U.S. patent application number 15/163208 was filed with the patent office on 2016-12-08 for functionalized polypeptides.
The applicant listed for this patent is ESBATech, an Alcon Biomedical Research Unit LLC. Invention is credited to David Urech.
Application Number | 20160354479 15/163208 |
Document ID | / |
Family ID | 41139486 |
Filed Date | 2016-12-08 |
United States Patent
Application |
20160354479 |
Kind Code |
A1 |
Urech; David |
December 8, 2016 |
FUNCTIONALIZED POLYPEPTIDES
Abstract
The invention provides functionalized polypeptides, especially
therapeutic polypeptides (e.g., scFv), comprising a linker sequence
that can be rapidly and specifically functionalized by the addition
of one or functional moieties (e.g., PEG) or binding specificities
(e.g., an amino acid sequence with a particular binding
specificity). Such functionalized polypeptides are advantageous in
that they have improved pharmacokinetic properties (e.g., improved
in vivo half-life, tissue penetration and tissue residency time)
over non-functionalized polypeptides. Methods for the rapid and
reproducible generation of functionalized polypeptides are also
provided.
Inventors: |
Urech; David;
(Hombrechtikon, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ESBATech, an Alcon Biomedical Research Unit LLC |
Schlieren |
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CH |
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|
Family ID: |
41139486 |
Appl. No.: |
15/163208 |
Filed: |
May 24, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14134988 |
Dec 19, 2013 |
9371525 |
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15163208 |
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13000499 |
Dec 21, 2010 |
8637022 |
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PCT/CH2009/000225 |
Jun 30, 2009 |
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14134988 |
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61076775 |
Jun 30, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1062 20130101;
C07K 16/241 20130101; A61K 47/60 20170801; C07K 2319/31 20130101;
C07K 2317/622 20130101; C12N 15/1037 20130101; C07K 16/00 20130101;
A61K 47/65 20170801; C07K 2317/40 20130101; C07K 2317/41 20130101;
A61P 31/00 20180101; A61P 37/00 20180101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C12N 15/10 20060101 C12N015/10; C07K 16/24 20060101
C07K016/24 |
Claims
1. A polypeptide comprising two domains connected by an amino acid
linker; wherein the linker comprises two cysteines capable of
forming an intrachain disulphide bond, and wherein the amino acids
between the cysteines in the linker sequence form a loop when the
two cysteines are disulphide bonded to one another.
2. The polypeptide of claim 1, wherein the linker comprises the
amino acid sequence set forth in SEQ ID No. 1.
3. The polypeptide of claim 1, wherein the linker comprises the
amino acid sequence set forth in SEQ ID No. 2.
4. The polypeptide of claim 1, wherein the loop binds to a target
molecule.
5. The polypeptide of claim 4, wherein the target molecule is a PK
modifier.
6. The polypeptide of claim 5, wherein the PK modifier is serum
albumin.
7. The polypeptide of claim 5, wherein the PK modifier is a
hyaluronic acid.
8. The polypeptide of claim 1, wherein the linker comprises the
amino acid sequence set forth in SEQ ID No. 3.
9. The polypeptide of claim 1, wherein the linker comprises the
amino acid sequence set forth in SEQ ID No. 4.
10. The polypeptide of claim 1, wherein the polypeptide is an
immunobinder.
11. The polypeptide of claim 10, which is a scFv.
12. The polypeptide of claim 1, comprising the amino acid sequence
set forth in SEQ ID No. 6 or 8.
13. A polypeptide of claim 1, wherein at least one cysteine residue
in the linker is covalently linked to a functional moiety.
14. The polypeptide of claim 13, wherein the functional moiety is
PEG.
15. A polypeptide of claim 1, wherein the two cysteine residues in
the linker are covalently linked to the same functional moiety.
16. The polypeptide of claim 15, wherein the functional moiety is
PEG.
17. A composition comprising the polypeptide of claim 1 and a
pharmaceutically acceptable carrier.
18. An isolated nucleic acid molecule encoding the polypeptide of
claim 1.
19. An expression vector comprising the nucleic acid molecule of
claim 18.
