor, plasminogen receptor KT (PLGR-KT), endothelial
protein C receptor thrombomodulin, n-cadherin, ICAM-1 and VCAM-1;
and, v) a targeting agent that binds to a membrane marker for
activated or injured endothelium, wherein the membrane marker is
one or more of anionic phospholipids, phosphatidylserine and
phosphatldylethanoiamine, whereby, optionally the targeting agents
are linked by linker amino acid sequences; b) optionally a linker
amino acid sequence; and, c) the plasminogen activator or
plasminogen-derived protease domain.
18. A nucleic acid molecule comprising a nucleotide sequence
encoding a fusion protein as defined claim 12, wherein the
nucleotide sequence encoding the fusion protein further preferably
comprises a nucleotide sequence encoding a signal peptide operably
linked to the fusion protein, and wherein nucleic acid molecule
further preferably comprises regulatory elements conducive to the
expression of the fusion protein, which regulatory elements are
operably linked to the nucleotide sequence.
19.-20. (canceled)
21. The method of claim 10, wherein the targeting agent
specifically binds to at least one of VWF, platelets and activated
or injured vascular endothelium.
22. The method of claim 10, wherein the targeting agent is one or
more of; a) a targeting, agent that at least binds unfolded VWF,
wherein preferably the targeting agent preferentially binds
unfolded VWF over globular VWF. b) a targeting agent that binds the
D3 domain of VWF; c) a targeting agent that binds the GP1B receptor
on platelets, d) a targeting agent that binds integrin
.alpha.IIb/.beta.III on platelets; e) a targeting agent that binds
to a receptor that is preferentially expressed by activated
endothelium, wherein preferably the receptor is selected from the
group consisting of E-setecan, P-selectin, uPAR, c1q receptor,
thrombomodulin, n-cadherin, ICAM-1 and VCAM-1; and, f) a targeting
agent that binds to a membrane marker for activated or injured
endothelium, wherein the membrane marker is one or more of anionic
phospholipids, phosphatidylserine and phosphatidylethanolamine.
23. The method of claim 10, wherein the fusion protein comprises
more than one targeting agent.
24. The method of claim 10, wherein the targeting agent comprises
at least one of: a) ao antibody variable domain that specifically
binds to at least one of VWF, platelets, and activated vascular
endothelium; and, b) a binding domain from a protein that naturally
binds VWF, platelets and activated or injured vascular endothelium,
which binding domain specifically binds to at least one of VWF,
platelets, and activated or injured vascular endothelium.
25. The method of claim 24, wherein the antibody variable domain is
a VHH, preferably a humanized VHH, or wherein the binding domain
comprises a binding domain selected front the group consisting of:
i) the platelet GP1B receptor-binding A1 domain from VWF; ii) a
VWF-binding domain from one of ADAMTS13, Factor XII, Factor H
(complement regulator), plasminogen and Factor VIII, and, iii) a
membrane binding domain selected from the vitamin K-dependent
carboxylation/gamma-carboxyglutamic (GLA) domain, the C-domain from
factor V and the C-domain from factor VIII.
26. The method of claim 10, wherein the plasminogen activator
comprises the protease domain of tissue plasminogen activator ((PAT
urokinase plasminogen activator (nPA), plasminogen, streptokinase
or staphylokinase, wherein preferably the plasminogen activator
further comprises at least the cysteine-containing part of the
connecting peptide that naturally occurs in the plasminogen
activator immediately upstream of its protease domain, and wherein
the fusion protein optionally comprises a linker amino acid
sequence linking the targeting agent and the plasminogen
activator
27. The method of claim 26. wherein the fusion protein comprises in
a N- to C-terminal order; a) the one or more targeting agents,
whereby, optionally the targeting agents axe linked by linker amino
acid sequences; b) optionally a linker amino acid sequence; and, c)
the plasminogen activator or plasminogen-derived protease domain.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of medicine and
pharmacy, in particular to the field of biopharmaceuticals for use
in the prevention or treatment of a disease or condition associated
with microvascular thrombosis, such as e.g. thrombotic
thrombocytopenic purpura. More specifically, the invention relates
to fusion proteins comprising a targeting agent and a plasminogen
activator, wherein the targeting agent targets the plasminogen
activator to at least one of VWF, platelets and activated vascular
endothelium with the aim to enzymatically degrade vascular
obstructions in a fibrin-independent manner. The invention further
relates to gene therapy vectors encoding such fusion proteins.
BACKGROUND ART
[0002] Microvascular thrombosis (MVT) is characterized by the
formation of microvascular platelet aggregates. They are minimally
composed of platelets and VWF. This becomes clear in thrombotic
thrombocytopenic purpura (TTP), where platelet- and VWF-rich, but
fibrin-poor microthrombi obstruct microvasculature with
life-threatening consequences. Thus, fibrin, which is seen in
macrovascular thrombosis, is not per se required for these
microvascular obstructions. MVT is a shared feature between several
disease states including thrombotic thrombocytopenic purpura,
hemolytic uremic syndrome, antiphospholipid antibody syndrome and
complement-mediated thrombotic microangiopathy (George et al.,
2014, N Engl J Med. 371(7):654-66). In severe cases of MVT,
multi-organ failure can occur with lethal consequences. Even in
less severe cases, organ damage may form and reducing both the
quality of life, as well as the life expectancy of the patient.
Recent studies suggest that microvascular disease underlies
cardiovascular disease/events in patients in the more generalized
population with cardiovascular disease, and that do not show overt
signs of macrovascular obstruction on radiological examination.
This is ultimately thought to cause heart failure, in particular in
females.
[0003] Patients with TTP experience attacks of microvascular
thrombosis, when platelets form complexes with ultra-large
multimers of von Willebrand Factor (VWF). This is the result of
severely decreased activity of the enzyme ADAMTS13 (a disintegrin
and metalloproteinase with a thrombospondin type I motif, member
13). ADAMTS13 normally regulates the thrombogenicity of VWF by
enzymatically reducing its multimer size. Hereto, VWF needs to
unfold from its globular form into an unrolled conformation,
thereby exposing its A2 domain for proteolysis. The majority of TTP
patients suffer from neutralizing autoantibodies against ADAMTS13.
For a small subgroup, mutations in ADAMTS13 have been described
that lead to deficiency (Upshaw-Shulman syndrome).
[0004] Current TTP therapy involves extensive plasma exchange to
deplete inhibitory antibodies and restore ADAMTS13 activity at the
same time. However, persistent autoantibodies impede elimination of
microthrombi. This makes therapy time-consuming and very costly
(Fijnheer et al., Ned Tijdsch Hematol 2016; 13 (1): 18-24).
[0005] Besides ADAMTS13, VWF can be cleaved by the enzyme plasmin
(Berkowitz et al., J Clin Invest 1987 Feb; 79(2):524-31). We
previously identified that systemic plasminogen activation (with
streptokinase) was therapeutic in a mouse model for TTP, suggesting
that plasmin can act as a functional alternative to ADAMTS13
(Tersteeg et al., 2014, Circulation, 129(12):1320-31). Although
plasmin(ogen) can directly bind to unrolled VWF, natural
plasminogen activators--such as tissue plasminogen activator (tPA)
and urokinase plasminogen activator (uPA)--cannot. The natural
targets of tPA and uPA are fibrin and the endothelial cell receptor
uPAR, respectively. Furthermore, microthrombi in TTP are
fibrin-poor and it is uncertain whether fibrin is a prerequisite
for other types of MVT. This renders these molecules (i.e. tPA,
uPA) that are generally used as thrombolytic agents in the
treatment of macrovascular disease ineffective in treating MVT.
Moreover, for safety reasons (i.e. low platelet counts) it is
desirable to avoid systemic plasminogen activation.
[0006] It is an object of the present invention to provide for
means and methods for treating MVT and associated conditions. The
invention therefore provides fusion proteins for targeted delivery
of plasminogen activators to platelet-VWF complexes, or
alternatively to the site where these are located, in a
fibrin-independent manner. The invention further provides
therapeutic methods for conditions that can be prevented or treated
by local delivery/stimulation of plasminogen activation to sites of
microvascular occlusion.
SUMMARY OF THE INVENTION
[0007] In a first aspect, the invention relates to a fusion protein
comprising a plasminogen activator and a targeting agent for
targeting the plasminogen activator to a site of a thrombus
comprising at least one of VWF and platelets. Preferably, the
targeting agent in the fusion protein of the invention specifically
binds to at least one of VWF, platelets, and activated or injured
vascular endothelium. It is further preferred that the targeting
agent in the fusion protein of the invention is not a targeting
agent that specifically binds to only the activated form of the
GPIlb/Illa receptor on platelets. More preferably, the targeting
agent in the fusion protein of the invention is one or more of: a)
a targeting agent that at least binds unfolded VWF, wherein
preferably the targeting agent preferentially binds unfolded VWF
over globular VWF; b) a targeting agent that binds the D3 domain of
VWF; c) a targeting agent that binds the GP1B receptor on
platelets; d) a targeting agent that binds integrin
.alpha.IIb/.beta.III on platelets; e) a targeting agent that binds
to a receptor that is preferentially expressed by activated
endothelium, wherein preferably the receptor is selected form the
group consisting of E-selectin, P-selectin, uPAR, c1q receptor,
kinin B1 receptor, plasminogen receptor KT (PLGR-KT), endothelial
protein C receptor, thrombomodulin, n-cadherin, ICAM-1 and VCAM-1;
and, f) a targeting agent that binds to a membrane marker for
activated or injured endothelium, wherein the membrane marker is
one or more of anionic phospholipids, phosphatidylserine and
phosphatidylethanolamine. In one embodiment, Preferably, the fusion
protein of the invention comprises more than one targeting
agent.
[0008] A fusion protein according to the invention preferably is a
fusion protein wherein the targeting agent comprises at least one
of: a) an antibody variable domain that specifically binds to at
least one of VWF, platelets, and activated vascular endothelium;
and, b) a binding domain from a protein that naturally binds VWF,
platelets and activated or injured vascular endothelium, which
binding domain specifically binds to at least one of VWF,
platelets, and activated or injured vascular endothelium. Thus, in
a fusion protein according to the invention, the antibody variable
domain preferably is a VHH, more preferably a humanized VHH.
Alternatively, in a fusion protein according to the invention,
binding domain from a protein that naturally binds VWF, platelets
and activated or injured vascular endothelium preferably comprises
a binding domain selected from the group consisting of: i) the
platelet GP1B receptor-binding Al domain from VWF; ii) a
VWF-binding domain from one of ADAMTS13, Factor XII, Factor H
(complement regulator), plasminogen and Factor VIII; and, iii) a
membrane binding domain selected from the vitamin K-dependent
carboxylation/gamma-carboxyglutamic (GLA) domain, the C-domain from
factor V and the C-domain from factor VIII.
[0009] In a fusion protein of the invention as defined above, the
plasminogen activator preferably comprises the protease domain of
tissue plasminogen activator (tPA), urokinase plasminogen activator
(uPA), plasminogen, streptokinase or staphylokinase. It is
preferred in a fusion protein of the invention that the plasminogen
activator further comprises at least the cysteine-containing part
of the connecting peptide that naturally occurs in the plasminogen
activator immediately upstream of its protease domain. Optionally,
a fusion protein of the invention comprises a linker amino acid
sequence linking the targeting agent and the plasminogen
activator.
[0010] Thus, a fusion protein in accordance with the invention
preferably comprises in a N- to C-terminal order: a) one or more
targeting agents as defined above, whereby, optionally the
targeting agents are linked by linker amino acid sequences; b)
optionally a linker amino acid sequence; and, c) a plasminogen
activator or plasminogen-derived protease domain as defined
above.
[0011] In a second aspect, the invention relates to a nucleic acid
molecule comprising a nucleotide sequence encoding a fusion protein
in accordance with the invention as defined above. Preferably, the
nucleotide sequence encoding the fusion protein further comprises a
nucleotide sequence encoding a signal peptide operably linked to
the fusion protein. The nucleic acid molecule further preferably
comprises regulatory elements conducive to the expression of the
fusion protein, which regulatory elements are operably linked to
the nucleotide sequence.
[0012] In a third aspect, the invention pertains to a gene therapy
vector comprising a nucleic acid molecule according to the
invention.
[0013] In a fourth aspect, the invention relates to a
pharmaceutical composition comprising a fusion protein according to
the invention or a gene therapy vector according to the invention
and a pharmaceutically acceptable excipient.
[0014] In a fifth aspect, the invention pertains a fusion protein
according to the invention, a gene therapy vector according to the
invention or a pharmaceutical composition according to the
invention, for use in the prevention or treatment of a disease or
condition associated with thrombi comprising at least one of VWF
and platelets, wherein preferably with the disease or condition
associated with microvascular thrombosis. More preferably, the
disease or condition associated with (micro)thrombi comprising at
least one of VWF and platelets is selected from the group
consisting of: acquired or hereditary thrombotic thrombocytopenic
purpura (TTP) complement-mediated thrombotic microangiopathy,
haemolytic uremic syndrome, antiphospholipid antibody syndrome,
non-occlusive thrombus, the formation of an occlusive thrombus,
arterial thrombus formation, acute coronary occlusion, peripheral
arterial occlusive disease, restenosis and disorders arising from
coronary by-pass graft, coronary artery valve replacement and
coronary interventions such angioplasty, stenting or atherectomy,
hyperplasia after angioplasty, atherectomy or arterial stenting,
occlusive syndrome in a vascular system or lack of patency of
diseased arteries, transient cerebral ischemic attack, unstable or
stable angina pectoris, cerebral infarction, HELLP syndrome,
carotid endarterectomy, carotid artery stenosis, critical limb
ischemia, cardioembolism, peripheral vascular disease, restenosis,
sickle cell disease and myocardial infarct.
[0015] In a sixth aspect, the invention pertains to a method for
treating or reducing the risk of a disease or condition associated
with thrombi comprising at least one of VWF and platelets, wherein
the method comprises the step of administering to a subject in need
thereof, an effective amount of fusion protein according to the
invention, a gene therapy vector according to the invention or a
pharmaceutical composition according to the invention, and wherein
preferably with the disease or condition associated with
microvascular thrombosis. More preferably, the disease or condition
associated with (micro)thrombi comprising at least one of VWF and
platelets is selected from the group consisting of: acquired or
hereditary thrombotic thrombocytopenic purpura (TTP)
complement-mediated thrombotic microangiopathy, haemolytic uremic
syndrome, antiphospholipid antibody syndrome, non-occlusive
thrombus, the formation of an occlusive thrombus, arterial thrombus
formation, acute coronary occlusion, peripheral arterial occlusive
disease, restenosis and disorders arising from coronary by-pass
graft, coronary artery valve replacement and coronary interventions
such angioplasty, stenting or atherectomy, hyperplasia after
angioplasty, atherectomy or arterial stenting, occlusive syndrome
in a vascular system or lack of patency of diseased arteries,
transient cerebral ischemic attack, unstable or stable angina
pectoris, cerebral infarction, HELLP syndrome, carotid
endarterectomy, carotid artery stenosis, critical limb ischemia,
cardioembolism, peripheral vascular disease, restenosis and
myocardial infarct.
DESCRIPTION OF THE INVENTION
Definitions
[0016] The terms "homology", "sequence identity" and the like are
used interchangeably herein. Sequence identity is herein defined as
a relationship between two or more amino acid (polypeptide or
protein) sequences or two or more nucleic acid (polynucleotide)
sequences, as determined by comparing the sequences. In the art,
"identity" also means the degree of sequence relatedness between
amino acid or nucleic acid sequences, as the case may be, as
determined by the match between strings of such sequences.
"Similarity" between two amino acid sequences is determined by
comparing the amino acid sequence and its conserved amino acid
substitutes of one polypeptide to the sequence of a second
polypeptide. "Identity" and "similarity" can be readily calculated
by known methods.
[0017] "Sequence identity" and "sequence similarity" can be
determined by alignment of two peptide or two nucleotide sequences
using global or local alignment algorithms, depending on the length
of the two sequences. Sequences of similar lengths are preferably
aligned using a global alignment algorithms (e.g. Needleman Wunsch)
which aligns the sequences optimally over the entire length, while
sequences of substantially different lengths are preferably aligned
using a local alignment algorithm (e.g. Smith Waterman). Sequences
may then be referred to as "substantially identical" or
"essentially similar" when they (when optimally aligned by for
example the programs GAP or BESTFIT using default parameters) share
at least a certain minimal percentage of sequence identity (as
defined below). GAP uses the Needleman and Wunsch global alignment
algorithm to align two sequences over their entire length (full
length), maximizing the number of matches and minimizing the number
of gaps. A global alignment is suitably used to determine sequence
identity when the two sequences have similar lengths. Generally,
the GAP default parameters are used, with a gap creation penalty=50
(nucleotides)/8 (proteins) and gap extension penalty=3
(nucleotides)/2 (proteins). For nucleotides the default scoring
matrix used is nwsgapdna and for proteins the default scoring
matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89,
915-919). Sequence alignments and scores for percentage sequence
identity may be determined using computer programs, such as the GCG
Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif. 92121-3752 USA, or using open
source software, such as the program "needle" (using the global
Needleman Wunsch algorithm) or "water" (using the local Smith
Waterman algorithm) in EmbossWlN version 2.10.0, using the same
parameters as for GAP above, or using the default settings (both
for `needle` and for `water` and both for protein and for DNA
alignments, the default Gap opening penalty is 10.0 and the default
gap extension penalty is 0.5; default scoring matrices are Blossum
62 for proteins and DNA Full for DNA). When sequences have a
substantially different overall lengths, local alignments, such as
those using the Smith Waterman algorithm, are preferred.
[0018] Alternatively percentage similarity or identity may be
determined by searching against public databases, using algorithms
such as FASTA, BLAST, etc. Thus, the nucleic acid and protein
sequences of the present invention can further be used as a "query
sequence" to perform a search against public databases to, for
example, identify other family members or related sequences. Such
searches can be performed using the BLASTn and BLASTx programs
(version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to oxidoreductase nucleic acid molecules of the invention. BLAST
protein searches can be performed with the BLASTx program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., BLASTx and BLASTn) can be used.
See the homepage of the National Center for Biotechnology
Information at http://www.ncbi.nlm.nih.gov/.
[0019] Optionally, in determining the degree of amino acid
similarity, the skilled person may also take into account so-called
"conservative" amino acid substitutions, as will be clear to the
skilled person. Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. Examples
of classes of amino acid residues for conservative substitutions
are given in the Tables below.
TABLE-US-00001 Acidic Residues Asp (D) and Glu (E) Basic Residues
Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser
(S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged Residues Gly
(G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Uncharged
Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F),
Tyr (Y), and Trp (W)
[0020] Alternative conservative amino acid residue substitution
classes.
TABLE-US-00002 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W
[0021] Alternative Physical and Functional Classifications of Amino
Acid Residues.
TABLE-US-00003 Alcohol group-containing residues S and T Aliphatic
residues I, L, V, and M Cycloalkenyl-associated residues F, H, W,
and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and
Y Negatively charged residues D and E Polar residues C, D, E, H, K,
N, Q, R, S, and T Positively charged residues H, K, and R Small
residues A, C, D, G, N, P, S, T, and V Very small residues A, G,
and S Residues involved in turn formation A, C, D, E, G, H, K, N,
Q, R, S, P and T Flexible residues Q, T, K, S, G, P, D, E, and
R
[0022] Nucleotide sequences encoding fusion proteins of the
invention may also be defined by their capability to hybridize with
the nucleotide sequences of encoding fusion proteins as exemplified
herein, under moderate, or preferably under stringent hybridization
conditions. Stringent hybridization conditions are herein defined
as conditions that allow a nucleic acid sequence of at least about
25, preferably about 50 nucleotides, 75 or 100 and most preferably
of about 200 or more nucleotides, to hybridize at a temperature of
about 65.degree. C. in a solution comprising about 1 M salt,
preferably 6.times.SSC or any other solution having a comparable
ionic strength, and washing at 65.degree. C. in a solution
comprising about 0.1 M salt, or less, preferably 0.2.times.SSC or
any other solution having a comparable ionic strength. Preferably,
the hybridization is performed overnight, i.e. at least for 10
hours and preferably washing is performed for at least one hour
with at least two changes of the washing solution. These conditions
will usually allow the specific hybridization of sequences having
about 90% or more sequence identity.
[0023] Moderate conditions are herein defined as conditions that
allow a nucleic acid sequences of at least 50 nucleotides,
preferably of about 200 or more nucleotides, to hybridize at a
temperature of about 45.degree. C. in a solution comprising about 1
M salt, preferably 6.times.SSC or any other solution having a
comparable ionic strength, and washing at room temperature in a
solution comprising about 1 M salt, preferably 6.times.SSC or any
other solution having a comparable ionic strength. Preferably, the
hybridization is performed overnight, i.e. at least for 10 hours,
and preferably washing is performed for at least one hour with at
least two changes of the washing solution. These conditions will
usually allow the specific hybridization of sequences having up to
50% sequence identity. The person skilled in the art will be able
to modify these hybridization conditions in order to specifically
identify sequences varying in identity between 50% and 90%.
[0024] A "nucleic acid construct" or "nucleic acid vector" is
herein understood to mean a man-made nucleic acid molecule
resulting from the use of recombinant DNA technology. The term
"nucleic acid construct" therefore does not include naturally
occurring nucleic acid molecules although a nucleic acid construct
may comprise (parts of) naturally occurring nucleic acid molecules.
The terms "expression vector" or expression construct" refer to
nucleic acid molecules that are capable of effecting expression of
a nucleotide sequence or gene in host cells or host organisms
compatible with such expression vectors or constructs. These
expression vectors typically include regulatory sequence elements
that are operably linked to the nucleotide sequence to be expressed
to effect its expression. Such regulatory elements usually at least
include suitable transcription regulatory sequences and optionally,
3' transcription termination signals. Additional elements necessary
or helpful in effecting expression may also be present, such as
expression enhancer elements. The expression vector will be
introduced into a suitable host cell and be able to effect
expression of the coding sequence in an in vitro cell culture of
the host cell. The expression vector will be suitable for
replication in the host cell or organism of the invention whereas
an expression construct will usually integrate in the host cell's
genome for it to be maintained. Techniques for the introduction of
nucleic acid into cells are well established in the art and any
suitable technique may be employed, in accordance with the
particular circumstances. For eukaryotic cells, suitable techniques
may include calcium phosphate transfection, DEAE-Dextran,
electroporation, liposome-mediated transfection and transduction
using retrovirus or other virus, e.g. adenovirus, AAV, lentivirus
or vaccinia. For microbial, e.g. bacterial, cells, suitable
techniques may include calcium chloride transformation,
electroporation and transfection using bacteriophage. The
introduced nucleic acid may be on an extra-chromosomal vector
within the cell or the nucleic acid may be integrated into the
genome of the host cell. Integration may be promoted by inclusion
of sequences within the nucleic acid or vector which promote
recombination with the genome, in accordance with standard
techniques. The introduction may be followed by expression of the
nucleic acid to produce the encoded fusion protein. In some
embodiments, host cells (which may include cells actually
transformed although more likely the cells will be descendants of
the transformed cells) may be cultured in vitro under conditions
for expression of the nucleic acid, so that the encoded fusion
protein polypeptide is produced, when an inducible promoter is
used, expression may require the activation of the inducible
promoter.
