U.S. patent application number 13/516002 was filed with the patent office on 2012-11-29 for modified ubiquitin proteins having a specific binding activity for the extradomain b of fibronectin.
This patent application is currently assigned to Scil Proteins GmbH. Invention is credited to Erik Fiedler, Markus Fiedler, Manja Gloser, Thomas Goettler, Ilka Haenssgen, Anja Kunert, Joerg Nerkamp, Arnd Steuernagel.
Application Number | 20120301393 13/516002 |
Document ID | / |
Family ID | 43648750 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120301393 |
Kind Code |
A1 |
Steuernagel; Arnd ; et
al. |
November 29, 2012 |
MODIFIED UBIQUITIN PROTEINS HAVING A SPECIFIC BINDING ACTIVITY FOR
THE EXTRADOMAIN B OF FIBRONECTIN
Abstract
The present invention refers to novel recombinant proteins
obtained from modified ubiquitin capable of binding the extradomain
B of fibronectin (ED-B). Furthermore, the invention refers to
fusion proteins comprising said recombinant protein fused to a
pharmaceutically and/or diagnostically active component.
Inventors: |
Steuernagel; Arnd;
(Goettingen, DE) ; Fiedler; Erik; (Halle/Saale,
DE) ; Fiedler; Markus; (Halle/Saale, DE) ;
Kunert; Anja; (Dresden, DE) ; Nerkamp; Joerg;
(Kiefersfelden, DE) ; Goettler; Thomas;
(Halle/Saale, DE) ; Gloser; Manja; (Teutschenthal,
DE) ; Haenssgen; Ilka; (Halle/Saale, DE) |
Assignee: |
Scil Proteins GmbH
|
Family ID: |
43648750 |
Appl. No.: |
13/516002 |
Filed: |
December 14, 2010 |
PCT Filed: |
December 14, 2010 |
PCT NO: |
PCT/EP2010/069666 |
371 Date: |
August 14, 2012 |
Current U.S.
Class: |
424/1.11 ;
424/649; 435/188; 435/252.33; 435/320.1; 435/7.1; 435/7.92;
436/501; 514/9.3; 530/350; 530/351; 530/399; 530/402; 536/23.4 |
Current CPC
Class: |
C07K 2319/31 20130101;
G01N 33/6878 20130101; C07K 14/435 20130101; C07K 14/525 20130101;
C12N 15/1041 20130101; G01N 33/6845 20130101; C12N 15/1037
20130101; C12N 15/1034 20130101; A61P 35/00 20180101; C07K 2319/95
20130101; C12N 15/1044 20130101; C07K 14/47 20130101 |
Class at
Publication: |
424/1.11 ;
530/350; 530/399; 530/351; 435/188; 530/402; 514/9.3; 424/649;
536/23.4; 435/320.1; 435/252.33; 436/501; 435/7.1; 435/7.92 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C12N 9/96 20060101 C12N009/96; A61K 33/24 20060101
A61K033/24; G01N 33/53 20060101 G01N033/53; C12N 15/62 20060101
C12N015/62; C12N 15/63 20060101 C12N015/63; C12N 1/21 20060101
C12N001/21; A61P 35/00 20060101 A61P035/00; C07K 14/00 20060101
C07K014/00; A61K 51/00 20060101 A61K051/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 2009 |
EP |
09179147.5 |
May 7, 2010 |
EP |
10162264.5 |
Oct 8, 2010 |
EP |
10186980.8 |
Claims
1-31. (canceled)
32. A protein capable of binding the extradomain B (ED-B) of
fibronectin, comprising a modified multimeric, optionally dimeric
or trimeric ubiquitin protein wherein at least two monomeric
ubiquitin units are linked together in a head-to-tail arrangement,
wherein each monomer of said multimeric protein is modified by at
least substitutions of at least 3 or 4 amino acids in positions 2,
4, 6, 8, 62, 63, 64, 65, 66, or 68 of SEQ ID NO: 1, said modified
monomeric ubiquitin unit having an amino acid identity to SEQ ID
NO: 1 of at least one of the group of at least 60%, at least 85%
and at least 90%, said protein having a specific binding affinity
to said ED-B domain of fibronectin of Kd=10-5-10-12 M and exhibits
a monovalent binding activity with respect to said extradomain B
(ED-B) of fibronectin.
33. The protein of claim 32, wherein the multimer is formed of the
same or different modified ubiquitin protein.
34. The protein of claim 33, wherein the modified ubiquitin
proteins are genetically or post-translationally fused, optionally
wherein the fusion is directly or via linkers.
35. The protein of any one of claims 32-34, which is a hetero-dimer
of said ubiquitin protein having substitutions at least in
positions 6, 8, 63-66 of the first ubiquitin monomer and at least
in positions 6, 8, 62-66, and optionally in position 2 of the
second ubiquitin monomer, or having substitutions at least in
positions 2, 4, 6, 62, 63, 64, 65, 66 of the first ubiquitin
monomer and at least in positions 6, 8, 62, 63, 64, 65, or 66 and
optionally in position 2 of the second ubiquitin monomer.
36. The protein of claim 32, which is a hetero-dimer of said
ubiquitin protein comprising the sequence SEQ ID NO: 47.
37. The protein of claim 32, wherein both ubiquitin monomers are
linked by a linker, preferably the linker of SEQ ID NO: 32 or by
the sequence SGGGG or any other linker, optionally GIG, SGGGG,
SGGGGIG, SGGGGSGGGGIG or SGGGGSGGGG.
38. The protein of claim 36 or 37, which comprises the ubiquitin
hetero-dimer of SEQ ID NO: 33 or 34.
39. A fusion protein comprising a protein according to claim 32,
fused to a pharmaceutically and/or diagnostically active component,
wherein said pharmaceutically active component is optionally a
cytokine, preferably TNF-alpha, a chemokine, a cytotoxic compound,
or an enzyme, and wherein said diagnostically active component is
selected from a fluorescent compound, a photosensitizer, or a
radionuclide.
40. The fusion protein of claim 39, wherein the fusion protein
preferably has the sequence of SEQ ID NO: 35 or 36 or has an
identity of at least 90% with the sequence of SEQ ID NO: 35 or
36.
41. A pharmaceutical composition containing a protein of claim 32,
a fusion protein of claim 39 or 40, or a combination thereof, and a
pharmaceutically acceptable carrier.
42. The pharmaceutical composition of claim 41, further comprising
one or more chemotherapeutic agents, preferably selected from
melphalan, doxorubicin, cyclophosphamide, dactinomycin,
fluorodesoxyuracil, cisplatin, paclitaxel, and gemcitabine, or from
the group of kinase inhibitors or radiopharmaceuticals.
43. The pharmaceutical composition of claim 42, which is in the
form of a combined preparation or in the form of a kit of
parts.
44. A polynucleotide coding for a recombinant protein of claim 32
or fusion protein according to claim 39 or 40.
45. A vector comprising a polynucleotide according to claim 44.
46. A host cell comprising a multimeric protein according to claim
32 or a fusion protein according to claim 39 or 40.
47. A host cell comprising a polynucleotide according to claim
44.
48. A host cell comprising a vector according to claim 45.
49. A diagnostic agent comprising a multimeric protein according to
claim 32, or a fusion protein according claim 39 or 40, with a
diagnostically acceptable carrier.
50. A method for providing a hetero-multimeric protein capable of
binding the extra domain B (ED-B) of fibronectin according to claim
32, comprising the following steps: a) providing a multimeric
ubiquitin protein comprising two or more modified ubiquitin
monomers linked either directly or by a suitable linker, wherein
each monomer of said multimeric ubiquitin protein was modified in
order to obtain a protein having an amino acid sequence identity to
the amino acid sequence of SEQ ID NO: 1 of at least 60% wherein at
least 3 or 4 amino acids in each monomer are modified by at least
substitution of amino acids in positions 2, 4, 6, 62, 63, 64, 65,
66, and/or 68; b) providing the ED-B of fibronectin; c) contacting
said hetero-multimeric modified ubiquitin protein with said ED-B of
fibronectin; d) screening for modified ubiquitin proteins which
bind to said ED-B of fibronectin with a specific binding affinity
of 10-5-10-12 M, and optionally e) isolating said modified
hetero-multimeric ubiquitin proteins.
51. The method of claim 50, wherein the multimeric ubiquitin
protein is a hetero-dimeric ubiquitin protein.
52. A method of treating cancer comprising administering a
multimeric protein according to claim 32, or a fusion protein
according to claim 39 or 40, to a subject in need thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention refers to novel proteins, in
particular hetero-multimeric proteins, capable of binding the
extradomain B of fibronectin (ED-B). Furthermore, the invention
refers to fusion proteins comprising said binding protein fused to
a pharmaceutically and/or diagnostically active component. The
invention is further directed to a method for the generation of
such binding protein or fusion protein and to
pharmaceutical/diagnostic compositions containing the same. In
addition, the invention refers to libraries which are based on a
scaffold protein comprising linear polyubiquitin chains with at
least two interacting binding determining regions (BDR).
[0002] In further embodiments, the invention is directed
polynucleotides coding for said binding protein or fusion protein,
vectors comprising said polynucleotide and host cells comprising
said protein, fusion protein, vector and/or polynucleotide. In a
preferred embodiment, said binding protein or fusion protein is
included in a medicament or a diagnostic agent. Additionally,
methods for producing said recombinant protein or fusion protein as
well as use of said proteins in medical treatment methods are
described.
BACKGROUND OF THE INVENTION
[0003] There is a growing demand for binding molecules consisting
of amino acids which are not immunoglobulins. While until now
antibodies represent the best-established class of binding
molecules there is still a need for new binding molecules in order
to target ligands with high affinity and specificity since
immunoglobulin molecules suffer from major drawbacks. Although they
can be produced quite easily and may be directed to almost any
target, they have a quite complex molecular structure. There is an
ongoing need to substitute antibodies by smaller molecules which
can be handled in an easy way. These alternative binding agents can
be beneficially used for instance in the medical fields of
diagnosis, prophylaxis and treatment of diseases.
[0004] Proteins having relatively defined 3-dimensional structures,
commonly referred to as protein scaffolds, may be used as starting
material for the design of said alternative binding agents. These
scaffolds typically contain one or more regions which are amenable
to specific or random sequence variation, and such sequence
randomisation is often carried out to produce a library of proteins
from which the specific binding molecules may be selected.
Molecules with a smaller size than antibodies and a comparable or
even better affinity towards a target antigen are expected to be
superior to antibodies in terms of pharmacokinetic properties and
immunogenicity.
[0005] A number of previous approaches do use protein scaffolds as
starting material of binding proteins. For example, in WO 99/16873
modified proteins of the lipocalin family (so-called Anticalins)
exhibiting binding activity for certain ligands were developed. The
structure of peptides of the lipocalin family is modified by amino
acid replacements in their natural ligand binding pocket using
genetic engineering methods. Like immunoglobulins, the Anticalins
can be used to identify or bind molecular structures. In a manner
analogously to antibodies, flexible loop structures are modified;
these modifications enable the recognition of ligands different
from the natural ones.
[0006] WO 01/04144 describes the artificial generation of a binding
domain on the protein surface in beta sheet structural proteins per
se lacking a binding site. By means of this de novo generated
artificial binding domain e.g. variations in .gamma.-crystallin--an
eye lens structural protein--can be obtained which interact with
ligands with high affinity and specificity. In contrast to the
modification of binding sites which are already present and formed
from flexible loop structures as mentioned above for Anticalins,
these binding domains are generated de novo on the surface of beta
sheets. However, WO 01/04144 only describes the alteration of
relatively large proteins for the generation of novel binding
properties. Due to their size the proteins according to WO 01/04144
can be modified on the genetic engineering level only by methods
which require some effort. Furthermore, in the proteins disclosed
so far only a relatively small proportion by percentage of the
total amino acids was modified in order to maintain the overall
structure of the protein. Therefore, only a relatively small region
of the protein surface is available which can be utilized for the
generation of binding properties that did not exist previously.
Moreover, WO 01/04144 discloses only the generation of a binding
property to .gamma.-crystallin.
[0007] WO 04/106368 describes the generation of artificial binding
proteins on the basis of ubiquitin proteins. Ubiquitin is a small,
monomeric, and cytosolic protein which is highly conserved in
sequence and is present in all known eukaryotic cells from
protozoans to vertebrates. In the organism, it plays a crucial role
in the regulation of the controlled degradation of cellular
proteins. For this purpose, the proteins destined for degradation
are covalently linked to ubiquitin or polyubiquitin chains during
their passage through a cascade of enzymes and are selectively
degraded because of this label. According to recent results,
ubiquitin or the labelling of proteins by ubiquitin, respectively,
plays an important role also in other cellular processes such as
the import of several proteins or the gene regulation thereof.
[0008] Besides the clarification of its physiological function,
ubiquitin is a research object primarily because of its structural
and protein-chemical properties. The polypeptide chain of ubiquitin
consists of 76 amino acids folded in an extraordinarily compact
.alpha./.beta. structure (Vijay-Kumar, 1987): almost 87% of the
polypeptide chain is involved in the formation of the secondary
structural elements by means of hydrogen bonds. Secondary
structures are three and a half alpha-helical turns as well as an
antiparallel .beta. sheet consisting of four strands. The
characteristic arrangement of these elements--an antiparallel
.beta. sheet exposed of the protein surface onto the back side of
which an alpha helix is packed which lies vertically on top of
it--is generally considered as so-called ubiquitin-like folding
motif. A further structural feature is a marked hydrophobic region
in the protein interior between the alpha helix and the .beta.
sheet.
[0009] Because of its small size, artificial preparation of
ubiquitin can be carried out both by chemical synthesis and by
means of biotechnological methods. Due to the favourable folding
properties, ubiquitin can be produced by genetic engineering using
microorganisms such as Escherichia coli in relatively large amounts
either in the cytosol or in the periplasmic space. Because of the
oxidizing conditions predominating in the periplasm the latter
strategy generally is reserved for the production of secretory
proteins. Due to the simple and efficient bacterial preparation
ubiquitin can be used as a fusion partner for other foreign
proteins to be prepared for which the production is problematic. By
means of fusion to ubiquitin an improved solubility and thereby an
improved production yield can be achieved.
[0010] Compared to antibodies or other alternative scaffolds,
artificial binding proteins on the basis of ubiquitin proteins
(also referred to as Affilin.RTM.) have the advantages of a small
size and high stability, high affinity high specificity, cost
effective microbial manufacturing, and adjustment of serum half
life. However, there is still a need to further develop those
proteins in terms of new therapeutic approaches with high
affinities to specific targets. While WO 05/05730 generally
describes the use of ubiquitin scaffolds in order to obtain
artificial binding proteins, no solution is provided on how to
modify an ubiquitin protein in order to obtain a specific and high
affinity binding to the ED-B of fibronectin.
[0011] WO 2008/022759 describes recombinant binding protein wherein
the Src homology 3 domain (SH3) of the FYN kinase is used for
obtaining new binding proteins. It was found that the target
specificity can be designed by mutating the RT loop and/or the Src
loop in order to develop protein therapeutics and/or diagnostics.
Like in lipocalins used as scaffold, the amino acid residues to be
mutagenized lie within the variable and flexible loop regions
mimicking the principle underlying the antibody/antigen binding
function. This overall flexibility of the interaction site by which
antibodies bind the epitope is a mainly enthalpically driven
process; this process, however, leads to an unfavorable entropic
contribution by loss of mobility upon association of the flexible
complementarity determining region. Contrary thereto, using
ubiquitin as a scaffold, the present inventors did not change amino
acid residues primarily within the flexible loop regions but within
the rigid and inflexible .beta. strands of a .beta. sheet region or
closely adjacent to the beta strands. The advantage of selecting
amino acid residues within the inflexible and rigid .beta. strands
or closely adjacent to the beta strands of ubiquitin as binding
regions for ED-B is inter alia the following: The binding partners
are thought to already present a complementary geometry appropriate
for tight binding. Consequently, these interactions involve
complementarity in shape, charge and hydrophilic/hydrophobic
elements of the more rigid structures of the binding partners.
These rigid body interactions optimize the interface and
accommodate biological function.
[0012] Fibronectins (FN) are an important class of high molecular
weight extracellular matrix glycoproteins abundantly expressed in
healthy tissues and body fluids. Their main role consists in
facilitating the adhesion of cells to a number of different
extracellular matrices.
[0013] The presence of fibronectins on the surface of
non-transformed cells in culture as well as their absence in the
case of transformed cells resulted in the identification of
fibronectins as important adhesion proteins. They interact with
numerous various other molecules, e.g. collagen, heparan
sulphate-proteoglycans and fibrin and thus regulate the cell shape
and the creation of the cytoskeleton. In addition, they are
responsible for cell migration and cell differentiation during
embryogenesis. They also play an important role in wound healing,
in which they facilitate the migration of macrophages and other
immune cells and in the formation of blood clots by enabling the
adhesion of blood platelets to damaged regions of the blood
vessels.
[0014] The extra-domain B (ED-B) of fibronectin is a small domain
which is inserted by alternative splicing of the primary RNA
transcript into the fibronectin molecule. The molecule is either
present or omitted in fibronectin molecules of the extracellular
matrix and represents a one of the most selective markers
associated with angiogenesis and tissue remodeling, as it is
abundantly expressed around new blood vessels, but undetectable in
virtually all normal adult tissues (except for uterus and ovaries).
ED-B is known to be involved in cancer and in psoriasis. High
levels of ED-B expression were detected in almost all human solid
cancer entities, including breast, colorectal, pancreatic,
non-small cell lung, hepatocellular, intracraneal meningeoma, human
skin, and glioblastoma (Menrad u. Menssen, 2005). Furthermore, ED-B
can be bound to diagnostic agents and be favorably used as
diagnostic tool. One example is its use in molecular imaging of
e.g. atherosclerotic plaques and detection of cancer, e.g. by
immunoscintigraphy of cancer patients. Plenty of further diagnostic
uses are conceivable.
[0015] The amino acid sequence of 91 amino acids of human
extra-domain B (ED-B) of fibronectin is shown in SEQ ID NO: 2. For
expression of the protein, a start methionin has to be added. ED-B
is conserved in mammals, e.g. in rodents, cattle, primates,
carnivore, human etc. Examples of animals in which there is a 100%
sequence identity to human ED-B are Rattus norvegicus, Bos taurus,
Mus musculus, Equus caballus, Macaca mulatta, Canis lupus
familiaris, and Pan troglodytes.
[0016] ED-B specifically accumulates in neo-vascular structures and
represents a target for molecular intervention in cancer. A number
of antibodies or antibody fragments to the ED-B domain of
fibronectin are known in the art as potential therapeutics for
cancer and other indications (see, for example, WO 97/45544, WO
07/054,120, WO 99/58570, WO 01/62800). Human single chain Fv
antibody fragment ScFvL19 (also referred to as L19) is specific to
the ED-B domain of fibronectin and has been verified to selectively
target tumor neovasculature, both in experimental tumor models and
in patients with cancer. Furthermore, conjugates comprising an
anti-ED-B antibody or an anti-ED-B antibody fragment with cytokines
such as IL-12, IL-2, IL-10, IL-15, IL-24, or GM-CSF have been
described for targeting drugs for the manufacture of a medicament
for inhibiting particularly cancer, angiogenesis, or neoplastic
growth (see, for example, WO06/119897, WO07/128,563, WO01/62298).
The selective targeting of neovasculature of solid tumors with
anti-ED-B antibodies or anti-ED-B antibody fragments such as L19
conjugated to an appropriate effector function such as a cytotoxic
or an immunostimulating agent has proven to be successful in animal
experiments. For the therapy of pancreatic cancer, fusion proteins
comprising an Interleukin-2 part (IL-2) and an anti-ED-B antibody
part were combined with the small molecule Gemcitabine
(2'-deoxy-2',2'-difluorocytidine) (see for example WO
07/115,837).
[0017] The above-discussed prior art documents describe the use of
various protein scaffolds including antibodies to generate new ED-B
binding proteins.
[0018] Targeting ED-B with currently available compounds has
certain disadvantages. Smaller molecules (such as ubiquitin-based
ED-B binding proteins of this invention) with a comparable or even
higher affinity towards the ED-B antigen are expected to have
significant advantages to antibodies or other binding proteins.
[0019] Since cancer represents one of the leading causes for death
worldwide, there is a growing need for improved agents for treating
cancer. Current chemotherapeutic agents and radiation treatment
suffer from poor selectivity and most chemotherapeutic agents do
not accumulate at the tumor site and thus fail to achieve adequate
levels within the tumor. There is a strong medical need to
effectively treat cancer.
[0020] It is thus an object of the present invention to provide new
binding proteins based on ubiquitin being able to bind specifically
with very high affinity to the extracellular domain of fibronectin
(ED-B). It is a further object of the present invention to identify
and provide novel binding proteins with very high binding
specificity to ED-B for example, for use in the treatment of
cancer. Furthermore, a method shall be provided in order to produce
said binding molecules.
[0021] The above-described objects are solved by the subject-matter
of the enclosed independent claims. Preferred embodiments of the
invention are included in the dependent claims as well as in the
following description, examples and figures.
DESCRIPTION OF THE INVENTION
[0022] More specifically, the inventors provide a protein capable
of binding the ED-B of human fibronectin, comprising a modified
ubiquitin protein having an amino acid sequence identity to the
amino acid sequence of SEQ ID NO: 1 of at least 60%, wherein at
least 4 amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66,
and 68 of SEQ ID NO: 1 are modified in order to obtain a modified
ubiquitin protein with a detectable binding to said ED-B of
fibronectin with a specific binding affinity of
Kd=10.sup.-6-10.sup.-12 M.
[0023] In a preferred embodiment, the protein is recombinant.
[0024] In further embodiments of the invention, 4, 5, 6, 7, 8, 9 or
all of the amino acids in positions 2, 4, 6, 8, 62, 63, 64, 65, 66,
and 68 of SEQ ID NO: 1 are modified.
DEFINITIONS OF IMPORTANT TERMS USED IN THE APPLICATION
[0025] The term "extra-domain B of fibronectin" or briefly
designated as "ED-B" comprises all proteins which show a sequence
identity to SEQ ID NO: 2 of at least 70%, optionally 75%, further
optionally 80%, 85%, 90%, 95%, 96% or 97% or more, or 100% and
having the above defined functionality of ED-B.
[0026] The terms "protein capable of binding" or "binding protein"
refer to an ubiquitin protein comprising a binding domain to ED-B
as further defined below. Any such binding protein based on
ubiquitin may comprise additional protein domains that are not
binding domains, such as, for example, multimerization moieties,
polypeptide tags, polypeptide linkers and/or non-proteinaceous
polymer molecules. Some examples of non-proteinaceous polymer
molecules are hydroxyethyl starch, polyethylene glycol,
polypropylene glycol, or polyoxyalkylene.