20. A host cell comprising the expression vector of claim 19.
21. A method producing a functionalized polypeptide comprising: (a)
providing a library of peptide sequences; (b) identifying from the
library at least one peptide sequence that bind to a target
molecule; (c) modifying the loop region of a linker-containing
polypeptide to comprise at least one peptide sequence identified in
step (b), thereby producing a functionalized polypeptide.
22. The method of claim 21, wherein said identifying step is
performed using phage display, yeast display, or mRNA display.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 14/134,988 filed Dec. 19, 2013 (now allowed), which is a
divisional of U.S. Pat. No. 8,637,022 filed Dec. 21, 2010, which
claims priority from PCT/CH2009/000225, filed Jun. 30, 2009, which
claims priority to U.S. Provisional Application Ser. No.
61/076,775, filed Jun. 30, 2008, the contents of which are hereby
incorporated by reference in their entireties.
BACKGROUND INFORMATION
[0002] The efficacy of therapeutic polypeptides is often greatly
limited by their intrinsic pharmacokinetic properties. For example,
in the case of therapeutic antibodies, problems with tissue
penetration, tissue residency and serum half-life are frequently
reported. Improvements in the pharmacokinetic properties of a
therapeutic polypeptide can result in improved efficacy and reduced
dosing regimes. Current methods of modulating the pharmacokinetic
properties of therapeutic polypeptides are typically limited to
addressing the issue of serum half-life. Moreover these methods
typically involve time consuming or non-specific chemical
alteration of existing polypeptides.
[0003] Accordingly, there is a need in the art for improved methods
of modulating the pharmacokinetic properties of therapeutic
polypeptides that provide a rapid and specific means of amino acid
modification, and that address pharmacokinetic parameters in
addition to half-life.
SUMMARY OF THE INVENTION
[0004] The invention provides functionalized polypeptides,
especially therapeutic polypeptides (e.g., scFv). The polypeptides
of the invention comprise a linker sequence that can be rapidly and
specifically functionalized by the addition of one or functional
moieties (e.g., PEG) and/or binding specificities (e.g., an amino
acid sequence with a particular binding specificity). Such
functionalized polypeptides are advantageous in that they have
improved pharmacokinetic properties (e.g., improved in vivo
half-life, tissue penetration and tissue residency time) over
non-functionalized polypeptides. Methods for the rapid and
reproducible generation of functionalized polypeptides are also
provided.
[0005] Accordingly, in one aspect, the invention provides a
polypeptide, such as an immunobinder (e.g. a scFv), comprising two
domains connected by an amino acid linker. The linker generally
comprises two cysteines capable of forming an intrachain disulphide
bond, and the amino acids in the linker sequence between these two
cysteines form a loop when the two cysteines are disulphide bonded
to one another. In a particular embodiment, the linker comprises
sequence set forth in SEQ ID No:1 or 2.
[0006] In certain embodiments, the linker contains a loop that
binds to a target molecule, such as a PK modifier (e.g., hyaluronic
acid, and serum albumin). By binding to the target molecule, the
serum half-life and/or the penetration efficacy into tissues and/or
particular binding specificity of the linker comprising polypeptide
is enhanced. In a particular embodiment, the linker comprises the
amino acid sequence set forth in SEQ ID No:3 or 4. In a preferred
embodiment, the polypeptide comprises the amino acid sequence set
forth in SEQ ID No. 6 or 8.
[0007] In certain embodiments, at least one cysteine residue in the
linker is covalently linked to a functional moiety. In a particular
embodiment, two cysteine residues in the linker are covalently
linked to the same functional moiety. Suitable functional moieties
include, for example, PEG, carbohydrate molecules and hydroxyethyl
starch (HES). Upon covalent linkage of a functional moiety such as
PEG or HES to a polypeptide, the half-life of such polypeptide
within a subject is prolonged. In another aspect, the invention
provides compositions comprising one or more polypeptides of the
invention and a pharmaceutically acceptable carrier.
[0008] In other aspects, the invention provides nucleic acid
molecules (e.g. vectors) encoding the polypeptides of the
invention, and host cells containing such nucleic acid
molecules.