[0025] As used herein, the term "promoter" or "transcription
regulatory sequence" refers to a nucleic acid fragment that
functions to control the transcription of one or more coding
sequences, and is located upstream with respect to the direction of
transcription of the transcription initiation site of the coding
sequence, and is structurally identified by the presence of a
binding site for DNA-dependent RNA polymerase, transcription
initiation sites and any other DNA sequences, including, but not
limited to transcription factor binding sites, repressor and
activator protein binding sites, and any other sequences of
nucleotides known to one of skill in the art to act directly or
indirectly to regulate the amount of transcription from the
promoter. A "constitutive" promoter is a promoter that is active in
most tissues under most physiological and developmental conditions.
An "inducible" promoter is a promoter that is physiologically or
developmentally regulated, e.g. by the application of a chemical
inducer.
[0026] The term "selectable marker" is a term familiar to one of
ordinary skill in the art and is used herein to describe any
genetic entity which, when expressed, can be used to select for a
cell or cells containing the selectable marker. The term "reporter"
may be used interchangeably with marker, although it is mainly used
to refer to visible markers, such as green fluorescent protein
(GFP). Selectable markers may be dominant or recessive or
bidirectional.
[0027] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements in a functional relationship. A
nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a transcription regulatory sequence is operably linked to
a coding sequence if it affects the transcription of the coding
sequence. Operably linked means that the DNA sequences being linked
are typically contiguous and, where necessary to join two protein
encoding regions, contiguous and in reading frame.
[0028] The terms "protein" or "polypeptide" are used
interchangeably and refer to molecules consisting of a chain of
amino acids, without reference to a specific mode of action, size,
3-dimensional structure or origin.
[0029] The term "signal peptide" (sometimes referred to as signal
sequence) is a short peptide (usually 16-30 amino acids long)
present at the N-terminus of the majority of newly synthesized
proteins that are destined towards the secretory pathway. At the
end of the signal peptide there is usually a stretch of amino acids
that is recognized and cleaved by signal peptidase either during or
after completion of translocation (from the cytosol into the
secretory pathway, i.e. ER) to generate a free signal peptide and a
mature protein. Signal peptides are extremely heterogeneous and
many prokaryotic and eukaryotic signal peptides are functionally
interchangeable even between different species however the
efficiency of protein secretion may depend on the signal peptide.
Suitable signal peptides are generally known in the art e.g. from
KaII et al. (2004 J. Mol. Biol. 338: 1027-1036) and von Heijne
(1985, J Mol Biol. 184 (1): 99-105).
[0030] The term "gene" means a DNA fragment comprising a region
(transcribed region), which is transcribed into an RNA molecule
(e.g. an mRNA) in a cell, operably linked to suitable regulatory
regions (e.g. a promoter). A gene will usually comprise several
operably linked fragments, such as a promoter, a 5' leader
sequence, a coding region and a 3' non-translated sequence (3' end)
comprising a polyadenylation site. "Expression of a gene" refers to
the process wherein a DNA region which is operably linked to
appropriate regulatory regions, particularly a promoter, is
transcribed into an RNA, which is biologically active, i.e. which
is capable of being translated into a biologically active protein
or peptide.
[0031] The term "homologous" when used to indicate the relation
between a given (recombinant) nucleic acid or polypeptide molecule
and a given host organism or host cell, is understood to mean that
in nature the nucleic acid or polypeptide molecule is produced by a
host cell or organisms of the same species, preferably of the same
variety or strain. If homologous to a host cell, a nucleic acid
sequence encoding a polypeptide will typically (but not
necessarily) be operably linked to another (heterologous) promoter
sequence and, if applicable, another (heterologous) secretory
signal sequence and/or terminator sequence than in its natural
environment. It is understood that the regulatory sequences, signal
sequences, terminator sequences, etc. may also be homologous to the
host cell. When used to indicate the relatedness of two nucleic
acid sequences the term "homologous" means that one single-stranded
nucleic acid sequence may hybridize to a complementary
single-stranded nucleic acid sequence. The degree of hybridization
may depend on a number of factors including the amount of identity
between the sequences and the hybridization conditions such as
temperature and salt concentration as discussed later.
[0032] The term "heterologous" when used with respect to a nucleic
acid (DNA or RNA) or protein refers to a nucleic acid or protein
that does not occur naturally as part of the organism, cell, genome
or DNA or RNA sequence in which it is present, or that is found in
a cell or location or locations in the genome or DNA or RNA
sequence that differ from that in which it is found in nature.
Heterologous nucleic acids or proteins are not endogenous to the
cell into which it is introduced, but has been obtained from
another cell or synthetically or recombinantly produced. Generally,
though not necessarily, such nucleic acids encode proteins that are
not normally produced by the cell in which the DNA is transcribed
or expressed. Similarly exogenous RNA encodes for proteins not
normally expressed in the cell in which the exogenous RNA is
present. Heterologous nucleic acids and proteins may also be
referred to as foreign nucleic acids or proteins. Any nucleic acid
or protein that one of skill in the art would recognize as
heterologous or foreign to the cell in which it is expressed is
herein encompassed by the term heterologous nucleic acid or
protein. The term heterologous also applies to non-natural
combinations of nucleic acid or amino acid sequences, i.e.
combinations where at least two of the combined sequences are
foreign with respect to each other.
[0033] Unless indicated otherwise, the terms "immunoglobulin" and
"antibody" whether it used herein to refer to a heavy chain
antibody or to a conventional 4-chain antibody is used as a general
term to include both the full-size antibody, the individual chains
thereof, as well as all parts, domains or fragments thereof
(including but not limited to antigen-binding domains or fragments
such as VHH domains or V.sub.H/V.sub.L domains, respectively). In
addition, the term "sequence" as used herein (for example in terms
like "immunoglobulin sequence", "antibody sequence", "variable
domain sequence", "VHH sequence" or "protein sequence"), should
generally be understood to include both the relevant amino acid
sequence as well as nucleic acid sequences or nucleotide sequences
encoding the same, unless the context requires a more specific
interpretation.
[0034] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domain of the heavy chain may be
referred to as "VH", or to "VHH" in case of a heavy chain antibody
such as the camelid antibodies that consist of only heavy chains.
The variable domain of the light chain may be referred to as "VL."
These domains are generally the most variable parts of an antibody
and contain the antigen-binding sites. The term "variable" refers
to the fact that certain segments of the variable domains differ
extensively in sequence among antibodies. The V domain mediates
antigen binding and defines specificity of a particular antibody
for its particular antigen. However, the variability is not evenly
distributed across the average 110-amino acid span of the variable
domains. Instead, the V regions consist of relatively invariant
stretches called framework regions (FRs) of about 15-30 amino acids
separated by shorter regions of extreme variability called
"hypervariable regions" (HVRs) or complementarity determining
regions (CDRs) that are each about 9-12 amino acids long. The
variable domains of native heavy and light chains each comprise
four FRs, largely adopting a .beta.-sheet configuration, connected
by three hypervariable regions, which form loops connecting, and in
some cases forming part of, the .beta.-sheet structure. The
hypervariable regions in each chain are held together in close
proximity by the FRs and, with the hypervariable regions from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, MD. (1991)).
[0035] The terms "VHH", "VHH domain" and "nanobody" are
interchangeable herein and are used herein to refer to the variable
domain of a heavy chain antibody, i.e. an antibody consisting only
of heavy chains and devoid of light chains as are known e.g. from
Camelids. The amino acid sequence and structure of a VHH 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 below 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
"CDRs", 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. The total number of amino acid residues in
a VHH can be in the region of 110-120, is preferably 112-115, and
is most preferably 113. It should however be noted that parts,
fragments or analogs (as further described herein below) of a VHH
are not particularly limited as to their length and/or size, as
long as such parts, fragments or analogs meet the further
functional requirements outlined herein below and are also
preferably suitable for the purposes described herein.
[0036] The amino acid residues of a VHH (or conventional 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 VHH domains from Camelids
by Riechmann and Muyldermans (1999, J. Immunol. Methods; 231:
25-38; see for example FIG. 2 of said reference). According to this
numbering, FR1 of a VHH comprises the amino acid residues at
positions 1-30, CDR1 of a VHH comprises the amino acid residues at
positions 31-36, FR2 of a VHH comprises the amino acids at
positions 36-49, CDR2 of a VHH comprises the amino acid residues at
positions 50-65, FR3 of a VHH comprises the amino acid residues at
positions 66-94, CDR3 of a VHH comprises the amino acid residues at
positions 95-102, and FR4 of a VHH comprises the amino acid
residues at positions 103-113. In this respect, it should be noted
that as is well known in the art for VH 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. Generally, however, it can be said
that, according to the numbering of Kabat and irrespective of the
number of amino acid residues in the CDRs, position 1 according to
the Kabat numbering corresponds to the start of FR1 and visa versa,
position 36 according to the Kabat numbering corresponds to the
start of FR2 and visa versa, position 66 according to the Kabat
numbering corresponds to the start of FR3 and visa versa, and
position 103 according to the Kabat numbering corresponds to the
start of FR4.
[0037] Alternative methods for numbering the amino acid residues of
VH domains, which methods can also be applied in an analogous
manner to VHH domains from Camelids, are the method described by
Chothia et al.(1989, Nature 342, 877-883), the so-called "AbM
definition" and the so-called "contact definition". However, in the
present description, claims and figures, the numbering according to
Kabat as applied to VHH domains by Riechmann and Muyldermans will
be followed, unless indicated otherwise.
[0038] For a general description of heavy chain antibodies and the
variable VHH domains thereof, reference is inter alia made to the
following references, which are mentioned as general background
art: WO 94/04678, WO 95/04079, WO 96/34103, WO 94/25591, WO
99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO
01/44301, EP 1134231, WO 02/48193, WO 97/49805, WO 01/21817, WO
03/035694, WO 03/054016, WO 03/055527 WO 03/050531, WO 01/90190, WO
03/025020; WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863
and WO 04/062551 and Hassanzadeh-Ghassabeh et al. (2013,
Nanomedicine, 8(6)1 013-1026). For a more specific description of
single domain VHH antibodies against Von Willebrand Factor or
platelet receptor GP1b, reference is made to WO 2004/062551 and WO
2006/122825.
[0039] Generally, it should be noted that the term "VHH" (or
nanobody) as used herein in its broadest sense is not limited to a
specific biological source or to a specific method of preparation.
For example, VHHs as used in the invention can be obtained (1) by
isolating the VHH domain of a naturally occurring heavy chain
antibody; (2) by expression of a nucleotide sequence encoding a
naturally occurring VHH domain; (3) by "humanization" (as described
below) of a naturally occurring VHH domain or by expression of a
nucleic acid encoding a such humanized VHH domain; (4) by
"camelization" of a naturally occurring VH domain from any animal
species, in particular a species of mammal, such as from a human
being, or by expression of a nucleic acid encoding such a camelized
VH domain; (5) using synthetic or semi-synthetic techniques for
preparing proteins, polypeptides or other amino acid sequences; (6)
by preparing a nucleic acid encoding a VHH using techniques for
nucleic acid synthesis, followed by expression of the nucleic acid
thus obtained; and/or (7) by any combination of the foregoing.
Suitable methods and techniques for performing the foregoing are
state of the art and therefore known to the skilled person.
[0040] One particularly preferred class of VHHs for use in the
invention comprises VHHs with an amino acid sequence that
corresponds to the amino acid sequence of a naturally occurring VHH
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 VHH sequence 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.
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 prior art on
humanization including e.g. Jones et al. (Nature 321:522-525,
1986); Riechmann et al., (Nature 332:323-329, 1988); Presta (Curr.
Op. Struct. Biol. 2:593-596, 1992), Vaswani and Hamilton (Ann.
Allergy, Asthma and Immunol., 1:105-115 1998); Harris (Biochem.
Soc. Transactions, 23:1035-1038, 1995); Hurle and Gross (Curr. Op.
Biotech., 5:428-433, 1994), and specific prior art relating to
humanization of VHHs such as e.g. Vincke et al. (2009, J. Biol.
Chem. 284:3273-3284). Again, it should be noted that such humanized
VHHs 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 VHH domain as a starting material.
[0041] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
binds. Preferred blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen. An "agonist antibody", as used herein, is an antibody
which mimics at least one of the functional activities of a
polypeptide of interest.
[0042] "Binding affinity" generally refers to the strength of the
sum total of non-covalent interactions between a single binding
site of a molecule (e.g., an antibody) and its binding partner
(e.g., an antigen or target). Unless indicated otherwise, as used
herein, "binding affinity" refers to intrinsic binding affinity
which reflects a 1:1 interaction between members of a binding pair
(e.g., antibody and antigen/target). The affinity of a molecule X
for its partner Y can generally be represented by the dissociation
constant (K.sub.d). Affinity can be measured by common methods
known in the art, including those described herein. Low-affinity
antibodies generally bind antigen/target slowly and tend to
dissociate readily, whereas high-affinity antibodies generally bind
antigen faster and tend to remain bound longer. A variety of
methods of measuring binding affinity are known in the art, any of
which can be used for purposes of the present invention. Specific
illustrative embodiments are described in the following.
[0043] A "K.sub.d" or "K.sub.d value" can be measured by using an
ELISA as described in the Examples herein or by using surface
plasmon resonance assays using a BlAcore.TM.-2000 or a
BlAcore.TM.-3000 (BlAcore, Inc., Piscataway, N.J.) at 25.degree. C.
with immobilized antigen CM5 chips at .about.10-50 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BlAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate
of 5 .mu./minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of the antibody or Fab
(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST)
at 25.degree. C. at a flow rate of approximately 25 .mu./min.
Association rates (kon) and dissociation rates (k.sub.off) are
calculated using a simple one-to-one Langmuir binding model
(BlAcore Evaluation Software version 3.2) by simultaneous fitting
the association and dissociation sensorgram. The equilibrium
dissociation constant (Kd) is calculated as the ratio
k.sub.off/k.sub.on. See, e.g., Chen, Y., et al., (1999) J. Mol Biol
293:865-881. If the on-rate exceeds 10.sup.6 M.sup.-1 S.sup.-1 by
the surface plasmon resonance assay above, then the on-rate can be
determined by using a fluorescent quenching technique that measures
the increase or decrease in fluorescence emission intensity
(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25.degree.
C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in
the presence of increasing concentrations of antigen as measured in
a spectrometer, such as a stop-flow equipped spectrophometer (Aviv
Instruments) or a 8000-series SLM-Aminco spectrophotometer
(ThermoSpectronic) with a stir red cuvette.
[0044] An "on-rate" or "rate of association" or "association rate"
or "k.sub.on" according to this invention can also be determined
with the same surface plasmon resonance technique described above
using a BlAcore.TM.-2000 or a BlAcore.TM.-3000 (BlAcore, Inc.,
Piscataway, N.J.) as described above.
[0045] The phrases "pharmaceutical or pharmacologically acceptable"
refers to molecular entities and compositions that do not produce
or produce acceptable adverse, allergic or other untoward reaction
when administered to an animal, such as, for example, a human, as
appropriate. Whether certain adverse effects are acceptable is
determined based on the severity of the disease. The preparation of
a pharmaceutical composition that contains at least one chimeric
polypeptide or additional active ingredient will be known to those
of skill in the art in light of the present disclosure, as
exemplified by Remington: The Science and Practice of Pharmacy"
(Ed. Allen, L. V. 22nd edition, 2012, www.pharmpress.com),
incorporated herein by reference. Moreover, for animal (e.g.,
human) administration, it will be understood that preparations
should meet sterility, pyrogenicity, general safety and purity
standards as required by FDA Office of Biological Standards.
[0046] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington: The Science and Practice of
Pharmacy" (Ed. Allen, L. V. 22nd edition, 2012,
www.pharmpress.com), incorporated herein by reference). Except
insofar as any conventional carrier is incompatible with the active
ingredient, its use in the therapeutic or pharmaceutical
compositions is contemplated.
[0047] Any reference to nucleotide or amino acid sequences
accessible in public sequence databases herein refers to the
version of the sequence entry as available on the filing date of
this document.
DESCRIPTION OF EMBODIMENTS
[0048] The present inventors have surprisingly found that targeting
of plasminogen activation to sites of MVT is a feasible approach
for the treatment of TTP. Patients with TTP experience attacks of
MVT, when platelets form complexes with ultra-large VWF. We
previously reported that systemic plasminogen activation (with
streptokinase) was therapeutic in a mouse model for TTP, suggesting
that plasmin can act as a functional alternative to ADAMTS13
(Tersteeg et al., 2014, supra). Although plasmin(ogen) can directly
bind to VWF, natural plasminogen activators (tPA, uPA) cannot.
Furthermore, microthrombi are fibrin-poor and for safety reasons it
is desirable to avoid systemic plasminogen activation. To increase
therapeutic efficacy and safety, the present invention therefore
aims to stimulate cleavage of the thrombogenic multimeric protein
VWF by plasmin. More specifically, the invention relates to
modified plasminogen activators that have acquired the ability to
bind either VWF, platelets or activated/injured (micro)vascular
endothelial cells to locally induce plasmin activity for clearance
of MVT, in a fibrin-independent manner. The invention further
provides therapeutic methods for conditions that can be prevented
or treated by local delivery/stimulation of plasminogen activation
to sites of MVT.
[0049] In a first aspect the invention therefore pertains to a
fusion protein comprising a plasminogen activator and a targeting
agent for targeting the plasminogen activator to a site of a
thrombus comprising at least one of VWF and platelets. The
thrombus-site whereto the targeting agent targets the fusion
protein of the invention can in principle be any site where a
thrombus is present or developing, including sites of macrovascular
as well as microvascular thrombi (MVT) and sites of (yet)
non-occlusive macro- or microvascular thrombi. However, the fusion
proteins of the invention are (also) aimed at clearing site of MVT,
which are fibrin-poor, but which do contain VWF, platelets and
where the vascular endothelium can be activated or injured.
Preferably therefore, the targeting agent in the fusion protein of
the invention is a targeting agent that specifically binds to at
least one of VWF, platelets, and activated or injured vascular
endothelium.
[0050] The targeting agent can be any ligand or binding molecule
that specifically binds to at least one of VWF, platelets, and
activated or injured vascular endothelium. Preferably, however, the
targeting agent is a proteinaceous targeting agent. More
preferably, the proteinaceous targeting agent is a part of the
single amino acid chain of the fusion protein, which chain also
comprises the plasminogen activator.
[0051] A targeting agent "which binds" a target of interest, e.g.
VWF, platelets, or activated/ injured endothelium, is an agent that
binds the target with sufficient affinity such that the targeting
agent is useful as a therapeutic agent in targeting a structure,
e.g. an MVT, cell or tissue expressing or exposing the target, and
does not significantly cross-react with other proteins or
molecules. In such embodiments, the extent of binding of the
targeting agent to a "non-target" molecule (e.g. protein) will be
less than about 10% of the binding of the targeting agent to its
particular target molecule as determined by fluorescence activated
cell sorting (FACS) analysis or radioimmunoprecipitation
[0052] (RIA). With regard to the binding of an targeting agent to a
target molecule, the term "specific binding" or "specifically binds
to" or "binds to" or is "specific for" a particular target molecule
or polypeptide, e.g. an epitope on a particular polypeptide target,
means binding that is measurably different from a non-specific
interaction. Specific binding can be measured, for example, by
determining binding of a molecule compared to binding of a control
molecule, which generally is a molecule of similar structure that
does not have binding activity. For example, specific binding can
be determined by competition with a control molecule that is
similar to the target, for example, an excess of non-labelled
target. In this case, specific binding is indicated if the binding
of the labelled target to a probe is competitively inhibited by
excess unlabeled target. The term "specific binding" or
"specifically binds to" or is "specific for" a particular
polypeptide or an epitope on a particular polypeptide target as
used herein can be exhibited, for example, by a molecule having a
K.sub.d for the target (which may be determined as described above)
of at least about 10.sup.-4 M, alternatively at least about
10.sup.-5 M, alternatively at least about 10.sup.-6 M,
alternatively at least about 10.sup.-7 M, alternatively at least
about 10.sup.-8 M, alternatively at least about 10.sup.-9 M,
alternatively at least about 10.sup.-10 M, alternatively at least
about 10.sup.-11 M, alternatively at least about 10.sup.-12 M, or
greater. In one embodiment, the term "specific binding" refers to
binding where a targeting agent binds to a particular target
molecule, polypeptide or epitope on a particular polypeptide
without substantially binding to any other molecule, polypeptide or
epitope.
[0053] In one embodiment of the invention, the targeting agent in
the fusion protein specifically binds to Von Willebrand factor
(VWF), preferably to human VWF. The basic human VWF monomer is a
2050-amino acid protein. Every monomer contains a number of
specific domains with a specific function including e.g. the D'/D3
domain, which binds to factor VIII, the Al domain, which inter alia
binds to the platelet GPIb-receptor, the A2 domain (which must
partially unfold to expose the buried cleavage site for the
specific ADAMTS13 protease that inactivates VWF by making much
smaller multimers), the A3 domain, which binds to collagen, the Cl
domain, in which the RGD motif binds to platelet integrin
.alpha.IIb.beta.3 when this is activated and the "cysteine knot"
domain (at the C-terminal end of the protein). Multimers of VWF can
be extremely large, >20,000 kDa and can consist of over 80
subunits of 250 kDa each. VWF's primary function is binding to
other proteins, in particular factor VIII, and it is important in
platelet adhesion to wound sites. VWF is not an enzyme and, thus,
has no catalytic activity. VWF binds to a number of cells and
molecules, including e.g. to collagen (e.g.
[0054] when is exposed in endothelial cells due to damage occurring
to the blood vessel) and to the platelet GP1 B receptor. The latter
binding occurs under all circumstances, but is most efficient under
high shear stress (i.e. rapid blood flow in narrow blood vessels).
VWF binds to other platelet receptors when they are activated,
e.g., by thrombin (i.e., when coagulation has been stimulated).
[0055] A targeting agent in the fusion protein of the invention can
specifically bind to any and all forms, conformation, domains and
epitopes of VWF. The targeting agent can thus specifically bind to
at least one of the unfolded (activated) conformation of VWF and
the globular (circulating unactivated) conformation of VWF (sVWF).
In one embodiment, the targeting agent that at least binds unfolded
VWF, wherein, preferably the targeting agent preferentially binds
unfolded VWF over globular VWF (i.e. has a higher affinity for
unfolded VWF than for globular VWF), more preferably, the targeting
agent binds the unfolded (activated) conformation of VWF and does
not bind to circulating unactivated globular forms of VWF. More
specifically, the targeting agent in the fusion protein of the
invention specifically binds to at least one of the VWF Al domain,
the Al domain of activated VWF, VWF A2 domain, the A2 domain of
activated VWF, the VWF A3 domain and the VWF D3 domain. Suitable
examples of targeting agents that bind VWF are the VHH camelid
antibody fragments used in the Examples herein, as further detailed
below.
[0056] In another embodiment of the invention, the targeting agent
in the fusion protein specifically binds to platelets (also
referred to as thrombocytes), preferably to human platelets.
Together with VWF, platelets are the main component of MVT and
therefore also suitable targets for the targeting agent in the
fusion protein of the invention. A targeting agent in the fusion
protein of the invention can specifically bind to any and all forms
of platelets. The targeting agent can thus specifically bind to at
least one of activated platelets and to non-activated platelets.
Preferably, targeting agent (at least) binds to non-activated
platelets. Unlike macrovascular thrombosis or regular thrombosis,
MVT do necessarily comprise activated platelets, and especially
unactivated platelets (together with VWF) are causing the problems
in MVT in e.g. TTP. Therefore, the targeting agent in the fusion
protein preferably is not a targeting agent that specifically binds
to only activated platelets (and not to non-activated platelets).