[0027] Antibodies and fragments thereof are well known to the
person skilled in the art. The binding protein of the invention is
not an antibody or a fragment thereof, such as Fab or scFv
fragments. Further, the binding domain of the invention does not
comprise an immunoglobulin fold as present in antibodies.
[0028] In the present specification, the terms "ligand" and
"target" and "binding partner" are used synonymously and can be
exchanged. A ligand is any molecule capable of binding with an
affinity as defined herein to the hetero-multimeric modified
ubiquitin protein.
[0029] The term "ubiquitin protein" covers the ubiquitin in
accordance with SEQ ID NO: 1 and modifications thereof according to
the following definition. Ubiquitin is highly conserved in
eukaryotic organisms. For example, in all mammals investigated up
to now ubiquitin has the identical amino acid sequence.
Particularly preferred are ubiquitin molecules from humans,
rodents, pigs, and primates. Additionally, ubiquitin from any other
eukaryotic source can be used. For instance ubiquitin of yeast
differs only in three amino acids from the sequence of SEQ ID NO:
1. Generally, the ubiquitin proteins covered by said term
"ubiquitin protein" show an amino acid identity of more than 70%,
preferably more than 75% or more than 80%, of more than 85%, of
more than 90%, of more than 95%, of more than 96% or up to a
sequence identity of 97% to SEQ ID NO: 1.
[0030] The term "a modified ubiquitin protein" refers to
modifications of the ubiquitin protein by any one of substitutions,
insertions, or deletions of amino acids, or a combination thereof.
The modified ubiquitin proteins of the invention are engineered
proteins with novel binding affinities to targets.
[0031] For determining the extent of sequence identity of a
derivative of the ubiquitin to the amino acid sequence of SEQ ID
NO: 1, for example, the SIM Local similarity program (Xiaoquin
Huang and Webb Miller, "Advances in Applied Mathematics, vol. 12:
337-357, 1991) or Clustal, W. can be used (Thompson et al., Nucleic
Acids Res., 22(22): 4673-4680, 1994.). Preferably, the extent of
the sequence identity of the modified protein to SEQ ID NO: 1 is
determined relative to the complete sequence of SEQ ID NO: 1.
[0032] The "hetero-dimeric fusion protein" or "hetero-dimeric
protein" of the invention is considered as a protein which
comprises two differently modified monomeric ubiquitin proteins
with two interacting binding domain regions providing together a
monovalent binding property (binding domain) for ED-B as the
specific binding partner. A hetero-dimer is accomplished by fusing
two monomeric ubiquitin molecules wherein both of these molecules
are differently modified as described herein.
[0033] An advantage of multimerization of differently modified
ubiquitin monomers in order to generate hetero-multimeric binding
proteins (here: hetero-dimeric proteins) with monovalent binding
activity lies in the increase of the total number of amino acid
residues that can be modified to generate a new high affinity
binding property to ED-B. The main advantage is that while even
more amino acids are modified, the protein-chemical integrity is
maintained without decreasing the overall stability of the scaffold
of said newly created binding protein to ED-B. The total number of
residues which can be modified in order to generate a novel binding
site for ED-B is increased as the modified residues can be
allocated to two monomeric ubiquitin proteins. The number of
modifications can so be two corresponding to the number of modified
monomeric ubiquitin molecules. A modular structure of the
ubiquitin-based ED-B binding protein allows increasing the overall
number of modified amino acids as said modified amino acids are
included on two monomeric ubiquitin molecules. The present method
provides for the identification of hetero-dimeric ubiquitin
molecules having one monovalent specificity for ED-B.
[0034] Thus, the use of hetero-dimers having a common binding site
for binding partners opens up the possibility to introduce an
increased number of modified residues which do not unduly influence
the protein-chemical integrity of the final binding molecule, since
the overall amount of those modified residues is distributed over
the two monomeric units which form the dimer. Said hetero-dimeric
modified ubiquitin proteins binding to ED-B are present in a
library of proteins.
[0035] "Monovalent" has to be understood as the capability that
both binding regions created in the first and the second monomeric
unit of the modified dimeric ubiquitin together bind ED-B in a
synergistic and combined manner, i.e. both binding regions act
together to form a monovalent binding activity. Taking each binding
region of both the first and the second modified ubiquitin in said
hetero-dimeric molecule separately will apparently bind ED-B with a
much lower efficiency and affinity than the dimeric molecule. Both
binding regions form a unique binding site which is formed as a
contiguous region of amino acids on the surface of the
hetero-dimeric modified ubiquitin protein so that said modified
ubiquitin is feasible to bind much more efficient to ED-B than each
monomeric protein taken alone. It is particularly important that
according to the present invention the two monomeric proteins are
not linked together after having screened the most potent binding
ubiquitin molecules but that already the screening process is
performed in the presence of the hetero-dimeric ubiquitins. After
having received the sequence information on the most potent binding
ubiquitin molecules, these molecules may be obtained by any other
method, e.g. by chemical synthesis or by genetic engineering
methods, e.g. by linking the two already identified monomeric
ubiquitin units together.
[0036] According to the invention, the at least two differently
modified ubiquitin monomers which bind to one ligand are to be
linked by head-to-tail fusion to each other using e.g. genetic
methods. The differently modified fused ubiquitin monomers bind in
a monovalent manner and are only effective if both "binding domain
regions" ("BDR") act together. A "binding domain region" is defined
herein as region on a ubiquitin monomer that has modified amino
acids in at least 4, preferably 6 amino acids of positions 2, 4, 6,
8, 62, 63, 64, 65, 66, 68 of SEQ ID NO:1 which are involved in
binding the target.
[0037] The modified and linked ubiquitin monomers which form the
hetero-dimeric protein bind to the same epitope via a single
contiguous binding region. This contiguous region of the heteromer
is formed by both binding determining regions of the two modules
formed by two differently modified ubiquitin monomers.
[0038] A "head to-tail fusion" is to be understood as fusing two
proteins together by connecting them in the direction N--C--N--C--
depending on the number of units contained in the dimer. In this
head-to-tail fusion, the ubiquitin monomers may be connected
directly without any linker. Alternatively, the fusion of ubiquitin
monomers can be performed via linkers, for example, a linker having
at least the amino acid sequence GIG or having at least the amino
acid sequence SGGGG or any other linker, for example GIG, SGGGG,
SGGGGIG, SGGGGSGGGGIG or SGGGGSGGGG. Also other linkers for the
genetic fusion of two ubiquitin monomers are known in the art and
can be used.
[0039] The modified ubiquitin proteins of the invention are
engineered proteins with novel binding affinities to ED-B as target
or ligand (which expressions are used herein interchangeably). The
term "substitution" comprises also the chemical modification of
amino acids by e.g. substituting or adding chemical groups or
residues to the original amino acid. The substitution of amino
acids in at least one surface-exposed region of the protein
comprising amino acids located in at least one beta sheet strand of
the beta sheet region or positioned up to 3 amino acids adjacent to
the beta sheet strand is crucial.
[0040] The substitution of amino acids for the generation of the
novel binding domain specific to the ED-B can be performed
according to the invention with any desired amino acid, i.e. for
the modification to generate the novel binding property to ED-B it
is not mandatory to take care that the amino acids have a
particular chemical property or a side chain, respectively, which
is similar to that of the amino acids substituted so that any amino
acid desired can be used for this purpose.
[0041] The step of modification of the selected amino acids is
performed according to the invention preferably by mutagenesis on
the genetic level by random mutagenesis, i.e. a random substitution
of the selected amino acids. Preferably, the modification of
ubiquitin is carried out by means of methods of genetic engineering
for the alteration of a DNA belonging to the respective protein.
Preferably, expression of the ubiquitin protein is then carried out
in prokaryotic or eukaryotic organisms.
[0042] Substitutions are performed particularly in surface-exposed
amino acids of the four beta strands of the beta sheets or surface
exposed amino acids up to 3 amino acids adjacent to the beta sheet
strand of ubiquitin protein. Each beta strand consists usually of
5-7 amino acids.
[0043] With reference to SEQ ID NO: 1, for example, the beta
strands usually cover amino acid residues 2-7, 12-16, 41-45 and
65-71. Regions which may be additionally and preferably modified
include positions up to 3 amino acids (i.e. 1, 2, or 3) adjacent to
the beta sheet strand. The preferred regions which may be
additionally and preferably modified include in particular amino
acid residues 8-11, 62-64 and 72-75. The preferred regions include
beta turns which link two beta strands together. One preferred
beta-turn includes amino residues 62-64. A most preferred amino
acid which is closely adjacent to the beta sheet strand is the
amino acid in position 8. In addition, further preferred examples
for amino acid substitutions are positions 36, 44, 70, and/or 71.
For example, those regions which may be additionally and preferably
modified include amino acids 62, 63, and 64 (3 amino acids), or 72,
73 (2 amino acids), or 8 (1 amino acid).
[0044] In preferred embodiments, the amino acid residues are
altered by amino acid substitutions. However, also deletions and
insertions are allowable. The number of amino acids which may be
added or deleted is limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
amino acids in a monomeric ubiquitin subunit, and accordingly 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26
or 28 amino acids with respect to the dimeric ubiquitin protein,
generally x-times the number of modifications in the monomeric
protein. In one embodiment, no amino acid insertions are made. In a
still further embodiment, no deletions have been performed.
[0045] Provided that the modified ubiquitin protein of the present
invention comprises substitutions, deletions and/or additions of
one or more amino acids, the amino acid positions given for
wildtype human ubiquitin (SEQ ID NO: 1) have to be aligned with the
modified ubiquitin in order to allot the corresponding proteins to
each other. In case of fusion proteins (see below), the numbering
(and alignment) of each of the monomeric ubiquitin subunits is done
in the same way, i.e. an alignment of, for example, a dimer is
started at amino acid position 1 for each respective subunit.
[0046] In monomeric ubiquitin, preferably from mammals, e.g. human,
at least 10% of the amino acids present in beta strands or
positions up to 3 amino acids adjacent to the beta sheet strand,
preferably at least 20%, further preferably at least 25%, can be
modified, preferably substituted, according to the present
invention to generate a binding property that did not exist
previously. At a maximum, preferably about 50% of the amino acids
present in beta strands or positions up to 3 amino acids adjacent
to the beta sheet strand, further preferably at a maximum about 40%
or about 35% or up to about 30% or up to about 25% are modified,
preferably substituted. In one beta strand, generally one to four
amino acids are modified. In one embodiment, three of six amino
acids in preferably the first and the fourth beta strand, e.g.
region of amino acid residues 2-7 or 65-71, are modified.
[0047] A modified monomeric ubiquitin according to the invention
used as building unit for a hetero-dimer accounts for in total up
to 20% of amino acids. Considering this, there is a sequence
identity to SEQ ID NO:1 of the modified ubiquitin protein to at
least 60%. In further embodiments of the invention, the sequence
identity on amino acid level is at least 60%, 70%, at least 80% and
furthermore at least 90% or at least 95% sequence identity to the
amino acid sequence of SEQ ID NO: 1. The invention covers also
amino acid sequence identities of more than 65%, 75%, 85% or 97% of
the modified ubiquitin protein compared to the amino acid sequence
of SEQ ID NO: 1.
[0048] In a further embodiment of the invention, an ubiquitin is
modified in 3 or 4 or 5 or 6 or 7 amino acids in positions 2, 4, 6,
8, 62, 63, 64, 65, 66, and/or 68 of SEQ ID NO: 1. In another
embodiment, the ubiquitin to be modified in these positions, was
already pre-modified. For example, further modifications could
comprise modifications at amino acids 74 and 75 or at amino acid 45
to generate better stability or protein-chemical properties. A
modified ubiquitin monomer is obtainable wherein in total up to 9,
10, 11, 12, 13, 14 and a maximum of 15 amino acids of the ubiquitin
of SEQ ID NO: 1 are modified, preferably substituted. According to
an example, a modified monomeric ubiquitin could be obtained having
14 substitutions and a deletion. Based on the total number of amino
acids of ubiquitin this corresponds to a percentage of about 20%.
This was extraordinarily surprising and could not be expected since
usually a much lower percentage is already sufficient to disturb
the folding of the protein.
[0049] In one embodiment of the invention, those amino acids are
modified for the generation of a region having the novel binding
properties which form a contiguous region on the surface of the
protein. In this manner, a contiguous region can be generated which
has a binding property to the ED-B. "Contiguous region" according
to the invention refers to the following: due to the charge, the
spatial structure and the hydrophobicity/hydrophilicity of their
side chains, amino acids interact with their environment in the
corresponding manner. The environment can be the solvent, generally
water, or other molecules, e.g. spatially close amino acids. By
means of structural information about the protein as well as the
respective software the surface of the proteins can be
characterized. For example, the interface region between the atoms
of the protein and the solvent can be visualized in this way
including the information about how this interface region is
structured, which surface areas are accessible to the solvent or
how the charges are distributed on the surface. A contiguous region
can be revealed for example by visualization of this type using
suitable software. Such methods are known to those skilled in the
art. According to the invention, basically, also the whole
surface-exposed region can be used as the contiguous region on the
surface to be modified for the generation of novel binding
properties. In one embodiment, for this purpose a modification can
also comprise the .alpha.-helical region. In a hetero-dimeric
modified ubiquitin protein, a binding-determining region comprises
two of the surface-exposed regions forming together one contiguous
region which comprises two times the length of one binding
determining region.
[0050] The modification of amino acids in at least one
surface-exposed region of the protein comprising at least one beta
strand of the beta sheet region or positions up to 3 amino acids
adjacent to the beta sheet strand is crucial. The "beta sheet
structure" is defined by being essentially sheet-like and almost
completely stretched. In contrast to alpha helices which are formed
from an uninterrupted segment of the polypeptide chain, beta sheets
can be formed by different regions of the polypeptide chain. In
this way, regions spaced further apart in the primary structure can
get into close proximity with each other. A beta strand typically
has a length of 5-10 amino acids (usually 5-6 residues in
ubiquitin) and has an almost completely stretched conformation. The
beta strands come so close to each other that hydrogen bonds form
between the C--O group of one strand and the NH group of the other
strand and vice versa. Beta-sheets can be formed from several
strands and have a sheet-like structure wherein the position of the
C alpha atoms alternates between above or below the sheet-like
plane. The amino acid side chains follow this pattern and, thus,
alternatively point towards the top or towards the bottom.
Depending on the orientation of the beta strands the sheets are
classified into parallel and antiparallel sheets. According to the
invention both can be mutated and used for the preparation of the
proteins claimed.
[0051] For the mutagenesis of the beta strands and the beta-sheet
structure, a beta strand or positions up to 3 amino acids adjacent
to the beta strand (which is a strand of the beta sheet) are
selected in the ubiquitin that are close to the surface
Surface-exposed amino acids can be identified with respect to the
available x-ray crystallographic structure. If no crystal structure
is available attempts can be made by means of computer analysis to
predict surface-exposed beta sheet regions and the accessibility of
individual amino acid positions with respect to the available
primary structure or to model the 3d protein structure and to
obtain information about potential surface-exposed amino acids in
this manner. Further disclosure thereof can be taken e.g. from J.
Mol. Biol., 1987 Apr. 5; 194(3):531-44. Vijay-Kumar S, Bugg C. E.,
Cook W. J.
[0052] It is, however, also possible to carry out modifications in
the beta sheet or of positions up to 3 amino acids adjacent to the
beta strand for which the time-consuming pre-selection of amino
acid positions to be mutagenized can be omitted. Those DNA regions
encoding the beta sheet structures or up to 3 amino acids adjacent
to the beta sheet strand are isolated from their DNA environment,
subjected to random mutagenesis and are afterwards re-integrated
into the DNA coding for the protein from which they were removed
previously. This is followed by a selection process for mutants
with the desired binding properties.
[0053] In another embodiment of the invention the beta strands or
up to 3 amino acids adjacent to the beta strand close to the
surface are selected as already explained above and the amino acid
positions to be mutagenized within these selected regions are
identified. The amino acid positions selected in this way can then
be mutagenized on the DNA level either by site-directed
mutagenesis, i.e. a codon coding for a specific amino acid is
substituted by a codon encoding another previously selected
specific amino acid, or this substitution is carried out in the
context of a random mutagenesis wherein the amino acid position to
be substituted is defined but not the codon encoding the novel, not
yet determined amino acid.
[0054] Surface-exposed amino acids are amino acids that are
accessible to the surrounding solvent. If the accessibility of the
amino acids in the protein is more than 8% compared to the
accessibility of the amino acid in the model tripeptide Gly-X-Gly,
the amino acids are called "surface-exposed". These protein regions
or individual amino acid positions, respectively, are also
preferred binding sites for potential binding partners for which a
selection shall be carried out according to the invention. In
addition, reference is made to Caster et al., 1983 Science, 221,
709-713, and Shrake & Rupley, 1973 J. Mol. Biol. 79(2):351-371,
which for complete disclosure are incorporated by reference in this
application.
[0055] Variations of ubiquitin protein scaffold differing by amino
acid substitutions in the region of the de novo generated
artificial binding site from the parental protein and from each
other can be generated by a targeted mutagenesis of the respective
sequence segments. In this case, amino acids having certain
properties such as polarity, charge, solubility, hydrophobicity or
hydrophilicity can be replaced or substituted, respectively, by
amino acids with respective other properties. Besides
substitutions, the terms "mutagenesis" and "modified" and
"replaced" comprises also insertions and deletions. On the protein
level the modifications can also be carried out by chemical
alteration of the amino acid side chains according to methods known
to those skilled in the art.
Methods of Mutagenesis of Ubiquitin
[0056] As a starting point for the mutagenesis of the respective
sequence segments, for example the cDNA of ubiquitin which can be
prepared, altered, and amplified by methods known to those skilled
in the art can be used. For site-specific alteration of ubiquitin
in relatively small regions of the primary sequence (about 1-3
amino acids) commercially available reagents and methods are on
hand ("Quick Change", Stratagenc; "Mutagene Phagemid in vitro
Mutagenesis Kit", Biorad). For the site-directed mutagenesis of
larger regions specific embodiments of e.g. the polymerase chain
reaction (PCR) are available to those skilled in the art. For this
purpose a mixture of synthetic oligodeoxynucleotides having
degenerated base pair compositions at the desired positions can be
used for example for the introduction of the mutation. This can
also be achieved by using base pair analogs which do not naturally
occur in genomic DNA, such as e.g. inosine.
[0057] Starting point for the mutagenesis of one or more beta
strands of the beta sheet region or positions up to 3 amino acids
adjacent to the beta sheet strand can be for example the cDNA of
ubiquitin or also the genomic DNA. Furthermore, the gene coding for
the ubiquitin protein can also be prepared synthetically.
[0058] Different methods known per se are available for
mutagenesis, e.g. methods for site-specific mutagenesis, methods
for random mutagenesis, mutagenesis using PCR or similar
methods.
[0059] In a preferred embodiment of the invention the amino acid
positions to be mutagenized are predetermined. The selection of
amino acids to be modified is carried out to meet the limitations
of present claim 1 with respect to those amino acids which have to
be modified. In each case, a library of different mutants is
generally established which is screened using methods known per se.
Generally, a pre-selection of the amino acids to be modified can be
particularly easily performed as sufficient structural information
is available for the ubiquitin protein to be modified.
[0060] Methods for targeted mutagenesis as well as mutagenesis of
longer sequence segments, for example by means of PCR, by chemical
mutagenesis or using bacterial mutator strains also belong to the
prior art and can be used according to the invention.
[0061] In one embodiment of the invention the mutagenesis is
carried out by assembly of DNA oligonucleotides carrying the amino
acid codon NNK. It should be understood, however, that also other
codons (triplets) can be used. The mutations are performed in a way
that the beta sheet structure is preferably maintained. Generally,
the mutagenesis takes place on the outside of a stable beta sheet
region exposed on the surface of the protein. It comprises both
site-specific and random mutagenesis. Site-specific mutagenesis
comprising a relatively small region in the primary structure
(about 3-5 amino acids) can be generated with the commercially
available kits of Stratagene (QuickChange) or Bio-Rad (Mutagene
phagemid in vitro mutagenesis kit) (cf. U.S. Pat. No. 5,789,166;
U.S. Pat. No. 4,873,192).
[0062] If more extended regions are subjected to site-specific
mutagenesis a DNA cassette must be prepared wherein the region to
be mutagenized is obtained by the assembly of oligonucleotides
containing the mutated and the unchanged positions (Nord et al.,
1997 Nat. Biotechnol. 8, 772-777; McConell and Hoess, 1995 J. Mol.
Biol. 250, 460-470.). Random mutagenesis can be introduced by
propagation of the DNA in mutator strains or by PCR amplification
(error-prone PCR) (e.g. Pannekoek et al., 1993 Gene 128, 135 140).
For this purpose, a polymerase with an increased error rate is
used. To enhance the degree of the mutagenesis introduced or to
combine different mutations, respectively, the mutations in the PCR
fragments can be combined by means of DNA shuffling (Stemmer, 1994
Nature 370, 389-391). A review of these mutagenesis strategies with
respect to enzymes is provided in the review of Kuchner and Arnold
(1997) TIBTECH 15, 523-530. To carry out this random mutagenesis in
a selected DNA region also a DNA cassette must be constructed which
is used for mutagenesis.
[0063] Random modification is performed by methods well-established
and well-known in the art. A "randomly modified nucleotide or amino
acid sequence" is a nucleotide or amino acid sequence which in a
number of positions has been subjected to insertion, deletion or
substitution by nucleotides or amino acids, the nature of which
cannot be predicted. In many cases the random nucleotides (amino
acids) or nucleotide (amino acid) sequences inserted will be
"completely random" (e.g. as a consequence of randomized synthesis
or PCR-mediated mutagenesis). However, the random sequences can
also include sequences which have a common functional feature (e.g.
reactivity with a ligand of the expression product) or the random
sequences can be random in the sense that the ultimate expression
product is of completely random sequence with e.g. an even
distribution of the different amino acids.
[0064] In order to introduce the randomized fragments properly into
the vectors, it is according to the invention preferred that the
random nucleotides are introduced into the expression vector by the
principle of site directed PCR-mediated mutagenesis. However, other
options are known to the skilled person, and it is e.g. possible to
insert synthetic random sequence libraries into the vectors as
well.
[0065] To generate mutants or libraries by fusion PCR, for example
three PCR reactions may carried out. Two PCR reactions are
performed to generate partially overlapping intermediate fragments.
A third PCR reaction is carried out to fuse the intermediate
fragments.
[0066] The method for construction the library or mutant variants
may include constructing a first set of primers around a desired
restriction site (restriction site primer), a forward and reverse
restriction primer and a second set of primers around, e.g.,
upstream and downstream of the codon of interest (the mutagenic
primers), a forward and reverse mutagenic primer. In one
embodiment, the primers are constructed immediately upstream and
downstream respectively of the codon of interest. The restriction
and mutagenic primers are used to construct the first intermediate
and second intermediate fragments. Two PCR reactions produce these
linear intermediate fragments. Each of these linear intermediate
fragments comprises at least one mutated codon of interest, a
flanking nucleotide sequence and a digestion site. The third PCR
reaction uses the two intermediate fragments and the forward and
reverse restriction primers to produce a fused linear product. The
opposite, here to for unattached ends of the linear product are
digested with a restriction enzyme to create cohesive ends on the
linear product. The cohesive ends of the linear product are fused
by use of a DNA ligase to produce a circular product, e.g. a
circular polynucleotide sequence.