[0009] In another aspect, the invention provides methods producing
a functionalized polypeptide. Such methods generally involve
providing a library of peptide sequences, identifying from the
library at least one peptide sequence that bind to a target
molecule, and modifying the loop region of a linker-containing
polypeptide to comprise at least one peptide sequence identified.
Desired peptide sequences can be identified by any art recognized
means, including, for example, phage display, yeast display, or
mRNA display.
DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 depicts an SDS page of variously treated ESBA105
molecules (lanes 3-10). Lane 1: Marker; lane 2: not specified; lane
3: reduced with DTT; lane 4: reduced and dialyzed; lane 5: reduced,
cysteine-PEGylated; lane 6: cysteine-PEGylated, dialyzed; lane 7:
Control; lane 8: Lysine-PEGylated; lane 9: Laysine-PEGylated,
dialysed; lane: 10 control. The molecular size of PEG was aprox.
0.7 kDa/PEG.
[0011] FIG. 2A shows ELISA analysis of the TNFalpha
binding-activity of cysteine-PEGylated ESBA105 The EC50 for ESBA105
was 0.9833; for reduced ESBA105: 1.291; and for Cys pegylated
ESBA105: 1.164. R.sup.2 for ESBA105 was 0.9814; for reduced
ESBA105: 0.9891; Cys pegylated ESBA105: 0.9857.
[0012] FIG. 2B shows ELISA analysis of the TNFalpha
binding-activity of lysine-PEGylated ESBA105. The EC50 for ESBA105
was 0.8073 and for Lys pegylated ESBA105: 1.326. R.sup.2 for
ESBA105 was 0.9870 and for pegylated ESBA105 0.9640.
[0013] FIG. 3A shows a schematic of unmodified, oxidized
ESBA105-SS-linker, FIG. 3B shows ESBA105-SS-linker PEGylated
(ESBA105-S.sub.2-PEG.sub.2) using a monofunctional PEGylating
reagent 1, and FIG. 3C shows ESBA105-SS-linker PEGylated
(ESBA105-S.sub.2-PEG) using a bifunctional PEGylating reagent
2.
[0014] FIG. 4A shows: ELISA analysis of the TNFalpha
binding-activity of ESBA105 and ESBA105-SS-linker;
[0015] FIG. 4B shows: an SDS-PAGE gel of ESBA105 subjected to
various treatments; lane 1: ESBA105 SS linker
reduced+dialyzed+up-concentrated; lane 2: ESBA105 SS linker
reduced+dialyzed; lane 3: ESBA105 SS linker reduced+pegylated; lane
4: ESBA105 SS linker reduced+pegylated+dialyzed; lane 5: ESBA105 SS
linker; and
[0016] FIG. 4C shows: a Western blot assessing the extent of
PEGylation of ESBA105 and ESBA105-SS-linker using a rabbit anti-PEG
antibody.
[0017] FIG. 5 shows a schematic illustrating the presentation of
the peptide loop of an scFv molecule 1 on the surface of a
bacteriophage 2 for the purposes of phage display (the scFv
molecule and the phage are not to scale). In the depicted case, the
loop comprises 7 amino acids which may interact with a target
molecule 3 such as serum albumin (half live prolongation), FcRx
(placenta transport), Mucin (epithelial residence time
prolongation), Integrin (RGD peptides), Complement, Tight junction
proteins, Multimerization, etc.
[0018] FIG. 6 shows ELISA analysis of the TNFalpha binding-activity
of ESBA105 and cysteine-PEGylated ESBA105-SS-linker. The EC50 for
ESBA105 was 0.9980; and for ESBA105 with pegylated SS linker:
1,181. R.sup.2 was for ESBA105 0.9397; and for ESBA105 with
pegylated SS linker: 0.9851.
[0019] FIG. 7A shows the VEGF binding kinetics of ESBA903. Fit: 1:1
binding; ka (1/Ms): 7,68E+5; kd (1/s): 4,310E-5; KD (M): 5,608E-11.