More specifically, the targeting agent preferably is not a
targeting agent that specifically binds to only the activated form
of the GPIIb/IIIa receptor on platelets, such as e.g. the
single-chain antibody SCE5 described by Schwarz et al. (2004, FASEB
J. 18:1704 1706). The targeting agent thus preferably specifically
binds to the non-active form of integrin .alpha.IIb/.beta.III. In a
preferred embodiment, the targeting agent in the fusion protein of
the invention specifically binds to at least one of: a) the
platelet GP1B receptor, and b) integrin .alpha.IIb/.beta.III on
platelets. A targeting agent that specifically binds to the
platelet GP1B receptor is however preferred. Suitable targeting
agents that bind platelets are known in the art, as e.g.
exemplified by the anti-GPlba antibody 6B4 described by Fontayne et
al. (2006, Thromb Haemost. 96(5):671-84) or by the anti-GP1B the
VHH antibody fragment used in the Examples herein.
[0057] In a further embodiment of the invention, the targeting
agent in the fusion protein specifically binds to activated,
injured and/or distressed endothelium (herein further collectively
referred to as activated endothelium. The activated endothelium
preferably is activated vascular endothelium, more preferably
activated microvascular endothelium. The targeting agent can thus
specifically bind to at least one of a receptor that is
preferentially expressed by activated endothelium and a membrane
marker for activated endothelium. Preferably, the receptor that is
preferentially expressed by activated endothelium the receptor is
selected form the group consisting of E-selectin, P-selectin, uPAR,
c1q receptor, kinin B1 receptor, plasminogen receptor KT (PLGR-KT),
endothelial protein C receptor, thrombomodulin, n-cadherin, ICAM-1
and VCAM-1. Preferably, the membrane marker for activated
endothelium is one or more of anionic phospholipids,
phosphatidylserine and phosphatidylethanolamine.
[0058] In one embodiment of a fusion protein of the invention, the
targeting agent that specifically binds to one of the above-defined
targets, preferably comprises at least one of: a) an antibody
variable domain that specifically binds to one of said targets;
and, b) a binding domain from a protein that naturally binds to one
of said targets.
[0059] A preferred antibody variable domain that is present as
targeting agent in a fusion protein of the invention is a VHH as
defined herein above, more preferably the antibody variable domain
is a humanized VHH.
[0060] In a preferred embodiment, the VHH that is present as
targeting agent in a fusion protein of the invention is a VHH that
specifically binds VWF. Suitable examples of VHHs that bind VWF and
that are part of a fusion protein of the invention as targeting
agent in are the VWF-binding VHHs that are part of the fusion
protein in the Examples herein or that are described in
US2013/0136736-A1, which is incorporated by reference herein.
[0061] A preferred VHH that binds soluble globular VWF and that is
present as targeting agent in a fusion protein of the invention is
VHH-sVWF, having the amino acid sequence of positions 55-178 of SEQ
ID NO:7 or a VHH having an amino acid sequence with at least 80,
85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity
with positions 55-178 of SEQ ID NO:7 and having a Kd for VWF of
less than 1, 0.5, 0.2, 0.1, 0.05 or 0.0306 nM.
[0062] A preferred VHH that binds the D3 domain of VWF and that is
present as targeting agent in a fusion protein of the invention is
VHH-D3, having the amino acid sequence of positions 55-178 of SEQ
ID NO:8 or a VHH having an amino acid sequence with at least 80,
85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity
with positions 55-178 of SEQ ID NO:8 and having a Kd for VWF of
less than 1, 0.5, 0.4, 0.35, or 0.33 nM.
[0063] A preferred VHH that binds the Al domain of VWF and that is
present as targeting agent in a fusion protein of the invention is
VHH-Al2, having the amino acid sequence of positions 55-179 of SEQ
ID NO:11 or a VHH having an amino acid sequence with at least 80,
85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity
with positions 55-179 of SEQ ID NO:11 and having a Kd for VWF of
less than 25, 20, 18, 15 or 13 nM. Another preferred of VHH that
binds the Al domain of VWF and that is present as targeting agent
in a fusion protein of the invention is the VHH having the amino
acid sequence of SEQ ID NO:13 or its humanized version having the
amino acid sequence of SEQ ID NO:14, or a VHH having an amino acid
sequence with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98
or 99% sequence identity with the SEQ ID NO:13 or 14 and having a
Kd for VWF of less than 10, 5 or 2 nM.
[0064] A preferred VHH that binds the platelet GP1B receptor and
that is present as targeting agent in a fusion protein of the
invention is VHH-GP1B17, having the amino acid sequence of
positions 55-178 of SEQ ID NO:12 or a VHH having an amino acid
sequence with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98
or 99% sequence identity with positions 55-178 of SEQ ID NO:12 and
having a Kd for the platelet GP1B receptor of less than 20, 15, 10,
5, 2, 1, 0.5, 0.2 or 0.1 nM. Other preferred VHHs that bind the
platelet GP1B receptor and that are present as targeting agent in
fusion proteins of the invention have an amino acid sequence
selected from the group consisting of amino acid sequences of
positions 55-175 of SEQ ID NO:20, positions 55-178 of SEQ ID NO:21,
positions 55-172 of SEQ ID NO:22, positions 55-171 of SEQ ID NO:23,
positions 55-174 of SEQ ID NO:24, positions 55-176 of SEQ ID NO:25;
positions 55-178 of SEQ ID NO:26, positions 55-178 of SEQ ID NO:27,
positions 55-178 of SEQ ID NO:28, positions 55-166 of SEQ ID NO:29,
positions 55-180 of SEQ ID NO:30, positions 55-176 of SEQ ID NO:31,
positions 55-176 of SEQ ID NO:32, positions 55-179 of SEQ ID NO:33,
positions 55-179 of SEQ ID NO:34, positions 55-177 of SEQ ID NO:35,
positions 55-178 of SEQ ID NO:36 and positions 55-181 of SEQ ID
NO:37, or have an amino acid sequence with at least 80, 85, 90, 91,
92, 93, 94, 95, 96, 97, 98 or 99% sequence identity with the amino
acid sequence in this group and having a Kd for the platelet GP1B
receptor of less than 20, 15, 10, 5, 2, 1, 0.5, 0.2 or 0.1 nM.
[0065] Another preferred VHH that binds the Al domain of VWF
comprises an amino acid sequence with at least 80, 85, 90, 91, 92,
93, 94, 95, 96, 97, 98 or 99% sequence identity with SEQ ID NO: 46.
Preferably a VHH that binds the Al domain of VWF and that comprises
the amino acid sequence of SEQ ID NO:46 is present as targeting
agent in a fusion protein of the invention is the VHH having the
amino acid sequence of SEQ ID NO:45 or its humanized version having
the amino acid sequence of SEQ ID NO:44, or a VHH having an amino
acid sequence with at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,
98 or 99% sequence identity with the SEQ ID NO:44 or 45 and having
a Kd for VWF of less than 10, 5 or 2 nM.
[0066] In another embodiment of a fusion protein of the invention,
the targeting agent that specifically binds to at least one of VWF,
platelets, and activated or injured vascular endothelium comprises
a binding domain from a protein that naturally binds to at least
one of VWF, platelets, and activated or injured vascular
endothelium. Preferably, the binding domain comprises a binding
domain selected from the group consisting of: i) the A1 domain from
VWF, or at least a part of the VWF A1 domain that binds the
platelet GP1B receptor; ii) a VWF-binding domain from one of
ADAMTS13, Factor XII, Factor H (complement regulator), plasminogen
and Factor VIII, at least a part of these domains that binds VWF;
and, iii) a domain that binds to membrane(s) of activated or
injured vascular endothelium, which domain is selected from the
vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA)
domain, the C-domain from factor V and the C-domain from factor
VIII.
[0067] In one embodiment of the invention, the fusion protein
comprises more than one targeting agent. The fusion protein can
thus comprises e.g. two, three, four, five, six or more targeting
agents. If more than one targeting agent is present in the fusion
protein, there can be more than one copy of the same targeting
agent present in the fusion protein. Alternatively, the fusion
protein can comprises at least two different targeting agents. For
example, the fusion protein can comprise at least two different
targeting agents that (each) bind to at least two different domains
of VWF or to at least two different receptors on platelets. Or the
fusion protein can comprises at least two different targeting
agents, of which at least one binds to VWF and at least one other
binds to platelets. An advantage of incorporating more than one
targeting agent in the fusion protein is the avidity of the
multivalent binding at the site of MVT, i.e. the accumulated
strength of multiple affinities of individual binding interactions
by the individual targeting agents. When more than one targeting
agent is present in the fusion protein, the individual targeting
agents are preferably arranged in tandem, and preferably with
suitable (flexible) spacer- or linker-amino acid sequences between
the individual targeting agents.
[0068] Suitable flexible linker-amino acid sequences are known in
the art (e.g. from Chen et al., 2013, Adv Drug Deliv Rev. 65(10):
1357-1369). Flexible linkers are usually applied when the joined
domains require a certain degree of movement or interaction. They
are generally composed of small, non-polar (e.g. Gly) or polar
(e.g. Ser or Thr) amino acids. The small size of these amino acids
provides flexibility, and allows for mobility of the connecting
functional domains. The incorporation of Ser or Thr can maintain
the stability of the linker in aqueous solutions by forming
hydrogen bonds with the water molecules, and therefore reduces the
unfavorable interaction between the linker and the protein
moieties. Preferred flexible linkers have sequences consisting
primarily of stretches of Gly and Ser residues ("GS" linker). An
example of preferred (and widely used) flexible linker has the
sequence of (Gly-Gly-Gly-Gly-Ser).sub.n. By adjusting the copy
number "n", the length of this GS linker can be optimized to
achieve appropriate separation of the functional domains, or to
maintain necessary inter-domain interactions. Besides the GS
linkers, many other flexible linkers have been designed for
recombinant fusion proteins. These flexible linkers are also rich
in small or polar amino acids such as Gly and Ser, but can contain
additional amino acids such as Thr and Ala to maintain flexibility,
as well as polar amino acids such as Lys and Glu to improve
solubility, such as e.g. the flexible linkers KESGSVSSEQLAQFRSLD
(SEQ ID NO:38) and EGKSSGSGSESKST (SEQ ID NO:38), that have been
applied for the construction of a bioactive scFv's.
[0069] In addition to the above described one or more targeting
agents for targeting to an MVT, a fusion protein of the invention
further at least comprises a plasminogen activator. Plasminogen
activators are serine proteases that catalyze the activation of
plasmin via proteolytic cleavage of its zymogen form plasminogen.
While plasmin is an important factor in fibrinolysis, in the
context of the present invention plasminogen activation is relied
upon for its fibrin-independent thrombolytic activity towards MVT.
Therefore, a fusion protein according to the invention comprises a
plasminogen activator, which plasminogen activation preferably
comprises a (catalytic) protease domain from tissue plasminogen
activator (tPA), urokinase plasminogen activator (uPA),
plasminogen, streptokinase or staphylokinase. The catalytic
protease domains of tPA, uPA, plasminogen, streptokinase and
staphylokinase are well known in the art. A preferred
plasminogen-activating catalytic protease domain for incorporation
in a fusion protein of the invention is the catalytic protease
domain of uPA, preferably of human uPA, which comprises the amino
acid sequence of positions 16-268 of SEQ ID NO:1, or the catalytic
protease domain of tPA, preferably of human tPA, which comprises
the amino acid sequence of positions 15-266 of SEQ ID NO:19.
[0070] In one embodiment, wherein the catalytic domain protease
domain for incorporation in a fusion protein of the invention is
the catalytic protease domain of uPA, preferably of human uPA, the
(human) uPA domain comprises a mutation in its sequence that
stabilizes the (human) uPA. Stabilizing mutations have been
described in U.S. Pat. No. 5,472,692 and Sun et al (J Biol Chem
1997 Sept. 19; 272 (38): 23818-23823) which incorporated herein in
their entirety. In one preferred embodiment, the catalytic domain
protease comprises and/or consists of SEQ ID NO: 1 wherein the
Lysin (K) located at position 157 of SEQ ID NO:1 is mutated to
Histidine (H). In one preferred embodiment, the catalytic domain
protease comprises and/or consists of SEQ ID NO: 2 wherein the
Lysin (K) located at position 158 of SEQ ID NO:2 is mutated to
Histidine (H).
[0071] In one embodiment, the plasminogen activator that is
incorporated in a fusion protein of the invention is comprises a
variant of a catalytic protease domain with reduced susceptibility
to natural inhibitors of plasminogen activators, such as
plasminogen activator inhibitor-1 (PAI-1). Such variants include
e.g. variants of the protease domains of uPA or tPA with one or
more modifications in their exosite loops that are involved in the
interaction with PAI-1, such as e.g. the 37- and 147-loops in the
protease domains of uPA or analogous loops in the protease domain
of tPA, e.g. the 37-, 60-, 97-, 147- and 217-loops (see Lin et al.
J Biol Chem. 2011 Mar 4; 286(9): 7027-7032). Examples of such
modifications are deletions of exosites that are specific to
interaction with PAI-1 (tPA del 296-302) or replacement of specific
amino acids that specifically mediate the interaction with PAI-1
(tPA Arg->Glu 304 or Arg ->Ser 304) (Madison et al. Nature.
1989 Jun 29;339(6227):721-4.).
[0072] In a preferred embodiment the plasminogen activator that is
incorporated in a fusion protein of the invention further comprises
at least the cysteine-containing part of the connecting peptide
that naturally occurs in the plasminogen activator immediately
upstream (N-terminally) of its protease domain. The presence of the
connecting peptide including its cysteine avoids that the
counterpart cysteine in the protease domain forms unwanted
disulfide bridges.
[0073] A preferred connecting peptides for inclusion in the
plasminogen activator that is incorporated in a fusion protein of
the invention is: a) a connecting peptide from uPA that at least
includes amino acids 17-27 of SEQ ID NO:16, more preferably the
connecting peptide at least includes amino acids 13-27 of SEQ ID
NO:16; b) a connecting peptide from tPA that at least includes
amino acids 3-14 of SEQ ID NO:17, more preferably the connecting
peptide at least includes amino acids 1-14 of SEQ ID NO:17; or c) a
connecting peptide from plasminogen that at least includes amino
acids 5-18 of SEQ ID NO:18, more preferably the connecting peptide
at least includes amino acids 1-18 of SEQ ID NO:18.
[0074] In another preferred embodiment, a fusion protein of the
invention preferably comprises a linker-amino acid sequence located
between the one or more targeting agents on the one hand, and the
plasminogen activator on the other hand. It is thereby understood
that if the plasminogen activator includes a connecting peptide
e.g. as described above, that the linker-amino acid sequence then
is located upstream of the plasminogen activator's connecting
peptide. The linker-amino acid sequence linking the targeting
agents and the plasminogen activator preferably is a flexible
linker-amino acid sequence as described above. A preferred
linker-amino acid sequence for linking the targeting agents and the
plasminogen activator has the sequence of
(Gly-Gly-Gly-Gly-Ser).sub.n, whereby n is 1-4, more preferably 2 or
3 and most preferably 2. A particularly preferred linker-amino acid
sequence has the amino acid sequence of SEQ ID NO:6.
[0075] Thus, a preferred fusion protein in accordance with the
invention is a fusion protein that comprises in a N- to C-terminal
order: a) one or more targeting agents as defined hereinabove,
whereby, optionally the targeting agents are linked by linker amino
acid sequences as defined hereinabove; b) optionally a linker amino
acid sequence as defined hereinabove; and, c) a plasminogen
activator as defined hereinabove. The fusion protein can further
comprise additional functional elements, including e.g. isolation
tags, such as e.g. a His-tag or a STREP isolation tag, or a
cleavage site recognized only by a specific protease such as e.g. a
Tobacco Etch Virus cleavage site, so to be able to cleave off
undesirable amino acid sequences (e.g. the isolation tag) from the
fusion protein. An example of the configuration of a preferred
fusion protein in accordance with the invention is shown in FIG. 1,
whereby the nucleotide sequence encoding the fusion protein also
encodes a (IN signal sequence upstream of the fusion protein to
direct its secretion from the cell in which the protein is
produced.
[0076] In a second aspect, the invention relates to a nucleic acid
molecule comprising a nucleotide sequence encoding a fusion protein
of the invention as defined hereinabove. The nucleotide sequence
encoding the fusion protein further preferably comprises a
nucleotide sequence encoding a signal peptide operably linked to
the fusion protein. A preferred signal peptide for directing
secretion of the fusion proteins of the invention is the Igic
signal peptide, having the amino acid sequence of SEQ ID NO: 3. A
nucleic acid molecule comprising a nucleotide sequence encoding a
fusion protein of the invention, further preferably comprises
regulatory elements conducive to the expression of the fusion
protein in an appropriate host cell, which regulatory elements are
operably linked to the nucleotide sequence.
[0077] In a third aspect, the invention relates to a vector
comprising a nucleic acid molecule according to the invention.
Optionally, the vector according to the invention is a gene therapy
vector.
[0078] Preferably, the a gene therapy vector is a viral gene
therapy vector, e.g. a viral gene therapy vector selected from gene
therapy vectors based on an adenovirus, an adeno-associated virus
(AAV), a herpes virus, a pox virus, an oncolytic virus vector and a
retrovirus. A preferred viral gene therapy vector is an AAV or
Lentiviral vector.
[0079] In a fourth aspect, the invention relates to a host cell
comprising a vector according to the invention, which host cell
expresses a fusion protein according to invention.
[0080] The cell preferably is an isolated cell or a cultured cell.
Among the host cells that may be employed are prokaryotes, yeast or
higher eukaryotic cells. Prokaryotes include gram negative or gram
positive organisms, for example Escherichia coli or bacilli. Higher
eukaryotic cells include insect cells and established cell lines of
mammalian origin. Examples of suitable mammalian host cell lines
include the COS-7 line of monkey kidney cells (Gluzman et al.,
1981, Cell 23:175), L cells, HEK 293 cells, C127 cells, 3T3 cells,
Chinese hamster ovary (CHO) cells, HeLa cells, BHK cell lines, and
the CVI/EBNA cell line derived from the African green monkey kidney
cell line CVI as described by McMahan et al., 1991, EMBO J. 10:
2821. Appropriate cloning and expression vectors for use with
bacterial, fungal, yeast, and mammalian cellular hosts are
described by Pouwels et al. (Cloning Vectors: A Laboratory Manual,
Elsevier, New York, 1985).
[0081] The transformed cells can be cultured under conditions that
promote which host cell expresses a fusion protein according to
invention. Thus in one aspect the invention relates to a method for
producing a fusion protein according to invention, the method
comprising the step of cultivating a cell comprising at least one
expression vector as defined herein, under conditions conducive to
expression of a fusion protein according to invention, optionally,
recovering the fusion protein according to invention.
[0082] A fusion protein according to invention can be recovered by
conventional protein purification procedures, including e.g.
protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography, using e.g.
strepavidin/biotin (see e.g. Low et al., 2007, J. Chromatography B,
848:48-63; Shukla et al., 2007, J. Chromatography B,
848:28-39).
[0083] In a fifth aspect, the invention relates to a pharmaceutical
composition comprising and/or consisting of a fusion protein
according to the invention, a nucleic acid according to the
invention, a vector or gene therapy vector according to the
invention, or a host cell according to the invention and a
pharmaceutically acceptable excipient.
[0084] The pharmaceutical composition further preferably comprises
at least one pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier such as an adjuvant, or
vehicle, is for administration of the antibody or antibody fragment
to a subject. Said pharmaceutical composition can be used in the
methods of treatment described herein below by administration of an
effective amount of the composition to a subject in need thereof.
The term "subject", as used herein, refers to all animals
classified as mammals and includes, but is not restricted to,
primates and humans. The subject is preferably a male or female
human of any age or race.
[0085] The term "pharmaceutically acceptable carrier", as used
herein, is intended to include any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration (see e.g. "Handbook of Pharmaceutical
Excipients", Rowe et al eds. 7.sup.th edition, 2012,
wkApirv.pharrnpress.com). The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the compositions is contemplated.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter ions such as sodium; metal complexes
(e.g. Zn.sup.2+-protein complexes); and/or non-ionic surfactants
such as TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0086] Supplementary active compounds can also be incorporated into
the pharmaceutical composition of the invention. Thus, in a
particular embodiment, the pharmaceutical composition of the
invention may also contain more than one active compound as
necessary for the particular indication being treated, preferably
those with complementary activities that do not adversely affect
each other. For example, it may be desirable to further provide a
chemotherapeutic agent, a cytokine, an analgesic agent, a
thrombolytic or an immunomodulating agent, e.g. an
immunosuppressive agent or an immunostimulating agent. The
effective amount of such other active agents depends, among other
things, on the amount of antibody of the invention present in the
pharmaceutical composition, the type of disease or disorder or
treatment, etc.
[0087] In an embodiment, the fusion protein according to the
invention is prepared with carriers that will protect said compound
against rapid elimination from the body, such as a controlled
release formulation, including implants and microencapsulated
delivery systems, e.g. liposomes. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. Liposomal suspensions,
including targeted liposomes can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811 or WO2010/095940.
[0088] The administration route of the fusion protein according to
the invention can be parenteral. The term "parenteral" as used
herein includes intravenous, intra-arterial, intralymphatic,
intraperitoneal, intramuscular, subcutaneous, rectal or vaginal
administration. The intravenous forms of parenteral administration
are preferred. By "systemic administration" is meant oral,
intravenous, intraperitoneal and intramuscular administration. The
amount of the fusion protein required for therapeutic or
prophylactic effect will, of course, vary with the fusion protein
chosen, the nature and severity of the condition being treated and
the patient. In addition, the fusion protein may suitably be
administered by pulse infusion, e.g., with declining doses of the
fusion protein. Preferably the dosing is given by injections, most
preferably intravenous or subcutaneous injections, depending in
part on whether the administration is brief or chronic.
[0089] Thus, in a particular embodiment, the pharmaceutical
composition of the invention may be in a form suitable for
parenteral administration, such as sterile solutions, suspensions
or lyophilized products in the appropriate unit dosage form.
Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, CremophorEM (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, a pharmaceutically acceptable polyol like
glycerol, propylene glycol, liquid polyetheylene glycol, and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol or sodium chloride in the
composition.
[0090] Prolonged absorption of the injectable compositions can be
brought about by including in the composition an agent which delays
absorption, for example, aluminium monostearate and gelatin.
[0091] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g a fusion protein) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0092] In a particular embodiment, said pharmaceutical composition
is administered via intravenous (IV) or subcutaneous (SC). Adequate
excipients can be used, such as bulking agents, buffering agents or
surfactants. The mentioned formulations will be prepared using
standard methods for preparing parenterally administrable
compositions as are well known in the art and described in more
detail in various sources, including, for example, "Remington: The
Science and Practice of Pharmacy" (Ed. Allen, L. V. 22nd edition,
2012, www,pharmpress.com).
[0093] It is especially advantageous to formulate the
pharmaceutical compositions, namely parenteral compositions, in
dosage unit form for ease administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units
suited as unitary dosages for the subject to be treated; each unit
containing a predetermined quantity of active compound (antibody of
the invention) calculated to produce the desired therapeutic effect
in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of individuals.