[0067] To construct the intermediate fragments, the design and
synthesis of two sets of forward and reverse primers are performed
by providing a first set containing a restriction enzymes digestion
site together with its flanking nucleotide sequence, and a second
set containing at least one variant codon of interest (mutagenic
primers). Those skilled in the art will recognize that the number
of variants will depend upon the number of variant amino acid
modifications desired. It is contemplated by the inventor that if
other restriction enzymes are used in the process, the exact
location of this digestion site and the corresponding sequence of
the forward and reverse primers may be altered accordingly. Other
methods are available in the art and may be used instead.
[0068] Apart from having the randomized fragment of the expression
product introduced into a scaffold in accordance with the present
invention, it is often necessary to couple the random sequence to a
fusion partner by having the randomized nucleotide sequence fused
to a nucleotide sequence encoding at least one fusion partner. Such
a fusion partner can e.g. facilitate expression and/or
purification/isolation and/or further stabilization of the
expression product.
[0069] Random substitution of amino acids according to one example
of the present invention of at least 3 or 4 amino acids at
positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and/or 68 of monomeric
ubiquitin can be performed particularly easily by means of PCR
since the positions mentioned are localized close to the amino or
the carboxy terminus of the protein. Accordingly, the codons to be
manipulated are at the 5' and 3' end of the corresponding cDNA
strand. Thus, the first oligodeoxynucleotide used for a mutagenic
PCR reaction apart from the codons at positions 2, 4, 6, and/or 8
to be mutated--corresponds in sequence to the coding strand for the
amino terminus of ubiquitin. Accordingly, the second
oligodeoxynucleotide--apart from the codons of positions 62, 63,
64, 65, 66, and/or 68 to be mutated--at least partially corresponds
to the non-coding strand of the polypeptide sequence of the carboxy
terminus. By means of both oligodeoxynucleotides a polymerase chain
reaction can be performed using the DNA sequence encoding the
monomeric ubiquitin as a template.
[0070] Furthermore, the amplification product obtained can be added
to another polymerase chain reaction using flanking
oligodeoxynucleotides which introduce for example recognition
sequences for restriction endonucleases. It is preferred according
to the invention to introduce the gene cassette obtained into a
vector system suitable for use in the subsequent selection
procedure for the isolation of ubiquitin variations having binding
properties to a predetermined hapten or antigen.
Regions to be Modified in Ubiquitin
[0071] The regions for modification can be basically selected as to
whether they can be accessible for ED-B as binding partner and
whether the overall structure of the protein will presumably show
tolerance to a modification.
[0072] Besides modifications in surface-exposed beta strands also
modifications in other surface-exposed regions of the protein can
be carried out, preferably in positions up to 3 amino acids
adjacent to the beta strand. These modified regions are involved in
the newly generated binding with high affinity to ED-B.
[0073] According to another preferred embodiment of the present
invention at least 3 or 4 or 6, optionally at least 8, 10, 12 and
maximal 15 surface-exposed amino acids of ubiquitin, preferably
mammalian or human ubiquitin, can be modified wherein a
substitution is preferred as the modification. This comprises the
modification of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15
surface-exposed amino acids of ubiquitin. These at least 4 and
maximal 15 surface-exposed modified amino acids then form the
region with binding affinity to the predetermined binding partner.
This region is defined herein as "binding domain region" (BDR). In
this respect, it is particularly preferred that at least 2,
optionally at least 4, further optionally at least 6, 8, 10, 12 and
maximal 15 of the surface-exposed amino acids are in a beta sheet
region, i.e. in a beta sheet strand or distributed on several beta
strands or positions up to 3 amino acids adjacent to a beta sheet
strand. It is further preferred that at least 3 of all modified,
preferably substituted, amino acids are directly adjacent to each
other in the primary sequence.
[0074] In another optional embodiment of the present invention
amino acids in one or two, preferably two of the four beta strands
in the protein or positions up to 3 amino acids adjacent to
preferably two of the four beta strands are modified to generate a
novel binding property. Also optional is a modification in three or
four of the four beta strands or positions up to 3 amino acids
adjacent to three or four of the beta strands for the generation of
an ED-B binding.
[0075] It is particularly preferred that amino acids in the
amino-terminal and carboxy-terminal strand or in positions up to 3
amino acids adjacent to the amino-terminal and carboxy-terminal
strand are modified, preferably substituted, to generate a novel
binding site to ED-B. In this respect, it is particularly preferred
that up to 4 amino acids adjacent to the carboxy-terminal beta
sheet strand are modified, preferably substituted, and up to 1
amino acid adjacent to the amino-terminal beta sheet strand is
modified, preferably substituted.
[0076] Particularly preferred is a modification, preferably a
substitution, in at least three surface-exposed amino acids of the
following positions of a mammalian ubiquitin, preferably human
ubiquitin: 2, 4, 6, 8, 62, 63, 64, 65, 66, 68. These at least four
amino acids from said group of amino acids form a contiguous
surface-exposed region on the surface of ubiquitin which was found
to be particularly suitable for the generation of modified proteins
having a binding affinity that did not exist previously with
respect to the ED-B as binding partner. At least 3 of these amino
acid residues have to be modified. Optionally 3, 4, 5, 6, 7, 8, 9
or 10 of said amino acid residues are modified, optionally in
combination with additional amino acid residues.
[0077] After having made the modifications above, the inventors
have found the amino acid modified ubiquitin sequences described in
the examples which bind ED-B with very high affinity (Kd values up
to 10.sup.-9).
Fusion Proteins
[0078] In another preferred embodiment, the invention relates to a
fusion protein comprising a binding protein of the invention fused
to a pharmaceutically and/or diagnostically active component.
[0079] In a still further aspect, the invention relates to a fusion
protein comprising a hetero-dimericbinding protein of the invention
fused to a pharmaceutically and/or diagnostically active component.
A fusion protein of the invention may comprise non-polypeptide
components, e.g. non-peptidic linkers, non-peptidic ligands, e.g.
for therapeutically or diagnostically relevant radionuclides. It
may also comprise small organic or non-amino acid based compounds,
e.g. a sugar, oligo- or polysaccharide, fatty acid, etc. In one
preferred embodiment of the invention, the heteromeric
ubiquitin-based ED-B binding molecule is covalently or
non-covalently conjugated to a protein or peptide having
therapeutically or diagnostically relevant properties.
[0080] The following gives some examples on how to obtain
ubiquitin-based fusion proteins with ED-B binding capacity. [0081]
a) conjugation of the protein via Lysine residues present in
ubiquitin; [0082] b) conjugation of the heterodimeric
ubiquitin-based binding protein via Cysteine residues--can be
located C-terminal, or at any other position (e.g. amino acid
residue 24 or 57); conjugation with maleimid selectable components;
[0083] c) peptidic or proteinogenic conjugations genetic fusions
(preferred C- or N-terminal); [0084] d) "Tag"-based fusions--A
protein or a peptide located either at the C- or N-terminus of the
target protein ED-B. Fusion "tags", e.g. poly-histidine
(particularly relevant for radiolabeling).
[0085] These and other methods for covalently and non-covalently
attaching a protein of interest to a support are well known in the
art, and are thus not described in further detail here.
[0086] Optionally, said active component is a cytokine, preferably
a cytokine selected from the group consisting of tumor necrosis
factors (e.g. TNF alpha, TNF beta), interleukins (e.g. IL-2, IL-12,
IL-10, IL-15, IL-24, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11,
IL-13, IL-8, IL-1 alpha, IL-1beta), interferons (e.g. IFN alpha,
IFN beta, IFN gamma), GM-CSF, GRO (GRO alpha, GRO beta, GRO
gamma,), MIP (MIP-1-alpha, MIP-1 beta, MIP-3 alpha, MIP-3 beta),
TGF-beta LIF1 CD80, CD-40 ligand, B70, LT-beta, Fas-ligand, ENA-78,
LDGF-PBP, GCP-2, PF4, Mig, IP-10, SDF-1 alpha/beta, BUNZO/STRC33,
I-TAC, BLC/BCA-1, MDC, TECK, TARC, RANTES, HCC-1, HCC-4, DC-CK1,
MCP-1-5, Eotaxin, Eotaxin-2, 1-309, MPIF-1, 6Ckine, CTACK, MEC,
Lymphotactin, Fractalkine, and others.
[0087] One of the most preferred cytokines for use in the present
invention is TNF. The inflammatory cytokine TNF has multiple
activities in the mammalian body including an anti-tumor effect
that is currently clinically irrelevant due to unacceptable
toxicity of effective doses in humans. Currently, TNF is
therapeutically used in combination with cytostatic substances like
Melphalan.
[0088] Further optionally, said active component that can be
conjugated to the hetero-multimeric ubiquitin-based binding protein
is a toxic compound, preferably a small organic compound or a
polypeptide, optionally a toxic compound, for example, selected
from the group consisting of saporin, truncated Pseudomonas
exotoxin A, recombinant gelonin, Ricin-A chain, calicheamicin,
neocarzinostatin, esperamicin, dynemicin, kedarcidin, maduropeptin,
doxorubicin, daunorubicin, auristatin, cholera toxin, modeccin,
diphtheria toxin.
[0089] In a further embodiment of the invention the ubiquitin-based
binding protein according to the invention may contain artificial
amino acids.
[0090] In further embodiments of the fusion protein of the present
invention said active component is a fluorescent dye, preferably a
component selected from the groups of a radionuclide either from
the group of gamma-emitting isotopes, preferably 99.sub.Tc,
123.sub.I, 111.sub.In, or from the group of positron emitters,
preferably 18.sub.F, 64.sub.Cu, 68.sub.Ga, 86.sub.Y, 124.sub.I, or
from the group of beta-emitter, preferably 131.sub.I, 90.sub.Y,
177.sub.Lu, 67.sub.Cu, or from the group of alpha-emitter,
preferably 213.sub.Bi, 211.sub.Al; Alexa Fluor or Cy dyes (Berlier
et al., J. Histochem. Cytochem. 51 (12): 1699-1712, 2003.); a
photosensitizer; a pro-coagulant factor, preferably tissue factor
(e.g. tTF truncated tissue factor); an enzyme for pro-drug
activation, preferably an enzyme selected from the group consisting
of carboxy-peptidases, glucuronidases and glucosidases; and/or a
functional Fc domain, preferably a human functional Fc domain.
[0091] A further embodiment relates to fusion proteins according to
the invention, further comprising a component modulating serum
half-life, preferably a component selected from the group
consisting of polyethylene glycol albumin-binding peptides, and
immunoglobulin.
Binding Specificities
[0092] The binding specificities (dissociation constants) of the
fusion proteins according to the invention are as defined above for
the non-fusion protein given in Kd. In accordance with the
invention, the term "Kd" defines the specific binding affinity
which is in accordance with the invention in the range of
10.sup.-5-10.sup.-12 M. A value of 10.sup.-5 M and below can be
considered as a quantifiable binding affinity. Depending on the
application a value of 10.sup.-6 M to 10.sup.-12 M is preferred,
further preferably 10.sup.-6 M to 10.sup.-11 M for e.g.
chromatographic applications or 10.sup.-9 to 10.sup.-12 M for e.g.
diagnostic or therapeutic applications. Further preferred binding
affinities are in the range of 10.sup.-7 to 10.sup.-1.degree. M,
preferably to 10.sup.-11 M.
[0093] The methods for determining the binding affinities are known
per se and can be selected for instance from the following methods:
ELISA, Surface Plasmon Resonance (SPR) based technology (offered
for instance by Biacore.RTM.), fluorescence spectroscopy,
isothermal titration calorimetry (ITC), analytical
ultracentrifugation, FACS. Other methods available in the art can
be used also by the expert within his general knowledge.
[0094] After having made the modifications above, the inventors
have found the amino acid modified ubiquitin sequences described in
the examples which bind their targets with very high affinity (Kd
values up to 10.sup.-12 M).
Multimerization of Ubiquitin
[0095] In a further embodiment of the invention said ubiquitin
protein capable of binding ED-B is fused with at least one second
ubiquitin protein capable of binding ED-B to obtain a multimer,
optionally a dimer or trimer of said ubiquitin protein which is
optionally fused with a pharmaceutically active component,
optionally a cytokine, or a diagnostic component. Alternatively,
said ubiquitin protein capable of binding ED-B is fused with at
least one second ubiquitin protein capable of binding ED-B to
obtain a multimer, optionally a dimer or trimer of said ubiquitin
protein wherein multimer, dimer or trimer is formed via said
pharmaceutically active component which is optionally TNF-alpha, or
via said diagnostic component.
[0096] The "multimer" is considered as a protein herein which might
comprise the same or one or more different monomeric ubiquitin
proteins. If the multimer contains the same monomeric ubiquitin
units, then a homomer or homo-multimer (for example homodimer or
-trimer) will be formed. If the dimer comprises two differently
modified monomers, it is called a "heteromeric-dimer" or
"hetero-dimer". If more than one different monomeric ubiquitin
units are present, a heteromer or hetero-multimer will be formed.
In a most preferred embodiment of this invention, the heteromer
with ED-B binding capability consists of at least two different
modified ubiquitin monomers. A "hetero-dimer" of the invention is
considered as a fusion of modified ubiquitin proteins consisting
out of two different monomeric ubiquitin proteins having each a
monovalent binding property for a specific binding partner.
[0097] Thus, the "hetero-multimer", optionally "hetero-dimer" of
the invention is considered as a fusion of a least two differently
modified (or at least one modified and one not modified) monomeric
ubiquitin proteins exhibiting a combined monovalent binding
property for the specific binding partner ED-B. It is emphasized
that the modified hetero-dimeric ED-B binding ubiquitin protein of
the invention is not obtained by separately screening each
monomeric ubiquitin protein and combining two of them afterwards
but by screening for hetero-dimeric proteins consisting of a first
and a second monomeric unit which exhibit together a monovalent
binding activity of said ED-B ligand. It is to be expected that
each of said subunits exhibit a quite limited binding affinity
towards ED-B while only the combined dimeric modified ubiquitin
protein will have the excellent binding properties described herein
(see, for example, FIG. 4).
[0098] According to the invention two differently modified
ubiquitin monomers (or at least one modified and one not modified)
genetically linked by head-to-tail fusion bind to the same epitope
of ED-B and are only effective if both binding domain regions act
together. The BDRs of the monomers form a single contiguous binding
region.
[0099] In one embodiment of the invention the modified ubiquitin
protein provided according to the invention can be linked to an
ubiquitin protein of a different specificity in a preferably
site-specific manner thereby obtaining a modularly composed
ubiquitin with monovalent binding properties, respectively, with
respect to a binding partner.
[0100] Thus, for example two variations of ubiquitin obtained by
the procedure described above can be linked to each other in a
site-specific manner. Thus, if multimers are formed, this can be
done by producing the monomeric ubiquitin units and linking them in
a head-to-tail-manner. That is to say, the monomeric units are
linked N--C--N--C-- depending on the number of units contained in
the multimer. The multimerization can be done either directly using
no linkers or via linkers.
[0101] In another embodiment of the invention, two variations of
modified ubiquitin monomers which bind to ED-B can be genetically
linked to each other by appropriate methods so that exclusively
highly specific binding molecules are obtained. Most preferred, two
variations of modified ubiquitin monomers genetically linked by
head-to-tail fusion bind to the same epitope and are only effective
if both binding domain regions act together. Or in other words,
they bind to the same epitope via a single contiguous binding
region which is formed by the acting together of both binding
regions of the two modules.
[0102] Thus, the ubiquitin protein modified in accordance with the
invention to efficiently bind ED-B of fibronectin may be
multimerized, particularly dimerized or trimerized. The fusion of
ubiquitin monomers can be performed via linkers, for example, a
linker having at least the sequence GIG or any other linker, for
example SGGGGIG or SGGGGSGGGGIG. Another linker for the genetic
fusion of two or more ubiquitin monomers is possible. Preferably,
such an ubiquitin protein consists of at least two binding
determining regions (BDR) wherein each BDR shows modifications in
at least one of amino acids 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 and
wherein the BDR are genetically fused to each other. The binding to
the target is mediated by both BDRs in collaboration.
[0103] A further multimerization of the modified ubiquitin protein
can be performed for example by fusing the modified ubiquitin
protein to effector molecules having a multimerization domain like
a cytokine for example TNF-.alpha.. A fusion of one or more
modified ubiquitin monomers, such as dimers, preferably
hetero-dimers, with TNF-alpha can be done via linkers. In a still
further embodiment, said multimerization is performed by
genetically fusing two or more modified ubiquitin molecules or by
using a polyethylene glycol (PEG) linker. In a still further
embodiment said multimerization domain also acts as
pharmaceutically active component; one example is TNF-alpha acting
both as multimerization domain and pharmaceutical component.
[0104] In a further embodiment of the invention, the multimerized
modified ubiquitin protein of the invention consists of heteromers.
The term "heteromer" means that ubiquitin monomers of different
compositions are included in the multimerized molecule.
Particularly preferred are hetero-dimers consisting of two
genetically fused ubiquitin monomers with different binding domain
regions but having one binding site. These hetero-dimers can be
multimerized by the use of for example TNF, most preferred,
TNFalpha. One advantage of using ubiquitin hetero-dimers is to
target ED-Bs with high binding specificities.
[0105] A further advantage of dimerization or generally
multimerization lies in the increase of the number of amino acid
residues to be modifiable maintaining at the same time
protein-chemical integrity without decreasing the overall stability
of the scaffold of said newly created binding protein to ED-B. On
the one hand the total number of residues which can be modified in
order to generate the binding site for ED-B is increased as the
modified residues can be allocated to the scaffolds of two or three
ubiquitin proteins; on the other hand the number of residues on the
scaffold of one ubiquitin protein is decreased maintaining at the
same time the protein-chemical stability of the modified binding
molecule. Summarizing, a modular structure of the ubiquitin-based
ED-B binding protein allows increasing the overall number of
modified amino acids as said modified amino acids are included on
two or three ubiquitin molecules. The number of modifications as
described above is given per one molecule of modified
ubiquitin.
[0106] Further, multimerisation may provide modified ubiquitin
molecules having two (or more) binding sites for target substances,
having one monovalent specificity (for one single epitope), but may
also provide bi- or multispecific (thus recognizing two or more
different epitopes at one time).
[0107] If two or more modified ubiquitin molecules are fused,
preferably as genetic head-to-tail fusion, the number of
modifications can be two times or x-times corresponding to the
number of modified ubiquitin molecules. As an alternative, the
fusion can be post-translationally.
[0108] Regarding the use of monomeric ubiquitin proteins in
modified form, the advantage of using multimeric structures such as
dimers, in particular homo- or hetero-dimers, resides in the
increase of the number of modifiable residues which do not affect
the proteinchemical integrity of the underlying scaffold.
Modified Ubiquitin Proteins Bind to ED-B
[0109] According to the invention, modified ubiquitin monomers were
identified that bind to ED-B. Preferred modified ubiquitin
hetero-dimers with specific binding to ED-B are as follows:
1. The binding determining region (BDR1) of the first ubiquitin
molecule: modified amino acids 2, 4, 6, 62 to 68, preferably
genetically fused to a second ubiquitin molecule with modified
amino acids 6, 8, 62 to 68 defining the BDR2. 2. The binding
determining region (BDR1) of the first ubiquitin molecule: modified
amino acids 6, 8, 62 to 68, preferably genetically fused to a
second ubiquitin molecule: modified amino acids 6, 8, 62 to 68
defining the BDR2. 3. The binding determining region (BDR1) of the
first ubiquitin molecule: modified amino acids 2, 4, 6, 8, 62 to
68, preferably genetically fused to a second ubiquitin molecule:
modified amino acids 2, 4, 6, 8, 62 to 68 defining the BDR2. 4. The
binding determining region (BDR1) of the first ubiquitin molecule:
modified amino acids 6, 8, 63 to 66, preferably genetically fused
to a second ubiquitin molecule: modified amino acids 2, 6, 8, 62 to
68 defining the BDR2.
[0110] In an embodiment, the fusion protein is a genetically fused
dimer of said ubiquitin protein having amino acids substitutions in
positions 6, 8, 63-66 of the first ubiquitin monomer and
substitutions in amino acid residues in positions 6, 8, 62-66, and
optionally in position 2 of the second ubiquitin monomer,
preferably
Lysine (K) to Tryptophane (W) or Phenylalanine (F) in position 6,
Leucine (L) to Tryptophane or Phenylalanine (W, F) in position
8,
Lysine (K) to Arginine (R) or Histidine (H) in Position 63,
[0111] Glutamic acid (E) to Lysine (K), Arginine (R) or Histidine
(H) in position 64, Serine (S) to Phenylalanine (F) or Tryptophane
(W) in position 65 and Threonine (T) to Proline (P) in position 66;
[0112] in the second ubiquitin monomer, the substitutions Lysine
(K) to Threonine (T), Asparagine (N), Serine (S) or Glutamine (Q)
in position 6, Leucine (L) to Glutamine (Q) or Threonine (T) or
Asparagine (N) or Serine (S) in position 8, Glutamine (Q) to
Trytophane (W) or Phenylalanine (F) in position 62, Lysine (K) to
Serine (S), Threonine (T), Asparagine (N) or Glutamine (Q) in
position 63, Glutamic acid (E) to Asparagine (N), Serine (S),
Threonine (T), or Glutamine (Q) in position 64, Serine (S) to
Phenylalanine (F) or Tryptophane (W) in position 65, and Threonine
(T) to Glutamic acid (E) or Aspartic acid (D) in position 66, and
Optionally Glutamine (Q) to Arginine (R), Histidine (H) or Lysine
(K) in position 2 are preferred.
[0113] Most preferred are the following substitutions:
(1) in the first monomeric unit at least--K6W, LBW, K63R, E64K,
S65F, and T66P; and in the second monomeric unit at least--K6T,
L8Q, Q62W, K63S, E64N, S65W, and T66E; optionally additionally Q2R,
or (2) in the first monomeric unit at least Q2T, F4W, K6H, Q62N,
K63F, E64K, S65L, and T66S; and in the second monomeric unit at
least K6X, L8X, Q62X, K63X, E64X, S65X, and T66X; optionally
additionally Q2X, wherein X can be any amino acid.
[0114] Particularly preferred are the following substitutions in
the first ubiquitin monomer to generate binding proteins for
ED-B:
2: Q.fwdarw.T, 4: F.fwdarw.W, 6: K.fwdarw.H, 62: Q.fwdarw.N, 63:
K.fwdarw.F, 64: E.fwdarw.K, 65: S.fwdarw.L, 66: T.fwdarw.S
[0115] Either no linker or any linker can be used to connect the
two monomers head-to-tail. Preferred linkers are those of SEQ ID
NO: 32 or the sequence GIG or SGGGGIG or SGGGGSGGGGIG.