FIG. 7B shows the binding kinetics of ESBA903-Pep1. Fit: 1:1
binding; ka (1/Ms): 1,133E+6; kd (1/s): 5,026E-5. For both, FIGS.
7A and 7B, the values of the X-axis are given in seconds, the
values of the Y-axis are given in resonance units (RU).
DETAILED DESCRIPTION
Definitions
[0020] The term "domain", with respect to a polypeptide, takes its
art recognized meaning and refers a discrete unit of tertiary
structure. Examples of domains include, without limitation,
antibody VH or VL domains, fibronectin domains and ankyrin-repeat
domains.
[0021] The term "linker" refers to a linear amino acid sequence
linking two domains. Linkers of the invention can be genetically
and/or chemically fused to a domain. In certain embodiments,
linkers contain a loop.
[0022] The term "loop" refers to a cyclical amino acid sequence
formed by an intrachain disulphide bond within a linker.
[0023] The term "polyethylene glycol" or "PEG" refers to a linear
or branched neutral polyether with the chemical formula
HO--(CH.sub.2CH.sub.2O).sub.n--H, and reactive derivatives
thereof.
[0024] Reactive PEG derivatives are well known in the art and
include, without limitation, PEG coupled to methyl-PEO12-maleimide,
N-hydroxy succinimidyl carbonate, N-hydroxy succinimidyl
propionate, p-nitrophenyl-carbonate, benzotriazole-carbonate, and
an aldehyde. In a particular embodiment, the amino-reactive PEG
derivative is a bis-sulfone-coupled PEG capable of reacting
specifically with disulphide bonds (see e.g., Brocchini et al,
Nature Protocols, 2006: 1(5), 241). Suitable PEG molecules are of a
molecular size between 0.5 kDa and 50 kDa.
[0025] The term "target molecule" refers to any molecule
specifically bound by the loop region of a polypeptide of the
invention. Target molecules, include, for example, sugars, proteins
and lipids.
[0026] The term "functional moiety" refers to a biological or
chemical entity that imparts additional functionality to a molecule
to which it is attached (e.g. a PEG molecule, one or more
carbohydrate molecules or hydroxyethyl starch (HES)).
[0027] The term "PK modifier" refers to any molecule that alters
the pharmacokinetic profile of a protein when bound to that
protein. A PK modifier is typically, but not necessarily, a
naturally occurring, endogenous molecule present in a subject (e.g.
a patient). The localization and abundance of such PK modifier
within a subject can be physiological or pathological (e.g.
overexpressed on the surface of cancer cells, or at an inflamed
site). Suitable PK modifiers include, for example, hyaluronic acid,
collagen type II, serum albumin, antibody Fc receptors (e.g.,
FcRx), antibody Fc regions, mucins, integrins, tight junction
proteins, transferrin, and complement factors.
[0028] The term "modified" or "modifying", with respect to the
amino acid sequence of a polypeptide, refers to both the addition
of amino acids into the polypeptide sequence or the substitution of
existing amino acids in the polypeptide sequence. Amino acids
suitable for modifying a polypeptide include all known natural
amino acids, unnatural amino acids, and functionalized derivatives
thereof (see. e.g., U.S. Pat. Nos. 7,045,337 and 7,083,970 which
are hereby incorporate by reference in their entireties).
[0029] The term "immunobinder" refers to a molecule that contains
all or a part of the antigen binding site of an antibody, e.g., all
or part of the heavy and/or light chain variable domain, such that
the immunobinder specifically recognizes a target antigen.
Non-limiting examples of immunobinders include full-length
immunoglobulin molecules and scFvs, as well as antibody fragments,
including but not limited to (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; (ii) a F(ab').sub.2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fab' fragment, which is essentially a Fab
with part of the hinge region (see, Fundamental Immunology (Paul
ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the
V.sub.H and C.sub.H1 domains; (v) a Fv fragment consisting of the
V.sub.L and V.sub.H domains of a single arm of an antibody,; and
(vii) a nanobody, a heavy chain variable region containing a single
variable domain and two constant domains.