[0094] Generally, for the prevention and/or treatment of the
diseases and disorders mentioned herein and depending on the
specific disease or condition to be treated and its severity, the
potency of the specific fusion protein of the invention to be used,
the specific route of administration and the specific
pharmaceutical formulation or composition used, the fusion proteins
of the invention will generally be administered in the range of
from 0.001 to 1,000 mg/kg body weight/day, preferably about 0.01 to
about 100 mg/kg body weight/day, most preferably from about 0.05 to
10 mg/kg body weight/day, such as about 1, 10, 100 or 1000
microgram per kg body weight per day, 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.
[0095] Aside from administration of a fusion protein according to
the invention to the patient, the present application contemplates
administration of a fusion proteins by gene therapy.
[0096] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0097] The fusion proteins and pharmaceutical compositions of this
invention may be used with other drugs to provide a combination
therapy. The other drugs may form part of the same composition, or
be provided as a separate composition for administration at the
same time or at different time.
[0098] In a sixth aspect, the invention pertains to at least one of
a fusion protein, a gene therapy vector and a pharmaceutical
composition, each as defined hereinabove, for use in the treatment
or prevention of a disease or condition associated with thrombosis.
The disease or condition to be prevented or treated can be any
disease or condition associated with or involving thrombi
comprising at least one of VWF and platelets. Such a disease or
condition associated with thrombi comprising at least one of VWF
and platelets can be any disease or condition involving
macrovascular and/or microvascular thrombi (MVT), and/or involving
sites of (yet) non-occlusive macro- or microvascular thrombi.
Preferably, however, the disease or condition to be prevented or
treated is a disease or condition associated with at least
microvascular thrombosis (MVT). It is to be understood herein that
the term "to prevent a disease or condition" includes and/or can be
equal to reducing the risk of that disease or condition to occur or
develop.
[0099] In a preferred embodiment the disease or condition
associated with thrombi comprising at least one of VWF and
platelets is a disease or condition is selected from the group
consisting of: acquired or hereditary thrombotic thrombocytopenic
purpura (TTP) complement-mediated thrombotic microangiopathy (as
reviewed by George et al., 2014, N Engl J Med. 371(7):654-66)
haemolytic uremic syndrome, antiphospholipid antibody syndrome, the
formation of an occlusive thrombus, arterial thrombus formation,
acute coronary occlusion, peripheral arterial occlusive disease,
restenosis and disorders arising from coronary by-pass graft,
coronary artery valve replacement and coronary interventions such
angioplasty, stenting or atherectomy, hyperplasia after
angioplasty, atherectomy or arterial stenting, occlusive syndrome
in a vascular system or lack of patency of diseased arteries,
transient cerebral ischemic attack, unstable or stable angina
pectoris, cerebral infarction, HELLP syndrome (HELLP is Hemolysis,
Elevated Liver enzymes, and Low Platelet count), carotid
endarterectomy, carotid artery stenosis, critical limb ischemia,
cardioembolism, peripheral vascular disease, restenosis, sickle
cell disease and myocardial infarct.
[0100] Further diseases or conditions associated with thrombi
comprising at least one of VWF and platelets to be treated or
prevented in accordance with a method of the invention are diseases
or conditions is selected from the group consisting of unstable
angina, stable angina, angina pectoris, embolus formation, deep
vein thrombosis, hemolytic anemia, acute renal failure,
thrombolytic complications, disseminated intravascular coagulopathy
(DIC), thrombosis, coronary heart disease, thromboembolic
complications, myocardial infarction, restenosis, and atrial
thrombosis formation in atrial fibrillation, chronic unstable
angina, transient ischemic attacks and strokes, peripheral vascular
disease, arterial thrombosis, pre-eclampsia, embolism, restenosis
and/or thrombosis following angioplasty, anastomosis of vascular
grafts, and chronic exposure to cardiovascular devices. Such
conditions may also result from thromboembolism and reocculsion
during and after thrombolytic therapy, after angioplasty, and after
coronary artery bypass.
[0101] In a further embodiment a fusion protein, a gene therapy
vector and a pharmaceutical composition, each as defined
hereinabove, for use in the treatment or prevention of a plaque or
thrombus in an individual. Said plaque or thrombus formation may be
under conditions of high shear. In both thrombosis and reocclusion,
the reversible adhesion or tethering of the platelets at high shear
rate is followed by a firm adhesion through the collagen receptor
on platelets resulting in platelet activation; the tethering of
platelets by VWF to collagen exposed in the damaged vessel wall is
especially important under high shear conditions. A fusion protein
of the present invention performs well under high shear
conditions.
[0102] In a seventh aspect the invention relates to method for
treating or reducing the risk of a disease or condition associated
with thrombi comprising at least one of VWF and platelets, wherein
the method comprises the step of administering to a subject in need
thereof, an effective amount of fusion protein as defined
hereinabove, a gene therapy vector as defined hereinabove or a
pharmaceutical composition as defined hereinabove, and wherein
preferably with the disease or condition associated with
microvascular thrombosis. Preferably, in the method the disease or
condition associated with thrombi comprising at least one of VWF
and platelets is selected from the group consisting of: acquired or
hereditary thrombotic thrombocytopenic purpura (TTP),
complement-mediated thrombotic microangiopathy, haemolytic uremic
syndrome, antiphospholipid antibody syndrome, non-occlusive
thrombus, the formation of an occlusive thrombus, arterial thrombus
formation, acute coronary occlusion, peripheral arterial occlusive
disease, restenosis and disorders arising from coronary by-pass
graft, coronary artery valve replacement and coronary interventions
such angioplasty, stenting or atherectomy, hyperplasia after
angioplasty, atherectomy or arterial stenting, occlusive syndrome
in a vascular system or lack of patency of diseased arteries,
transient cerebral ischemic attack, unstable or stable angina
pectoris, cerebral infarction, HELLP syndrome, carotid
endarterectomy, carotid artery stenosis, critical limb ischemia,
cardioembolism, peripheral vascular disease, restenosis, sickle
cell disease and myocardial infarct, or a further diseases or
conditions defined hereinabove.
[0103] In an eighth aspect the invention relates to method for
treating or reducing the risk of at least one of microvascular
thrombi, fibrin-independent thrombi and thrombi comprising at least
one of VWF and platelets. Preferably, in the method at least one
these thrombi is treated or the risk of its occurrence is reduced
in a disease or condition is selected from the group consisting of:
acquired or hereditary thrombotic thrombocytopenic purpura (TTP),
complement-mediated thrombotic microangiopathy, haemolytic uremic
syndrome, antiphospholipid antibody syndrome, non-occlusive
thrombus, the formation of an occlusive thrombus, arterial thrombus
formation, acute coronary occlusion, peripheral arterial occlusive
disease, restenosis and disorders arising from coronary by-pass
graft, coronary artery valve replacement and coronary interventions
such angioplasty, stenting or atherectomy, hyperplasia after
angioplasty, atherectomy or arterial stenting, occlusive syndrome
in a vascular system or lack of patency of diseased arteries,
transient cerebral ischemic attack, unstable or stable angina
pectoris, cerebral infarction, HELLP syndrome, carotid
endarterectomy, carotid artery stenosis, critical limb ischemia,
cardioembolism, peripheral vascular disease, restenosis, sickle
cell disease and myocardial infarct, or a further diseases or
conditions defined hereinabove.
[0104] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition, reference to an element by
the indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one".
[0105] The word "about" or "approximately" when used in association
with a numerical value (e.g. about 10) preferably means that the
value may be the given value (of 10) more or less 0.1% of the
value.
[0106] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
[0107] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
DESCRIPTION OF THE FIGURES
[0108] FIG. 1: VHH-miniUPA (mUPA) construct A) Schematic
representation of an mUPA construct B) Schematic representation of
an mUPA construct comprising a VHH coding sequence (VHH-mUPA).
[0109] FIG. 2: Western Blot of purified VHH-mUPA constructs.
[0110] FIG. 3: VHH-mUPA construct activity after activation by
plasmin.
[0111] FIG. 4: Plasminogen activation by VHH-mUPA constructs.
[0112] FIG. 5: Plasminogen activation by VHH-mUPA constructs in the
presence of globular or open VWF. A) Plasminogen activation by
VHH-sVWF, B) Plasminogen activation by VHH-D3, C) Plasminogen
activation by VHH-GP1B17, D) Plasminogen activation by VHH-R2, E)
Plasminogen activation by VHH-A12.
[0113] FIG. 6: Plasmin substrate conversion after 5 minutes
incubation at 37.degree. C. of the constructs VHH-sVWF, VHH-D3,
VHH-GP1B17, VHH-R2, and VHH-A12.
[0114] FIG. 7: Micro-thrombolysis (i.e. enzymatic breakdown) of
VWF-platelet agglutinates. Lysis of the agglutinates was monitored
overtime A) Microthrombolysis induced by VHH-sVWF, B)
Microthrombolysis induced by VHH-D3, C) Microthrombolysis induced
by VHH-R2, D) Microthrombolysis induced by VHH-GP1B17.
[0115] FIG. 8: A) Graphic example of analytical method to determine
time points by which 50% microthrombus degradation has occurred. B)
50% microthrombus degradation of the constructs VHH-sVWF, VHH-D3,
VHH-GP1B17, VHH-R2, and VHH-A12.
[0116] FIG. 9: Micro-thrombolysis of VWF-platelet complexes adhered
to human vascular endothelial cells as analyzed in flow perfusion
experiments. VWF-platelet complexes are visible as strings of
platelets and the number of visible string are counted as a
function of time in response to the addition of fusion proteins as
indicated.
[0117] FIG. 10: Micro-thrombolysis (i.e. enzymatic breakdown) of
VWF-platelet agglutinates. Lysis of the agglutinates was monitored
overtime on a light transmission aggregometer. A) Microthrombolysis
induced by (154.3 nM) Caplacizumab or (154.3 nM) VHH-D3 fusion
protein with UPA in the presence of (100 .mu.g/mL) plasminogen. B)
Microthrombolysis induced by (154.3 nM) Caplacizumab or (154.3 nM)
VHH-GP1B17 fusion protein with UPA in the presence of (100
.mu.g/mL) plasminogen.
EXAMPLES
Example 1
Methods and Materials
[0118] Nanobody-mUPA construction
[0119] The cDNA sequence for both human and mouse urokinase (PLAU)
was obtained from the NCBI database (NM_002658.4 and NM_008873.3
respectively). The sequence for the signal peptide, EGF-like and
Kringle domain were removed as well as the first part of the
connecting peptide. To the remaining connecting peptide and S1
peptidase domain (Catalytic domain) a N-terminal sequence coding
for a Tobacco Etch Virus cleavage site followed by an GGGGS linker
was added. In the GGGGS linked a Pstl and BamHI digestion site were
incorporated without disturbing the amino acid sequence. At the 5'
side an EcoRl digestion site was added and at the 3' side and Notl
digestion was added after the STOP codon of PLAU. The construct was
obtained from IDT (Integrated DNA Technologies, Leuven, Belgium) as
a custom gene construct.
[0120] Coding sequences for nanobodies (also known as VHH) were
codon optimized via IDT for expression in a human host cells. At
the N-terminal side of the VHH coding sequence, a sequence coding
for a Tobacco Etch Virus cleavage site was placed and at the
C-terminal side a GGGGS linker (encoding a Pstl and BamHl digestion
site). These DNA segments were obtained from IDT as Double stranded
DNA fragments (gBLocks).
[0121] The custom gene construct was propagated in E.coli TOP10 and
selected by ampicillin resistance. Obtained plasmid DNA was
digested by EcoRl and Notl. The resulting insert (886) was
separated on and isolated from agarose gel and ligated into a
modified pcDNA6 expression vector (pSM2) (De Maat et al, 2016 J
Allergy Clin Immunol Nov;30;138(5):1414-23)). pSM2 encodes a
N-terminal murine IgK secretion signal and a double STREP isolation
tag whereafter the modified UPA construct is ligated.
[0122] The gBlocks were ligated into the pJET1.2 cloning vector
according to manufacturer instructions (CloneJET PCR Cloning Kit;
Thermo Fisher). The constructs were propagated in E.coli TOP10 and
selected by ampicillin resistance. Obtained plasmid DNA was
digested by EcoRl and BamHl. The resulting insert was separated on
and isolated from agarose gel and ligated into the pSM2 vector
containing the miniUPA construct. The gene constructs are
schematically depicted in in FIG. 1. Table 1 lists the
nanobody-mUPA fusion protein constructs that were prepared,
including the targets of their nanobodies and their names.
TABLE-US-00004 TABLE 1 Description of nanobody-mUPA fusion protein
constructs Specificity Name Target VWF sVWF-mUPA globular and
unfolded VWF D3-mUPA D3 domain of VWF A11-mUPA A1 domain of VWF
A12-mUPA A1 domain of VWF Platelets GP1B17-mUPA platelet GP1B
receptor Negative control R2-mUPA no binding to VWF or
platelets
Nanobody-mUPA Production
[0123] The nanobody/mUPA-pSM2 constructs were transfected into
HEK293 FreeStyle.TM. cells using 239Fectin as instructed by the
manufacturer (ThermoFisher). After 1 day cells were expanded to a
20 mL. 2 Days hereafter the cells were placed under blasticidin (5
.mu.g/mL) selection. Transfected cells were further cultured
according to manufacturer instructions until the constructs were
stably integrated into the HEK genome. Cell were expanded (to
1.1*10.sup.6 cells/mL) and after 7 days of protein production, the
cells were spun down at 2000.times.g for 5 minutes. Hereafter the
supernatant was collected and benzamine (0.174 mg/mL) was added,
where after the supernatant was stored at -20.degree. C. until
further use.
[0124] Nanobody-mUPA purification
[0125] Collected supernatant (400 mL) was concentrated on a
Quixstand to 150 mL using a 10 kDA cutoff membrane (GE healthcare).
Hereafter the concentrate was dialyzed against 2L of 1.times. STREP
buffer containing benzamidine (100 mM Tris, 150 mM NaCl, 0.174
mg/mL Benzamidine, pH 8.0). The concentrate was flowed over a
column containing 8 mL Strep-Tactin superflow beads (IBA). After
washing the column with 20 mL of 1.times. STREP buffer, the protein
was eluted via d-Desthiobiotin (2.5 mM; Sigma Aldrich) in 1.times.
STREP buffer. The purified proteins were dialyzed against 2L sodium
acetate (4 mM sodium acetate, 150 mM NaCl, pH 5.4) and stored at
-80.degree. C.
Results
Purified Nanobody-mUPA Constructs on Western Blot
[0126] The nanobody-mUPA constructs were diluted in sample buffer
(25 mM DTT) to a concentration of 100 .mu.g/mL. 10 .mu.L sample was
loaded onto a 4-12% gradient Bis-Tris gel in MES buffer. Sample
separation was performed at 165 Volt for 50 minutes. The gel is
transferred onto a Immobilon-FL membrane in blotting buffer for 1
hour at 125 Volt. The membrane is blocked with 0.5.times. Odyssey
blocking buffer where after the constructs are detected with a
rabbit polyclonal anti human UPA antibody in combination with an
1R800 labeled Goat-anti-Rabbit antibody. Results were analyzed via
the near-infrared odyssey scanner (Licor) according to manufacturer
instructions. FIG. 2 shows that all fusion proteins except for
A11-mUPA were expressed at near-equal levels.
Urokinase Activity of Nanobody-mUPA Constructs
[0127] The nanobody-mUPA constructs should display no spontaneous
(i.e. non-induced) activity but should be activatable through
molecular cleavage by plasmin.
[0128] To verify that none of the nanobody-mUPA constructs shows
any spontaneous activity, the constructs (1 .mu.g/mL) were
incubated in 0.2% BSA-HBS in the presence of a 0.5 mM urokinase
substrate (11140; Bachem). Substrate conversion was measured
according to manufacturer instruction at 37.degree. C. None of the
fusion proteins showed any detectable spontaneous activity towards
the urokinase substrate (data not shown).
[0129] To test whether nanobody-mUPA constructs are activatable by
plasmin, plasminogen was pre-activated by streptokinase for 15
minutes at 37.degree. C. (hereafter called plasmin). The
nanobody-mUPA fusion constructs (1 .mu.g/mL final concentration)
were diluted in in 0.2% BSA-HBS and incubated for 12 minutes with
plasmin (1 .mu.g/mL final concentration). Subsequently, urokinase
substrate (0.5 mM 11140, Bachem) was added and its conversion was
measured according to manufacturer's instructions at 37.degree. C.
FIG. 3 shows that the fusion proteins are normally activatable.
Plasminogen Activation
[0130] The basis of plasminogen activation by urokinase depends on
the reciprocal cleavage between urokinase and plasminogen. To test
plasminogen activation by the nanobody-mUPA constructs, the
constructs (1 .mu.g/mL) were incubated in 0.2% BSA-HBS in the
presence of 100 .mu.g/mL plasminogen and 0.2 mM plasmin substrate
(11390; Bachem). Substrate conversion was measured according to
manufacturer instruction at 37.degree. C. FIG. 4 shows that all
constructs showed development of comparable enzyme activity.
Binding of Anti-VWF Constructs to VWF as Determined by ELISA
[0131] A nunc maxisorp plate (Thermo) was coated overnight with 1
.mu.g/mL VWF in PBS. The following day the plate was blocked with
1% BSA-PBS. The anti-VWF nanobody-mUPA fusion constructs were
diluted in 1% BSA-PBS at various concentrations after which 50 uL
is added to each well and incubated for 1 hours. Hereafter the
plate is washed with PBS-Tween 20 (PBST; 0.05% v/v). Bound
construct was detected via rabbit anti-UPA polyclonal antibody in
combination Goat-anti-rabbit-HRP secondary antibody (Abcam). Wells
were rinsed with PBST, after which 100 .mu.l TMB was added at room
temperature. Substrate was developed for 5 minutes after which 50
.mu.l H.sub.2SO.sub.4 (0.3 M) was added. Results were analyzed at
450 nm by absorption. Results were analyzed by Graphpad Prism 7.02
and the K.sub.d (in nM) was determined via non-linear regression
curve fit. Binding affinities are listed in Table 2.
TABLE-US-00005 TABLE 2 Binding affinities of anti-VWF nanobody-mUPA
fusion constructs to VWF as determined by ELISA. Construct K.sub.d
(nM) Standard deviation sVWF-mUPA 0.03055 0.02491 D3-mUPA 0.3279
0.2005 A12-mUPA 12.84 6.571 R2-mUPA 1039010 1798731
Plasminogen Activation in the Presence of Globular or Open VWF
[0132] VWF is the scaffold for microthrombus formation. It also has
plasmin(ogen) binding properties that are dependent on protein
conformation (Tersteeg et al., 2014, supra). VWF unfolds under
shear stress (as well as during immobilization on microtiter plates
see previous experiment). This can be mimicked by incubation with
the small molecule ristocetin. We asked whether plasminogen
activation by our targeted fusion proteins is influenced by the
conformation of VWF. The nanobody fusion constructs (0.25 .mu.g/mL)
and VWF (5 .mu.g/mL) are diluted in 0.2% BSA-HBS. Hereafter,
ristocetin (0.6 mg/mL) or buffer is added to open up the VWF or
keep it globular, respectively, while incubating at 37.degree. C.
for 5 minutes. Hereafter plasminogen is added (100 .mu.g/mL)
followed by plasmin substrate (11390 Bachem; 0.2 mM). Substrate
conversion was measured according to manufacturer's instructions at
37.degree. C. For comparison, the substrate at conversion after 5
minutes was shown for the different constructs. In the bar graph
below. Data was processed in Graphpad Prism 7.02 and analyzed by
one-way Anova. * P<0.05. FIGS. 5 and 6 show that the time to
development of plasmin activity is significantly shorted by open
VWF (in the presence of ristocetin) as compared to closed VWF.
Micro-Thrombolysis of VWF-Platelet Agglutinates.
[0133] Blood platelets were isolated from citrated whole blood
according to earlier described methods (Tersteeg et al., 2014,
supra). Isolated blood platelets (200.000 /mL) were incubated with
VWF (5 .mu.g/mL), plasminogen (100 .mu.g/mL) and the aggregation
inhibitors RGDW (200 .mu.M) and Iloprost (0.4 .mu.g/mL) for 15
minutes at 37.degree. C. in a light transmission aggregometer.
Agglutination was induced by the addition of ristocetin (0.6
mg/mL). 6 minutes hereafter, the nanobody-mUPA constructs (1
.mu.g/mL) were added and lysis of the agglutinates was monitored
overtime (FIG. 7). For all samples, time points were determined by
which 50% microthrombus degradation has occurred (graphic example
of analytical method is shown in FIG. 8A). Results were processed
in Graphpad Prism 7.02 and analyzed by one-way Anova (* P<0.05)
and are shown in FIG. 8B. Clearly, the targeting of plasminogen
activation by at least the fusion proteins sVWF-mUPA, D3-mUPA and
GP1B17-mUPA accelerates microthrombolysis.
Example 2
Micro-Thrombolysis of VWF-Platelet on Endothelial Cells in Flow
Perfusion Materials and Methods
Human Vascular Endothelial Cell (Huvecs) Culture on Cover
Glasses
[0134] Huvecs (passage 0) stored in liquid nitrogen were thawed at
37.degree. C. and added to medium 1:10 (EBM-2 Lonza or Promocel
supplemented with huvec growth factors EGM2) and spun down 100 g
for 5 minutes. Supernatant was discarded and cells were taken up in
5 mL medium and cultured in T25 flasks at 37 degrees Celsius, 5%
CO.sub.2. The following day the cells were passed to 3.times. T75
flasks. On day 6 the cells are passed 1:6 to the cover glasses
pre-treated with 1,25% glutaraldehyde in HT-buffer pH 7.4 (HEPES
Tyrode buffer: 10 mM HEPES, 0.5 mM Na.sub.2HPO4, 145 mM NaCl, 5 mM
KCl, 1 mM MgSO4).
[0135] To coat cover glasses with glutaraldehyde, coverglasses were
rinsed with demi water and with ethanol. Cover glasses were then
incubated in HCL 37% : methanol (1:1) for 30 minutes followed by a
rinse with demi water for 5 minutes. Next cover glasses were
incubated in aminopropyltriethoxysilane : ethanol (1:100) for 30
seconds followed by a rinse with demi water and with ethanol. Cover
glasses were then dried and incubated with glutaraldehyde 20% :
HT-buffer pH 7.4 (1:20) for 1 hour followed by a rinse with demi
water and glasses are stored in ethanol until use.
[0136] Huvecs were cultured on the cover glasses for approximately
10-15 days prior to use.
Heat Inactivated Plasma Preparation
[0137] Heat inactivated plasma was prepared by mixing two bags of
plasma (Ominplasma from octapharma both bloodtype AB Lot No:
C442A9521, bags: X000214223782; X000214223577). 200 mL of this mix
was divided over 10 falcon tubes (50mL, 20mL each) and were
incubated in a water bath set at exactly 56.degree. C. for 30
minutes (the 20 mL was completely submerged) and halfway through
(15 min) the falcon tubes were mixed. Following the 30 min
incubation the falcon tubes were covered in ice and kept on ice
until centrifuging (note: when removing the tubes from ice for
centrifugation the tubes were still rather warm). The tubes were
centrifuged 5 minutes (without cooling) at 15.000 g. The
supernatant was combined and kept on ice until aliquoting in 1 mL
aliquots.