[0116] In a preferred embodiment, a ubiquitin hetero-dimer with two
binding determining regions
[0117] (BDR) acting together for binding ED-B comprises the amino
acid sequence of SEQ ID NO: 33 or 34. A preferred fusion protein of
the invention comprising TNF-alpha as a pharmaceutically active
component has the sequence of SEQ ID NO: 35 or 36. A further
preferred protein is provided by the following sequence wherein
XXXX may be any amino acid (SEQ ID NO: 47). As linker, SGGGGSGGGGIG
was used here (shown in italics). It is to be understood that also
other kind of linkers or no linker are feasible alternatives.
TABLE-US-00001 ##STR00001##
[0118] The consensus sequences of examples of proteins with these
sequences are shown in FIG. 19.
[0119] In a still further embodiment, the fusion protein of the
present invention is a trimer of a fusion protein of a ubiquitin
hetero-dimer fused to TNF-alpha, wherein the fusion protein
preferably has the sequence of SEQ ID NO: 35 or 36 or has an
identity of at least 90%, preferably of more than 95%, of more than
96% or up to a sequence identity of 97% to SEQ SEQ ID NO: 35 or 36,
and, at the same time, maintains or improves the capability of the
original fusion protein of high affinity binding the ED-B domain of
fibronectin.
[0120] In a further aspect of the invention, the present invention
covers also polynucleotides which encode for a protein or fusion
protein as described before. Additionally, vectors comprising said
polynucleotide are covered by the invention.
[0121] In an additional aspect of the present invention, host cells
are covered which comprise a protein or a fusion protein described
herein and/or a polynucleotide coding for said recombinant protein
or fusion protein of the invention or a vector containing said
polynucleotide.
Uses of the Proteins of the Invention, e.g. Hetero-Dimeric
Ubiquitin Based Binding Proteins Specifically for ED-B Fused to an
Effector Such as TNF Alpha
[0122] The modified ubiquitin ED-B binding proteins of the
invention are to be used for instance for preparing diagnostic
means for in vitro or in vivo use as well as therapeutic means. The
proteins according to the invention can be used e.g. as direct
effector molecules (modulator, antagonist, agonist) or
antigen-recognizing domains. Examples of tumors with abundant
appearance of ED-B antigen are shown in the table of FIG. 1.
[0123] Depending on the selected fusion partner the pharmaceutical
composition of the invention is adapted to be directed to the
treatment of cancer, e.g. breast and colorectal cancers, or any
other tumor diseases in which ED-B is abundant (cf examples thereof
listed in FIG. 1).
[0124] The compositions are adapted to contain a therapeutically
effective dose. The quantity of the dose to be administered depends
on the organism to be treated, the type of disease, the age and
weight of the patient and further factors known per se.
[0125] The compositions contain a pharmaceutically or
diagnostically acceptable carrier and optionally can contain
further auxiliary agents known per se. These include for example
but not limited to stabilizing agents, surface-active agents,
salts, buffers, colouring agents etc.
[0126] The pharmaceutical composition can be in the form of a
liquid preparation, a cream, a lotion for topical application, an
aerosol, in the form of powders, granules, tablets, suppositories,
or capsules, in the form of an emulsion or a liposomal preparation.
The compositions are preferably sterile, non-pyrogenic and isotonic
and contain the pharmaceutically conventional and acceptable
additives known per se. Additionally, reference is made to the
regulations of the U.S. Pharmacopoeia or Remington's Pharmaceutical
Sciences, Mac Publishing Company (1990).
[0127] In the field of human and veterinary medical therapy and
prophylaxis pharmaceutically effective medicaments containing at
least one ED-B binding ubiquitin protein modified in accordance
with the invention can be prepared by methods known per se.
Depending on the galenic preparation these compositions can be
administered intravenously, intraperitoneally, intramuscularly,
subcutaneously, transdermally or by other methods of application.
The type of pharmaceutical preparation depends on the type of
disease to be treated, the severity of the disease, the patient to
be treated and other factors known to those skilled in the art of
medicine. The administration can either be parentally by injection
or infusion, systemically, rectally, transdermally or by any other
methods conventionally employed.
[0128] In an embodiment, the pharmaceutical composition contains a
protein or a fusion protein of the invention or a combination
thereof and further comprises one or more chemotherapeutic agents,
preferably selected from the following table:
TABLE-US-00002 Substance Class Examples Alkylating agents (ATC
L01A) melphalan, cyclophosphamide Antimetabolites (ATC L01B)
5-fluorouracil, gemcitabine Taxanes (ATC L01CD) Paclitaxel
Cytotoxic antibiotics (ATC L01D) doxorubicin, liposomal doxorubicin
Platinum compounds (ATC L01XA) Cisplatin
[0129] In a preferred embodiment, the chemotherapeutic agent is
selected from melphalan, doxorubicin, cyclophosphamide,
dactinomycin, fluorodesoxyuracil, cisplatin, paclitaxel, and
gemcitabine; or from the group of kinase inhibitors.
[0130] A "pharmaceutical composition" according to the invention
may be present in the form of a composition, wherein the different
active ingredients and diluents and/or carriers are in admixed with
each other, or may take the form of a combined preparation, where
the active ingredients are present in partially or totally distinct
form. An example for such a combination or combined preparation is
a kit-of-parts.
[0131] A "composition" according to the present invention comprises
at least two pharmaceutically active compounds. These compounds can
be administered simultaneously or separately with a time gap of one
minute to several days. The compounds can be administered via the
same route or differently; e.g. oral administration of one active
compound and parenteral administration of another are possible.
Also, the active compounds may be formulated in one medicament,
e.g. in one infusion solution or as a kit comprising both compounds
formulated separately. Also, it is possible that both compounds are
present in two or more packages.
[0132] A particularly preferred combination is a fusion protein
according to the invention and melphalan, and/or (liposomal)
doxorubicin. Apart from antineoplastic agents from the ATC class
L01, the TNF-fusion protein of the invention can be combined with
other antineoplastic substances including cytokines and derivatives
thereof, radiopharmaceuticals, cell based therapeutics and
nanoparticles.
[0133] Due to its tumor permeabilisation activity, the TNF-fusion
protein of the invention (but also the other recombinant
proteins/fusion proteins of the present invention) can be combined
with all antineoplastic agents as listed under L01 in the
Anatomical Therapeutic Chemical Classification System (ATC)
provided by the World Health Organisation. Here, it surprisingly
turned out that a fusion protein of a ubiquitin hetero-dimer fused
to TNF-alpha, wherein the fusion protein preferably has the
sequence of SEQ ID NO: 35 or 36, can be advantageously applied in
therapy. TNF-alpha is highly toxic and, thus, may only be
administered in low dosages, which usually lie below the minimum
therapeutic threshold (and thus are therapeutically inactive). Due
to this toxicity of TNF-alpha, in order to reach a therapeutically
effective concentration, the isolated limb perfusion approach is
presently selected when using TNF-alpha. Limb perfusion is a
medical technique that may be used to deliver anticancer drugs
directly to an arm or leg. The flow of blood to and from the limb
is temporarily stopped with a tourniquet, and anticancer drugs are
put directly into the blood of the limb. This allows the patient to
receive a high dose of TNF-alpha in the area where the cancer
occurred.
[0134] However, by applying the TNF-alpha fusion proteins of the
present invention, it is possible to administer TNF-alpha in a
non-toxic, but still therapeutically effective concentration. Since
TNF-alpha is coupled to the (binding) fusion protein of the present
invention, it can be directly active at the disease site (for
example, tumor site) and, thus, the amount of "free" TNF-alpha can
be drastically reduced.
[0135] Systemic side effects of TNF-alpha can be remarkably reduced
by administering TNF-alpha as a fusion protein according to the
present invention. By using a TNF-alpha fusion protein of the
invention, the overall dosage of TNF-alpha to reach a therapeutic
effect thus can be reduced to a large extent and can be
advantageously used for systemic tumor treatment (without the
necessity and restrictions of limb perfusion) in particular in
combination with chemotherapeutic agents (see above).
[0136] In a further embodiment, the pharmaceutical composition is
in the form of a kit of parts, providing separated entities for the
recombinant protein/fusion protein of the invention and for the one
or more chemotherapeutic agents.
Method of Production of the ED-B Binding Proteins of the
Invention
[0137] ED-B binding proteins according to the invention may be
prepared by any of the many conventional and well known techniques
such as plain organic synthetic strategies, solid phase-assisted
synthesis techniques or by commercially available automated
synthesizers. On the other hand, they may also be prepared by
conventional recombinant techniques alone or in combination with
conventional synthetic techniques.
[0138] In another aspect of the present invention, a method for
generating a recombinant modified protein is provided. The method
comprises at least the following steps: [0139] a) providing an
ubiquitin protein; [0140] b) providing the ED-B of fibronectin;
[0141] c) modifying said ubiquitin protein in order to obtain a
protein having an amino acid sequence identity to the amino acid
sequence of SEQ ID NO: 1 of at least 60% wherein at leak 4 amino
acids are modified by substitution, deletion or addition of amino
acids in positions 2, 4, 6, 62, 63, 64, 65, 66, and/or 68; [0142]
d) contacting said modified ubiquitin protein with said ED-B of
fibronectin; [0143] e) screening for modified ubiquitin proteins
which bind to said ED-B of fibronectin with a specific binding
affinity of 10.sup.-5-10.sup.-12M, and optionally [0144] f)
isolating said modified ubiquitin proteins.
[0145] Optionally, the modification may be performed by genetic
engineering on the DNA level and expression of the modified protein
in prokaryotic or eukaryotic organisms or in vitro.
[0146] In a further embodiment, said modification step includes a
chemical synthesis step.
[0147] In a still further embodiment, said method is adapted in
order that said modified ubiquitin protein is fused with a
pharmaceutically active component, optionally a cytokine,
preferably TNF-alpha, or a diagnostic component,
or wherein said recombinant protein is fused with at least one
second recombinant ubiquitin protein to obtain a multimer,
optionally a dimer or trimer of said recombinant ubiquitin protein
which is optionally fused with a pharmaceutically active component,
optionally a cytokine, or a diagnostic component, or [0148] wherein
said recombinant protein is fused with at least one second
recombinant ubiquitin protein to obtain a multimer, optionally a
dimer or trimer of said recombinant ubiquitin protein wherein said
multimer, dimer or trimer is formed via said pharmaceutically
active component which is optionally TNF-alpha, or via said
diagnostic component.
[0149] According to the invention, a modified protein can further
be prepared by chemical synthesis. In this embodiment the steps c)
to d) of claim 1 are then performed in one step.
[0150] In a further aspect of the invention, a method for
generating a hetero-multimeric fusion protein is provided,
comprising the following steps: [0151] a) providing a multimeric
ubiquitin protein comprising two or more modified ubiquitin
monomers linked by a suitable linker, wherein each monomer of said
multimeric ubiquitin protein was modified in order to obtain a
protein having an amino acid sequence identity to the amino acid
sequence of SEQ ID NO: 1 of at least 60% wherein at least 4 amino
acids in each monomer are modified by substitution, deletion or
addition of amino acids in positions 2, 4, 6, 62, 63, 64, 65, 66,
and/or 68; [0152] b) providing the ED-B of fibronectin; [0153] c)
contacting said hetero-multimeric modified ubiquitin protein with
said ED-B of fibronectin; [0154] d) screening for modified
ubiquitin proteins which bind to said ED-B of fibronectin with a
specific binding affinity of 10.sup.-5-10.sup.-12 M, and optionally
[0155] e) isolating said modified hetero-multimeric ubiquitin
proteins.
[0156] In another aspect of the present invention, a method for
generating a recombinant modified ubiquitin protein is provided.
The method comprises at least the following steps: [0157] a)
providing a population of differently modified dimeric ubiquitin
proteins originating from monomeric ubiquitin proteins, said
population comprising dimeric ubiquitin proteins comprising two
modified ubiquitin monomers linked together in a head-to-tail
arrangement wherein each monomer of said dimeric protein is
differently modified by substitutions of at least 6 amino acids in
positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 of SEQ ID NO: 1
[0158] wherein said substitutions comprise [0159] (1) in the first
monomeric unit substitutions at least in amino acid positions 6, 8,
63, 64, 65, and 66; and [0160] in the second monomeric unit
substitutions at least in amino acid positions 6, 8, 62, 63, 64,
65, and 66; optionally additionally 2; or [0161] (2) in the first
monomeric unit substitutions at least in amino acid positions 2, 4,
6, 62, 63, 64, 65, and 66; and [0162] in the second monomeric unit
substitutions at least in amino acid positions 6, 8, 62, 63, 64,
65, and 66; optionally additionally 2 [0163] b) providing the
extradomain B (ED-B) of fibronectin as potential ligand; [0164] c)
contacting said population of differently modified proteins with
said extradomain B (ED-B) of fibronectin; [0165] d) identifying a
modified dimeric ubiquitin protein by a screening process, wherein
said modified dimeric ubiquitin protein binds to said the
extradomain B (ED-B) of fibronectin with a specific binding
affinity of Kd in a range of 10.sup.-7-10.sup.-12 M and exhibits a
monovalent binding activity with respect to said extradomain B
(ED-B) of fibronectin, and optionally [0166] e) isolating said
modified dimeric ubiquitin protein with said binding affinity.
[0167] In said preferred embodiment, the hetero-multimeric
ubiquitin protein is a hetero-dimeric ubiquitin protein.
[0168] In a further aspect, the present invention is directed to a
library based on linear polyubiquitin chains as mentioned above and
being randomised at least in two BDR's.
[0169] In a still further aspect of the invention, a fusion library
containing DNA obtained by fusing two libraries as specified above
is provided each library encoding for differently modified
monomeric ubiquitin protein units in order to obtain hetero-dimeric
ubiquitin fusion proteins, the monomeric units thereof being linked
together in a head-to-tail arrangement, said library encoding for
hetero-dimeric fusion proteins of ubiquitin exhibiting a monovalent
binding activity with respect to said extradomain B (ED-B) of
fibronectin. Said linking together is performed either by using
anyone of the linkers known by the skilled artisan or a linker
described herein. In one embodiment of the invention TNF-alpha is
used as linker acting simultaneously as pharmaceutically active
compound.
[0170] Example 1 outlines the production of a complex library.
However, care must be taken as regards the quality of such a
library. The quality of a library in the antibody or scaffold
technology is in the first place dependent from its complexity
(number of individual variants) as well as functionality
(structural and protein-chemical integrity of the resulting
candidates). Both characteristics, however, may exert negative
influences on each other: enhancing the complexity of a library by
increasing the number of modified positions on the scaffold might
lead to a deterioration of the protein-chemical characteristics of
the variants. This might result in a decreased solubility,
aggregation and/or low yields. A reason for this is the larger
deviation from native scaffolds having an energetically favourable
protein packaging.
[0171] Therefore, it is a balancing act to construct such a
scaffold library suitably between the extreme positions of
introducing as many variations as possible into the original
sequence in order to optimize it for a target and, on the other
hand, of conserving the original primary sequence as much as
possible in order to avoid negative protein-chemical effects.
[0172] It is noted that the present disclosure encompasses also
each conceivable combination of the features described herein in
view of the aspects or embodiments of the invention.
Selection of the Modified Ubiquitin Proteins with Binding Affinity
with Respect to the Target ED-B and Determination of the Modified
Amino Acids Responsible for the Binding Affinity
[0173] After e.g. at least two different DNA libraries encoding for
hetero-dimeric modified ubiquitin proteins have been established by
differently modifying selected amino acids in each of the monomeric
ubiquitin units, these libraries are genetically fused by e.g.
linker technology to obtain DNA molecules encoding for
hetero-dimeric modified ubiquitin proteins. The DNA of these
libraries is expressed into proteins and the modified dimeric
proteins obtained thereby are contacted according to the invention
with the ED-B to optionally enable binding of the partners to each
other if a binding affinity does exist.
[0174] It is a crucial aspect of the invention that the contacting
and screening process is performed already with respect to the
hetero-dimeric ubiquitin protein. This process enables screening on
those ubiquitin proteins which provide a monovalent binding
activity to ED-B.
[0175] Contacting according to the invention is preferably
performed by means of a suitable presentation and selection method
such as the phage display, ribosomal display, mRNA display or cell
surface display, yeast surface display or bacterial surface display
methods, preferably by means of the phage display method. For
complete disclosure, reference is made also to the following
references: Hoess, Curr. Opin. Struct. Biol. 3 (1993), 572-579;
Wells and Lowmann, Curr. Opin. Struct. Biol. 2 (1992), 597-604; Kay
et al., Phage Display of Peptides and Proteins--A Laboratory Manual
(1996), Academic Press. The methods mentioned above are known to
those skilled in the art and can be used according to the invention
including modifications thereof.
[0176] The determination whether the modified protein has a
quantifiable binding affinity with respect to a predetermined
binding partner can be performed according to the invention
preferably by one or more of the following methods: ELISA, plasmon
surface resonance spectroscopy, fluorescence spectroscopy, FACS,
isothermal titration calorimetry and analytical
ultracentrifugation.
Phage Display Selection Method
[0177] One type of phage display procedure adapted to this
application is described in the following as an example for a
selection procedure according to the invention with respect to
variations of ubiquitin which show binding properties. In the same
manner e.g. methods for the presentation on bacteria (bacterial
surface display; Daugherty et al., 1998, Protein Eng.
11(9):825-832) or yeast cells (yeast surface display; Kieke et al.,
1997 Protein Eng. 10(10:1303-10) or cell-free selection systems
such as the ribosome display (Hanes and Pluckthun, 1997 Proc Natl
Acad Sci USA. 94(10):4937-4942; He and Taussig, 1997 Nucleic Acids
Res. 25(24):5132-5134) or the cis display (Odegrip et al., 2004
Proc Natl Acad Sci U S A. 101(9):2806-2810) or the mRNA display can
be applied. In the latter case a transient physical linkage of
genotype and phenotype is achieved by coupling of the protein
variation to the appropriate mRNA via the ribosome.
[0178] In the phage display procedure described herein recombinant
variations of ubiquitin are presented on a filamentous phage while
the coding DNA of the presented variation is present at the same
time packed in a single-stranded form in the phage envelope. Thus,
in the frame of an affinity enrichment variations having certain
properties can be selected from a library and their genetic
information can be amplified by infection of suitable bacteria or
added to another cycle of enrichment, respectively. Presentation of
the mutated ubiquitin on the phage surface is achieved by genetic
fusion to an amino-terminal signal sequence--preferably the PelB
signal sequence--and a capsid or surface protein of the
phage-preferred is the carboxyterminal fusion to the capsid protein
pIII or a fragment thereof. Furthermore, the encoded fusion protein
can contain further functional elements such as e.g. an affinity
tag or an antibody epitope for detection and/or purification by
affinity chromatography or a protease recognition sequence for
specific cleavage of the fusion protein in the course of the
affinity enrichment. Furthermore, an amber stop codon can be
present for example between the gene for the ubiquitin variation
and the coding region of the phage capsid protein or the fragment
thereof which is not recognized during translation in a suitable
suppressor strain partially due to the introduction of one amino
acid.
[0179] The bacterial vector suitable for the selection procedure in
the context of the isolation of ubiquitin variations with binding
properties to a predetermined hapten or antigen and into which the
gene cassette for the fusion protein described is inserted is
referred to as phagemid Among others, it contains the intergenic
region of a filamentous phage (e.g. M13 or f1) or a portion thereof
which in the case of a superinfection of the bacterial cell
carrying the phagemid by means of helper phages such as e.g. M13K07
results in the packaging of a closed strand of phagemid DNA into a
phage capsid. The phagemids generated in this manner are secreted
by the bacterium and present the respective ubiquitin variation
encoded--due to its fusion to the capsid protein pIII or the
fragment thereof--on their surface. Native pIII capsid proteins are
present in the phagemid so that its ability to re-infect suitable
bacterial strains and therefore the possibility to amplify the
corresponding DNA is retained. Thus, the physical linkage between
the phenotype of the ubiquitin variation--i.e. its potential
binding property--and its genotype is ensured.
[0180] Phagemids obtained can be selected with respect to the
binding of the ubiquitin variation presented thereon to
predetermined haptens or antigens by means of methods known to
those skilled in the art. For this purpose, the presented ubiquitin
variations can be transiently immobilized to target substance bound
e.g. on microtiter plates and can be specifically eluted after
non-binding variations have been separated. The elution is
preferably performed by basic solutions such as e.g. 100 mM
triethylamine. Alternatively, the elution can be performed under
acidic conditions, by proteolysis or direct addition of infected
bacteria. The phagemids obtained in this manner can be re-amplified
and enriched by successive cycles of selection and amplification of
ubiquitin variations with binding properties to a predetermined
hapten or antigen.
[0181] Further characterization of the ubiquitin variations
obtained in this way can be performed in the form of the phagemid,
i.e. fused to the phage, or after cloning of the corresponding gene
cassette into a suitable expression vector in the form of a soluble
protein. The appropriate methods are known to those skilled in the
art or described in the literature. The characterization can
comprise e.g. the determination of the DNA sequence and thus of the
primary sequence of the variations isolated. Furthermore, the
affinity and specificity of the variations isolated can be detected
e.g. by means of biochemical standard methods such as ELISA or
plasmon surface resonance spectroscopy, fluorescence spectroscopy,
FACS, isothermal titration calorimetry, analytical
ultracentrifugation or others. In view of the stability analysis,
for example spectroscopic methods in connection with chemical or
physical unfolding are known to those skilled in the art.
Ribosomal Display Selection Method
[0182] In a further embodiment of the invention ribosomal display
procedure variations of ubiquitin are prepared by means of a
cell-free transcription/translation system and presented as a
complex with the corresponding mRNA as well as the ribosome. For
this purpose, a DNA library as described above is used as a basis
in which the genes of variations are present in form of fusions
with the corresponding regulatory sequences for expression and
protein biosynthesis. Due to the deletion of the stop codon at the
3' end of the gene library as well as suitable experimental
conditions (low temperature, high Mg.sup.2+ concentration) the
ternary complex consisting of the nascent protein, the mRNA and the
ribosome is maintained during in vitro
transcription/translation.
[0183] After a protein library containing hetero-dimeric modified
ubiquitin proteins has been established by differently modifying of
selected amino acids in each of the monomeric ubiquitin units, the
modified dimeric proteins are contacted according to the invention
with the ED-B to enable binding of the partners to each other if a
binding affinity does exist. These protein libraries may be in the
form of a display method library displaying or using any other
method presenting the modified proteins in a manner enabling the
contact between the modified proteins and the ED-B target protein,
wherein said display method is optionally a phage display,
ribosomal display, TAT phage display, yeast display, bacterial
display or mRNA display method.