[0030] The term "antibody" as used herein is a synonym for
"immunoglobulin." Antibodies according to the present invention may
be whole immunoglobulins or fragments thereof, comprising at least
one variable domain of an immunoglobulin, such as single variable
domains, Fv (Skerra A. and Pluckthun, A. (1988) Science
240:1038-41), scFv (Bird, R. E. et al. (1988) Science 242:423-26;
Huston, J. S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83),
Fab, (Fab')2 or other fragments well known to a person skilled in
the art.
[0031] The term "single chain antibody" or "scFv" refers to a
molecule comprising an antibody heavy chain variable region
(V.sub.H) and an antibody light chain variable region (V.sub.L)
connected by a linker. Such scFv molecules can have the general
structures: NH.sub.2--V.sub.L-linker-V.sub.H--COOH or
NH.sub.2--V.sub.H-linker-V.sub.L--COOH.
[0032] As used herein, the term "functional property" is a property
of a polypeptide (e.g., an immunobinder) includes, without
limitation, the stability (e.g., thermal stability), solubility
(e.g., in vivo and in cell culture), and antigen binding
affinity.
[0033] The terms "specific binding," "selective binding,"
"selectively binds," and "specifically binds," refer to antibody
binding to an epitope on a predetermined antigen. Typically, the
antibody binds with an affinity (K.sub.D) of approximately less
than about 10.sup.-7 M, such as approximately less than about 10
.sup.-8 M, 10.sup.-9 M or 10.sup.-10 M.
[0034] The term "nucleic acid molecule," refers to DNA molecules
and RNA molecules. A nucleic acid molecule may be single-stranded
or double-stranded, but preferably is double- stranded DNA. A
nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence.
[0035] The term "vector," refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
One type of vector is a "plasmid," which refers to a circular
double stranded DNA loop into which additional DNA segments may be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) can be
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome.
[0036] The term "host cell" refers to a cell into which an
expression vector has been introduced. Host cells can include
bacterial, microbial, plant or animal cells. Bacteria, which are
susceptible to transformation, include members of the
enterobacteriaceae, such as strains of Escherichia coli or
Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus;
Streptococcus, and Haemophilus influenzae. Suitable microbes
include Saccharomyces cerevisiae and Pichia pastoris. Suitable
animal host cell lines include CHO (Chinese Hamster Ovary lines)
and NS0 cells.
[0037] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the present specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
[0038] Various aspects of the invention are described in further
detail in the following subsections. It is understood that the
various embodiments may be combined at will.
Polypeptide Linkers
[0039] In one aspect, the invention provides polypeptides
comprising two domains, wherein the domains are joined by an amino
acid linker, and wherein the linker contains two cysteine residues
that are capable of forming an intrachain disulphide bond. Such
linkers are particularly advantageous in that they allow the site
specific addition of a functional moiety (e.g., PEG), and/or the
addition of one or more binding affinities to the polypeptide. The
linkers of the invention are preferably genetically joined to a
domain by genetic engineering, more preferably between two domains,
thereby linking them to form a single polypeptide. Alternatively,
the linkers of the invention can be chemically joined to a domain
using any art recognized chemistry for effecting the joining of
amino acids. Functional moieties can be connected to the linker by
any art recognized chemistry. Preferably, they are connected to one
or more cysteines being present in the linker.
[0040] In certain embodiments, the amino acid linker is of the
general formula: (X).sub.a-C-(X).sub.b-C-(X).sub.c, where C is
cysteine; X is any amino acid, including natural, non-natural amino
acids, and chemical derivatives thereof; and, a, b, and c
correspond the number of amino acids and can be any natural number.
Preferably, a and c are numbers between 1-25, b is a number between
3-250. More preferably, a and c are numbers between 1-20, b is a
number between 3-100.
[0041] In one embodiment, the linker comprises a peptide sequence
in the loop forming region X.sub.b. In said case, b is preferably
any number of 3-30 amino acids, i.e. 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29 or 30. Most preferably b is 7 or 12. Preferably, the loop
forming region X.sub.b does not comprise a cysteine residue.