Flow Chamber Setup
[0138] A laminar-flow perfusion chamber was filled with pre-warmed
medium to remove all air from the tubing prior to placing the
coverslips. The inlet tube was cut to a length that corresponds
with a volume of 90,6 .mu.L so that during the perfusion the fluid
that enters the inlet arrives into the perfusion chamber after
exactly 1 minute. The syringe that is used has a diameter of 16 mm
(only for braun 12 ml syringes) and the syringe pump is set to 90,6
.mu.L per minute as this (with 3 mm tubing) results in a shear rate
of 300 s.sup.-1. The huvec coverslip is placed on the medium,
attached with the vacuum set at 10 bar and the perfusion chamber is
placed under an inverted microscope (Zeiss observer Z.1, Carl
Zeiss) with heating module that keeps the perfusion chamber at
37.degree. C. Any remaining air bubbles are removed by perfusing
medium.
Washed Platelets in Heat Inactivated Plasma
[0139] Blood from healthy consenting volunteers was collected into
0.1 volume 3.2% 10.9 mM trisodium citrate. Platelet-rich plasma
(PRP) was obtained by centrifugation (160 g for 15 min at RT). PRP
supplemented with 10% (v/v) acid citrate dextrose, 85 mM
tri-sodiumcitrate, 71 mM citric acid, 111 mM D-glucose, was
centrifuged (400 g for 15 min at RT) and platelets were resuspended
in HEPES tyrode buffer pH 6.5 containing 0.145M NaCl, 5mM KCl,
0.5mM Na.sub.2HPO.sub.4, 1mM MgSO.sub.4, 10mM HEPES and 5.5 mM
D-glucose. 10 .mu.g/mL PGl.sub.2 was added to the platelet
suspension prior to another centrifugation step (400 g for 15 min
at room temperature) and platelets were resuspended in heat
inactivated plasma and platelet count was adjusted to a final count
of 200 G/L.
Perfusion Experiment Setup
[0140] The heat inactivated plasma containing 200 G/L platelets
(that is pre-warmed in 37.degree. C. waterbath) is divided over 2
mL eppendorfcups (40 min experiment.fwdarw.2.times.2mL required,
etc). and prior to the start to all eppendorfcups illoprost (8
.mu.L (250 fold dilution) of 0.1 mg/mL stock is added, final
concentration 0.4 .mu.g/mL, Bayer Schering Pharma AG) is added
first, followed by histamine (4 .mu.L (500 fold dilution) of 500
.mu.M stock in medium, final concentration 100 .mu.M). The
perfusion is started immediately after the addition of Iloprost and
histamine by transferring the inlet tube from the 2 mL eppendorfcup
containing medium to the eppendorfcup containing heat inactivated
plasma with platelets by squeezing the tube to ensure no air is
introduced. The experiment is started with frame 1 after
approximately 1 minute, when the first platelets enter the flow
chamber (visible change). After 7 minutes the constructs are added
to the remaining (2000-634.2=1365.8 .mu.L because 7*90.6=634 .mu.L
is used at this point) by pipetting the required volume directly
into the eppendorfcup and mixing with a plastic pipette, final
concentration is 10 .mu.g/mL. At this point the construct is also
added to the additional Eppendorf cups to make sure the
pre-incubation time with plasma remains equal. During the perfusion
the 2mL Eppendorf cup needs to be re-filled by carefully adding
plasma with a plastic pipette drop by drop. This refill is done at
frames 150, 250, etc. so that potential disturbances due to the
refilling can be matched to the corresponding frames/time points.
During the experiment every 5 seconds a DIC image is made and at
the end of the experiment 5 screenshots are made of other regions
in the perfusion chamber and the number of platelet-VWF complexes,
visible in the form of "platelet-strings" are counted.
Results
[0141] The results are shown in FIG. 9, which shows that targeted
plasminogen activation by at least the fusion proteins GP1B17-mUPA,
sVWF-mUPA and D3-mUPA accelerates thrombolysis of platelet-VWF
complexes bound to Huvecs, as compared to the control fusion
protein R2-mUPA. In particular fusion protein GP1B17-mUPA, rapidly
clears platelet-VWF complexes, which demonstrates that in
particular targeting of platelets is efficient in clearing
platelet-VWF complexes in microthrombi.
Example 3
Methods and Materials
Caplacizumab Production
Cloning
[0142] Caplacizumab (a bi-valent variant of the Cablivi VHH) was
produced in E.coli and purified via HIS-tag affinity
chromatography. The Caplacizumab protein sequence was derived from
its EMA assessment report (EMA/490172/2018; Procedure No.
EMEA/H/C/004426/0000) and codon optimized for E.coli expression via
the integrated DNA technologies (IDT) codon optimized tool.
N-terminal BamHl and C-terminal Notl digestion sites were added to
the constructs and ordered as a double stranded DNA fragment from
IDT (SEQ ID NO:40).
[0143] The DNA fragment were dissolved in 5 mM Tris-buffer (pH=8.5)
and heated at 50.degree. C. for 20 minutes. Hereafter the DNA
fragment was ligated into the pJET1.2 vector (CloneJET PCR Cloning
Kit; Thermo Fisher) according to manufacturer instructions. The
ligated product was transformed into chemically competent E.coli
TOP10 (Thermo Fisher) via heat shock according to manufacturer
instructions. Transformed bacteria were cultured in 10 mL
2.times.YT media (containing 100 .mu.g/mL ampicillin) and grown
overnight at 37.degree. C. Plasmid DNA was isolated via the plasmid
isolation kit (M&N) according to manufacturer instructions. The
insert was digested via BamHl-HF and Notl-HF (NEB) in Cutsmart
buffer and separated on 0.7% (w/v) agarose gel (1.times.TBE buffer;
1:10.00 gel red) at 130V for 1 hours. The insert was excised from
gel and purified via the PCR&Gel cleanup kit (M&N)
according to manufacturer instructions.
[0144] The purified insert was ligated into the pTH4.0 vector. The
pTH4.0 vector is a modified pET32a(+) vector encoding an N-terminal
PeIB signal peptide; a His6 tag for purification purposes and a
sequence encoding a cleavage site for the tobacco etch virus (TEV)
protease followed by a BamHl digestion site. After the C-terminal
Notl digestion a myc-tag was placed for detection purposes followed
by a stop-codon (Table 1). The pTH4.0 vector was digested via
BamHI-HF and Notl-HF and subsequently purified as described for the
fragments. The fragment were ligated into the digested pTH4.0
vector in a 3:1 ratio via T4 ligase in 1.times. T4 ligate buffer
according to manufacturer instructions. The ligation mixture was
transformed in TOP10 bacteria as described before, and the
transformed bacteria were grown on YT-agar plate (100 .mu.g/mL
ampicillin, 2% (w/v) glucose) overnight at 37.degree. C. Colonies
were picked, and grown in 10 mL 2.times.YT media (containing 100
.mu.g/mL ampicillin, 2% (w/v) glucose). Plasmid DNA was isolated as
described before. The DNA sequence was confirmed by
sanger-sequencing via Macrogen (SEQ ID NO: 41).
Production
[0145] Caplacizumab in pTH4.0 plasmid DNA was transformed into
chemically competent BL21 pLysS E.coli (Thermo) according to
manufacturer instructions. Transformed bacteria were cultured
overnight at 37.degree. C. in 10 mL 2.times.YT media (containing
100 .mu.g/mL ampicillin, 34 .mu.g/mL chloramphenicol and 2% (w/v)
glucose). The overnight was diluted 1:10 in 2.times.YT media
(containing 100 .mu.g/mL ampicillin, 34 .mu.g/mL chloramphenicol
and 2% (w/v) glucose) and grown for 3 hours at 37.degree. C.
Hereafter the culture was diluted 1:100 in 2.times.YT media
(containing 100 .mu.g/mL ampicillin, 34 .mu.g/mL chloramphenicol)
and grown at 37.degree. C. for 3 hours. When the bacteria reached
OD600nm=0.6, protein production was induced by the addition of
Isopropyl .beta.-D-1-thiogalactopyranoside (0.1 mM final
concentration). Protein production was performed overnight at
24.degree. C. Bacteria were pelleted at 5000.times.g for 15 minutes
and the supernatant was discarded. The bacteria pellet from 400 ml
culture was resuspended in 25 mL dulbecco's phosphate buffer saline
(PBS; 137 mM NaCl, 2.7mM KCl, 1.5 mM KH2PO4, 8.2 mM Na2HPO4,
pH=7.4) and frozen at -20.degree. C.
Purification
[0146] The frozen bacteria were thawed at 37.degree. C. and
pelleted at 10.000.times.g for 15 min by centrifugation. The
supernatant was transferred to new tubes. 5 mL of Cobalt-sepharose
beads (TALON Superflow, G&E Heatlhcare; 50% solution;) were
washed by PBS according to manufacturer instructions, added to the
supernatant and incubated for 2 hours at room temperature on a
roller bench. The TALON was pelleted at 1000.times.g for 5 minutes
by centrifugation. Supernatant was discarded and the pellets were
dissolved in 20 mL of PBS. The TALON washing was repeated three
times in total. After the last step, the TALON was dissolved in 8
mL of PBS and loaded into a PD-10 column. The column was rinsed
with an excess of PBS. The column was eluted with Imidazole (150 mM
in PBS) and 0.5 mL fractions were collected. Protein containing
fraction were pooled and dialyzed overnight against HEPES-buffered
saline (HBS: 10mM HEPES, 150 mM NaCl, pH=7.4) via a 3.500 MWCO
dialysis membrane (3 RC tubing; Spectra/Por). Protein concentration
was determined by absorption at 280 nm on the DeNovix
Spectrophotometer (DS-11), where after the concentrations were
corrected for their extinction coefficient (calculated via
ProtParam). Purity was assessed by SDS-PAGE with Coomassie Page
Blue staining.
Microthrombolysis of VWF-Platelet Agglutinates.
[0147] Blood platelets were isolated from citrated whole blood
according to earlier described methods (Tersteeg et al., 2014,
supra). Isolated blood platelets (200.000 /.mu.L) were incubated
with VWF (5 .mu.g/mL), plasma-purified plasminogen (100 .mu.g/mL)
and the aggregation inhibitors RGDW (200 .mu.M) and Iloprost (0.4
.mu.g/mL) for 15 minutes at 37.degree. C. in a light transmission
aggregometer. Agglutination was induced by the addition of
ristocetin (0.6 mg/mL). 6 minutes hereafter, the nanobody-mUPA
constructs or Caplacizumab were added and lysis of the agglutinates
was monitored overtime. For all samples, time points were
determined by which 50% microthrombus degradation has occurred
(graphic example of analytical method is shown in FIG. 8A). Results
were processed in Graphpad Prism 7.02 and analyzed by one-way ANOVA
(* P<0.05) and are shown in FIG. 10.
Results
[0148] Results are shown in FIG. 10 wherein it can be clearly seen
that the targeting of plasminogen activation by at least the fusion
proteins, D3-mUPA and GP1 B17-mUPA accelerates microthrombolysis as
compared to the microthrombolysis induced by Caplacizumab.
TABLE-US-00006 TABLE 3 Description of the sequences SEQ ID NO: Seq
name Sequence 1 hmUPA
LKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLIS
PCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSA
DTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFG
KENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKT
DSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPW IRSHTKEENGLAL 2
mmUPA QGFQCGQKALRPRFKIVGGEFTEVENQPWFAAIYQKNKGGSPPSFKCGGS
LISPCWVASAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLILHEY
YREDSLAYHNDIALLKIRTSTGQCAQPSRSIQTICLPPRFTDAPFGSDCEITGF
GKESESDYLYPKNLKMSVVKLVSHEQCMQPHYYGSEINYKMLCAADPEW
KTDSCKGDSGGPLICNIEGRPTLSGIVSWGRGCAEKNKPGVYTRVSHFLD WIQSHIGEEKGLAF 3
secretion signal METDTLLLWVLLLWVPGSTGD peptide 4 2xSTREP
GSSAWSHPQFEKGSSAWSHPQFEK 5 TEV EFENLYFQS 6 LINKER SAAGGGGSGGGGSAAA
7 sVWF-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRTFSSNAMGWFRQAPGKE
REFVAAISWSGGSTYYLDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY
CAGSAGLGYVGDPDAMDYWGKGTQVTVSSSAAGGGGSGGGGSAAALK
FQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 8
D3-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGQTLSNYVMGWFRQAPGKE
REFVAVISRVGGSTSYADSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYY
CAAAYTIAVVTAMREYDFWGQGTQVTVSSSAAGGGGSGGGGSAAALKF
QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 9
R2-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSQVQLQESGGGLVQAGGSLRLSCAASGRATSGHGHYGMGWFRQV
PGKEREFVAAIRWSGKETWYKDSVKGRFTISRDNAKTTVYLQMNSLKPED
TAVYYCAARPVRVDDISLPVGFDYWGQGTQVTVSSSAAGGGGSGGGGSA
AALKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSL
ISPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYS
ADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGF
GKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQW
KTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLP WIRSHTKEENGLAL
10 A11-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSAVQLVESGGRLVKAGASLRLSCAASGRTFSSLPMAWFRQAPGKER
EFVAFIGSDSSTLYTSSVRGRFTISRDNGKNTVYLQMMNLKPEDTAVYYCA
ARSSAFSSGIYYREGSYAYWGQGTQVTVSSSAAGGGGSGGGGSAAALKF
QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 11
A12-hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSQVQLVESGGGLVQAGGSLRLSCTASGRTFSTYALGWFRQVPGKGR
EFIAVIYWRDGSSLYSDSVKGRFTISKDNAKNTVYLQMNSLKPEDTAVYYC
ANRHDSRGTYYSSRGYDYWGQGTQVTVSSSAAGGGGSGGGGSAAALKF
QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 12
GP1B-17 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASDIFSINAMGWYRQAPGKQRE
LVASITRGGDPWYADSVKGRFTISRDGAKNARNTVYLQMNSLKPEDTAVY
YCNAMGIRGSGGDYAREAGGQGTQVTVSSSAAGGGGSGGGGSAAALKF
QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 13
12A5 AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVAT
ITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQ
GSYGYRFNDYWGQGTQVTVSS 14 12A5H1
EVQLVESGGGLVQPGGSLRLSCAASGRIFSIGAMGMYRQAPGKGRELVAT
ITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLRAEDTAVYYCYANLKQ
GSYGYRFNDYWGQGTQVTVSS 15 R2-mmUPA
METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSQVQLQESGGGLVQAGGSLRLSCAASGRATSGHGHYGMGWFRQV
PGKEREFVAAIRWSGKETWYKDSVKGRFTISRDNAKTTVYLQMNSLKPED
TAVYYCAARPVRVDDISLPVGFDYWGQGTQVTVSSSAAGGGGSGGGGSA
AAQGFQCGQKALRPRFKIVGGEFTEVENQPWFAAIYQKNKGGSPPSFKC
GGSLISPCWVASAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLIL
HEYYREDSLAYHNDIALLKIRTSTGQCAQPSRSIQTICLPPRFTDAPFGSDCEI
TGFGKESESDYLYPKNLKMSVVKLVSHEQCMQPHYYGSEINYKMLCAADP
EWKTDSCKGDSGGPLICNIEGRPTLSGIVSWGRGCAEKNKPGVYTRVSHFL DWIQSHIGEEKGLAF
16 uPA connecting ADGKKPSSPPEELKFQCGQKTLRPRFK peptide 17 tPA
connecting STCGLRQYSQPQFR peptide 18 Plasmin PSFDCGKPQVEPKKCPGR
connecting peptide 19 tPA calatytic
STCGLRQYSQPQFRIKGGLFADIASHPWQAAIFAKHRRSPGERFLCGGILIS domain
sequence SCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEEEQKFEVEKYIVHKEFDD
DTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDWTECELSGYGK
HEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSGGPQ
ANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVT NYLDWIRDNMRP 20
GPB1-1- hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQAPGKG
LEWVSAINTGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYY
CAKDLPNSDSLGYDYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCG
QKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS
ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH
NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDY
LYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGD
SGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKE ENGLAL 21
GPB1-2- hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLICVGSDRIFSNYSMGWFRQAPGKE
RQFVSTISRHGTSTAYADSVRGRFTISRDNAENIVYLQMNSLEPEDTAVYYC
AARPHTQHYVRVESYGVWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQ
CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV
ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH
HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST
DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ
GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 22
GPB1-3- hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGSINSIRAMGWYRQPPGKQR
ELVATITRDGRTNYPDSVKGQFTISIDNARNTVSLQRNSLKPEDTAVYYCVA
DWGEGYLTRVWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCGQKTL
RPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHC
FIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIA
LLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPE
QLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGG
PLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENG LAL 23 GPB1-4
hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASETFSIRAMGWYRQAPGKQRE
LVAYITSGGSTNYADSVKGRFTISRDNDRNTVSLQMNSLKPEDTAVYYCYQ
APRSGYDPVYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCGQKTLR
PRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFI
DYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALL
KIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQL
KMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLV
CSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL 24 GPB1-5 hmUPA
METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASEFTFSKHWMYWVRQAPGKG
LEWVSGINLGGDSTYYADSVKGRFTISRDNAKNTLYLQMDSLKSEDTAVYY
CAKGASSWFGDFGSWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCG
QKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS
ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH
NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDY
LYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGD
SGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKE ENGLAL 25 GPB1-6
hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFAMNWVRQAPGKG
LEWVSFINRGGGSTGYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVY
YCAKFSRSVPPYYGMDYWGKGTLVTVSSSAAGGGGSGGGGSAAALKFQC
GQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVI
SATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH
HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST
DYLYPEQLKMTVVKLISHRECQQPHYYGSEVITKMLCAADPQWKTDSCQ
GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 26
GPB1-7 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRVDSMAWFRQAPGKEREF
VATITWSDSKIYYADSVKGRFTISGERAKNTMYLQMNTLRPEDTAVYYCAA
AHRPYRSGYYYMQSRYDYWGQGTQVTVSSSAAGGGGSGGGGSAAALKF
QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 27
GPB1-8 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAAPSMFSINAMGWYRQAPGRQR
ELVATITSGDSTYYADSVKGRFTISRDNAKYTKNTVYLQMNSLKPEDTAVYY
CNAAHIRGSGGDYAREAWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQ
CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV
ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH
HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST
DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ
GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 28
GPB1-9 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGPTVSNYYMGWFRQAPGKE
RDFVAGISRSGVEKYYADSVKGRFTISRDNALNTVYLQMNSLKPEDTAAYY
CAARERVGITFAHSTVDYWGKGTLVTVSSSAAGGGGSGGGGSAAALKFQ
CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV
ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH
HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST
DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ
GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 29
GPB1-10 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSKYGMSWVRQAPGKGL
EWVSIIDSGGGAIGYADAVKGRFTISRDNVKNTLYLQMNSLKPEDTAVYHC
VFGDYKGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCGQKTLRPRFKIIG
GEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVISATHCFIDYPKKE
DYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHHNDIALLKIRSKE
GRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDYLYPEQLKMTV
VKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQGDSGGPLVCSLQ
GRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKEENGLAL 30 GPB1-11 hmUPA
METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSSAMTWVRQAPGKGL
EWVSAINSGGSGTRYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYY
CAKRRDGQNWYPGISYESMYRGQGTQVTVSSSAAGGGGSGGGGSAAAL
KFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISP
CWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSAD
TLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWI R SHTKEENGLAL 31
GPB1-12 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRTFSSYTMAWFRQAPGKER
EFVGLISWNAKSTYVTDSVKGRFTITRENAKDMVYLQMNSLKPEDSATYYC
AANRYGSSVPGAYNYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQCG
QKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVIS
ATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAHH
NDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENSTDY
LYPEQLKMTVVKLISHRECQQPHYYGSEVITKMLCAADPQWKTDSCQGD
SGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHTKE ENGLAL 32
GPB1-13 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGL
EWVSAINMGGGSTYYADSVKGRFTISRDNAKNTLYLQMSGLKPEDTALYY
CVRGGSAYSVRYEYAYWGQGTQVTVSSSAAGGGGSGGGGSAAALKFQC
GQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWVI
SATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH
HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST
DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ
GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 33
GPB1-14 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAAAASWFSIYAMGWYRQAPGKQ
RELVAIILSDGDTDYADSVKGRFTISRDNAKNTKNTVYLQMNSLKPEDTAV
YYCNARGIRGSGGDYAREAWGQGTQVTVSSSAAGGGGSGGGGSAAALK
FQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 34
GPB1-15 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGSMFSINDMGWYRQAPGK
QRELVATITRGGNTYYADSVKGRFTISRDNATYTKNTVYLQMNSLKPEDTA
VYYCNARHIRGSGGDYAREAWGQGTQVTVSSSAAGGGGSGGGGSAAAL
KFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISP
CWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSAD
TLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 35
GPB1-16 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASRRTFSNYVMGWFRQAPGKE
RESVTAIGRSGTILYADSMKGRITISRDNAKNTVYLQMNSLTPDDTAVYYC
AASSGSMQQFWRMEYDYEGQGTQVTVSSSAAGGGGSGGGGSAAALKF
QCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPC
WVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADT
LAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKE
NSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDS
CQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIR SHTKEENGLAL 36
GPB1-19 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQAGGSLRLSCAASGRTFGSYVMGWFRQAPGKE
REFVAAIGRSGTTYYLDSVKGRFTISRDNAKNTVYLQMNSLKSEDTAVYYC
GASLKGTVLGIARYEYDVRGQGTQVTVSSSAAGGGGSGGGGSAAALKFQ
CGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLISPCWV
ISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYSADTLAH
HNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGFGKENST
DYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQWKTDSCQ
GDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLPWIRSHT KEENGLAL 37
GPB1-20 hmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGSVQAGGSLRLSCAASGRTLSSLAMGWFRQAPGKER
EFVAADRRNGGYTVVADYTDSVKGRFTIFRDNAKNTVYLQMNNLKPEDT
AVYYCAADSDRTMSLRSTDYDYWGQGTQVIVSSSAAGGGGSGGGGSAA
ALKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLI
SPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYS
ADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGF
GKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQW
KTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLP WIRSHTKEENGLAL
38 Flexible linker KESGSVSSEQLAQFRSLD 39 Flexible linker
EGKSSGSGSESKST 40 Caplacizumab
GGATCCGAAGTCCAGCTTGTAGAATCAGGAGGAGGCCTTGTCCAGCCA double stranded
GGTGGTAGCCTTCGTCTGTCGTGTGCTGCTTCGGGCCGCACATTTTCGT DNA fragment