[0184] Selection of the modified ubiquitin variations with respect
to their binding activities to ED-B with a specific binding
affinity of Kd in a range of 10.sup.-7-10.sup.-12M can be performed
by means of methods known to those skilled in the art. For this
purpose, the ubiquitin variations presented e.g. on the ribosomal
complexes can be transiently immobilized to target substance bound
e.g. on microtiter plates or can be bound to magnetic particles
after binding in solution, respectively. Following separation of
non-binding variations the genetic information of variations with
binding activity can be specifically eluted in the form of the mRNA
by destruction of the ribosomal complex. The elution is preferably
carried out with 50 mM EDTA. The mRNA obtained in this manner can
be isolated and reverse transcribed into DNA using suitable methods
(reverse transcriptase reaction), and the DNA obtained in this
manner can be re-amplified.
[0185] By means of successive cycles of in vitro
transcription/translation, selection, and amplification ubiquitin
variations with binding properties for a predetermined hapten or
antigen can be enriched.
Characterization of the EDB-Binding Proteins
[0186] The further characterization of the ubiquitin variations
obtained in this manner can be performed in the form of a soluble
protein as detailed above after cloning of the corresponding gene
cassette into a suitable expression vector. The appropriate methods
are known to those skilled in the art or described in the
literature.
[0187] Preferably, the step of detection of the proteins having a
binding affinity with respect to a predetermined binding partner is
followed by a step of isolation and/or enrichment of the detected
protein.
[0188] Following the expression of the ubiquitin protein modified
according to the invention, it can be further purified and enriched
by methods known per se. The selected methods depend on several
factors known per se to those skilled in the art, for example the
expression vector used, the host organism, the intended field of
use, the size of the protein and other factors. For simplified
purification the protein modified according to the invention can be
fused to other peptide sequences having an increased affinity to
separation materials. Preferably, such fusions are selected that do
not have a detrimental effect on the functionality of the ubiquitin
protein or can be separated after the purification due to the
introduction of specific protease cleavage sites. Such methods are
also known per se to those skilled in the art.
BRIEF DESCRIPTION OF THE FIGURES
[0189] FIG. 1 shows a Table listing the occurrence of ED-B in
various tumors.
[0190] FIG. 2A shows a concentration dependent ELISA of the binding
of modified ubiquitin ED-B binder 5E1 to human ED-B (filled
circles) with an affinity of 23 nM compared to no binding to BSA
(open circles).
[0191] FIG. 2B shows a concentration dependent ELISA of the binding
of modified ubiquitin ED-B binder 1H4 to human ED-B (filled
circles) with an affinity of 7.8 nM. The binding to BSA is plotted
as a control (open circles).
[0192] FIG. 3 shows that tetramerization leads to an increase in
affinity. The table shows the Kd values of modified ubiquitin
monomers compared to tetramers consisting of modified ubiquitin
monomers. Shown are ubiquitin variants 5E1 and 1H4 as examples. The
ED-B binding is compared to binding to c-FN (cellular fibronectin).
The figures demonstrate the significant higher affinity in binding
of the tetrameric variant (for example, 56 nM for 5E1 or 1.4 nM for
1H4) to the target ED-B compared to the monomer (4.51 microM for
5E1 or 9.98 microM for 1H4).
[0193] FIG. 4 shows that the recombination of a front (first)
modified ubiquitin monomer (having a BDR1) with a different
modified rear (second) ubiquitin monomer (having a BDR2) to
generate a hetero-dimer results in an significant increase of
affinity as well as specificity. The modified ubiquitin molecules
are analyzed via Biacore, fluorescence anisotropy, binding on cells
and tissue sections. Shown are concentration dependent ELISAs
(cont-ELISA) of the binding of several variants to human ED-B.
[0194] FIG. 4 A shows a binding affinity of Kd=9.45 .mu.M for the
monomer 41B10.
[0195] FIG. 4 B shows that a binding affinity of a Kd=131 nM for
41B10 combined with a different second monomer resulting in
46H9.
[0196] FIG. 5 shows specific variants fused to a cytokine (for
example, TNFalpha). The fusion proteins trimerize the modified
ubiquitin monomer and are biologically active molecules.
[0197] FIG. 5 A is a schematic drawing of a modified ubiquitin
based ED-B binding-effector-fusion protein; in green (structure on
top)--effector, e.g. a cytokine, preferably TNF-alpha; brown: light
brown: structure of the modified ubiquitin monomers
(Affilin.RTM.).
[0198] FIG. 5 B shows that the modified ubiquitin effector
conjugate 5E1-TNF-conjugate has pro-apoptotic activity (as measured
in an L929 apoptosis assay).
[0199] FIG. 5 C shows high affinity binding of 1H4-TNFalpha-fusion
to ED-B (Kd=15.1 nM) (closed circles connected by a fitted line).
The binding to BSA is plotted as a control (closed circles not
connected by a line).
[0200] FIG. 6 shows the affinity and activity of a modified
ubiquitin based ED-B binding hetero-dimer molecule fused to a
cytokine, for example, TNFalpha. [0201] Apotosis inducing activity
of modified ubiquitin based ED-B binding cytokine fusion: EC.sub.50
0.78.+-.0.24 pM [0202] Apoptosis inducing activity of free
cytokine: EC.sub.50 3.14.+-.3.59 pM
[0203] FIG. 6A shows the affinity of modified ubiquitin based ED-B
binding hetero-dimer 24H12 (Kd 50.7 nM).
[0204] FIG. 6B shows the affinity of modified ubiquitin based ED-B
binding heterodimer 24H12 genetically fused to cytokine TNFalpha to
result in a multimerisation of the hetero-dimer 24H12 (Kd=5.6
nM
[0205] FIG. 6C shows an analysis of exemplary candidates from a
hetero-dimeric modified ubiquitin library selection, for example
hetero-dimer clones 9E12, 22D1, 24H12, 41B10. The Kd ELISA values
are increased for the target ED-B compared to cytosolic fibronectin
used as control, confirming a specific binding to the target.
[0206] FIG. 6D shows results of an analysis of the modified
hetero-dimeric ubiquitin molecule 9E12 via label-free interaction
assays using Biacore.RTM.. Different concentrations of the
hetero-dimeric ubiquitin variants were analyzed (see figure legend:
0-15 microM of 9E12) for binding to ED-B immobilized on a chip
(Biacore) to analyze the interaction between the hetero-dimeric
variant 9E12 and ED-B. A Kd could not be determined from analyzing
the association and dissociation curves.
[0207] FIG. 6E shows results of an analysis of the modified
hetero-dimeric ubiquitin molecule 41B10 via label-free interaction
assays using Biacore.RTM.. Different concentrations of the
hetero-dimeric ubiquitin variants were analyzed (see figure legend:
0-15 microM of 41B10) for binding to ED-B immobilized on a chip
(Biacore) to analyze the interaction between the hetero-dimeric
variant 41B10 and ED-B. Analyzing the association and dissociation
curves resulted in a Kd of 623 nM (623.times.10.sup.-9 M,
6.2.times.10.sup.-7 M).
[0208] FIG. 7 shows the contribution of different modified
ubiquitin based variants to binding affinity and specificity. The
different variants share common sequence modules which are marked
with lower case letters. The variants were analyzed with respect to
their ED-B binding. FIG. 3 shows different combinations of monomers
resulting in modified ubiquitin-heterodimers. Hetero-dimeric
variants 46-A5, 50-G11 and 46-H4 have all the same first (front)
modified monomer with BDR1 (labeled with the letter "a" in the
figure), but a second (rear) ubiquitin monomer modified in
different positions with BDR2. Variants 52-D10 and 52-B3 have a
different first (front) modified monomer compared to 46-H9 with
BDR1, but the same second (rear) ubiquitin monomer with BDR2
(labeled with the letter "e").
[0209] The modified ubiquitin hetero-dimers have the following
sequences:
46-H4: SEQ ID NO: 25, 45-H9: SEQ ID NO: 26, 46-A5: SEQ ID NO: 27,
50-G11: SEQ ID NO: 28, 52-B3: SEQ ID NO: 29, 52-D10: SEQ ID NO:
30
[0210] The above described sequences were modified in the course of
the experiments by adding a His-Tag with the sequence LEHHHHHH (SEQ
ID NO: 31).
[0211] As can be seen from FIG. 7, 46-H4 has an excellent binding
affinity to ED-B (Kd=189 nM); 46-A5 and 52-D10 have no binding
activity while other modified ubiquitin proteins provide a minor
binding activity compared 46-H4 to ED-B. Thus it can be concluded
that both monomers in a hetero-dimeric variant are required for a
high affinity binding to a target; both monomers show a monovalent
binding to the target.
[0212] The modified ubiquitin hetero-dimer with high ED-B binding
activity named 46 H9 is identified by the following amino acid
replacements in both binding domain region in the two monomers as
compared to wild type ubiquitin monomers:
in the first module (BDR1) (a) Q2G, F4V, K6R, Q62P, K63H, E64A,
S65T, T66L in the second module (BDR2) (c) K6H, LBM, Q62K, K63P,
E64I, S65A, T66E
50G11
[0213] in the first module (46H9)(a) Q2G, F4V, K6R, Q62P, K63H,
E64P, S65T, T66L in the second module (c) K6M L8R, Q62M, K63N,
E64A, S65R, T66L
46H4
[0214] in the first module (46H9)(a) Q2G, F4V, K6R, Q62P, K63H,
E64P, S65T, T66L in the second module (d) K6G, L8W, Q62T, K63Q,
E64Q, S65T, T66R
52B3
[0215] in the first module (g) Q2R, F4P, K6Y, Q62P, K63P, E64F,
S65A, T66R in the second module (46H9) K6H, L8M, Q62K, K63P, E64I,
S65A, T66E 52D10 (non-ED-B binder) in the first module Q2V, F4C,
K6R, Q62T, K63A, E64P, S65G, T66D in the second module (46H9) (e)
K6H, L8M, Q62K, K63P, E64I, S65A, T66E 46A5 (non-ED-B binder) in
the first module (46H9)(a) Q2G, F4V, K6R, Q62P, K63H, E64P, S65T,
T66L in the second module (b) K6L, L8M, Q62L, K63A, E64F, S65A,
[0216] FIG. 8 shows a sequence alignment. Line 1: Two monomers of
the wild type ubiquitin protein (1.sup.st line) are linked with a
12-amino acid linker SGGGGSGGGGIG starting at Position 77 and
ending at Position 88; the second monomer with BDR2 starts at
position 89 with a Methionine. This dimeric wild-type ubiquitin
protein is aligned with the modified ubiquitin hetero-dimeric
variant 46-H9 (2.sup.nd line) with different modifications in the
first and in the second monomer resulting in two BDR's. Both BDRs
act together in the binding of the target due to a monovalent
binding to the target.
[0217] FIG. 9 shows a sequence alignment of modified ubiquitin
hetero-dimeric variant 1041-D11 (1.sup.st line) to "Ub2_TsX9"
(ubiquitin modified in position 45 in both monomers to Tryptophane,
showing the linker GIG between the two monomers (position 77 to 79;
the second monomer starts with a Methionine at Position 80), and an
exchange from Glycine to Alanine at the last c-terminal amino acids
of the 2.sup.nd monomer. The third line shows "Ubi-Dimer wt", the
wildtype ubiquitin as dimer; showing no linker alignment (thus, the
second monomer starts at position 77 with a Methionin). The
4.sup.th line shows the "Ubi-Monomer wt" which is the human wild
type ubiquitin.
[0218] FIG. 10 shows a concentration dependent ELISA of the binding
of the hetero-dimeric ubiquitin variant 1041-D11 to human ED-B.
Variant 1041-D11 shows very high affinity binding to ED-B (Kd=6.9
nM=6.9.times.10.sup.-9 M). The closed dots show the affinity of the
binding of hetero-dimeric ubiquitin variant 1041-D11 to an ED-B
containing fibronectin fragment (referred to as 67B8940) compared
to no binding of this variant to negative control (referred to as
678940) (open circles).
[0219] FIG. 11 shows competitive concentration dependent ELISAs of
the binding of hetero-dimeric ubiquitin variant 1041-D11 to
immobilized ED-B containing fibronectin fragment (67B89) in the
presence of increasing amounts of free target. Hetero-dimeric
ubiquitin variant 1041-D11 shows a very high affinity binding to
ED-B (IC.sub.50=140 nM).
[0220] FIG. 12 shows a result of an analysis of the modified
hetero-dimeric ubiquitin molecule 1041-D11 in label-free
interaction assays using Biacore.RTM.. Different concentrations of
the hetero-dimeric ubiquitin variant were analyzed (see figure
legend: 0-200 nM of 1041-D11) for binding to an ED-B containing
fibronectin fragment (referred to as 67B89) immobilized on a
SA-chip (Biacore). Analyzing the association and dissociation
curves resulted in a Kd of 1 nM (1.times.10.sup.-9 M) and a
k.sub.off rate of 7.7.times.10.sup.-4 s.sup.-1 which indicates a
long half time of an complex of 1041-D11 and ED-B.
[0221] FIG. 13 shows the binding of hetero-dimeric ubiquitin
variant 1041-D11 to ED-B in a concentration dependent ELISA
simultaneously analyzing the serum-stability of binding activity.
Shown are different conditions, such as pre-incubation for 1 h at
37.degree. C. of the variant in mouse or rat serum or in PBST as
control. The Kd-values are all between 10 and 20 nM. Thus, it can
be concluded that the binding of the hetero-dimer 1041-D11 to ED-B
is not significantly influenced by blood serum.
[0222] FIG. 14 shows an analysis of the complex-formation of
hetero-dimeric ubiquitin variant 1041-D11 with fibronectin
fragments by SE-HPLC.
[0223] FIG. 14 A shows the complex formation of 1041-D11 with ED-B.
Three HPLC runs are overlaid: the blue peak with a retention time
of 21.651 min originates from pure 1041-D11; the black peak with a
retention time of 26.289 min represents the fibronectin fragment
67B89; a mixture of 1041-D11 and 67B89 results in the red peak with
a retention time of 21.407 min after SE-HPLC. The shift of the
1041-D11 peak to a lower retention time as well as the
disappearance of the 67B89 peak indicates formation of a complex of
1041-D11 and soluble ED-B.
[0224] FIG. 14 B shows the overlay of three SE-HPLC runs of
1041-D11 (blue, 21.944 min), fibronectin fragment 6789 without ED-B
(black, 26.289 min) and a mixture of 1041-D11 and 6789 (red line
with peaks at 21.929 min and 26.289 min). Almost no shift of the
1041-D11 peak is observed. This fact together with a lack of
disappearance of the 6789 peak indicates no significant binding of
the ED-B free fibronectin fragment 6789.
[0225] FIG. 15 shows the binding of hetero-dimeric ubiquitin
variant 1041-D11 to cell culture cells.
[0226] FIG. 15A shows binding of the hetero-dimeric ubiquitin
variant 1041-D11 on human fetal lung fibroblast cells (Wi38) which
were fixed. The first column in FIG. 15 shows the control using
anti-ED-B antibodies; the second column shows the incubation of the
variant at a protein concentration of 58.7 nM, the third column a
ten-fold higher concentration of 1041-D11 protein (587 nM), and the
fourth column is a negative control with PBS. In the first row,
human Wi38 fibroblast cells are shown in phase contrast; the second
row shows the immunofluorescence and the third row a DAPI staining
the nuclei. It can be concluded that the variant 1041-D11 binds to
Wi38 with high specificity to ED-B containing extracellular matrix.
A control using NHDF cells which express low level of EDB was
performed (data not shown). The variants do not bind to those
cells.
[0227] FIG. 15B shows the binding on vital human fetal lung
fibroblast cells (Wi38). The negative control cells type NHDF are
primary normal fibroblast cells, which express low levels of
EDB-fibronectin. The first and third line shows the variant at
different protein concentration and the negative control. The
second and fourth line shows the incubation of the control using
EDB antibodies. The first 2 lines show the variant and positive
control on Wi38-cell line. The third and fourth line shows the
incubation of NHDF-cells. It can be seen that the variant 1041-D11
binds to Wi38 with high specificity to ED-B containing
extracellular matrix.
[0228] FIG. 15C shows the binding on fixed murine Balb 3T3-cells.
Three different protein concentrations (1, 10, 50 nM) of the
variant were tested. The first rows shows the variant
(SPVF-28-1041-411-TsX9) on cells, the second row shows the positive
control (Fv28-EDB-Antibodies), the third row shows the incubation
with the negative control (UB2_TsS9; unmodified ubiquitin
corresponding to SEQ ID NO:1). It can be seen that the variant
1041-D11 binds to murine Balb 3T3 cells with high specificity to
ED-B containing extracellular matrix.
[0229] FIG. 15D shows the binding on fixed murine ST-2-cells. Three
different protein concentrations (1, 10, 50 nM) of the variant were
tested. The first rows shows the variant (SPVF-28-1041-411-TsX9) on
cells, the second row shows the positive control
(Fv28-EDB-Antibodies), the third row shows the incubation with the
negative control (UB2_TsS9; unmodified ubiquitin corresponding to
SEQ ID NO: 1). It can be seen that the hetero-dimeric ubiquitin
variant 1041-D11 binds to murine Balb ST-2 cells with high
specificity to ED-B containing extracellular matrix.
[0230] FIG. 16 A shows the specificity of hetero-dimeric ubiquitin
variant 1041-D11 to the target in mammalian tissue sections. F9
tumor tissues from seven samples were evaluated.
Immunohistochemistry with different concentrations between 10 nM
and 100 nM of hetero-dimeric ubiquitin variant 1041-D11 resulted in
ED-B specific vascular staining on F9 tumors from mice. ED-B is a
highly specific marker for tumor vasculature. The target protein
ED-B is located on the abluminal side of the vessels. Variant
1041-D11 specifically decorates the vasculature in tissue sections
from F9 tumors. The obtained results are comparable to the antibody
fragment L19. In addition, 48 tissues were tested; no unspecific
staining in any out of 48 tissues in FDA relevant panel was
observed.
[0231] FIG. 16 B shows the accumulation of 1041-D11 in tumor tissue
in comparison to wild type ubiquitin (in the figure, Ub2 (NCP2). F9
tumor tissues were analyzed for the presence of 1041-D11 and
wildtype ubiquitin at different time points between 30 min and 16
h. The highest accumulation of 1041-D11 in tumor tissue is observed
30 min and 16 h after administration whereas the accumulation of
wildtype ubiquitin in F9 tumor tissues is low. The variant is
enriched in tumors expressing ED-B when compared to wildtype
ubiquitin. This is an evidence for the directed targeting of
1041-D11 to tumor tissues. Further, the tumour to blood-ratio of
1041-D11 in a cancer model clearly demonstrates in vivo activity of
1041-D11 variant in animals (data not shown).
[0232] FIG. 17 shows the high selectivity and specificity
1041-D11-TNF-alpha fusion protein for ED-B.
[0233] FIGS. 17A and 17B: Apoptosis inducing TNF-alpha activity of
the 1041-D11-TNF.alpha. fusion protein was tested in a cell based
assay (L929 cells). The figures clearly show that the
1041-D11-TNF-alpha fusion protein (FIG. 17B) is as active as free
TNF-alpha (FIG. 17B) in cell culture.
[0234] FIG. 17C demonstrates the high selectivity of the
hetero-dimeric ubiquitin 1041-D11 TNF-alpha fusion protein to the
target ED-B. The human ED-B fibronectin domain 67B89 is bound with
an apparent KD value of 1.8 nM to variant 1041-D11 (closed
circles), showing the high affinity for the target. Human
fibronectin lacking the ED-B domain (h6789) is not bound by
1041-D11 TNFalpha (open circles).
[0235] FIG. 17D shows the binding analysis of modified
ubiquitin-based ED-B binding 1041-D11-TNF-alpha fusion protein by
Biacore assays. The results demonstrate the high affinity of
1041-D11 TNF-alpha fusion protein with a KD value of 1.13 nM.
[0236] FIG. 17E shows the high binding specificity observed with
variant 1041-D11 in cell culture is preserved when the 1041-D11 is
fused to TNF-alpha. The fusion protein specifically binds to EDB
expressing cells. Thus, 1041-D11 TNF-alpha fusion protein binds
with very high affinity and specificity to the target ED-B
("target(+)"). In serum without ED-B ("target(-)"), no cross
reaction can be observed.
[0237] FIG. 18 shows the relative tumor growth in vivo during the
time of treatment of mice for 7 days with variant 1041-D11 fused to
TNFalpha in combination with Melphalan. The data clearly show that
1041-D11-TNFalpha in combination with the cytostatic agent
Melphalan reduces the relative tumor growth more efficiently that
mTNF-alpha in combination with Melphalan or Melphalan alone. The
tumor growth kinetic 7 days after treatment shows the efficient
reduction of tumors by 1041-D11-mTNFa. This is a clear evidence for
the efficacy of a treatment of tumors with fusion protein
1041-D11-TNF-alpha in combination with Melphalan. ED-B is identical
in several mammalian species, including mice and human, and thus,
the results are predictive of the performance of variant
1041-D11-TNFalpha in humans.
[0238] FIG. 19 shows the consensus positions and amino acid
substitutions of 16 further sequences which have been found to have
surprisingly strong binding affinities to ED-B. The consensus amino
acid positions are in the first monomeric binding determining
region 2, 4, 6, 62, 63, 64, 65, 66 while the consensus amino acid
substitutions are Q2T, F4W, K6H, Q62N, K63F, E64K, S65L, and T66S.
As can be taken from FIG. 2, 4 families of sequences could be
enriched (consensus sequences, seize of the letters correspond to
the frequency of occurrence of the amino acids). Positions 85 and
87 are positions in the hetero-dimeric protein; with reference to
the second monomer, the corresponding positions are 6 and 8;
141-145 correspond to positions 62-64). TWH NFKLS depicted in
dark-blue colour originates from 1071-C12. Residues marked with red
colour belong to one of the said four families of sequences.
Residues marked in red have been enriched predominantly (178/457
sequences) and include according to HIT ELISAs the strongest
binding molecules.
EXAMPLES
[0239] The following Examples are provided for further illustration
of the invention. The invention is particularly demonstrated with
respect to the modification of ubiquitin as an example. The
invention, however, is not limited thereto, and the following
Examples merely show the practicability of the invention on the
basis of the above description. For a complete disclosure of the
invention reference is made also to the literature cited in the
application and in the annex which are all incorporated in their
entirety into the application by reference.
Example 1
Identification of ED-B Binding Proteins Based on Modified Ubiquitin
Proteins
Library Construction for Monomeric Binding Proteins and Cloning
[0240] Unless otherwise indicated, established recombinant genetic
methods were used, for example as described in Sambrook et al. A
random library of human ubiquitin monomers with high complexity was
prepared by concerted mutagenesis of in total up to 10 selected
amino acid positions. The modified amino acids, which were
substituted by NNK triplets, comprised at least 3 amino acids
selected from positions 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 within
the ubiquitin monomer.