[0042] In certain embodiments, the linker comprises a polypeptide
domain which is folded. In the general formula:
(X).sub.a-C-(X).sub.b-C-(X).sub.c, b is preferably a number between
30 and 250, e.g. 50-200, 100-200, 125-225, 75-225, 125-225, such as
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 120, 130, 140, 150, 160,
170, 180, 190, 200, 210, 220, 230, 240 or 250. It is to be
understood that any natural number in the indicated range is
disclosed herewith. Accordingly, the amino acid linker is of the
general formula (X).sub.a-C-domain-C-(X).sub.b, where C is
cysteine; X is any amino acid, including natural, non-natural amino
acids, and chemical derivatives thereof; domain is a domain as
defined above; and, a, b correspond the number of amino acids and
can be any natural number.
[0043] In certain embodiments, the amino acid linker is of the
general formula:
(X).sub.n=3-15--C--(X).sub.n=3-50--C--(X).sub.n=3-15 (SEQ ID No:1
in Table 1), where C is cysteine; X is any amino acid, including
natural, non-natural amino acids, and chemical derivatives thereof;
and, n is the number of amino acids. The number of amino acid
residues in each region of the linker may be varied according to
the structure of the polypeptide to which it is attached, provided
that the cysteine residues in the linker are able to form an
intrachain disulphide bond under non-reducing conditions. Moreover,
the linker length and sequence must be such that the linker does
not substantially impair one or more functional properties of the
polypeptide to which it is a part. In a particular embodiment, the
amino acid linker comprises the amino acid sequence set forth in
any one of SEQ ID No:2, 3, and 4 (see Table 1).
[0044] The linkers of the invention are particularly well suited
for joining VH and VL domains in scFv, especially those scFv that
are highly stable and soluble, such as those described in
WO09/000098, the contents of which are incorporated herein by
reference. The amino sequences of exemplary scFv polypeptides
comprising the linkers of the invention are set forth in SEQ ID
No:6 and 8 (see Table 1).
Polypeptides
[0045] The invention provides functionalized polypeptides,
especially therapeutic polypeptides (e.g., scFv). The polypeptides
of the invention comprise a linker sequence that can be rapidly and
specifically functionalized by the addition of one or more
functional moieties (e.g., PEG) and/or binding specificities (e.g.,
an amino acid sequence with a particular binding specificity).
[0046] Any polypeptide can be functionalized using the methods and
compositions of the invention, including without limitation,
polypeptides comprises an immunoglobulin domain (e.g., a VH or VL
domain). In a particular embodiment, the polypeptide is an
immunobinder, (e.g., an scFv).
[0047] In a preferred embodiment, polypeptides of the present
invention are of the following general formula: domain
1-(X).sub.a-C-(X).sub.b-C-(X).sub.c-domain 2, where C is cysteine;
domain 1 and 2 are domains as defined above; X is any amino acid,
including natural, non-natural amino acids, and chemical
derivatives thereof; and, a, b, and c are the number of amino
acids. Preferably, domain 1 and 2 are V.sub.H and V.sub.L domains,
or V.sub.L and V.sub.H domains, respectively.
Addition of Functional Moieties to Linkers
[0048] The linkers of the invention contain at least two cysteine
residues that under non-reducing conditions form an intrachain
disulphide bond (see FIG. 3). This disulphide bond allows the site
specific addition of a functional moiety (e.g., PEG) to the linker
using the methods described herein. Specifically, the cysteine
residues in the disulphide bond can be covalently linked to a
functional moiety (e.g., PEG). As the exposed cysteines allow for a
much better directed pegylation, a manufacturing advantage over
conventional PEG-attachment sites is achieved which often times
yield a inhomogeneous population regarding the sites of
PEG-attachment. If a functional moiety containing a monofunctional
reactive group is employed, the functional moiety will become
linked to a single cysteine residue. However, if a functional
moiety containing a disulphide bond-specific bifunctional reactive
group is employed, the functional moiety will become linked to both
cysteine residues of an intrachain disulphide bond. Such
bifunctional reagents are well known in the art and include,
bis-sulfones (see e.g., Brocchini et al, Nature Protocols, 2006:
1(5), 241).