ATAACCCTATGGGTTGGTTTCGCCAAGCACCCGGCAAGGGACGCGAGT
TGGTGGCCGCGATTAGTCGTACGGGTGGTTCCACCTACTACCCGGATTC
AGTGGAAGGACGCTTTACGATTAGCCGTGATAACGCGAAGCGTATGGT
CTACTTACAGATGAATAGCTTGCGCGCGGAAGACACCGCGGTATACTA
TTGTGCTGCAGCAGGAGTCCGTGCTGAGGATGGACGCGTCCGCACGTT
ACCTAGTGAGTATACATTCTGGGGCCAGGGCACCCAAGTTACCGTATCC
AGTGCAGCAGCGGAAGTACAACTGGTCGAATCTGGAGGAGGACTTGT
ACAACCAGGGGGTTCCTTACGTTTGTCATGTGCGGCAAGTGGGCGCAC
ATTTAGTTACAACCCTATGGGCTGGTTCCGTCAAGCCCCGGGAAAAGG
GCGCGAACTTGTAGCCGCCATTTCGCGTACAGGGGGAAGTACCTATTA
CCCGGACTCAGTAGAGGGACGCTTCACGATTTCTCGTGACAACGCAAA
GCGCATGGTTTATCTGCAAATGAATAGTTTACGCGCCGAAGATACAGC
AGTTTACTATTGCGCCGCAGCTGGAGTCCGCGCCGAAGACGGCCGTGT
ACGCACCTTGCCTTCTGAATACACTTTTTGGGGTCAAGGAACACAGGTG
ACCGTGTCATCTGCGGCCGC 41 Caplacizumab in
MKYLLPTAAAGLLLLAAQPAMAQSGHHHHHHHHDYDIPSSENLYFQGSE pTH4.0 protein
VQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAA sequence
ISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAG
VRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSL
RLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRF
TISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTF
WGQGTQVTVSSAAAEQKLISEEDL 42 Primer Fw TAATACGACTCACTATAGGG 43
Primer Rv GCTAGTTATTGCTCAGCGG 44 Cablivi-hmUPA
METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKG
RELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYY
CAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSSAAGGGGSGGGGSAA
ALKFQCGQKTLRPRFKIIGGEFTTIENQPWFAAIYRRHRGGSVTYVCGGSLI
SPCWVISATHCFIDYPKKEDYIVYLGRSRLNSNTQGEMKFEVENLILHKDYS
ADTLAHHNDIALLKIRSKEGRCAQPSRTIQTICLPSMYNDPQFGTSCEITGF
GKENSTDYLYPEQLKMTVVKLISHRECQQPHYYGSEVTTKMLCAADPQW
KTDSCQGDSGGPLVCSLQGRMTLTGIVSWGRGCALKDKPGVYTRVSHFLP WIRSHTKEENGLAL
45 Cablivi-mmUPA METDTLLLWVLLLWVPGSTGDGSSAWSHPQFEKGSSAWSHPQFEKEFEN
LYFQSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKG
RELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYY
CAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSSAAGGGGSGGGGSAA
AQGFQCGQKALRPRFKIVGGEFTEVENQPWFAAIYQKNKGGSPPSFKCG
GSLISPCWVASAAHCFIQLPKKENYVVYLGQSKESSYNPGEMKFEVEQLIL 46 Cablivi
EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVA
AISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAA
GVRAEDGRVRTLPSEYTFWGQGTQVTVSS
Sequence CWU 1
1
461268PRTArtificial SequencehmUPA 1Leu Lys Phe Gln Cys Gly Gln Lys
Thr Leu Arg Pro Arg Phe Lys Ile1 5 10 15Ile Gly Gly Glu Phe Thr Thr
Ile Glu Asn Gln Pro Trp Phe Ala Ala 20 25 30Ile Tyr Arg Arg His Arg
Gly Gly Ser Val Thr Tyr Val Cys Gly Gly 35 40 45Ser Leu Ile Ser Pro
Cys Trp Val Ile Ser Ala Thr His Cys Phe Ile 50 55 60Asp Tyr Pro Lys
Lys Glu Asp Tyr Ile Val Tyr Leu Gly Arg Ser Arg65 70 75 80Leu Asn
Ser Asn Thr Gln Gly Glu Met Lys Phe Glu Val Glu Asn Leu 85 90 95Ile
Leu His Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His Asn Asp 100 105
110Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro
115 120 125Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn
Asp Pro 130 135 140Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly
Lys Glu Asn Ser145 150 155 160Thr Asp Tyr Leu Tyr Pro Glu Gln Leu
Lys Met Thr Val Val Lys Leu 165 170 175Ile Ser His Arg Glu Cys Gln
Gln Pro His Tyr Tyr Gly Ser Glu Val 180 185 190Thr Thr Lys Met Leu
Cys Ala Ala Asp Pro Gln Trp Lys Thr Asp Ser 195 200 205Cys Gln Gly
Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg 210 215 220Met
Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys225 230
235 240Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp
Ile 245 250 255Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 260
2652269PRTArtificial SequencemmUPA 2Gln Gly Phe Gln Cys Gly Gln Lys
Ala Leu Arg Pro Arg Phe Lys Ile1 5 10 15Val Gly Gly Glu Phe Thr Glu
Val Glu Asn Gln Pro Trp Phe Ala Ala 20 25 30Ile Tyr Gln Lys Asn Lys
Gly Gly Ser Pro Pro Ser Phe Lys Cys Gly 35 40 45Gly Ser Leu Ile Ser
Pro Cys Trp Val Ala Ser Ala Ala His Cys Phe 50 55 60Ile Gln Leu Pro
Lys Lys Glu Asn Tyr Val Val Tyr Leu Gly Gln Ser65 70 75 80Lys Glu
Ser Ser Tyr Asn Pro Gly Glu Met Lys Phe Glu Val Glu Gln 85 90 95Leu
Ile Leu His Glu Tyr Tyr Arg Glu Asp Ser Leu Ala Tyr His Asn 100 105
110Asp Ile Ala Leu Leu Lys Ile Arg Thr Ser Thr Gly Gln Cys Ala Gln
115 120 125Pro Ser Arg Ser Ile Gln Thr Ile Cys Leu Pro Pro Arg Phe
Thr Asp 130 135 140Ala Pro Phe Gly Ser Asp Cys Glu Ile Thr Gly Phe
Gly Lys Glu Ser145 150 155 160Glu Ser Asp Tyr Leu Tyr Pro Lys Asn
Leu Lys Met Ser Val Val Lys 165 170 175Leu Val Ser His Glu Gln Cys
Met Gln Pro His Tyr Tyr Gly Ser Glu 180 185 190Ile Asn Tyr Lys Met
Leu Cys Ala Ala Asp Pro Glu Trp Lys Thr Asp 195 200 205Ser Cys Lys
Gly Asp Ser Gly Gly Pro Leu Ile Cys Asn Ile Glu Gly 210 215 220Arg
Pro Thr Leu Ser Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Glu225 230
235 240Lys Asn Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Asp
Trp 245 250 255Ile Gln Ser His Ile Gly Glu Glu Lys Gly Leu Ala Phe
260 265321PRTArtificial Sequencesecretion signal peptide 3Met Glu
Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly
Ser Thr Gly Asp 20424PRTArtificial SequenceSTREP 4Gly Ser Ser Ala
Trp Ser His Pro Gln Phe Glu Lys Gly Ser Ser Ala1 5 10 15Trp Ser His
Pro Gln Phe Glu Lys 2059PRTArtificial SequenceTEV 5Glu Phe Glu Asn
Leu Tyr Phe Gln Ser1 5616PRTArtificial Sequencelinker 6Ser Ala Ala
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala1 5 10
157462PRTArtificial SequencesVWF-hmUPA 7Met Glu Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Gly
Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser Ser Ala
Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu Tyr Phe
Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu Val Gln
Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75 80Arg
Thr Phe Ser Ser Asn Ala Met Gly Trp Phe Arg Gln Ala Pro Gly 85 90
95Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr
100 105 110Tyr Tyr Leu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn 115 120 125Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
Lys Pro Glu Asp 130 135 140Thr Ala Val Tyr Tyr Cys Ala Gly Ser Ala
Gly Leu Gly Tyr Val Gly145 150 155 160Asp Pro Asp Ala Met Asp Tyr
Trp Gly Lys Gly Thr Gln Val Thr Val 165 170 175Ser Ser Ser Ala Ala
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala Leu
Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200 205Lys
Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe 210 215
220Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val
Cys225 230 235 240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser
Ala Thr His Cys 245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr
Ile Val Tyr Leu Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr Gln
Gly Glu Met Lys Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His Lys
Asp Tyr Ser Ala Asp Thr Leu Ala His His 290 295 300Asn Asp Ile Ala
Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala305 310 315 320Gln
Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn 325 330
335Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu
340 345 350Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr
Val Val 355 360 365Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His
Tyr Tyr Gly Ser 370 375 380Glu Val Thr Thr Lys Met Leu Cys Ala Ala
Asp Pro Gln Trp Lys Thr385 390 395 400Asp Ser Cys Gln Gly Asp Ser
Gly Gly Pro Leu Val Cys Ser Leu Gln 405 410 415Gly Arg Met Thr Leu
Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala 420 425 430Leu Lys Asp
Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro 435 440 445Trp
Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
4608462PRTArtificial SequenceD3-hmUPA 8Met Glu Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Gly
Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser Ser Ala
Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu Tyr Phe
Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu Val Gln
Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75 80Gln
Thr Leu Ser Asn Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly 85 90
95Lys Glu Arg Glu Phe Val Ala Val Ile Ser Arg Val Gly Gly Ser Thr
100 105 110Ser Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn 115 120 125Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
Lys Pro Glu Asp 130 135 140Thr Ala Val Tyr Tyr Cys Ala Ala Ala Tyr
Thr Ile Ala Val Val Thr145 150 155 160Ala Met Arg Glu Tyr Asp Phe
Trp Gly Gln Gly Thr Gln Val Thr Val 165 170 175Ser Ser Ser Ala Ala
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala Leu
Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200 205Lys
Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe 210 215
220Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val
Cys225 230 235 240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser
Ala Thr His Cys 245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr
Ile Val Tyr Leu Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr Gln
Gly Glu Met Lys Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His Lys
Asp Tyr Ser Ala Asp Thr Leu Ala His His 290 295 300Asn Asp Ile Ala
Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala305 310 315 320Gln
Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn 325 330
335Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu
340 345 350Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr
Val Val 355 360 365Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His
Tyr Tyr Gly Ser 370 375 380Glu Val Thr Thr Lys Met Leu Cys Ala Ala
Asp Pro Gln Trp Lys Thr385 390 395 400Asp Ser Cys Gln Gly Asp Ser
Gly Gly Pro Leu Val Cys Ser Leu Gln 405 410 415Gly Arg Met Thr Leu
Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala 420 425 430Leu Lys Asp
Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro 435 440 445Trp
Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
4609466PRTArtificial SequenceR2-hmUPA 9Met Glu Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Gly
Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser Ser Ala
Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu Tyr Phe
Gln Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Gly 50 55 60Leu Val Gln
Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75 80Arg
Ala Thr Ser Gly His Gly His Tyr Gly Met Gly Trp Phe Arg Gln 85 90
95Val Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Arg Trp Ser Gly
100 105 110Lys Glu Thr Trp Tyr Lys Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser 115 120 125Arg Asp Asn Ala Lys Thr Thr Val Tyr Leu Gln Met
Asn Ser Leu Lys 130 135 140Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Ala Arg Pro Val Arg Val145 150 155 160Asp Asp Ile Ser Leu Pro Val
Gly Phe Asp Tyr Trp Gly Gln Gly Thr 165 170 175Gln Val Thr Val Ser
Ser Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly 180 185 190Gly Gly Ser
Ala Ala Ala Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu 195 200 205Arg
Pro Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn 210 215
220Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser
Val225 230 235 240Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Cys
Trp Val Ile Ser 245 250 255Ala Thr His Cys Phe Ile Asp Tyr Pro Lys
Lys Glu Asp Tyr Ile Val 260 265 270Tyr Leu Gly Arg Ser Arg Leu Asn
Ser Asn Thr Gln Gly Glu Met Lys 275 280 285Phe Glu Val Glu Asn Leu
Ile Leu His Lys Asp Tyr Ser Ala Asp Thr 290 295 300Leu Ala His His
Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu305 310 315 320Gly
Arg Cys Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro 325 330
335Ser Met Tyr Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly
340 345 350Phe Gly Lys Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln
Leu Lys 355 360 365Met Thr Val Val Lys Leu Ile Ser His Arg Glu Cys
Gln Gln Pro His 370 375 380Tyr Tyr Gly Ser Glu Val Thr Thr Lys Met
Leu Cys Ala Ala Asp Pro385 390 395 400Gln Trp Lys Thr Asp Ser Cys
Gln Gly Asp Ser Gly Gly Pro Leu Val 405 410 415Cys Ser Leu Gln Gly
Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly 420 425 430Arg Gly Cys
Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser 435 440 445His
Phe Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu 450 455
460Ala Leu46510464PRTArtificial SequenceA11-hmUPA 10Met Glu Thr Asp
Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr
Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly
Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn
Leu Tyr Phe Gln Ser Ala Val Gln Leu Val Glu Ser Gly Gly Arg 50 55
60Leu Val Lys Ala Gly Ala Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65
70 75 80Arg Thr Phe Ser Ser Leu Pro Met Ala Trp Phe Arg Gln Ala Pro
Gly 85 90 95Lys Glu Arg Glu Phe Val Ala Phe Ile Gly Ser Asp Ser Ser
Thr Leu 100 105 110Tyr Thr Ser Ser Val Arg Gly Arg Phe Thr Ile Ser
Arg Asp Asn Gly 115 120 125Lys Asn Thr Val Tyr Leu Gln Met Met Asn
Leu Lys Pro Glu Asp Thr 130 135 140Ala Val Tyr Tyr Cys Ala Ala Arg
Ser Ser Ala Phe Ser Ser Gly Ile145 150 155 160Tyr Tyr Arg Glu Gly
Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val 165 170 175Thr Val Ser
Ser Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly 180 185 190Ser
Ala Ala Ala Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro 195 200
205Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro
210 215 220Trp Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val
Thr Tyr225 230 235 240Val Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp
Val Ile Ser Ala Thr 245 250 255His Cys Phe Ile Asp Tyr Pro Lys Lys
Glu Asp Tyr Ile Val Tyr Leu 260 265 270Gly Arg Ser Arg Leu Asn Ser
Asn Thr Gln Gly Glu Met Lys Phe Glu 275 280 285Val Glu Asn Leu Ile
Leu His Lys Asp Tyr Ser Ala Asp Thr Leu Ala 290 295 300His His Asn
Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg305 310 315
320Cys Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met
325 330 335Tyr Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly
Phe Gly 340 345 350Lys Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln
Leu Lys Met Thr 355 360 365Val Val Lys Leu Ile Ser His Arg Glu Cys
Gln Gln Pro His Tyr Tyr 370 375 380Gly Ser Glu Val Thr Thr Lys Met
Leu Cys Ala Ala Asp Pro Gln Trp385 390 395 400Lys Thr Asp Ser Cys
Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Ser 405 410 415Leu Gln Gly
Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly 420 425 430Cys
Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr
Arg Val Ser His Phe 435 440 445Leu Pro Trp Ile Arg Ser His Thr Lys
Glu Glu Asn Gly Leu Ala Leu 450 455 46011463PRTArtificial
SequenceA12-hmUPA 11Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu
Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Gly Ser Ser Ala Trp Ser
His Pro Gln Phe Glu 20 25 30Lys Gly Ser Ser Ala Trp Ser His Pro Gln
Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu Tyr Phe Gln Ser Gln Val Gln
Leu Val Glu Ser Gly Gly Gly 50 55 60Leu Val Gln Ala Gly Gly Ser Leu
Arg Leu Ser Cys Thr Ala Ser Gly65 70 75 80Arg Thr Phe Ser Thr Tyr
Ala Leu Gly Trp Phe Arg Gln Val Pro Gly 85 90 95Lys Gly Arg Glu Phe
Ile Ala Val Ile Tyr Trp Arg Asp Gly Ser Ser 100 105 110Leu Tyr Ser
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn 115 120 125Ala
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 130 135
140Thr Ala Val Tyr Tyr Cys Ala Asn Arg His Asp Ser Arg Gly Thr
Tyr145 150 155 160Tyr Ser Ser Arg Gly Tyr Asp Tyr Trp Gly Gln Gly
Thr Gln Val Thr 165 170 175Val Ser Ser Ser Ala Ala Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 180 185 190Ala Ala Ala Leu Lys Phe Gln Cys
Gly Gln Lys Thr Leu Arg Pro Arg 195 200 205Phe Lys Ile Ile Gly Gly
Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp 210 215 220Phe Ala Ala Ile
Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val225 230 235 240Cys
Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His 245 250
255Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly
260 265 270Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys Phe
Glu Val 275 280 285Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp
Thr Leu Ala His 290 295 300His Asn Asp Ile Ala Leu Leu Lys Ile Arg
Ser Lys Glu Gly Arg Cys305 310 315 320Ala Gln Pro Ser Arg Thr Ile
Gln Thr Ile Cys Leu Pro Ser Met Tyr 325 330 335Asn Asp Pro Gln Phe
Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys 340 345 350Glu Asn Ser
Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val 355 360 365Val
Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His Tyr Tyr Gly 370 375
380Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln Trp
Lys385 390 395 400Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu
Val Cys Ser Leu 405 410 415Gln Gly Arg Met Thr Leu Thr Gly Ile Val
Ser Trp Gly Arg Gly Cys 420 425 430Ala Leu Lys Asp Lys Pro Gly Val
Tyr Thr Arg Val Ser His Phe Leu 435 440 445Pro Trp Ile Arg Ser His
Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455 46012462PRTArtificial
SequenceGP1B-17 hmUPA 12Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Gly Ser Ser Ala Trp
Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser Ser Ala Trp Ser His Pro
Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu Tyr Phe Gln Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu Val Gln Ala Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Asp65 70 75 80Ile Phe Ser Ile Asn
Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 85 90 95Gln Arg Glu Leu
Val Ala Ser Ile Thr Arg Gly Gly Asp Pro Trp Tyr 100 105 110Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Lys 115 120
125Asn Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
130 135 140Asp Thr Ala Val Tyr Tyr Cys Asn Ala Met Gly Ile Arg Gly
Ser Gly145 150 155 160Gly Asp Tyr Ala Arg Glu Ala Gly Gly Gln Gly
Thr Gln Val Thr Val 165 170 175Ser Ser Ser Ala Ala Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala Leu Lys Phe Gln Cys
Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200 205Lys Ile Ile Gly Gly
Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe 210 215 220Ala Ala Ile
Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys225 230 235
240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His Cys
245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu
Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys
Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His Lys Asp Tyr Ser Ala
Asp Thr Leu Ala His His 290 295 300Asn Asp Ile Ala Leu Leu Lys Ile
Arg Ser Lys Glu Gly Arg Cys Ala305 310 315 320Gln Pro Ser Arg Thr
Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn 325 330 335Asp Pro Gln
Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu 340 345 350Asn
Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val Val 355 360
365Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His Tyr Tyr Gly Ser
370 375 380Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln Trp
Lys Thr385 390 395 400Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu
Val Cys Ser Leu Gln 405 410 415Gly Arg Met Thr Leu Thr Gly Ile Val
Ser Trp Gly Arg Gly Cys Ala 420 425 430Leu Lys Asp Lys Pro Gly Val
Tyr Thr Arg Val Ser His Phe Leu Pro 435 440 445Trp Ile Arg Ser His
Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455 46013122PRTArtificial
Sequence12A5 13Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Leu Ala Ser Gly Arg Ile
Phe Ser Ile Gly 20 25 30Ala Met Gly Met Tyr Arg Gln Ala Pro Gly Lys
Gln Arg Glu Leu Val 35 40 45Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn
Tyr Ala Asp Pro Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Gly
Pro Lys Asn Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Lys Pro
Glu Asp Thr Ala Val Tyr Tyr Cys Tyr 85 90 95Ala Asn Leu Lys Gln Gly
Ser Tyr Gly Tyr Arg Phe Asn Asp Tyr Trp 100 105 110Gly Gln Gly Thr
Gln Val Thr Val Ser Ser 115 12014122PRTArtificial Sequence12A5H1
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 Arg Ile Phe Ser Ile
Gly 20 25 30Ala Met Gly Met Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu
Leu Val 35 40 45Ala Thr Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp
Pro Val Lys 50 55 60Gly Arg Phe Thr Ile Ser Arg Asp Gly Pro Lys Asn
Thr Val Tyr Leu65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Tyr 85 90 95Ala Asn Leu Lys Gln Gly Ser Tyr Gly
Tyr Arg Phe Asn Asp Tyr Trp 100 105 110Gly Gln Gly Thr Gln Val Thr
Val Ser Ser 115 12015467PRTArtificial SequenceR2-mmUPA 15Met Glu
Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly
Ser Thr Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25
30Lys Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu
35 40 45Asn Leu Tyr Phe Gln Ser Gln Val Gln Leu Gln Glu Ser Gly Gly
Gly 50 55 60Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly65 70 75 80Arg Ala Thr Ser Gly His Gly His Tyr Gly Met Gly
Trp Phe Arg Gln 85 90 95Val Pro Gly Lys Glu Arg Glu Phe Val Ala Ala
Ile Arg Trp Ser Gly 100 105 110Lys Glu Thr Trp Tyr Lys Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser 115 120 125Arg Asp Asn Ala Lys Thr Thr
Val Tyr Leu Gln Met Asn Ser Leu Lys 130 135 140Pro Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Ala Arg Pro Val Arg Val145 150 155 160Asp Asp
Ile Ser Leu Pro Val Gly Phe Asp Tyr Trp Gly Gln Gly Thr 165 170
175Gln Val Thr Val Ser Ser Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly
180 185 190Gly Gly Ser Ala Ala Ala Gln Gly Phe Gln Cys Gly Gln Lys
Ala Leu 195 200 205Arg Pro Arg Phe Lys Ile Val Gly Gly Glu Phe Thr
Glu Val Glu Asn 210 215 220Gln Pro Trp Phe Ala Ala Ile Tyr Gln Lys
Asn Lys Gly Gly Ser Pro225 230 235 240Pro Ser Phe Lys Cys Gly Gly
Ser Leu Ile Ser Pro Cys Trp Val Ala 245 250 255Ser Ala Ala His Cys
Phe Ile Gln Leu Pro Lys Lys Glu Asn Tyr Val 260 265 270Val Tyr Leu
Gly Gln Ser Lys Glu Ser Ser Tyr Asn Pro Gly Glu Met 275 280 285Lys
Phe Glu Val Glu Gln Leu Ile Leu His Glu Tyr Tyr Arg Glu Asp 290 295
300Ser Leu Ala Tyr His Asn Asp Ile Ala Leu Leu Lys Ile Arg Thr
Ser305 310 315 320Thr Gly Gln Cys Ala Gln Pro Ser Arg Ser Ile Gln
Thr Ile Cys Leu 325 330 335Pro Pro Arg Phe Thr Asp Ala Pro Phe Gly
Ser Asp Cys Glu Ile Thr 340 345 350Gly Phe Gly Lys Glu Ser Glu Ser
Asp Tyr Leu Tyr Pro Lys Asn Leu 355 360 365Lys Met Ser Val Val Lys
Leu Val Ser His Glu Gln Cys Met Gln Pro 370 375 380His Tyr Tyr Gly
Ser Glu Ile Asn Tyr Lys Met Leu Cys Ala Ala Asp385 390 395 400Pro
Glu Trp Lys Thr Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu 405 410
415Ile Cys Asn Ile Glu Gly Arg Pro Thr Leu Ser Gly Ile Val Ser Trp
420 425 430Gly Arg Gly Cys Ala Glu Lys Asn Lys Pro Gly Val Tyr Thr
Arg Val 435 440 445Ser His Phe Leu Asp Trp Ile Gln Ser His Ile Gly
Glu Glu Lys Gly 450 455 460Leu Ala Phe4651627PRTArtificial
Sequencehuman uPA connecting peptide 16Ala Asp Gly Lys Lys Pro Ser
Ser Pro Pro Glu Glu Leu Lys Phe Gln1 5 10 15Cys Gly Gln Lys Thr Leu
Arg Pro Arg Phe Lys 20 251714PRTArtificial Sequencehuman tPA
connecting peptide 17Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro
Gln Phe Arg1 5 101818PRTArtificial Sequencehuman plasminogen
connecting peptide 18Pro Ser Phe Asp Cys Gly Lys Pro Gln Val Glu
Pro Lys Lys Cys Pro1 5 10 15Gly Arg19266PRTArtificial SequencetPA
catalytic domain 19Ser Thr Cys Gly Leu Arg Gln Tyr Ser Gln Pro Gln
Phe Arg Ile Lys1 5 10 15Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro
Trp Gln Ala Ala Ile 20 25 30Phe Ala Lys His Arg Arg Ser Pro Gly Glu