Library Construction for Hetero-Dimeric Binding Proteins and
Cloning
[0241] Unless otherwise indicated, established recombinant genetic
methods were used, for example as described in Sambrook et al. A
random library of human ubiquitin hetero-dimers with high
complexity was prepared by concerted mutagenesis of in total 15
selected amino acid positions. The modified amino acids, which were
substituted by NNK triplets, comprised at least 3 amino acids
selected from positions 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 within
the proximal (first) ubiquitin monomer and at least 3 amino acids
selected from positions 2, 4, 6, 8, 62, 63, 64, 65, 66, 68 within
the distal (second) ubiquitin monomer. Both ubiquitin monomers were
genetically linked (head to tail) by a Glycine/Serine linker with
at least the sequence GIG or by Glycine/Serine linker with at least
the sequence SGGGG, for example GIG, SGGGG, SGGGGIG, SGGGGSGGGGIG
(SEQ ID NO: 32) or SGGGGSGGGG, but any other linker is
possible.
TAT Phage Display Selection
[0242] The ubiquitin library was enriched against the target using,
for example, TAT phage display as selection system. Other selection
methods known in the art can be used. The target can be immobilized
nonspecifically onto protein binding surfaces or via biotinylated
residues which were covalently coupled to the protein. The
immobilization via biotin onto streptavidin beads or neutravidin
strips is preferred. The target-binding phages are selected either
in solution or on immobilized target; for example, the biotinylated
and immobilized target with phage was incubated followed by washing
of the phages bound to the matrix and by elution of matrix-bound
phages. In each cycle following target incubation, the beads were
magnetically separated from solution and washed several times. In
the first selection cycle the biotinylated target was immobilized
to neutravidin strips whereas in cycles two to four selections in
solution was performed followed by immobilization of target-phage
complexes on Streptavidin-coated Dynabeads.RTM. (Invitrogen). After
washing in the first two selection cycles the phages of
target-binding modified ubiquitin molecules were released by
elution with acidic solution. In selection cycles three and four
elution of phages was carried out by competitive elution with
excess target. The eluted phages were reamplified. To direct
specificty of binders a protein similar to the target can be
included during selection.
Alternatively to TAT Phage Display Selection: Ribosome Display
Selection
[0243] The ubiquitin library was enriched against the target using,
for example, ribosome display as selection system (Zahnd et al.,
2007), Ohashi et al., 2007). Other selection methods known in the
art can be used. The target was biotinylated according to standard
methods and immobilized on Streptavidin-coated Dynabeads.RTM.
(Invitrogen). Ternary complexes comprising ribosomes, mRNA and
nascent ubiquitin polypeptide were assembled using the
PURExpress.TM. In Vitro Protein Synthesis Kit (NEB). Two primary
rounds of selection were performed, wherein ternary complexes were
incubated followed by two similar rounds of selection. In each
cycle following target incubation, the beads were magnetically
separated from solution and washed with ribosome display buffer
with increasing stringency. After washing in the first two
selection cycles, the beads were again magnetically separated from
solution and mRNA of target-binding modified ubiquitin molecules
was released from ribosomes by addition of 50 mM EDTA. In selection
cycles three and four elution of mRNA was carried out by
competitive elution with excess target (Lipovsek and Pluckthun,
2004). After each cycle, RNA purification and cDNA synthesis were
performed using RNeasy MinElute Cleanup Kit (Qiagen, Germany),
Turbo DNA-free Kit (Applied Biosystems, USA) and Transcriptor
Reverse Transcriptase (Roche, Germany).
Cloning of Enriched Pools
[0244] After the fourth selection cycle the synthesized cDNA was
amplified by PCR via primers F1
(GGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGA
TATACATATG) (SEQ ID NO: 9) and WUBI(co)RD_xho
(AAAAAAAAACTCGAGACCGCCACGCAGACGCAGAACCAG) (SEQ ID NO: 10), cut with
restriction nucleases NdeI and XhoI (Promega, USA) and ligated into
expression vector pET-20b(+) (Merck, Germany) via compatible
cohesive ends.
Single Colony Hit Analysis
[0245] After transformation into NovaBlue (DE3) cells (Merck,
Germany) ampicillin-resistant single colonies were grown for 6 h at
37.degree. C. in 200 .mu.l SOBAG medium (SOB medium containing 100
.mu.g/ml ampicilin and 20 g/l glucose). expression of the
ED-B-binding modified ubiquitin was achieved by cultivation for 16
h at 37.degree. C. in 96-well deep well plates (Genetix, UK) using
500 .mu.l auto induction medium ZYM-5052 (Studier, 2005). Cells
were harvested by 15 min of centrifugation at 4.degree. C. and 3600
g and subsequently lysed by incubation for 30 min at 37.degree. C.
with 300 .mu.l lysis buffer per well, containing 0.2.times.
BugBuster.RTM. (Merck, Germany), 0.3 mg/ml lysozyme (VWR, Germany)
0.2 mM PMSF (Roth, Germany), 3 mM MgCl.sub.2 and 0.2 U/ml Benzonase
(VWR, Germany) in 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, pH8. After
centrifugation for 30 min at 4.degree. C. and 3600 g the resulting
supernatants were screened by ELISA using Nunc MediSorp plates
(Thermo Fisher Scientific, USA) coated with 4 .mu.g/m lED-B and a
ubiquitin-specific Fab fragment conjugated with horseradish
peroxidase (POD). As detecting reagent TMB-Plus (Biotrend, Germany)
was used and the yellow colour was developed using 50 .mu.l/well
0.2 M H.sub.2SO.sub.4 solution and measured in a plate reader at
450 nm versus 620 nm.
[0246] Several cycles of selection display versus ED-B were carried
out. In the last two cycles of selection binding molecules were
eluted with an excess of free ED-B. These ED-B-binding variants
were identified, among others:
TABLE-US-00003 1H4: (SEQ ID NO: 3)
MWIKVHTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQ
LEDGRTLSDYNITLSRSLHLVLRLRGG 4B10: (SEQ ID NO: 4)
MLILVLTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQ
LEDGRTLSDYNIATKPILHLVLRLRGG 5E1: (SEQ ID NO: 5)
MVINVFTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQ
LEDGRTLSDYNIRSTSKLHLVLRLRGG
Sequence of 46H9 (linker between the different monomers shown in
italics)
TABLE-US-00004 (SEQ ID NO: 6)
MGIVVRTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQ
LEDGRTLSDYNIPHPTLLHLVLRLRGGSGGGGSGGGGIGMQIFVHTMTG
KTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDY
NIKPIAELHLVLRLRGG
Sequence of 9E12 (linker between the different monomers shown in
italics)
TABLE-US-00005 (SEQ ID NO: 7)
MRIPVYTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQ
LEDGRTLSDYNIPPFARLHLVLRLRGGSGGGGSGGGGIGMQIFVMTRTG
KTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDY
NIMNARLLHLVLRLRGG
Sequence of 22D1 (linker between the different monomers shown in
italics)
TABLE-US-00006 (SEQ ID NO: 8)
MLILVRTLTDKTITLEVEPSDTIGNVKAKIQDKEGIPPDQQRLIWAGKQ
LEDGRTLSDYNISVGAMLHLVLRLRGGSGGGGSGGGGIGMQIFVLTWTG
KTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGKQLEDGRTLSDY
NIRRLPPLHLVLRLRGG
[0247] A sequence alignment of wild type ubiquitin monomer (Ubi
monomer wt), with wild type ubiquitin dimer (ubi dimer wt) and wild
type ubiquitin protein (Ub2-TsX in FIG. 9, with an exchange in
Position 45 of each monomer and with two substitutions at the
C-terminus) with the modified ubiquitin hetero-dimeric variant
1041-D11 is shown in FIG. 9. In Ub2-TsX the substitutions at the
C-terminus (GG to AA) of the monomer increase the stability in
serum because deubiquitinases cleave behind the GG of ubiquitin but
not behind the AA. The secondary structure of the wild type
ubiquitin compared to the ubiquitin with these substitutions at the
C-terminus is almost identical.
[0248] The modified ubiquitins with superior ED-B binding activity
referred to as 1041-D11 (shown in FIG. 9; SEQ ID NO: 36) or
1045-D10 are identified by the following amino acid replacements as
compared to the wild type: in the first module: K6W, LBW, K63R,
E64K, S65F, T66P; in the second module: K6T, L8Q, Q62W, K63S, E64N,
S65W, T66E; optionally Q2R (in variant 1041-D11, but not in variant
1045-D10). Suitable preferred linkers for the fusion protein are
those of SEQ ID NO: 32 or the sequence GIG. However, there are many
conceivable linkers which can be used instead.
[0249] As a further preferred example a protein is provided by the
following sequence wherein X may be any amino acid (SEQ ID NO: 47)
As linker, SGGGGSGGGGIG was used here (shown in italics). It is to
be understood that also other kind of linkers or no linker are
feasible alternatives.
TABLE-US-00007 ##STR00002##
[0250] Examples of proteins with these sequences are shown in FIG.
19.
Example 2
Production of Fusion Proteins from ED-B-Binding Modified Ubiquitin
Variants and TNFalpha (for Example, Human TNF.alpha., Referred to
as TNF.alpha.)
[0251] The variants can be expressed as fusion proteins between the
modified ubiquitins, for example heterodimeric variant1041-D11, and
mammalian, for example mouse or human, TNF.alpha. in E. coli.
Protein analysis of the fusion protein includes: protein expression
and purity, no aggregation potential, TNF.alpha. activity in cell
culture, affinity for target protein ED-B, Selectivity, specific
binding in cell culture can be anaylzed. A prerequisite for an
animal experiment to induce tumor shrinkage in F9 tumor bearing
mice is a fusion with mouse TNF.alpha.
Step 1: Production of a Vector for Cloning of Fusion Proteins
(pETSUMO-- TNF.alpha.)
[0252] pETSUMOadapt is a modified vector pETSUMO (Invitrogen),
which was modified by insertion of an additional multiple cloning
site (MCS). Starting from TNF.alpha. cloned in pETSUMOadapt,
restriction sites for the insertion of modified ubiquitin variants
binding ED-B--were introduced. The resulting construct has the
structure His.sub.6-SUMO-TNF.alpha. with the following DNA-sequence
(SEQ ID NO: 11):
TABLE-US-00008 ATGGGCAGCAGCCATCATCATCATCATCACGGCAGCGGCCTGGTGCCGC
GCGGCAGCGCTAGCATGTCGGACTCAGAAGTCAATCAAGAAGCTAAGCC
AGAGGTCAAGCCAGAAGTCAAGCCTGAGACTCACATCAATTTAAAGGTG
TCCGATGGATCTTCAGAGATCTTCTTCAAGATCAAAAAGACCACTCCTT
TAAGAAGGCTGATGGAAGCGTTCGCTAAAAGACAGGGTAAGGAAATGGA
CTCCTTAAGATTCTTGTACGACGGTATTAGAATTCAAGCTGATCAGACC
CCTGAAGATTTGGACATGGAGGATAACGATATTATTGAGGCTCACAGAG
AACAGATTGGTGGTGTGCGTAGCAGCAGCCGTACCCCGAGCGATAAACC
GGTGGCGCATGTGGTGGCGAATCCGCAGGCGGAAGGCCAGCTGCAGTGG
CTGAACCGTCGTGCGAATGCGCTGCTGGCCAACGGCGTGGAACTGCGTG
ATAATCAGCTGGTTGTGCCGAGCGAAGGCCTGTATCTGATTTATAGCCA
GGTGCTGTTTAAAGGCCAGGGCTGCCCGAGCACCCATGTGCTGCTGACC
CATACCATTAGCCGTATTGCGGTGAGCTATCAGACCAAAGTGAACCTGC
TGTCTGCGATTAAAAGCCCGTGCCAGCGTGAAACCCCGGAAGGCGCGGA
AGCGAAACCGTGGTATGAACCGATTTATCTGGGCGGCGTGTTTCAGCTG
GAAAAAGGCGATCGTCTGAGCGCGGAAATTAACCGTCCGGATTATCTGG
ATTTTGCGGAAAGCGGCCAGGTGTATTTTGGCATTATTGCGCTGTAATA A
[0253] The TNF.alpha. sequence was amplified via PCR by introducing
a BamHI- and XhoI-site. Primers used:
TABLE-US-00009 SUMO-EDB- TNF.alpha.-fw (SEQ ID NO: 12): TTT TTT GGA
TCC GTG CGT AGC AGC AGC SUMO-EDB- TNF.alpha.-rev (SEQ ID NO: 13):
CTT GTC TCT CGA GGC GGC CGC TTA TTA C
[0254] The fw-primer (SEQ ID NO: 12) recognizes the first 15 base
pairs of TNF.alpha. (underlined region) and has a BamHI-sequence
(shown in bold). The rev-primer (SEQ ID NO: 13) contains the last
base pair of TNF.alpha., theestop codons (underlined) and a
XhoI-restriction site (bold).
PCR Reaction Mix (100 .mu.l):
[0255] 84.5 .mu.l H.sub.2O; 10 .mu.l 10.times. Pwo buffer+Mg; 2
.mu.l 10 mM dNTPs (=200 .mu.M); each 0.5 .mu.l 100 .mu.M primer
fw/rev (=each 0.5 .mu.M); 2 .mu.l DNA (=0.25 .mu.g); 0.5 .mu.l Pwo
polymerase (=2.5 U; Roche)
PCR-Program:
[0256] 3 min 94.degree. C., 30 s 94.degree. C., 30 s 60.degree. C.,
2 min 72.degree. C. (steps 2-4: 30 cycles), 5 min 72.degree. C.,
followed by 4.degree. C. followed by purification of the PCR
product with the Qiagen-MinElute-Kit (elution in 10 .mu.l EB). The
PCR product is introduced in the MCS of the vector pETSUMOadapt via
BamHI-XhoI-restriction and ligation.
Restriction Mix (100 .mu.l):
[0257] Vector: 83 .mu.l H.sub.2O; 10 .mu.l 10.times.NE buffer 3; 1
.mu.l 100.times.BSA; 3 .mu.l BamHI (=30 U; NEB), 1.5 .mu.l XhoI
(=30 U; NEB); 1.65 .mu.l vector; 3 h 37.degree. C. incubation. PCR
product: 76.5 .mu.l H.sub.2O; 10 pa 10.times.NE buffer 3; 1 .mu.l
100.times.BSA; 3 .mu.l BamHI (=30 U; NEB), 1.5 .mu.l XhoI (=30 U;
NEB); 8 .mu.l insert; 3 h 37.degree. C. incubation Separation of
Restriction in 1% Agarosegel (100 V 60 min run); cut vector
fragment (5659 bp) and insert (491 bp); Purification with Qiagen
gel extraction kit (elution in 30 .mu.l EB).
Ligation (20 .mu.l):
[0258] 15.2 .mu.l H.sub.2O; 2 .mu.l 10.times. T4-DNA-ligasebuffer;
2.26 .mu.l Vector (200 ng); 0.54 .mu.l insert (40 ng) 5 min
65.degree. C. incubation; cool to 16.degree. C.; add 1 .mu.l
T4-DNA-ligase (=3 U; NEB); 16 h 16.degree. C. incubation.
NaAc/Isopropanol-Precipitation:
[0259] Ligation-mixture (20 .mu.l)+2.2 .mu.l 3 M NaAc (pH 5.0)+22.2
.mu.l Isopropanol; 30 min -20.degree. C.; 15 min 4.degree. C. 13000
Upm; resuspend Pellet in 500 .mu.l 70% EtOH; spin; resuspend pellet
in 10 .mu.l H.sub.2O.
Transformation:
[0260] Mix electro-competent Novablue (DE3)-cells (40
.mu.l-aliquot) with 10 .mu.l Ligationsproduct; transfer to
0.1-cm-elektroporation cuvette; puts in elektroporator (1.8 kV, 50
.mu.F, 100 Ohm); incubate solution with 1 ml SOC-medium 45 min
37.degree. C. 220 Upm; 100 .mu.l on LB-plate with Kanamycin;
incubation overnight 37.degree. C.
Step 2: Cloning of Modified Ubiquitin-Based EDB-Fusion Proteins
[0261] For the production of fusions of EDB-binding modified
ubiquitin-based variants and TNF.alpha., the EDB-modified
ubiquitin-based sequence of interest in amplified from a
pET20b-vector via PCR; BsaI- and BamHI-restriction sites are
introduced. The method is suitable for monomeric and for dimeric
EDB-modified ubiquitin-based variants. Primer for monomeric
WT-Ubiquitin (Wubi):
TABLE-US-00010 SUMO-EDB-WUBI-fw (SEQ ID NO: 14):: GTT CCA AGG TCT
CAT GGT ATG CAG ATC TTC GTG SUMO-EDB-Linker-rev (SEQ ID NO: 15)::
GTG GTG GGA TCC ACC GCC ACC ACC AGA ACC GCC ACG CAG ACG
[0262] The fw-primer (SEQ ID NO: 14) recognizes the first 15 base
pairs of modified ubiquitin (underlined region) and has a
BsaI-sequence (shown in bold). The rev-Primer (SEQ ID NO: 15)
recognizes the last 15 base pairs of modified ubiquitin and inserts
an amino acid linker (sequence SGGGG) and a BamHI-restriction site
(bold). For each modified ubiquitin-based-variant, a specific
fw-primer is used. Primers monomeric EDB-modified ubiquitin-based
variants 1H4, 5E1 and 4B10:
TABLE-US-00011 1H4 (MWIKV . . . ): Primer (SUMO-EDB-1H4-fw) (SEQ ID
NO: 16): GTT CCA AGG TCT CAT GGT ATG TGG ATC AAG GTG 4B10 (MLILV):
Primer (SUMO-EDB-4B10-fw) (SEQ ID NO: 17): GTT CCA AGG TCT CAT GGT
ATG TTG ATC CTG GTG 5E1 (MVINV . . . ): Primer (SUMO-EDB-5E1-fw)
(SEQ ID NO: 18): GTT CCA AGG TCT CAT GGT ATG GTT ATC AAT GTG
[0263] The rev-primer is used for all monomeric modified
ubiquitin-based variants. Rev-Primer for dimeric modified
ubiquitin-based variants:
TABLE-US-00012 Dimer-t0a-rev (SEQ ID NO: 19): GTG GTG GGA TCC ACC
GCC ACC ACC AGA ACC ACC ACG TAA ACG
[0264] fw-primer for the cloning of dimeric WT-ubiquitins
(WubiHubi) and for dimeric EDB-modified ubiquitin-based
variants:
TABLE-US-00013 WT (MQIFV . . . ) Primer (SUMO-EDB-WUBI-fw) (SEQ ID
NO: 20): GTT CCA AGG TCT CAT GGT ATG CAG ATC TTC GTG
[0265] (note: fw-Primer for dimerics WT-ubiquitin is identical to
fw-Primer for monomeric WT-ubiquitin.)
TABLE-US-00014 9E12 (MRIPV . . . ): Primer (9E12-t0a-fw) (SEQ ID
NO: 21): GTT CCA AGG TCT CAT GGT ATG CGT ATC CCT GTG 24H12 (MVIKV .
. . ): Primer (24H12-t0a-fw) (SEQ ID NO: 22): GTT CCA AGG TCT CAT
GGT ATG GTT ATC AAG GTG 15G7 (MEIGV . . . ): Primer (15G7-t0a-fw)
(SEQ ID NO: 23): GTT CCA AGG TCT CAT GGT ATG GAG ATC GGT GTG 22D1
(MLILV . . . ): Primer (22D1-t0a-fw) (SEQ ID NO: 24): GTT CCA AGG
TCT CAT GGT ATG CTT ATC TTG GTG
PCR-Mixture (100 .mu.l):
[0266] 84.5 .mu.l H.sub.2O; 10 .mu.l 10.times. Pwo-buffer+Mg; 2
.mu.l 10 mM dNTPs (=200 .mu.M); each 0.5 .mu.l 100 .mu.M Primer
fw/rev (=je 0.5 .mu.M); 2 .mu.l DNA (dependent on the variant); 0.5
.mu.l Pwo-Polymerase (=2.5 U; Roche)
PCR-Program:
[0267] 1. 3 min 94.degree. C. [0268] 2. 30 s 94.degree. C. [0269]
3. 30 s 60.degree. C. [0270] 4. 2 min 72.degree. C. (steps 2-4: 30
cycles) [0271] 5. 5 min 72.degree. C., followed by 4.degree. C.
Purification of the PCR-products in agarose gel, cut required band
and purify with Qiagen-gel extraction kit. Cloning of the
PCR-product via BsaI-BamHI-restriction (in pETSUMO-TNFa)
Restriction (100 .mu.l):
[0272] 75 .mu.l H.sub.2O; 10 .mu.l 10.times. NEBuffer 3; 1 .mu.l
100.times.BSA; 3 .mu.l BsaI (=30 U; NEB); 8 .mu.l DNA (Vector or
PCR-Product).sub.2 h 50.degree. C. incubation, 10 min 65.degree.
C., add 3 .mu.l BamHI (=30 U; NEB), 2 h 37.degree. C. Separation of
restriction in 1% agarose gel; cut vector fragment and insert;
purification with Qiagen-gel extraction kit (elution in 30 .mu.l
EB).
Ligation (20 .mu.l):
[0273] 12.5 .mu.l H.sub.2O; 2 .mu.l 10.times. T4-DNA-ligase buffer;
5 .mu.l vector (66 ng); 0.5 .mu.l Insert (variabel) 5 min
65.degree. C. incubation; cool to 16.degree. C.; add 1 .mu.l
T4-DNA-ligase (=3 U; NEB); 16 h 16.degree. C. incubation
NaAc/Isopropanol-Precipitation (See Step 1)
[0274] Transformation in elektrocompetent Novablue (DE3)-cells as
described above. The result is the following fusion construct:
EDB-modified ubiquitin and TNF.alpha. in pETSUMOadapt with der
His.sub.6-SUMO-modified ubiquitin-SGGGG-TNF.alpha. (359 amino acids
with monomeric modified ubiquitin447 amino acids with dimeric
modified ubiquitin)
Example 3
Expression and Purification of Ubiquitin-Based-TNF.alpha. Fusion
Proteins
[0275] DNA sequence analysis showed the correct sequences of the
SUMO-TNF.alpha. fusion proteins. For expression of the variants the
clones were cultivated in a shaker flask by diluting a preculture
1:100 with LB/Kanamycin and agitating the culture at 200 rpm and
37.degree. C. up to an optical density at 600 nm (OD.sub.600) of
0.5. Expression was induced by adding IPTG (final concentration 1
mM). Culturing was continued for 4 hours at 30.degree. C. and 200
rpm. The bacteria cells were harvested by centrifugation at
4.degree. C., 6000.times.g for 20 min. The cell pellet was
suspended in 30 ml of NPI-20 buffer including benzonase and
lysozyme. Cells were disrupted by ultrasonication (3.times.20 sec)
on ice. The supernatant containing the soluble proteins was
obtained after centrifugation of the suspension at 4.degree. C. and
40000.times.g for 30 min. Both proteins were purified by affinity
chromatography at room temperature. One column of Ni-Agarose (5 ml,
GE Healthcare) was equilibrated with 50 ml of NPI-20. The
supernatant containing the soluble proteins was applied to the
column, followed by a washing step with NPI-20. The bound protein
was eluted with a linear gradient to 50% NPI-500 in 100 ml.
Fractions were analyzed by SDS-PAGE with respect to their purity.
Suitable fractions were pooled and applied to a gel filtration
column (Superdex 75, 1.6.times.60 cm, GE Healthcare) equilibrated
with SUMO-hydrolase cleavage buffer (50 mM Tris, 300 mM NaCl, pH
8.0) at a flow rate of 1 ml/min.
[0276] The cleavage reaction was done according to the manufactures
instruction (Invitrogen). After cleavage the protein was applied to
a Ni-Agarose column (5 ml, GE Healthcare). His-tagged
SUMO-hydrolase and His-tagged SUMO were bound to the column and the
correct fusion protein passed the column (His-tag free). Purity of
the proteins was proofed by rpHPLC analysis and gel
electrophoresis. The correct molecular mass of the trimer (via
TNFa) was confirmed using analytical SEC analysis (10/30 Superdex
G75, GE Healthcare).
Example 4
Binding Analysis of Modified Ubiquitin-Based ED-B binding Variants
to Human ED-B
Example 4A
Binding Analysis of Modified Ubiquitin-Based ED-B Binding Variants
by Concentration Dependent ELISA
[0277] Binding of ubiquitin-based variants to human ED-B was
assayed by a concentration dependent ELISA. Increasing amounts of
purified protein applied to NUNC-medisorp plates coated with human
ED-B, BSA and cellular fibronectin (cFN). Antigen coating with 50
.mu.l (10 .mu.g/ml) per well was performed at 4.degree. C.
overnight. After washing the plates with PBS, 0.1% Tween 20 pH 7.4
(PBST) the wells were blocked using blocking solution (PBS pH 7.4;
3% BSA; 0.5% Tween 20) at 37.degree. C. for 2 h. Wells were washed
again three times with PBST.
[0278] Different concentrations of modified ubiquitin based ED-B
binding protein were then incubated in the wells at RT for 1 h (50
.mu.l volume)(in FIG. 10, as start concentration, 500 nM of
1041-D11 protein was used). After washing the wells with PBST, the
anti-Ubi fab fragment (AbyD) POD conjugate was applied in an
appropriate dilution (for example, 1:2000 or 1:6500) in PBST. The
plate was washed three times with 300 .mu.l buffer PBST/well. 50
.mu.l TMB substrate solution (KEM-EN-Tec) were added to each well
and was incubated for 15 min. The reaction was stopped by adding 50
.mu.l 0.2 M H.sub.2SO.sub.4 per well. The ELISA plates were read
out using the TECAN Sunrise ELISA-Reader. The photometric
absorbance measurements were done at 450 nm using 620 nm as a
reference wavelength. FIG. 1 shows clearly the specific binding of
the 1H4 to ED-B with an apparent KD value of 11 nM. The variant 5E1
shows an apparent KD value of 7.7 .mu.M and 4B10 of 280 nM
respectively. FIG. 10 shows very high affinity binding of variant
1041-D11 to ED-B (KD=6.9 nM). Thus, only a few modifications (up to
8 substitutions in each monomer) in the ubiquitin-wildtype result
in a very higher affinity binding to ED-B.
Example 4B
Binding Analysis of Modified Ubiquitin-Based ED-B Binding Variants
by Competitive Concentration Dependent ELISA
[0279] Competitive concentration dependent ELISAs analyzed the
binding of ubiquitin variant 1041-D11 to immobilized ED-B
containing fibronectin fragment (67889) in the presence of
increasing amounts of free target. Conditions of the ELISA were as
described for Example 5A, except that 1041-D11 protein was
preincubated with ED-B (67B89) (0 .mu.M-10 .mu.M) or also with
negative control 6789 (0 .mu.M-10 .mu.M) for 1 h and subsequently
the mixture was given to the target 67B89 that was placed on a
Medisorp-plate; following this, the variant was detected by the
corresponding antibody (anti-Ubiquitin-Fab-POD; dilution
1:6500).
[0280] FIG. 11 shows that variant 1041-D11 has a very high affinity
binding to ED-B (IC50=140 nM). The result shown in FIG. 10 is
confirmed; only a few modifications (up to 8 substitutions in each
monomer) in the ubiquitin-wildtype result in a very higher affinity
binding to ED-B.
Example 4C
Binding Analysis of Modified Ubiquitin-Based ED-B Binding Variants
by Concentration Dependent ELISA Simultaneously Analyzing the
Serum-Stability of Binding Activity
[0281] The ELISA is performed using procedures well known in the
art and as described above (Example 5A and 5B). ED-B (here referred
to as 67B89) is coated to microtiter plates, the variant is bound
to ED-B and detected by a specific ubiquitin-antibody
(Anti-Ubi-Fab-POD). The variant in this assay is treated in
different ways: the variant is incubated in mouse serum for 1 h at
37.degree. C. (see in FIG. 13, circles in blue); the variant is
incubated in rat serum for 1 h at 37.degree. C. (in FIG. 13,
circles in red); or the variant is incubated PBS for 1 h at
37.degree. C. (in FIG. 13, circles in black). FIG. 13 shows that
all KDs of variant 1041-D11 are between 10.3 nM (in PBS) to 20.74
nM (in mouse-serum).
Example 4D
Binding Analysis of Modified Ubiquitin-Based ED-B Binding Variants
by Biacore Assays
[0282] Different concentrations of the variant were analyzed (for
example, 0-200 nM of the variant, preferably 1041-D11) for binding
to an ED-B containing fibronectin fragment (referred to as 67B89)
immobilized on a CM5-chip (Biacore) using methods known to those
skilled in the art. The obtained data were processed via the
BIAevaluation software and 1:1-Langmuir-fitting. The K.sub.D of
variant 1041-D11 was 1.0 nM, as shown in FIG. 12. The kinetic
binding constants were k.sub.on=7.6*10.sup.5 M.sup.-1 s.sup.-1;
k.sub.off=7.7*10.sup.-4 s.sup.-1. The K.sub.D of the fusion protein
1041-D11-TNF.alpha. was 1.13 nM, as shown in FIG. 17D. The kinetic
binding constants were k.sub.on=4.5*10.sup.5 M.sup.-1 s.sup.-1;
k.sub.off=5.0*10.sup.-4 s.sup.-1.
Example 4E
Complex-Formation Analysis of Modified Ubiquitin-Based ED-B Binding
Variants by SE-HPLC
[0283] For the analysis of complex formation, Tricorn Superdex 75
5/150 GL columns (GE-Healthcare) (V=3 ml) was used, protein amount
of 50 .mu.l was applied. Further conditions: buffer: 1.times.PBS,
pH 7.3, flow-rate: 0.3 ml/min, run: 45 min (injection of sample:
after 15 min). Condition: 0.72 nmol 1041-D11 protein+0.72 nmol ED-B
(herein referred to 67B89 or also as a negative control 6789)
incubated for 1 h at RT; then applied to column for analysis of
complex-formation. In FIG. 14, only the variant is shown in black,
only the target ED-B is shown in blue, the variant binding building
a complex with ED-B in pink. FIG. 14 A shows ED-B with the variant;
FIG. 14 B is the variant without ED-B. The figure shows that
variant 1041-D11 builds a complex together with ED-B (67B89), but
it builds no complex with 6789.
Example 5
Biological Assay of TNF.alpha.
[0284] The physiological TNF.alpha.-activity of TNF.alpha.-modified
ubiquitin based ED-B binding fusions has been determined using the
L929 apoptosis assay (Flick et al., 1984 J. Immunol. Methods.
68:167-175). In this assay, TNF-alpha efficiently stimulates cell
death in actinomycinD sensitized cells at EC.sub.50 values in the
picomolar range.
[0285] Cells have been resuspended in medium containing FBS and
antibiotics. A cell suspension of 100 .mu.l of a densitiy of
3.5.times.10.sup.5 cells/ml has been seeded into the wells of a 96
well standard cell culture plate followed by over night incubation
in a humidified CO.sub.2 incubator. Thereafter, the culture medium
has been removed and 50 .mu.l of medium containing FBS,
ActinomycinD and antibiotics has been added to each well followed
by a further 30 min incubation time. Thereafter, 50 .mu.l of the
test items, TNF.alpha.-modified ubiquitin based ED-B
binding-fusions or the human recombinant TNF.alpha.control, at an
appropriate concentration range of between 10.sup.-7 and 10.sup.-18
M, have been added. After a further 48 h incubation time the
metabolic activity as a measure of cell survival was determined
using WST-1 reagent (Roche).
[0286] Per test item at least three independent experiments have
been conducted, each of them in triplicates. Each testing of
TNF.alpha.-modified ubiquitin based ED-B binding-fusion proteins
was paralleled by testing a dose range of human recombinant
TNF.alpha. to get information on the inter assay variability.
[0287] The quantitative evaluation is based on the EC.sub.50-value,
i.e. the value according to the test item concentration promoting
the survival of half of the cells.
TABLE-US-00015 TABLE 2 EC.sub.50 value mub .RTM.- Corresponding
TNF.alpha.-mub-Fusion TNF.alpha.Fusion TNF.alpha. Wubi-TNF-alpha
5.18 .+-. 2.84 pM 7.97 .+-. 12.18 pM Wubi-Hubi-TNF-alpha 32.58 .+-.
11.26 pM 5.02 .+-. 3.70 pM SPWF-28_22-D1_TNF-alpha 26.15 .+-. 14.41
pM 2.32 .+-. 2.07 pM SPWF-28_24-H12_TNF- 0.78 .+-. 0.24 pM 3.01
.+-. 4.18 pM alpha mub: modified ubiquitin based ED-B binding
[0288] Of the TNF.alpha.-modified ubiquitin based ED-B
binding-fusion one ubiquitin monomer (Wubi) and three ubiquitin
dimer constructs have been analyzed. Depending on the modified
ubiquitin based ED-B binding variant coupled to the TNF-alpha
moiety the TNF-alpha associated activity has been increased
(SPWF-28.sub.--24-H12_TNF-alpha) or decreased
(SPWF-28.sub.--22-D1_TNF-alpha, Wubi-Hubi-TNF-alpha) by about one
order of magnitude. See FIG. 17 for variant 1041-D11 TNFalpha
analysis.
Example 6
Binding Analysis of Ubiquitin Variants in Cell Culture Assays
[0289] The binding of, for example, variant 1041-D11 to cell
culture cells was tested. Different cell culture cells were
analysed, including normal human fetal lung fibroblast cells having
high expression levels of ED-B (Wi38 cells), a mouse embryonic
fibroblast cell line (Balb 3T3); a stromal cell line, derived from
mouse bone marrow (ST-2) monocytes/macrophages (RAW 264.7), NHDF
cells and murine fibroblast cells (LM).
[0290] The variant 1041-D11 (different concentrations) or an ED-B
specific antibody (500 nM FV28 CH4/F1 1.times.PBS were incubated (1
h, 37.degree. C.) with Wi38 cells (60,000 cells/ml; from ATCC),
followed by fixation with methanol (5 min, -20.degree. C.),
blocking (5% Horse/PBS, 1 h); incubation with
rabbit-a-Strep-Tag-IgG (obtained from GenScript A00875, 1:500) for
1 h and incubation with a-rabbit-IgG*Alexa488-AK (obtained from
Invitrogen A11008, 1:1000) for 1 h. The nuclei were stained with
DAPI. The first column in FIG. 15A shows the control using EDB
antibodies, the second column shows the incubation of the variant
at a protein concentration of 58.7 nM, the third column a ten-fold
higher concentration of 1041-D11 protein (587 nM), the fourth
column is a negative control with PBS. In the first row, human Wi38
fibroblast cells are shown in phase contrast, the second row shows
the immunofluorescence and the third row a DAPI staining. It can be
concluded from the pictures that the variant 1041-D11 binds to
fixed Wi38 cells with high specificity to ED-B containing
extracellular matrix. The negative control cell type NHDF are
primary normal fibroblast cells, which express low levels of
EDB-fibronectin (data not shown). The variants do not bind to those
cells.
[0291] FIG. 15 B shows the analysis of variant 1041-D11 on vital
Wi38 cells. The negative control cell type NHDF is primary normal
fibroblast cells, which express low levels of EDB-fibronectin. The
cells were plated in chamber-slides (NUNC, 60000 cells/ml). To
analyses the binding potential the cells were fixed with 100% MeOH
for 5 min at -20.degree. C. To block unspecific binding, the cells
were incubated with 5% Horse-serum 1 h 37.degree. C. The cells were
tested with the variant 1041-D11, an ED-B specific antibody FV28
CH4/F1 as positive control or UB.sub.--2 as negative control with
different concentrations 1 h RT. The proving occurred about an
incubation with rabbit-a-Strep-Tag-IgG (obtained from GenScript
A00875, 1:500) for 1 h and incubation with a-rabbit-IgG*Alexa488-AK
(obtained from Invitrogen A11008, 1:1000) for 1 h. The nuclei were
stained with DAPI. The first and third line in FIG. 15B shows the
variant at different protein concentration and the negative
control. The second and fourth line shows the incubation of the
control using EDB antibodies. The first 2 lines show the variant
and positive control on Wi38-cell line. The third and fourth line
shows the incubation of NHDF-cells. It can be seen from the
pictures that the variant 1041-D11 binds to vital Wi38 cells with
high specificity to ED-B containing extracellular matrix. A control
using NHDF cells which do not contain low EDB was performed (data
not shown). The variants do not bind to those cells.
[0292] Similar experiments were performed using different cells
types, for example Balb3T3 (ATCC, Kat-Nr. 30-2002), Raw (Lonza,
Kat-Nr. BE12-115F/U1), ST-2 (Lonza, Kat-Nr. BE12-115F/U1). FIGS.
15C and D show that the binding of ED-B is highly specific to
murine Balb3T3 and ST-2 cells. No binding was observed to
monocytes/macrophages (Raw)(data not shown).
[0293] As outlined above, FIG. 16 A shows the specificity of
1041-D11 in tissue sections. F9 tumor tissues from seven samples
were evaluated. Immune-histochemistry with 500 nM 1041-D11 resulted
in ED-B specific vascular staining on F9 tumors from mice. ED-B is
a highly specific marker for tumor vasculature. The target protein
EDB is located on the abluminal side of the vessels. 1041-D11
specifically decorates the vasculature in tissue sections from F9
tumors. The obtained results are comparable to tissue specificity
of the antibody fragment L19. In addition, 48 tissues were tested;
no unspecific staining in any out of 48 tissues in FDA relevant
panel was observed. FIG. 16 B shows the accumulation of 1041-D11 in
tumor cells in comparison to wild type Ubiquitin. Thus, fusion
proteins based on modified ubiquitin specifically binding to ED-B
are suitable an ED-B based targeted therapy for cancer.
Example 7
Efficacy In Vivo Study of 1041D11-TNF.alpha.
[0294] To establish the therapeutic efficacy of 1041-D11-TNFalpha,
the compound was tested on F9 teratoma (see Borsi et al., 2003
Blood 102, 4384-4392) in mouse models. The ED-B expression in mice
is comparable to the human in vivo situation and is suitable for an
evaluation of the therapeutic impact of 1041-D11-mTNFalpha on
cancer, preferably in combination with a cytotoxic compound such as
Melphalan. F9 teratoma is an aggressive tumor with high vascular
density. Borsi et al described that targeting of mouse TNFalpha via
EDB-antibodies improve the efficacy of Melphalan which is
demonstrated by retardation in tumor growth. The experimental
schedule for the efficacy study was adapted from Borsi, 2003.
[0295] Stage 1 defined the pharmacologic active and tolerable dose
with endpoints relating to the ratio of tumor vs. body weight,
weight loss and survival. The inventors found that 1041D11-TNFalpha
is tolerated at highest dose (6.75 .mu.mol/g) but has no
suppressing effect on tumor growth (>10% body weight after 3, 4
and 8 days.fwdarw.animals were killed), whereas 1041D11-TNFalpha at
lowest dose (0.25 pmol/g) seems to retard tumor growth. Dosing
groups further used were descending from 2.25 pmol/g
1041D11-TNFalpha.
[0296] Stage 2 of the study defined the dose-dependent efficacy
with Melphalan having as endpoint the retardation of tumor growth
(animal weight loss >10%, tumor >10% body weight, ulceration
of tumor). In the study, 1041D11/mTNFa, murine TNFa, in combination
with melphalan were tested. 168 animals were used, 14 Dosing groups
(8 mice per group recruited when bearing F9 tumors of 300-400
mm.sup.3); Administration of test sample i. v. followed by i. p.
injection of Melphalan 24 h later Table 1 shows the dosing
schedule:
TABLE-US-00016 Dose Melphalan ** TNF-.alpha. proteins Animals Group
Test item (mg/kg) (pmol/g) Route Appl. vol (n) * 1 PBS 0 0 iv 10
ml/kg 8 2 mouse TNF-.alpha. 0 2.25 iv 10 ml/kg 8 fusion protein 3
mouse TNF-.alpha. 0 0.75 iv 10 ml/kg 8 fusion protein 4 mouse
TNF-.alpha. 0 0.25 iv 10 ml/kg 8 fusion protein 5 mouse TNF-.alpha.
0 0.025 iv 10 ml/kg 8 fusion protein 6 mouse TNF-.alpha. 0 0.0025
iv 10 ml/kg 8 fusion protein 7 Melphalan 4.5 0 ip 10 ml/kg 8 8
Melphalan/ 4.5 2.25 ip/iv 10/10 ml/kg 8 mouse TNF-.alpha. fusion
protein * 9 Melphalan/ 4.5 0.75 ip/iv 10/10 ml/kg 8 mouse
TNF-.alpha. fusion protein * 10 Melphalan/ 4.5 0.25 ip/iv 10/10
ml/kg 8 mouse TNF-.alpha. fusion protein * 11 Mephalan/ 4.5 0.025
ip/iv 10/10 ml/kg 8 mouse TNF-.alpha. fusion protein * 12
Melphalan/ 4.5 0.0025 ip/iv 10/10 ml/kg 8 mouse TNF-.alpha. fusion
protein * 13 mouse TNF-.alpha. 0 0.25 iv 10 ml/kg 8 14 Melphatan/
4.5 0.25 ip/iv 10/10 ml/kg 8 mouse TNF-.alpha. * animals with
subcutaneous tumors of 300-400 mm3 ** Melphalan is applied 24 hours
after mouse TNF-.alpha. protein injection # the MTD will be
determined in study P10.0164
[0297] FIG. 18 shows the relative tumor growth during the time of
treatment (7 days). FIG. 18a clearly shows that our compound
1041-D11-TNFalpha in combination with Melphalan reduces the
relative tumor growth more efficiently that mTNFalpha in
combination with Melphalan or Melphalan alone. The tumor growth
kinetic 7 days after treatment shows the significant reduction of
tumors by 1041-D11-mTNFa. This is a clear evidence for efficacy in
combination with Melphalan.
PUBLICATIONS
[0298] 1. Birchler, M., F. Viti, L. Zardi, B. Spiess, and D. Neri.
1999. Selective targeting and photocoagulation of ocular
angiogenesis mediated by a phage-derived human antibody fragment.
Nat Biotechnol 17:984-8. [0299] 2. Brenmoehl, J., M. Lang, M.
Hausmann, S, N. Leeb, W. Falk, J. Scholmerich, M. Goke, and G.
Rogler. 2007. Evidence for a differential expression of fibronectin
splice forms ED-A and ED-B in Crohn's disease (CD) mucosa. Int J
Colorectal Dis 22:611-23. [0300] 3. Dubin, D., J. H. Peters, L. F.
Brown, B. Logan, K. C. Kent, B. Berse, S. Berven, B. Cercek, B. G.
Sharifi, R. E. Pratt, and et al. 1995. Balloon catheterization
induced arterial expression of embryonic fibronectins. Arterioscler
Thromb Vasc Biol 15:1958-67. [0301] 4. Goodsell, D. S. 2001.
FUNDAMENTALS OF CANCER MEDICINE: The Molecular Perspective:
Antibodies. The Oncologist 6:547-548. [0302] 5. Kaczmarek, J., P.
Castellani, G. Nicolo, B. Spina, G. Allemanni, and L. Zardi. 1994.
Distribution of oncofetal fibronectin isoforms in normal,
hyperplastic and neoplastic human breast tissues. Int J Cancer
59:11-6. [0303] 6. Menrad, A., and H. D. Menssen. 2005. ED-B
fibronectin as a target for antibody-based cancer treatments.
Expert Opin Ther Targets 9:491-500. [0304] 7. Pujuguet, P., A.
Hammann, M. Moutet, J. L. Samuel, F. Martin, and M. Martin. 1996.
Expression of fibronectin ED-A+ and ED-B+ isoforms by human and
experimental colorectal cancer. Contribution of cancer cells and
tumor-associated myofibroblasts. Am J Pathol 148:579-92. [0305] 8.
Trachsel, E., M. Kaspar, F. Bootz, M. Detmar, and D. Neri. 2007. A
human mAb specific to oncofetal fibronectin selectively targets
chronic skin inflammation in vivo. J Invest Dermatol 127:881-6.
[0306] 9. Van Vliet, A., H. J. Baelde, L. J. Vleming, E. de Heer,
and J. A. Bruijn. 2001. Distribution of fibronectin isoforms in
human renal disease. J Pathol 193:256-62. [0307] 10. Lipovsek, D.,
and Pluckthun, A. (2004). In-vitro protein evolution by ribosome
display and mRNA display. J. Immunol. Methods 290, 51-67. [0308]
11. Ohashi, H., Shimizu, Y., Ying, B. W., and Ueda, T. (2007).
Efficient protein selection based on ribosome display system with
purified components. Biochem Biophys. Res. Commun. 352, 270-276.
[0309] 12. Studier, F. W. (2005). Protein production by
auto-induction in high density shaking cultures. Protein Expr Purif
41, 207-234. [0310] 13. Zahnd, C., Amstutz, P., and Pluckthun, A.
(2007). Ribosome display: selecting and evolving proteins in vitro
that specifically bind to a target. Nat. Methods 4, 269-279.