Arg Phe Leu Cys Gly Gly 35 40 45Ile Leu Ile Ser Ser Cys Trp Ile Leu
Ser Ala Ala His Cys Phe Gln 50 55 60Glu Arg Phe Pro Pro His His Leu
Thr Val Ile Leu Gly Arg Thr Tyr65 70 75 80Arg Val Val Pro Gly Glu
Glu Glu Gln Lys Phe Glu Val Glu Lys Tyr 85 90 95Ile Val His Lys Glu
Phe Asp Asp Asp Thr Tyr Asp Asn Asp Ile Ala 100 105 110Leu Leu Gln
Leu Lys Ser Asp Ser Ser Arg Cys Ala Gln Glu Ser Ser 115 120 125Val
Val Arg Thr Val Cys Leu Pro Pro Ala Asp Leu Gln Leu Pro Asp 130 135
140Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys His Glu Ala Leu
Ser145 150 155 160Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His Val
Arg Leu Tyr Pro 165 170 175Ser Ser Arg Cys Thr Ser Gln His Leu Leu
Asn Arg Thr Val Thr Asp 180 185 190Asn Met Leu Cys Ala Gly Asp Thr
Arg Ser Gly Gly Pro Gln Ala Asn 195 200 205Leu His Asp Ala Cys Gln
Gly Asp Ser Gly Gly Pro Leu Val Cys Leu 210 215 220Asn Asp Gly Arg
Met Thr Leu Val Gly Ile Ile Ser Trp Gly Leu Gly225 230 235 240Cys
Gly Gln Lys Asp Val Pro Gly Val Tyr Thr Lys Val Thr Asn Tyr 245 250
255Leu Asp Trp Ile Arg Asp Asn Met Arg Pro 260
26520459PRTArtificial SequenceGPB1-1 - hmUPA 20Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Phe Thr Phe Ser Ser Tyr Trp Met Tyr Trp Val Arg Gln Ala Pro Gly
85 90 95Lys Gly Leu Glu Trp Val Ser Ala Ile Asn Thr Gly Gly Gly Ser
Thr 100 105 110Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn 115 120 125Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Lys Ser Glu Asp 130 135 140Thr Ala Val Tyr Tyr Cys Ala Lys Asp
Leu Pro Asn Ser Asp Ser Leu145 150 155 160Gly Tyr Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val Ser Ser Ser 165 170 175Ala Ala Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala Leu 180 185 190Lys Phe
Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile Ile 195 200
205Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala Ile
210 215 220Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys Gly
Gly Ser225 230 235 240Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr
His Cys Phe Ile Asp 245 250 255Tyr Pro Lys Lys Glu Asp Tyr Ile Val
Tyr Leu Gly Arg Ser Arg Leu 260 265 270Asn Ser Asn Thr Gln Gly Glu
Met Lys Phe Glu Val Glu Asn Leu Ile 275 280 285Leu His Lys Asp Tyr
Ser Ala Asp Thr Leu Ala His His Asn Asp Ile 290 295 300Ala Leu Leu
Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro Ser305 310 315
320Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp Pro Gln
325 330 335Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu Asn
Ser Thr 340 345 350Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val
Val Lys Leu Ile 355 360 365Ser His Arg Glu Cys Gln Gln Pro His Tyr
Tyr Gly Ser Glu Val Thr 370 375 380Thr Lys Met Leu Cys Ala Ala Asp
Pro Gln Trp Lys Thr Asp Ser Cys385 390 395 400Gln Gly Asp Ser Gly
Gly Pro Leu Val Cys Ser Leu Gln Gly Arg Met 405 410 415Thr Leu Thr
Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp 420 425
430Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile Arg
435 440 445Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450
45521462PRTArtificial SequenceGPB1-2-hmUPA 21Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Thr Cys Val Gly Ser Asp65 70 75
80Arg Thr Phe Ser Asn Tyr Ser Met Gly Trp Phe Arg Gln Ala Pro Gly
85 90 95Lys Glu Arg Gln Phe Val Ser Thr Ile Ser Arg His Gly Thr Ser
Thr 100 105 110Ala Tyr Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser
Arg Asp Asn 115 120 125Ala Glu Asn Ile Val Tyr Leu Gln Met Asn Ser
Leu Glu Pro Glu Asp 130 135 140Thr Ala Val Tyr Tyr Cys Ala Ala Arg
Pro His Thr Gln His Tyr Val145 150 155 160Arg Val Glu Ser Tyr Gly
Val Trp Gly Gln Gly Thr Gln Val Thr Val 165 170 175Ser Ser Ser Ala
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala
Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200
205Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe
210 215 220Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr
Val Cys225 230 235 240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile
Ser Ala Thr His Cys 245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp
Tyr Ile Val Tyr Leu Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr
Gln Gly Glu Met Lys Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His
Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His 290 295 300Asn Asp Ile
Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala305 310 315
320Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn
325 330 335Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly
Lys Glu 340 345 350Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys
Met Thr Val Val 355 360 365Lys Leu Ile Ser His Arg Glu Cys Gln Gln
Pro His Tyr Tyr Gly Ser 370 375 380Glu Val Thr Thr Lys Met Leu Cys
Ala Ala Asp Pro Gln Trp Lys Thr385 390 395 400Asp Ser Cys Gln Gly
Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln 405 410 415Gly Arg Met
Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala 420 425 430Leu
Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro 435 440
445Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46022456PRTArtificial SequenceGPB1-3- hmUPA 22Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Ser Ile Asn Ser Ile Arg Ala Met Gly Trp Tyr Arg Gln Pro Pro Gly
85 90 95Lys Gln Arg Glu Leu Val Ala Thr Ile Thr Arg Asp Gly Arg Thr
Asn 100 105 110Tyr Pro Asp Ser Val Lys Gly Gln Phe Thr Ile Ser Ile
Asp Asn Ala 115 120 125Arg Asn Thr Val Ser Leu Gln Arg Asn Ser Leu
Lys Pro Glu Asp Thr 130 135 140Ala Val Tyr Tyr Cys Val Ala Asp Trp
Gly Glu Gly Tyr Leu Thr Arg145 150 155 160Val Trp Gly Gln Gly Thr
Gln Val Thr Val Ser Ser Ser Ala Ala Gly 165 170 175Gly Gly Gly Ser
Gly Gly Gly Gly Ser Ala Ala Ala Leu Lys Phe Gln 180 185 190Cys Gly
Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile Ile Gly Gly Glu 195 200
205Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg
210 215 220His Arg Gly Gly Ser Val Thr Tyr Val Cys Gly Gly Ser Leu
Ile Ser225 230 235 240Pro Cys Trp Val Ile Ser Ala Thr His Cys Phe
Ile Asp Tyr Pro Lys 245 250 255Lys Glu Asp Tyr Ile Val Tyr Leu Gly
Arg Ser Arg Leu Asn Ser Asn 260 265 270Thr Gln Gly Glu Met Lys Phe
Glu Val Glu Asn Leu Ile Leu His Lys 275 280 285Asp Tyr Ser Ala Asp
Thr Leu Ala His His Asn Asp Ile Ala Leu Leu 290 295 300Lys Ile Arg
Ser Lys Glu Gly Arg Cys Ala Gln Pro Ser Arg Thr Ile305 310 315
320Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp Pro Gln Phe Gly Thr
325 330 335Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu Asn Ser Thr Asp
Tyr Leu 340 345 350Tyr Pro Glu Gln Leu Lys Met Thr Val Val Lys Leu
Ile Ser His Arg 355 360 365Glu Cys Gln Gln Pro His Tyr Tyr Gly Ser
Glu Val Thr Thr Lys Met 370 375 380Leu Cys Ala Ala Asp Pro Gln Trp
Lys Thr Asp Ser Cys Gln Gly Asp385 390 395 400Ser Gly Gly Pro Leu
Val Cys Ser Leu Gln Gly Arg Met Thr Leu Thr 405 410 415Gly Ile Val
Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly 420 425 430Val
Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile Arg Ser His Thr 435 440
445Lys Glu Glu Asn Gly Leu Ala Leu 450 45523455PRTArtificial
SequenceGPB1-4 hmUPA 23Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Gly Ser Ser Ala Trp
Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser Ser Ala Trp Ser His Pro
Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu Tyr Phe Gln Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu Val Gln Ala Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Glu65 70 75 80Thr Phe Ser Ile Arg
Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 85 90 95Gln Arg Glu Leu
Val Ala Tyr Ile Thr Ser Gly Gly Ser Thr Asn Tyr 100 105 110Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Arg 115 120
125Asn Thr Val Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
130 135 140Val Tyr Tyr Cys Tyr Gln Ala Pro Arg Ser Gly Tyr Asp Pro
Val Tyr145 150 155 160Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
Ser Ala Ala Gly Gly 165 170 175Gly Gly Ser Gly Gly Gly Gly Ser Ala
Ala Ala Leu Lys Phe Gln Cys 180 185 190Gly Gln Lys Thr Leu Arg Pro
Arg Phe Lys Ile Ile Gly Gly Glu Phe 195 200 205Thr Thr Ile Glu Asn
Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His 210 215 220Arg Gly Gly
Ser Val Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro225 230 235
240Cys Trp Val Ile Ser Ala Thr His Cys Phe Ile Asp Tyr Pro Lys Lys
245 250 255Glu Asp Tyr Ile Val Tyr Leu Gly Arg Ser Arg Leu Asn Ser
Asn Thr 260 265 270Gln Gly Glu Met Lys Phe Glu Val Glu Asn Leu Ile
Leu His Lys Asp 275 280 285Tyr Ser Ala Asp Thr Leu Ala His His Asn
Asp Ile Ala Leu Leu Lys 290 295 300Ile Arg Ser Lys Glu Gly Arg Cys
Ala Gln Pro Ser Arg Thr Ile Gln305 310 315 320Thr Ile Cys Leu Pro
Ser Met Tyr Asn Asp Pro Gln Phe Gly Thr Ser 325 330 335Cys Glu Ile
Thr Gly Phe Gly Lys Glu Asn Ser Thr Asp Tyr Leu Tyr 340 345 350Pro
Glu Gln Leu Lys Met Thr Val Val Lys Leu Ile Ser His Arg Glu 355 360
365Cys Gln Gln Pro His Tyr Tyr Gly Ser Glu Val Thr Thr Lys Met Leu
370 375 380Cys Ala Ala Asp Pro Gln Trp Lys Thr Asp Ser Cys Gln Gly
Asp Ser385 390 395 400Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg
Met Thr Leu Thr Gly 405 410 415Ile Val Ser Trp Gly Arg Gly Cys Ala
Leu Lys Asp Lys Pro Gly Val 420 425 430Tyr Thr Arg Val Ser His Phe
Leu Pro Trp Ile Arg Ser His Thr Lys 435 440 445Glu Glu Asn Gly Leu
Ala Leu 450 45524458PRTArtificial SequenceGPB1-5 hmUPA 24Met Glu
Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly
Ser Thr Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25
30Lys Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu
35 40 45Asn Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly
Gly 50 55 60Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Glu65 70 75 80Phe Thr Phe Ser Lys His Trp Met Tyr Trp Val Arg
Gln Ala Pro Gly 85 90 95Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Leu
Gly Gly Asp Ser Thr 100 105 110Tyr Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn 115 120 125Ala Lys Asn Thr Leu Tyr Leu
Gln Met Asp Ser Leu Lys Ser Glu Asp 130 135 140Thr Ala Val Tyr Tyr
Cys Ala Lys Gly Ala Ser Ser Trp Phe Gly Asp145 150 155 160Phe Gly
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser Ala 165 170
175Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala Leu Lys
180 185 190Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile
Ile Gly 195 200 205Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe
Ala Ala Ile Tyr 210 215 220Arg Arg His Arg Gly Gly Ser Val Thr Tyr
Val Cys Gly Gly Ser Leu225 230 235 240Ile Ser Pro Cys Trp Val Ile
Ser Ala Thr His Cys Phe Ile Asp Tyr 245 250 255Pro Lys Lys Glu Asp
Tyr Ile Val Tyr Leu Gly Arg Ser Arg Leu Asn 260 265 270Ser Asn Thr
Gln Gly Glu Met Lys Phe Glu Val Glu Asn Leu Ile Leu 275 280 285His
Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His Asn Asp Ile Ala 290 295
300Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro Ser
Arg305 310 315 320Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn
Asp Pro Gln Phe 325 330 335Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly
Lys Glu Asn Ser Thr Asp 340 345 350Tyr Leu Tyr Pro Glu Gln Leu Lys
Met Thr Val Val Lys Leu Ile Ser 355 360 365His Arg Glu Cys Gln Gln
Pro His Tyr Tyr Gly Ser Glu Val Thr Thr 370 375 380Lys Met Leu Cys
Ala Ala Asp Pro Gln Trp Lys Thr Asp Ser Cys Gln385 390 395 400Gly
Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg Met Thr 405 410
415Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys Asp Lys
420 425 430Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile
Arg Ser 435 440 445His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450
45525460PRTArtificial SequenceGPB1-6 hmUPA 25Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Phe Thr Phe Ser Asn Phe Ala Met Asn Trp Val Arg Gln Ala Pro Gly
85 90 95Lys Gly Leu Glu Trp Val Ser Phe Ile Asn Arg Gly Gly Gly Ser
Thr 100 105 110Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn 115 120 125Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp 130 135 140Thr Ala Val Tyr Tyr Cys Ala Lys Phe
Ser Arg Ser Val Pro Pro Tyr145 150 155 160Tyr Gly Met Asp Tyr Trp
Gly Lys Gly Thr Leu Val Thr Val Ser Ser 165 170 175Ser Ala Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala 180 185 190Leu Lys
Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile 195 200
205Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala
210 215 220Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys
Gly Gly225 230 235 240Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala
Thr His Cys Phe Ile 245 250 255Asp Tyr Pro Lys Lys Glu Asp Tyr Ile
Val Tyr Leu Gly Arg Ser Arg 260 265 270Leu Asn Ser Asn Thr Gln Gly
Glu Met Lys Phe Glu Val Glu Asn Leu 275 280 285Ile Leu His Lys Asp
Tyr Ser Ala Asp Thr Leu Ala His His Asn Asp 290 295 300Ile Ala Leu
Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro305 310 315
320Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp Pro
325 330 335Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu
Asn Ser 340 345 350Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr
Val Val Lys Leu 355 360 365Ile Ser His Arg Glu Cys Gln Gln Pro His
Tyr Tyr Gly Ser Glu Val 370 375 380Thr Thr Lys Met Leu Cys Ala Ala
Asp Pro Gln Trp Lys Thr Asp Ser385 390 395 400Cys Gln Gly Asp Ser
Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg 405 410 415Met Thr Leu
Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys 420 425 430Asp
Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile 435 440
445Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46026462PRTArtificial SequenceGPB1 - 7 hmUPA 26Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Arg Val Asp Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
85 90 95Glu Phe Val Ala Thr Ile Thr Trp Ser Asp Ser Lys Ile Tyr Tyr
Ala 100 105 110Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gly Glu Arg
Ala Lys Asn
115 120 125Thr Met Tyr Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Thr
Ala Val 130 135 140Tyr Tyr Cys Ala Ala Ala His Arg Pro Tyr Arg Ser
Gly Tyr Tyr Tyr145 150 155 160Met Gln Ser Arg Tyr Asp Tyr Trp Gly
Gln Gly Thr Gln Val Thr Val 165 170 175Ser Ser Ser Ala Ala Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala Leu Lys Phe
Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200 205Lys Ile Ile
Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe 210 215 220Ala
Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys225 230
235 240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala Thr His
Cys 245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr
Leu Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met
Lys Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His Lys Asp Tyr Ser
Ala Asp Thr Leu Ala His His 290 295 300Asn Asp Ile Ala Leu Leu Lys
Ile Arg Ser Lys Glu Gly Arg Cys Ala305 310 315 320Gln Pro Ser Arg
Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn 325 330 335Asp Pro
Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu 340 345
350Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr Val Val
355 360 365Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His Tyr Tyr
Gly Ser 370 375 380Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp Pro
Gln Trp Lys Thr385 390 395 400Asp Ser Cys Gln Gly Asp Ser Gly Gly
Pro Leu Val Cys Ser Leu Gln 405 410 415Gly Arg Met Thr Leu Thr Gly
Ile Val Ser Trp Gly Arg Gly Cys Ala 420 425 430Leu Lys Asp Lys Pro
Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro 435 440 445Trp Ile Arg
Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46027462PRTArtificial SequenceGPB1-8 hmUPA 27Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Ser65 70 75
80Met Phe Ser Ile Asn Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Arg
85 90 95Gln Arg Glu Leu Val Ala Thr Ile Thr Ser Gly Asp Ser Thr Tyr
Tyr 100 105 110Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys 115 120 125Tyr Thr Lys Asn Thr Val Tyr Leu Gln Met Asn
Ser Leu Lys Pro Glu 130 135 140Asp Thr Ala Val Tyr Tyr Cys Asn Ala
Ala His Ile Arg Gly Ser Gly145 150 155 160Gly Asp Tyr Ala Arg Glu
Ala Trp Gly Gln Gly Thr Gln Val Thr Val 165 170 175Ser Ser Ser Ala
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala
Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200
205Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe
210 215 220Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr
Val Cys225 230 235 240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile
Ser Ala Thr His Cys 245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp
Tyr Ile Val Tyr Leu Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr
Gln Gly Glu Met Lys Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His
Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His 290 295 300Asn Asp Ile
Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala305 310 315
320Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn
325 330 335Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly
Lys Glu 340 345 350Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys
Met Thr Val Val 355 360 365Lys Leu Ile Ser His Arg Glu Cys Gln Gln
Pro His Tyr Tyr Gly Ser 370 375 380Glu Val Thr Thr Lys Met Leu Cys
Ala Ala Asp Pro Gln Trp Lys Thr385 390 395 400Asp Ser Cys Gln Gly
Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln 405 410 415Gly Arg Met
Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala 420 425 430Leu
Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro 435 440
445Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46028462PRTArtificial SequenceGPB1-9 hmUPA 28Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Pro Thr Val Ser Asn Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly
85 90 95Lys Glu Arg Asp Phe Val Ala Gly Ile Ser Arg Ser Gly Val Glu
Lys 100 105 110Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn 115 120 125Ala Leu Asn Thr Val Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp 130 135 140Thr Ala Ala Tyr Tyr Cys Ala Ala Arg
Glu Arg Val Gly Ile Thr Phe145 150 155 160Ala His Ser Thr Val Asp
Tyr Trp Gly Lys Gly Thr Leu Val Thr Val 165 170 175Ser Ser Ser Ala
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala
Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200
205Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe
210 215 220Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr
Val Cys225 230 235 240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile
Ser Ala Thr His Cys 245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp
Tyr Ile Val Tyr Leu Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr
Gln Gly Glu Met Lys Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His
Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His 290 295 300Asn Asp Ile
Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala305 310 315
320Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn
325 330 335Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly
Lys Glu 340 345 350Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys
Met Thr Val Val 355 360 365Lys Leu Ile Ser His Arg Glu Cys Gln Gln
Pro His Tyr Tyr Gly Ser 370 375 380Glu Val Thr Thr Lys Met Leu Cys
Ala Ala Asp Pro Gln Trp Lys Thr385 390 395 400Asp Ser Cys Gln Gly
Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln 405 410 415Gly Arg Met
Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala 420 425 430Leu
Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro 435 440
445Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46029450PRTArtificial SequenceGPB1-10 hmUPA 29Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Phe Thr Phe Ser Lys Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly
85 90 95Lys Gly Leu Glu Trp Val Ser Ile Ile Asp Ser Gly Gly Gly Ala
Ile 100 105 110Gly Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn 115 120 125Val Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Lys Pro Glu Asp 130 135 140Thr Ala Val Tyr His Cys Val Phe Gly
Asp Tyr Lys Gly Gln Gly Thr145 150 155 160Gln Val Thr Val Ser Ser
Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly 165 170 175Gly Gly Ser Ala
Ala Ala Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu 180 185 190Arg Pro
Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn 195 200
205Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val
210 215 220Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val
Ile Ser225 230 235 240Ala Thr His Cys Phe Ile Asp Tyr Pro Lys Lys
Glu Asp Tyr Ile Val 245 250 255Tyr Leu Gly Arg Ser Arg Leu Asn Ser
Asn Thr Gln Gly Glu Met Lys 260 265 270Phe Glu Val Glu Asn Leu Ile
Leu His Lys Asp Tyr Ser Ala Asp Thr 275 280 285Leu Ala His His Asn
Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu 290 295 300Gly Arg Cys
Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro305 310 315
320Ser Met Tyr Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly
325 330 335Phe Gly Lys Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln
Leu Lys 340 345 350Met Thr Val Val Lys Leu Ile Ser His Arg Glu Cys
Gln Gln Pro His 355 360 365Tyr Tyr Gly Ser Glu Val Thr Thr Lys Met
Leu Cys Ala Ala Asp Pro 370 375 380Gln Trp Lys Thr Asp Ser Cys Gln
Gly Asp Ser Gly Gly Pro Leu Val385 390 395 400Cys Ser Leu Gln Gly
Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly 405 410 415Arg Gly Cys
Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser 420 425 430His
Phe Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu 435 440
445Ala Leu 45030464PRTArtificial SequenceGPB1-11 hmUPA 30Met Glu
Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly
Ser Thr Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25
30Lys Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu
35 40 45Asn Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly
Gly 50 55 60Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly65 70 75 80Phe Thr Phe Ser Ser Ser Ala Met Thr Trp Val Arg
Gln Ala Pro Gly 85 90 95Lys Gly Leu Glu Trp Val Ser Ala Ile Asn Ser
Gly Gly Ser Gly Thr 100 105 110Arg Tyr Ala Asp Ser Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn 115 120 125Ala Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser Leu Lys Pro Glu Asp 130 135 140Thr Ala Val Tyr Tyr
Cys Ala Lys Arg Arg Asp Gly Gln Asn Trp Tyr145 150 155 160Pro Gly
Ile Ser Tyr Glu Ser Met Tyr Arg Gly Gln Gly Thr Gln Val 165 170
175Thr Val Ser Ser Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
180 185 190Ser Ala Ala Ala Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu
Arg Pro 195 200 205Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile
Glu Asn Gln Pro 210 215 220Trp Phe Ala Ala Ile Tyr Arg Arg His Arg
Gly Gly Ser Val Thr Tyr225 230 235 240Val Cys Gly Gly Ser Leu Ile
Ser Pro Cys Trp Val Ile Ser Ala Thr 245 250 255His Cys Phe Ile Asp
Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr Leu 260 265 270Gly Arg Ser
Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys Phe Glu 275 280 285Val
Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp Thr Leu Ala 290 295
300His His Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly
Arg305 310 315 320Cys Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys
Leu Pro Ser Met 325 330 335Tyr Asn Asp Pro Gln Phe Gly Thr Ser Cys
Glu Ile Thr Gly Phe Gly 340 345 350Lys Glu Asn Ser Thr Asp Tyr Leu
Tyr Pro Glu Gln Leu Lys Met Thr 355 360 365Val Val Lys Leu Ile Ser
His Arg Glu Cys Gln Gln Pro His Tyr Tyr 370 375 380Gly Ser Glu Val
Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln Trp385 390 395 400Lys
Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Ser 405 410
415Leu Gln Gly Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly
420 425 430Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser
His Phe 435 440 445Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn
Gly Leu Ala Leu 450 455 46031460PRTArtificial SequenceGPB1-12 hmUPA
31Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1
5 10 15Gly Ser Thr Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe
Glu 20 25 30Lys Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu
Phe Glu 35 40 45Asn Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser
Gly Gly Gly 50 55 60Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly65 70 75 80Arg Thr Phe Ser Ser Tyr Thr Met Ala Trp
Phe Arg Gln Ala Pro Gly 85 90 95Lys Glu Arg Glu Phe Val Gly Leu Ile
Ser Trp Asn Ala Lys Ser Thr 100 105 110Tyr Val Thr Asp Ser Val Lys
Gly Arg Phe Thr Ile Thr Arg Glu Asn 115 120 125Ala Lys Asp Met Val
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp 130 135 140Ser Ala Thr
Tyr Tyr Cys Ala Ala Asn Arg Tyr Gly Ser Ser Val Pro145 150 155
160Gly Ala Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
165 170 175Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
Ala Ala 180 185 190Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro
Arg Phe Lys Ile 195 200 205Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn
Gln Pro Trp Phe Ala Ala 210 215 220Ile Tyr Arg Arg His Arg Gly Gly
Ser Val Thr Tyr Val Cys Gly Gly225 230 235 240Ser Leu Ile Ser Pro
Cys Trp Val Ile Ser Ala Thr His Cys Phe Ile 245 250 255Asp Tyr Pro
Lys Lys Glu Asp Tyr Ile Val Tyr Leu Gly Arg Ser Arg 260 265 270Leu
Asn Ser Asn Thr
Gln Gly Glu Met Lys Phe Glu Val Glu Asn Leu 275 280 285Ile Leu His
Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His Asn Asp 290 295 300Ile
Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro305 310
315 320Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp
Pro 325 330 335Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys
Glu Asn Ser 340 345 350Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met
Thr Val Val Lys Leu 355 360 365Ile Ser His Arg Glu Cys Gln Gln Pro
His Tyr Tyr Gly Ser Glu Val 370 375 380Thr Thr Lys Met Leu Cys Ala
Ala Asp Pro Gln Trp Lys Thr Asp Ser385 390 395 400Cys Gln Gly Asp
Ser Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg 405 410 415Met Thr
Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys 420 425
430Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile
435 440 445Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46032460PRTArtificial SequenceGPB1-13 hmUPA 32Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Phe Thr Phe Ser Ser Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
85 90 95Lys Gly Leu Glu Trp Val Ser Ala Ile Asn Met Gly Gly Gly Ser
Thr 100 105 110Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn 115 120 125Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Gly
Leu Lys Pro Glu Asp 130 135 140Thr Ala Leu Tyr Tyr Cys Val Arg Gly
Gly Ser Ala Tyr Ser Val Arg145 150 155 160Tyr Glu Tyr Ala Tyr Trp
Gly Gln Gly Thr Gln Val Thr Val Ser Ser 165 170 175Ser Ala Ala Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala 180 185 190Leu Lys
Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys Ile 195 200
205Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala Ala
210 215 220Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val Cys
Gly Gly225 230 235 240Ser Leu Ile Ser Pro Cys Trp Val Ile Ser Ala
Thr His Cys Phe Ile 245 250 255Asp Tyr Pro Lys Lys Glu Asp Tyr Ile
Val Tyr Leu Gly Arg Ser Arg 260 265 270Leu Asn Ser Asn Thr Gln Gly
Glu Met Lys Phe Glu Val Glu Asn Leu 275 280 285Ile Leu His Lys Asp
Tyr Ser Ala Asp Thr Leu Ala His His Asn Asp 290 295 300Ile Ala Leu
Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln Pro305 310 315
320Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp Pro
325 330 335Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys Glu
Asn Ser 340 345 350Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met Thr
Val Val Lys Leu 355 360 365Ile Ser His Arg Glu Cys Gln Gln Pro His
Tyr Tyr Gly Ser Glu Val 370 375 380Thr Thr Lys Met Leu Cys Ala Ala
Asp Pro Gln Trp Lys Thr Asp Ser385 390 395 400Cys Gln Gly Asp Ser
Gly Gly Pro Leu Val Cys Ser Leu Gln Gly Arg 405 410 415Met Thr Leu
Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu Lys 420 425 430Asp
Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp Ile 435 440
445Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46033463PRTArtificial SequenceGPB1-14 hmUPA 33Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ala Ala65 70 75
80Ser Trp Phe Ser Ile Tyr Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly
85 90 95Lys Gln Arg Glu Leu Val Ala Ile Ile Leu Ser Asp Gly Asp Thr
Asp 100 105 110Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala 115 120 125Lys Asn Thr Lys Asn Thr Val Tyr Leu Gln Met
Asn Ser Leu Lys Pro 130 135 140Glu Asp Thr Ala Val Tyr Tyr Cys Asn
Ala Arg Gly Ile Arg Gly Ser145 150 155 160Gly Gly Asp Tyr Ala Arg
Glu Ala Trp Gly Gln Gly Thr Gln Val Thr 165 170 175Val Ser Ser Ser
Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 180 185 190Ala Ala
Ala Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg 195 200
205Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp
210 215 220Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr
Tyr Val225 230 235 240Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val
Ile Ser Ala Thr His 245 250 255Cys Phe Ile Asp Tyr Pro Lys Lys Glu
Asp Tyr Ile Val Tyr Leu Gly 260 265 270Arg Ser Arg Leu Asn Ser Asn
Thr Gln Gly Glu Met Lys Phe Glu Val 275 280 285Glu Asn Leu Ile Leu
His Lys Asp Tyr Ser Ala Asp Thr Leu Ala His 290 295 300His Asn Asp
Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys305 310 315
320Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr
325 330 335Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe
Gly Lys 340 345 350Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu
Lys Met Thr Val 355 360 365Val Lys Leu Ile Ser His Arg Glu Cys Gln
Gln Pro His Tyr Tyr Gly 370 375 380Ser Glu Val Thr Thr Lys Met Leu
Cys Ala Ala Asp Pro Gln Trp Lys385 390 395 400Thr Asp Ser Cys Gln
Gly Asp Ser Gly Gly Pro Leu Val Cys Ser Leu 405 410 415Gln Gly Arg
Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys 420 425 430Ala
Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu 435 440
445Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450
455 46034463PRTArtificial SequenceGPB1- 15 hmUPA 34Met Glu Thr Asp
Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr
Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly
Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn
Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55
60Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65
70 75 80Ser Met Phe Ser Ile Asn Asp Met Gly Trp Tyr Arg Gln Ala Pro
Gly 85 90 95Lys Gln Arg Glu Leu Val Ala Thr Ile Thr Arg Gly Gly Asn
Thr Tyr 100 105 110Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala 115 120 125Thr Tyr Thr Lys Asn Thr Val Tyr Leu Gln
Met Asn Ser Leu Lys Pro 130 135 140Glu Asp Thr Ala Val Tyr Tyr Cys
Asn Ala Arg His Ile Arg Gly Ser145 150 155 160Gly Gly Asp Tyr Ala
Arg Glu Ala Trp Gly Gln Gly Thr Gln Val Thr 165 170 175Val Ser Ser
Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 180 185 190Ala
Ala Ala Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg 195 200
205Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp
210 215 220Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr
Tyr Val225 230 235 240Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val
Ile Ser Ala Thr His 245 250 255Cys Phe Ile Asp Tyr Pro Lys Lys Glu
Asp Tyr Ile Val Tyr Leu Gly 260 265 270Arg Ser Arg Leu Asn Ser Asn
Thr Gln Gly Glu Met Lys Phe Glu Val 275 280 285Glu Asn Leu Ile Leu
His Lys Asp Tyr Ser Ala Asp Thr Leu Ala His 290 295 300His Asn Asp
Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys305 310 315
320Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr
325 330 335Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe
Gly Lys 340 345 350Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu
Lys Met Thr Val 355 360 365Val Lys Leu Ile Ser His Arg Glu Cys Gln
Gln Pro His Tyr Tyr Gly 370 375 380Ser Glu Val Thr Thr Lys Met Leu
Cys Ala Ala Asp Pro Gln Trp Lys385 390 395 400Thr Asp Ser Cys Gln
Gly Asp Ser Gly Gly Pro Leu Val Cys Ser Leu 405 410 415Gln Gly Arg
Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys 420 425 430Ala
Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu 435 440
445Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450
455 46035461PRTArtificial SequenceGPB1- 16 hmUPA 35Met Glu Thr Asp
Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr
Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly
Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn
Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55
60Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg65
70 75 80Arg Thr Phe Ser Asn Tyr Val Met Gly Trp Phe Arg Gln Ala Pro
Gly 85 90 95Lys Glu Arg Glu Ser Val Thr Ala Ile Gly Arg Ser Gly Thr
Ile Leu 100 105 110Tyr Ala Asp Ser Met Lys Gly Arg Ile Thr Ile Ser
Arg Asp Asn Ala 115 120 125Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
Leu Thr Pro Asp Asp Thr 130 135 140Ala Val Tyr Tyr Cys Ala Ala Ser
Ser Gly Ser Met Gln Gln Phe Trp145 150 155 160Arg Met Glu Tyr Asp
Tyr Glu Gly Gln Gly Thr Gln Val Thr Val Ser 165 170 175Ser Ser Ala
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala 180 185 190Ala
Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe Lys 195 200
205Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe Ala
210 215 220Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr Val
Cys Gly225 230 235 240Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser
Ala Thr His Cys Phe 245 250 255Ile Asp Tyr Pro Lys Lys Glu Asp Tyr
Ile Val Tyr Leu Gly Arg Ser 260 265 270Arg Leu Asn Ser Asn Thr Gln
Gly Glu Met Lys Phe Glu Val Glu Asn 275 280 285Leu Ile Leu His Lys
Asp Tyr Ser Ala Asp Thr Leu Ala His His Asn 290 295 300Asp Ile Ala
Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala Gln305 310 315
320Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn Asp
325 330 335Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly Lys
Glu Asn 340 345 350Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys Met
Thr Val Val Lys 355 360 365Leu Ile Ser His Arg Glu Cys Gln Gln Pro
His Tyr Tyr Gly Ser Glu 370 375 380Val Thr Thr Lys Met Leu Cys Ala
Ala Asp Pro Gln Trp Lys Thr Asp385 390 395 400Ser Cys Gln Gly Asp
Ser Gly Gly Pro Leu Val Cys Ser Leu Gln Gly 405 410 415Arg Met Thr
Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala Leu 420 425 430Lys
Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe Leu Pro Trp 435 440
445Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu 450 455
46036462PRTArtificial SequenceGPB1- 19 hmUPA 36Met Glu Thr Asp Thr
Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly
Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser
Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu
Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75
80Arg Thr Phe Gly Ser Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly
85 90 95Lys Glu Arg Glu Phe Val Ala Ala Ile Gly Arg Ser Gly Thr Thr
Tyr 100 105 110Tyr Leu Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala 115 120 125Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
Lys Ser Glu Asp Thr 130 135 140Ala Val Tyr Tyr Cys Gly Ala Ser Leu
Lys Gly Thr Val Leu Gly Ile145 150 155 160Ala Arg Tyr Glu Tyr Asp
Val Arg Gly Gln Gly Thr Gln Val Thr Val 165 170 175Ser Ser Ser Ala
Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 180 185 190Ala Ala
Leu Lys Phe Gln Cys Gly Gln Lys Thr Leu Arg Pro Arg Phe 195 200
205Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn Gln Pro Trp Phe
210 215 220Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val Thr Tyr
Val Cys225 230 235 240Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile
Ser Ala Thr His Cys 245 250 255Phe Ile Asp Tyr Pro Lys Lys Glu Asp
Tyr Ile Val Tyr Leu Gly Arg 260 265 270Ser Arg Leu Asn Ser Asn Thr
Gln Gly Glu Met Lys Phe Glu Val Glu 275 280 285Asn Leu Ile Leu His
Lys Asp Tyr Ser Ala Asp Thr Leu Ala His His 290 295 300Asn Asp Ile
Ala Leu Leu Lys Ile Arg Ser Lys Glu Gly Arg Cys Ala305 310 315
320Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro Ser Met Tyr Asn
325 330 335Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly Phe Gly
Lys Glu 340 345 350Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys
Met Thr Val Val 355 360 365Lys Leu Ile Ser His Arg Glu Cys Gln Gln
Pro His Tyr Tyr Gly Ser 370 375 380Glu Val Thr Thr Lys Met Leu Cys
Ala Ala Asp Pro Gln Trp Lys Thr385 390 395 400Asp Ser Cys Gln Gly
Asp Ser Gly Gly Pro Leu Val Cys Ser Leu Gln 405 410
415Gly Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg Gly Cys Ala
420 425 430Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val Ser His Phe
Leu Pro 435 440 445Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu
Ala Leu 450 455 46037465PRTArtificial SequenceGPB1- 20 hmUPA 37Met
Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10
15Gly Ser Thr Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu
20 25 30Lys Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe
Glu 35 40 45Asn Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly
Gly Gly 50 55 60Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly65 70 75 80Arg Thr Leu Ser Ser Leu Ala Met Gly Trp Phe
Arg Gln Ala Pro Gly 85 90 95Lys Glu Arg Glu Phe Val Ala Ala Asp Arg
Arg Asn Gly Gly Tyr Thr 100 105 110Val Val Ala Asp Tyr Thr Asp Ser
Val Lys Gly Arg Phe Thr Ile Phe 115 120 125Arg Asp Asn Ala Lys Asn
Thr Val Tyr Leu Gln Met Asn Asn Leu Lys 130 135 140Pro Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Ala Asp Ser Asp Arg Thr145 150 155 160Met
Ser Leu Arg Ser Thr Asp Tyr Asp Tyr Trp Gly Gln Gly Thr Gln 165 170
175Val Thr Val Ser Ser Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly
180 185 190Gly Ser Ala Ala Ala Leu Lys Phe Gln Cys Gly Gln Lys Thr
Leu Arg 195 200 205Pro Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr
Ile Glu Asn Gln 210 215 220Pro Trp Phe Ala Ala Ile Tyr Arg Arg His
Arg Gly Gly Ser Val Thr225 230 235 240Tyr Val Cys Gly Gly Ser Leu
Ile Ser Pro Cys Trp Val Ile Ser Ala 245 250 255Thr His Cys Phe Ile
Asp Tyr Pro Lys Lys Glu Asp Tyr Ile Val Tyr 260 265 270Leu Gly Arg
Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met Lys Phe 275 280 285Glu
Val Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp Thr Leu 290 295
300Ala His His Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys Glu
Gly305 310 315 320Arg Cys Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile
Cys Leu Pro Ser 325 330 335Met Tyr Asn Asp Pro Gln Phe Gly Thr Ser
Cys Glu Ile Thr Gly Phe 340 345 350Gly Lys Glu Asn Ser Thr Asp Tyr
Leu Tyr Pro Glu Gln Leu Lys Met 355 360 365Thr Val Val Lys Leu Ile
Ser His Arg Glu Cys Gln Gln Pro His Tyr 370 375 380Tyr Gly Ser Glu
Val Thr Thr Lys Met Leu Cys Ala Ala Asp Pro Gln385 390 395 400Trp
Lys Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys 405 410
415Ser Leu Gln Gly Arg Met Thr Leu Thr Gly Ile Val Ser Trp Gly Arg
420 425 430Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val
Ser His 435 440 445Phe Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu
Asn Gly Leu Ala 450 455 460Leu4653818PRTArtificial Sequenceflexible
linker 38Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe
Arg Ser1 5 10 15Leu Asp3914PRTArtificial Sequenceflexible linker
39Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr1 5
1040791DNAArtificial SequencedsDNA 40ggatccgaag tccagcttgt
agaatcagga ggaggccttg tccagccagg tggtagcctt 60cgtctgtcgt gtgctgcttc
gggccgcaca ttttcgtata accctatggg ttggtttcgc 120caagcacccg
gcaagggacg cgagttggtg gccgcgatta gtcgtacggg tggttccacc
180tactacccgg attcagtgga aggacgcttt acgattagcc gtgataacgc
gaagcgtatg 240gtctacttac agatgaatag cttgcgcgcg gaagacaccg
cggtatacta ttgtgctgca 300gcaggagtcc gtgctgagga tggacgcgtc
cgcacgttac ctagtgagta tacattctgg 360ggccagggca cccaagttac
cgtatccagt gcagcagcgg aagtacaact ggtcgaatct 420ggaggaggac
ttgtacaacc agggggttcc ttacgtttgt catgtgcggc aagtgggcgc
480acatttagtt acaaccctat gggctggttc cgtcaagccc cgggaaaagg
gcgcgaactt 540gtagccgcca tttcgcgtac agggggaagt acctattacc
cggactcagt agagggacgc 600ttcacgattt ctcgtgacaa cgcaaagcgc
atggtttatc tgcaaatgaa tagtttacgc 660gccgaagata cagcagttta
ctattgcgcc gcagctggag tccgcgccga agacggccgt 720gtacgcacct
tgccttctga atacactttt tggggtcaag gaacacaggt gaccgtgtca
780tctgcggccg c 79141320PRTArtificial SequenceCaplacizumab in
pTH4.0 protein sequence 41Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala
Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Gln Ser Gly
His His His His His His His 20 25 30His Asp Tyr Asp Ile Pro Ser Ser
Glu Asn Leu Tyr Phe Gln Gly Ser 35 40 45Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 50 55 60Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Arg Thr Phe Ser Tyr Asn65 70 75 80Pro Met Gly Trp
Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 85 90 95Ala Ala Ile
Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp Ser Val 100 105 110Glu
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 115 120
125Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
130 135 140Ala Ala Ala Gly Val Arg Ala Glu Asp Gly Arg Val Arg Thr
Leu Pro145 150 155 160Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr Gln
Val Thr Val Ser Ser 165 170 175Ala Ala Ala Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln 180 185 190Pro Gly Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Arg Thr Phe 195 200 205Ser Tyr Asn Pro Met
Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg 210 215 220Glu Leu Val
Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro225 230 235
240Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg
245 250 255Met Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val 260 265 270Tyr Tyr Cys Ala Ala Ala Gly Val Arg Ala Glu Asp
Gly Arg Val Arg 275 280 285Thr Leu Pro Ser Glu Tyr Thr Phe Trp Gly
Gln Gly Thr Gln Val Thr 290 295 300Val Ser Ser Ala Ala Ala Glu Gln
Lys Leu Ile Ser Glu Glu Asp Leu305 310 315 3204220DNAArtificial
Sequenceprimer 42taatacgact cactataggg 204319DNAArtificial
Sequenceprimer Rv 43gctagttatt gctcagcgg 1944466PRTArtificial
SequenceCablivi-hmUPA 44Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu
Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp Gly Ser Ser Ala Trp
Ser His Pro Gln Phe Glu 20 25 30Lys Gly Ser Ser Ala Trp Ser His Pro
Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn Leu Tyr Phe Gln Ser Glu Val
Gln Leu Val Glu Ser Gly Gly Gly 50 55 60Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly65 70 75 80Arg Thr Phe Ser Tyr
Asn Pro Met Gly Trp Phe Arg Gln Ala Pro Gly 85 90 95Lys Gly Arg Glu
Leu Val Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr 100 105 110Tyr Tyr
Pro Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn 115 120
125Ala Lys Arg Met Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
130 135 140Thr Ala Val Tyr Tyr Cys Ala Ala Ala Gly Val Arg Ala Glu
Asp Gly145 150 155 160Arg Val Arg Thr Leu Pro Ser Glu Tyr Thr Phe
Trp Gly Gln Gly Thr 165 170 175Gln Val Thr Val Ser Ser Ser Ala Ala
Gly Gly Gly Gly Ser Gly Gly 180 185 190Gly Gly Ser Ala Ala Ala Leu
Lys Phe Gln Cys Gly Gln Lys Thr Leu 195 200 205Arg Pro Arg Phe Lys
Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu Asn 210 215 220Gln Pro Trp
Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser Val225 230 235
240Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile Ser
245 250 255Ala Thr His Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr
Ile Val 260 265 270Tyr Leu Gly Arg Ser Arg Leu Asn Ser Asn Thr Gln
Gly Glu Met Lys 275 280 285Phe Glu Val Glu Asn Leu Ile Leu His Lys
Asp Tyr Ser Ala Asp Thr 290 295 300Leu Ala His His Asn Asp Ile Ala
Leu Leu Lys Ile Arg Ser Lys Glu305 310 315 320Gly Arg Cys Ala Gln
Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu Pro 325 330 335Ser Met Tyr
Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr Gly 340 345 350Phe
Gly Lys Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu Lys 355 360
365Met Thr Val Val Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro His
370 375 380Tyr Tyr Gly Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala
Asp Pro385 390 395 400Gln Trp Lys Thr Asp Ser Cys Gln Gly Asp Ser
Gly Gly Pro Leu Val 405 410 415Cys Ser Leu Gln Gly Arg Met Thr Leu
Thr Gly Ile Val Ser Trp Gly 420 425 430Arg Gly Cys Ala Leu Lys Asp
Lys Pro Gly Val Tyr Thr Arg Val Ser 435 440 445His Phe Leu Pro Trp
Ile Arg Ser His Thr Lys Glu Glu Asn Gly Leu 450 455 460Ala
Leu46545297PRTArtificial SequenceCablivi-mmUPA 45Met Glu Thr Asp
Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr
Gly Asp Gly Ser Ser Ala Trp Ser His Pro Gln Phe Glu 20 25 30Lys Gly
Ser Ser Ala Trp Ser His Pro Gln Phe Glu Lys Glu Phe Glu 35 40 45Asn
Leu Tyr Phe Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly 50 55
60Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly65
70 75 80Arg Thr Phe Ser Tyr Asn Pro Met Gly Trp Phe Arg Gln Ala Pro
Gly 85 90 95Lys Gly Arg Glu Leu Val Ala Ala Ile Ser Arg Thr Gly Gly
Ser Thr 100 105 110Tyr Tyr Pro Asp Ser Val Glu Gly Arg Phe Thr Ile
Ser Arg Asp Asn 115 120 125Ala Lys Arg Met Val Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 130 135 140Thr Ala Val Tyr Tyr Cys Ala Ala
Ala Gly Val Arg Ala Glu Asp Gly145 150 155 160Arg Val Arg Thr Leu
Pro Ser Glu Tyr Thr Phe Trp Gly Gln Gly Thr 165 170 175Gln Val Thr
Val Ser Ser Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly 180 185 190Gly
Gly Ser Ala Ala Ala Gln Gly Phe Gln Cys Gly Gln Lys Ala Leu 195 200
205Arg Pro Arg Phe Lys Ile Val Gly Gly Glu Phe Thr Glu Val Glu Asn
210 215 220Gln Pro Trp Phe Ala Ala Ile Tyr Gln Lys Asn Lys Gly Gly
Ser Pro225 230 235 240Pro Ser Phe Lys Cys Gly Gly Ser Leu Ile Ser
Pro Cys Trp Val Ala 245 250 255Ser Ala Ala His Cys Phe Ile Gln Leu
Pro Lys Lys Glu Asn Tyr Val 260 265 270Val Tyr Leu Gly Gln Ser Lys
Glu Ser Ser Tyr Asn Pro Gly Glu Met 275 280 285Lys Phe Glu Val Glu
Gln Leu Ile Leu 290 29546128PRTArtificial SequenceCablivi VHH 46Glu
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 Arg Thr Phe Ser Tyr Asn
20 25 30Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu
Val 35 40 45Ala Ala Ile Ser Arg Thr Gly Gly Ser Thr Tyr Tyr Pro Asp
Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg
Met Val Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Ala Ala Gly Val Arg Ala Glu Asp Gly
Arg Val Arg Thr Leu Pro 100 105 110Ser Glu Tyr Thr Phe Trp Gly Gln
Gly Thr Gln Val Thr Val Ser Ser 115 120 125
* * * * *
References