Sequence CWU 1
1
50176PRTartificialUbiquitin protein 1Met Gln Ile Phe Val Lys Thr
Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr
Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro
Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60Ser Thr Leu
His Leu Val Leu Arg Leu Arg Gly Gly65 70 75291PRTartificialED-B
domain of oncofetal fibronectin 2Glu Val Pro Gln Leu Thr Asp Leu
Ser Phe Val Asp Ile Thr Asp Ser1 5 10 15Ser Ile Gly Leu Arg Trp Thr
Pro Leu Asn Ser Ser Thr Ile Ile Gly 20 25 30Tyr Arg Ile Thr Val Val
Ala Ala Gly Glu Gly Ile Pro Ile Phe Glu 35 40 45Asp Phe Val Asp Ser
Ser Val Gly Tyr Tyr Thr Val Thr Gly Leu Glu 50 55 60Pro Gly Ile Asp
Tyr Asp Ile Ser Val Ile Thr Leu Ile Asn Gly Gly65 70 75 80Glu Ser
Ala Pro Thr Thr Leu Thr Gln Gln Thr 85 90376PRTartificialUbiquitin
variant 1H4 3Met Trp Ile Lys Val His Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala
Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp
Tyr Asn Ile Thr Leu Ser 50 55 60Arg Ser Leu His Leu Val Leu Arg Leu
Arg Gly Gly65 70 75476PRTartificialUbiquitin variant 4B10 4Met Leu
Ile Leu Val Leu Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val
Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25
30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys
35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ala Thr
Lys 50 55 60Pro Ile Leu His Leu Val Leu Arg Leu Arg Gly Gly65 70
75576PRTartificialUbiquitin variant 5E1 5Met Val Ile Asn Val Phe
Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp
Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile
Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Ser Thr 50 55 60Ser Lys
Leu His Leu Val Leu Arg Leu Arg Gly Gly65 70
756164PRTartificialUbiquitin dimer 46H9 6Met Gly Ile Val Val Arg
Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp
Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile
Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Pro His Pro 50 55 60Thr Leu
Leu His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75
80Gly Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val His Thr Met
85 90 95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile
Glu 100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro
Pro Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Lys Pro Ile Ala
Glu Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly7164PRTartificialUbiquitin dimer 9E12 7Met Arg Ile Pro Val Tyr
Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp
Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile
Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Pro Pro Phe 50 55 60Ala Arg
Leu His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75
80Gly Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val Met Thr Arg
85 90 95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile
Glu 100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro
Pro Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Met Asn Ala Arg
Leu Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly8164PRTartificialUbiquitin dimer 22D1 8Met Leu Ile Leu Val Arg
Thr Leu Thr Asp Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp
Thr Ile Gly Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile
Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ser Val Gly 50 55 60Ala Met
Leu His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75
80Gly Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val Leu Thr Trp
85 90 95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile
Glu 100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro
Pro Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu
Asp Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Arg Arg Leu Pro
Pro Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly965DNAartificialPrimer F1 9ggagaccaca acggtttccc tctagaaata
attttgttta actttaagaa ggagatatac 60atatg 651039DNAartificialPrimer
WUBI(co)RD_xho 10aaaaaaaaac tcgagaccgc cacgcagacg cagaaccag
3911834DNAartificialConstruct His6-SUMO-TNFa 11atgggcagca
gccatcatca tcatcatcac ggcagcggcc tggtgccgcg cggcagcgct 60agcatgtcgg
actcagaagt caatcaagaa gctaagccag aggtcaagcc agaagtcaag
120cctgagactc acatcaattt aaaggtgtcc gatggatctt cagagatctt
cttcaagatc 180aaaaagacca ctcctttaag aaggctgatg gaagcgttcg
ctaaaagaca gggtaaggaa 240atggactcct taagattctt gtacgacggt
attagaattc aagctgatca gacccctgaa 300gatttggaca tggaggataa
cgatattatt gaggctcaca gagaacagat tggtggtgtg 360cgtagcagca
gccgtacccc gagcgataaa ccggtggcgc atgtggtggc gaatccgcag
420gcggaaggcc agctgcagtg gctgaaccgt cgtgcgaatg cgctgctggc
caacggcgtg 480gaactgcgtg ataatcagct ggttgtgccg agcgaaggcc
tgtatctgat ttatagccag 540gtgctgttta aaggccaggg ctgcccgagc
acccatgtgc tgctgaccca taccattagc 600cgtattgcgg tgagctatca
gaccaaagtg aacctgctgt ctgcgattaa aagcccgtgc 660cagcgtgaaa
ccccggaagg cgcggaagcg aaaccgtggt atgaaccgat ttatctgggc
720ggcgtgtttc agctggaaaa aggcgatcgt ctgagcgcgg aaattaaccg
tccggattat 780ctggattttg cggaaagcgg ccaggtgtat tttggcatta
ttgcgctgta ataa 8341227DNAartificialPrimer SUMO-EDB-TNFa-fw
12ttttttggat ccgtgcgtag cagcagc 271328DNAartificialPrimer
SUMO-EDB-TNFa-rev 13cttgtctctc gaggcggccg cttattac
281433DNAartificialPrimer SUMO-EDB-WUBI-fw 14gttccaaggt ctcatggtat
gcagatcttc gtg 331542DNAartificialPrimer SUMO-EDB-Linker-rev
15gtggtgggat ccaccgccac caccagaacc gccacgcaga cg
421633DNAartificialPrimer SUMO-EDB-1H4-fw 16gttccaaggt ctcatggtat
gtggatcaag gtg 331733DNAartificialPrimer SUMO-EDB-4B10-fw
17gttccaaggt ctcatggtat gttgatcctg gtg 331833DNAartificialPrimer
SUMO-EDB-5E1-fw 18gttccaaggt ctcatggtat ggttatcaat gtg
331942DNAartificialPrimer Dimer-t0a-rev 19gtggtgggat ccaccgccac
caccagaacc accacgtaaa cg 422033DNAartificialPrimer SUMO-EDB-WUBI-fw
20gttccaaggt ctcatggtat gcagatcttc gtg 332133DNAartificialPrimer
9E12-t0a-fw 21gttccaaggt ctcatggtat gcgtatccct gtg
332233DNAartificialPrimer 24H12-t0a-fw 22gttccaaggt ctcatggtat
ggttatcaag gtg 332333DNAartificialPrimer 15G7-t0a-fw 23gttccaaggt
ctcatggtat ggagatcggt gtg 332433DNAartificialPrimer 22D1-t0a-fw
24gttccaaggt ctcatggtat gcttatcttg gtg 3325164PRTartificialVariant
46-H4 25Met Gly Ile Val Val Arg Thr Leu Thr Gly Lys Thr Ile Thr Leu
Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile
Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp
Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn
Ile Pro His Pro 50 55 60Thr Leu Leu His Leu Val Leu Arg Leu Arg Gly
Gly Ser Gly Gly Gly65 70 75 80Gly Ser Gly Gly Gly Gly Ile Gly Met
Gln Ile Phe Val Gly Thr Trp 85 90 95Thr Gly Lys Thr Ile Thr Leu Glu
Val Glu Pro Ser Asp Thr Ile Glu 100 105 110Asn Val Lys Ala Lys Ile
Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln 115 120 125Gln Arg Leu Ile
Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu 130 135 140Ser Asp
Tyr Asn Ile Thr Gln Ala Thr Arg Leu His Leu Val Leu Arg145 150 155
160Leu Arg Gly Gly26164PRTartificialVariant 45-H9 26Met Arg Ile Pro
Val Tyr Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro
Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu
Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln
Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Pro Pro Phe 50 55
60Ala Arg Leu His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65
70 75 80Gly Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val Leu Thr
Met 85 90 95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr
Ile Glu 100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile
Pro Pro Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu
Glu Asp Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Leu Ala Phe
Ala Thr Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly27164PRTartificialVariant 46-A5 27Met Gly Ile Val Val Arg Thr
Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr
Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro
Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Pro His Pro 50 55 60Thr Leu Leu
His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75 80Gly
Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val Leu Thr Met 85 90
95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu
100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro
Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp
Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Leu Ala Phe Ala Thr
Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly28164PRTartificialVariant 50-G11 28Met Gly Ile Val Val Arg Thr
Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr
Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro
Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Pro His Pro 50 55 60Thr Leu Leu
His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75 80Gly
Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val Met Thr Arg 85 90
95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu
100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro
Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp
Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Met Asn Ala Arg Leu
Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly29164PRTartificialVariant 52-B3 29Met Arg Ile Pro Val Tyr Thr
Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr
Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro
Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Pro Pro Phe 50 55 60Ala Arg Leu
His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75 80Gly
Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val His Thr Met 85 90
95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu
100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro
Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp
Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Lys Pro Ile Ala Glu
Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly30164PRTartificialVariant 52-D10 30Met Val Ile Cys Val Arg Thr
Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr
Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro
Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Thr Ala Pro 50 55 60Gly Asp Leu
His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75 80Gly
Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe Val His Thr Met 85 90
95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu
100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro
Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp
Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile Lys Pro Ile Ala Glu
Leu His Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly318PRTartificialHis-Tag 31Leu Glu His His His His His His1
53212PRTartificialGlycine/serine linker 32Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ile Gly1 5 1033155PRTartificialUbiquitin dimer
SPVF-28_1041-D11_TsX9 33Met Gln Ile Phe Val Trp Thr Trp Thr Gly Lys
Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu
Ser Asp Tyr Asn Ile Gln Arg Lys 50 55 60Phe Pro Leu His Leu Val Leu
Arg Leu Arg Gly Gly Gly Ile Gly Met65 70 75 80Arg Ile Phe Val Thr
Thr Gln Thr Gly Lys Thr Ile Thr Leu Glu Val 85 90 95Glu Pro Ser Asp
Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 100 105 110Glu Gly
Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln
115 120 125Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Ser
Asn Trp 130 135 140Glu Leu His Leu Val Leu Arg Leu Arg Ala Ala145
150 15534155PRTartificialUbiquitin dimer SPVF-28_1045-D10_TsX9
34Met Gln Ile Phe Val Trp Thr Trp Thr Gly Lys Thr Ile Thr Leu Glu1
5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala
Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile
Gln Arg Lys 50 55 60Phe Pro Leu His Leu Val Leu Arg Leu Arg Gly Gly
Gly Ile Gly Met65 70 75 80Gln Ile Phe Val Thr Thr Gln Thr Gly Lys
Thr Ile Thr Leu Glu Val 85 90 95Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp Lys 100 105 110Glu Gly Ile Pro Pro Asp Gln
Gln Arg Leu Ile Trp Ala Gly Lys Gln 115 120 125Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Trp Ser Asn Trp 130 135 140Glu Leu His
Leu Val Leu Arg Leu Arg Ala Ala145 150 15535319PRTartificialfusion
protein SPVF-28_1041-D11_T0aX9 35Met Gln Ile Phe Val Trp Thr Trp
Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro
Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Lys 50 55 60Phe Pro Leu His
Leu Val Leu Arg Leu Arg Gly Gly Gly Ile Gly Met65 70 75 80Arg Ile
Phe Val Thr Thr Gln Thr Gly Lys Thr Ile Thr Leu Glu Val 85 90 95Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 100 105
110Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln
115 120 125Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Ser
Asn Trp 130 135 140Glu Leu His Leu Val Leu Arg Leu Arg Ala Ala Ser
Gly Gly Gly Gly145 150 155 160Gly Ser Val Arg Ser Ser Ser Arg Thr
Pro Ser Asp Lys Pro Val Ala 165 170 175His Val Val Ala Asn Pro Gln
Ala Glu Gly Gln Leu Gln Trp Leu Asn 180 185 190Arg Arg Ala Asn Ala
Leu Leu Ala Asn Gly Val Glu Leu Arg Asp Asn 195 200 205Gln Leu Val
Val Pro Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val 210 215 220Leu
Phe Lys Gly Gln Gly Cys Pro Ser Thr His Val Leu Leu Thr His225 230
235 240Thr Ile Ser Arg Ile Ala Val Ser Tyr Gln Thr Lys Val Asn Leu
Leu 245 250 255Ser Ala Ile Lys Ser Pro Cys Gln Arg Glu Thr Pro Glu
Gly Ala Glu 260 265 270Ala Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly
Gly Val Phe Gln Leu 275 280 285Glu Lys Gly Asp Arg Leu Ser Ala Glu
Ile Asn Arg Pro Asp Tyr Leu 290 295 300Asp Phe Ala Glu Ser Gly Gln
Val Tyr Phe Gly Ile Ile Ala Leu305 310 31536318PRTartificialfusion
protein SPVF-28_1041-D11_T0uX9 36Met Gln Ile Phe Val Trp Thr Trp
Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro
Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Lys 50 55 60Phe Pro Leu His
Leu Val Leu Arg Leu Arg Gly Gly Gly Ile Gly Met65 70 75 80Arg Ile
Phe Val Thr Thr Gln Thr Gly Lys Thr Ile Thr Leu Glu Val 85 90 95Glu
Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 100 105
110Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln
115 120 125Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Trp Ser
Asn Trp 130 135 140Glu Leu His Leu Val Leu Arg Leu Arg Ala Ala Ser
Gly Gly Gly Gly145 150 155 160Gly Ser Leu Arg Ser Ser Ser Gln Asn
Ser Ser Asp Lys Pro Val Ala 165 170 175His Val Val Ala Asn His Gln
Val Glu Glu Gln Leu Glu Trp Leu Ser 180 185 190Gln Arg Ala Asn Ala
Leu Leu Ala Asn Gly Met Asp Leu Lys Asp Asn 195 200 205Gln Leu Val
Val Pro Ala Asp Gly Leu Tyr Leu Val Tyr Ser Gln Val 210 215 220Leu
Phe Lys Gly Gln Gly Cys Pro Asp Tyr Val Leu Leu Thr His Thr225 230
235 240Val Ser Arg Phe Ala Ile Ser Tyr Gln Glu Lys Val Asn Leu Leu
Ser 245 250 255Ala Val Lys Ser Pro Cys Pro Lys Asp Thr Pro Glu Gly
Ala Glu Leu 260 265 270Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly
Val Phe Gln Leu Glu 275 280 285Lys Gly Asp Gln Leu Ser Ala Glu Val
Asn Leu Pro Lys Tyr Leu Asp 290 295 300Phe Ala Glu Ser Gly Gln Val
Tyr Phe Gly Val Ile Ala Leu305 310 31537155PRTartificialUbiquitin
dimer SPVF-28_1048-E10_TsX9 37Met Gln Ile Phe Val Trp Thr His Thr
Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu
Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly His
Thr Leu Ser Asp Tyr Asn Ile Pro Arg Arg 50 55 60Ser Trp Leu His Leu
Val Leu Arg Leu Arg Gly Gly Gly Ile Gly Met65 70 75 80Gln Ile Phe
Val Ser Thr Thr Thr Gly Glu Thr Ile Thr Leu Glu Val 85 90 95Glu Pro
Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 100 105
110Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln
115 120 125Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Ala Asp
Pro Arg 130 135 140Trp Leu His Leu Val Leu Arg Leu Arg Ala Ala145
150 15538155PRTartificialUbiquitin dimer SPVF-28_1041-E6_TsX9 38Met
Gln Ile Phe Val Trp Thr His Thr Gly Lys Thr Ile Thr Leu Glu1 5 10
15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp
20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Pro
Arg Arg 50 55 60Ser Trp Leu His Leu Val Leu Arg Leu Arg Gly Gly Gly
Ile Gly Met65 70 75 80Gln Ile Phe Val Ser Thr Thr Thr Gly Lys Thr
Ile Thr Leu Glu Val 85 90 95Glu Pro Ser Asp Thr Ile Glu Asn Val Lys
Ala Lys Ile Gln Asp Lys 100 105 110Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu Ile Trp Ala Gly Lys Arg 115 120 125Leu Glu Asp Gly Arg Thr
Leu Ser Asp Tyr Asn Ile Ala Asp Pro Arg 130 135 140Trp Leu His Leu
Val Leu Arg Leu Arg Ala Ala145 150 15539155PRTartificialUbiquitin
dimer SPVF-28_1041-H10_TsX9 39Met Gln Ile Phe Val Trp Thr Asn Thr
Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu
Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Glu His Gly 50 55 60Lys Trp Leu His Leu
Val Leu Arg Leu Arg Gly Gly Gly Ile Gly Met65 70 75 80Gln Ile Phe
Val Asn Thr Thr Thr Gly Lys Thr Ile Thr Leu Glu Val 85 90 95Glu Pro
Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 100 105
110Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln
115 120 125Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Phe Ile
Gly His 130 135 140Trp Leu His Leu Val Leu Arg Leu Arg Ala Ala145
150 15540158PRTartificialUbiquitin dimer SPVF-28_1056-B6_TsX6 40Met
Gln Ile Phe Val His Thr His Thr Gly Lys Thr Ile Thr Leu Glu1 5 10
15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp
20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly
Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Asn
Arg Asp 50 55 60Lys Arg Leu His Leu Val Leu Arg Leu Arg Ala Ala Ser
Gly Gly Gly65 70 75 80Gly Ile Gly Met Gln Ile Phe Val Asn Thr Asn
Thr Gly Glu Thr Ile 85 90 95Thr Leu Glu Val Glu Pro Ser Asp Thr Ile
Glu Asn Val Lys Ala Lys 100 105 110Ile Gln Asp Lys Glu Gly Ile Pro
Pro Asp Gln Gln Arg Leu Ile Trp 115 120 125Ala Gly Lys Arg Leu Glu
Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile 130 135 140Asp Trp Arg Trp
Leu His Leu Val Leu Arg Leu Arg Ala Ala145 150
15541154PRTartificialUbiquitin dimer SPVF-28_1051-B7_TsX9 41Met Gln
Ile Phe Val His Thr Thr Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val
Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25
30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys
35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Thr Leu
Thr 50 55 60Pro Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Gly Ile
Gly Met65 70 75 80Gln Ile Phe Val Leu Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu Val 85 90 95Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala
Lys Ile Gln Asp Lys 100 105 110Glu Gly Ile Pro Pro Asp Gln Gln Arg
Leu Ile Trp Ala Gly Lys Gln 115 120 125Leu Glu Asp Gly Arg Thr Leu
Ser Asp Tyr Asn Ile Asp Trp Arg Trp 130 135 140Leu His Leu Val Leu
Arg Leu Arg Ala Ala145 15042155PRTartificialUbiquitin dimer
SPVF-28_1035-E6_TsX9 42Met Gln Ile Phe Val His Thr Phe Thr Gly Lys
Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu
Ser Asp Tyr Asn Ile His Glu Arg 50 55 60Glu Ile Leu His Leu Val Leu
Arg Leu Arg Gly Gly Gly Ile Gly Met65 70 75 80Gln Ile Phe Val Ser
Thr Pro Thr Gly Lys Thr Ile Thr Leu Glu Val 85 90 95Glu Pro Ser Asp
Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 100 105 110Glu Gly
Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys Gln 115 120
125Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gly Val Glu Met
130 135 140Leu Leu His Leu Val Leu Arg Leu Arg Ala Ala145 150
15543154PRTartificialUbiquitin dimer SPVF-28_1049-D4_TsX9 43Met Gln
Ile Phe Val His Thr Asp Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val
Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Glu Ile Gln Asp 20 25
30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys
35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile His Asn
Trp 50 55 60Arg Asn Leu His Leu Val Leu Arg Leu Arg Gly Gly Gly Ile
Gly Met65 70 75 80Gln Ile Phe Val Ile Thr Ile Thr Gly Lys Thr Ile
Thr Leu Glu Val 85 90 95Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala
Lys Ile Gln Asp Lys 100 105 110Glu Gly Ile Pro Pro Asp Gln Gln Arg
Leu Ile Trp Ala Gly Lys Gln 115 120 125Leu Lys Asp Gly Arg Thr Leu
Ser Asp Tyr Asn Ile Asp Trp Arg Trp 130 135 140Leu His Leu Val Leu
Arg Leu Arg Ala Ala145 15044164PRTartificialUbiquitin dimer
SPWF-28_1071-C8_TsX2 44Met Trp Ile Arg Val Pro Thr Leu Thr Gly Lys
Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu
Ser Asp Tyr Asn Ile His Met Pro 50 55 60Asp Ile Leu His Leu Val Leu
Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75 80Gly Ser Gly Gly Gly
Gly Ile Gly Met Gln Ile Phe Val Trp Thr Met 85 90 95Thr Gly Lys Thr
Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu 100 105 110Asn Val
Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln 115 120
125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu
130 135 140Ser Asp Tyr Asn Ile His Leu His Met Arg Leu His Leu Val
Leu Arg145 150 155 160Leu Arg Gly Gly45164PRTartificialUbiquitin
dimer SPWF-28_1071-C12_TsX2 45Met Thr Ile Trp Val His Thr Leu Thr
Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu
Asn Val Lys Ala Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp
Gln Gln Arg Leu Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Asn Phe Lys 50 55 60Leu Ser Leu His Leu
Val Leu Arg Leu Arg Gly Gly Ser Gly Gly Gly65 70 75 80Gly Ser Gly
Gly Gly Gly Ile Gly Met Gln Ile Phe Val Ser Thr Phe 85 90 95Thr Gly
Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu 100 105
110Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln
115 120 125Gln Arg Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg
Thr Leu 130 135 140Ser Asp Tyr Asn Ile His Tyr Leu Pro Lys Leu His
Leu Val Leu Arg145 150 155 160Leu Arg Gly
Gly46172PRTartificialUbiquitin dimer SPWF-28_1071-H7_TsX2 46Met Trp
Ile Arg Val Pro Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu1 5 10 15Val
Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25
30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Trp Ala Gly Lys
35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Arg Arg
Val 50 55 60Asn Tyr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ser Gly
Gly Gly65 70 75 80Gly Ser Gly Gly Gly Gly Ile Gly Met Gln Ile Phe
Val Trp Thr Ser 85 90 95Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro
Ser Asp Thr Ile Glu 100 105 110Asn Val Lys Ala Lys Ile Gln Asp Lys
Glu Gly Ile Pro Pro Asp Gln 115 120 125Gln Arg Leu Ile Trp Ala Gly
Lys Gln Leu Glu Asp Gly Arg Thr Leu 130 135 140Ser Asp Tyr Asn Ile
Tyr Thr Tyr Met Arg Leu His
Leu Val Leu Arg145 150 155 160Leu Arg Gly Gly Leu Glu His His His
His His His 165 17047164PRTartificialUbiquitin dimer with linker
SGGGGSGGGGIG 47Met Thr Ile Trp Val His Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu1 5 10 15Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala
Lys Ile Gln Asp 20 25 30Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu
Ile Trp Ala Gly Lys 35 40 45Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp
Tyr Asn Ile Asn Phe Lys 50 55 60Leu Ser Leu His Leu Val Leu Arg Leu
Arg Gly Gly Ser Gly Gly Gly65 70 75 80Gly Ser Gly Gly Gly Gly Ile
Gly Met Gln Ile Phe Val Xaa Thr Xaa 85 90 95Thr Gly Lys Thr Ile Thr
Leu Glu Val Glu Pro Ser Asp Thr Ile Glu 100 105 110Asn Val Lys Ala
Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln 115 120 125Gln Arg
Leu Ile Trp Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu 130 135
140Ser Asp Tyr Asn Ile Xaa Xaa Xaa Xaa Xaa Leu His Leu Val Leu
Arg145 150 155 160Leu Arg Gly Gly485PRTartificialGlycine/serine
linker 48Ser Gly Gly Gly Gly1 5497PRTartificialGlycine/serine
linker 49Ser Gly Gly Gly Gly Ile Gly1
55010PRTartificialGlycine/serine linker 50Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly1 5 10
* * * * *