[0049] In certain embodiments, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No:2, 3, 4,
6 and 8 (see Table 1), wherein at least one cysteine residue in the
linker is covalently linked to a functional moiety (e.g., PEG). In
other embodiments, the invention provides a polypeptide comprising
the amino acid sequence set forth in SEQ ID No:2, 3, 4, 6 and 8
(see Table 1), wherein both cysteine residues in the linker are
covalently linked to the same functional moiety (e.g., PEG) using a
bifunctional reactive group, (e.g., a bis-sulfone).
Functionalization of Loops
[0050] The formation of intrachain disulphide bonds by the cysteine
residues in the linkers of the invention results in the cyclisation
of the linker amino acid sequence between the cysteine residues to
form a loop (see FIGS. 3 and 5). This loop structure is
particularly advantageous in that it can be modified to comprise an
amino acid sequence with one or more particular binding
specificities, and hence add additional functionality to a
polypeptide of which the loop is a part.
[0051] The loop sequence can e.g. have a binding specificity for
any target molecule. Suitable target molecules include, without
limitation, PK modifiers such, as hyaluronic acid, serum albumin,
an integrin, an antibody Fc region, transferrin, and the like. For
example, binding to serum albumin prolongs the half live of the
polypeptide. Binding to FcRx improves the transport of the
polypeptide into the placenta, whereas binding to mucin prolongs
the residence time in the epithelium. In the case of an
immunobinder with specificity for an antigen, in certain
embodiments, the loop region between the VH and VL domains can be
engineered to bind to the antigen and thus improve the affinity of
the immunobinder. In another embodiment, the target molecule is the
polypeptide itself. In other words, the loop can provide a
multimerization function that causes the polypeptide to build
dimers, trimers or higher order multimers. Multimers have a
stronger avidity as compared to monomers. Furthermore, multimers
allow for cross-linking and eventually followed by activation e.g.
of receptors on the surface of a cell. In another embodiment, the
target molecule is another immunobinder displaying a complementary
loop in its linker i.e. the loops of the two molecules bind to each
other, whereas the loops might be identical or different. This
allows for the formation of bispecific or multispecific
immunobinder complexes. Bispecific or multispecific complexes allow
for recruiting molecules or cells to another cell or molecule, e.g.
the recruitment of T cells to cancer cells results in the killing
of the cancer cell (Cancer Res. volume 69 page 4941). Any short
amino acid sequence with a binding specificity for a desired target
molecule can be introduced into the loops of the invention. In a
preferred embodiment, the loop sequence consists of 5 to 15 amino
acids (i.e. b is 5-15 in the general formula
(X).sub.a-C-(X).sub.b-C-(X).sub.c) and contains no cysteines.
Suitable amino acid sequences that can be incorporated into a loop
include, without limitation, a hyaluronic acid binding peptide
(e.g., a peptide comprising residues 12 to 23 of SEQ ID No:4 in
Table 1), and an integrin binding peptide (e.g., an RGD
peptide).
[0052] New peptides with binding specificities to desired target
molecules, suitable for incorporation into a loop, can be
identified by art recognized means such as, for example, phage
display, yeast display and mRNA display. Such display systems are
well known in the art (see, for example, U.S. Pat. Nos. 66,258,558,
6,699,658; and 7,118,879, which are hereby incorporated by
reference). By way of example, to perform a phage display screen,
the loop formed by the two cysteines of the linker can be fused to
a phage capsid protein for display on a phage surface (see FIG. 5
for a schematic drawing). The loop sequence, which ideally
comprises between 5-15 amino acids and contains no cysteines other
than those required to form the loop, is randomized to provide a
library of loop sequences to screen. Suitable phage libraries are
commercially available, e.g. the disulfide-constrained heptapeptide
(Ph.D.-C7C) library from New England Biolab. Target molecules for
such phage screens include, for example, PK modifiers, such as,
collagen type II, albumin, antibody Fc regions, antibody Fc
receptors (e.g., FcRx), mucins, integrins, tight junction proteins
and complement factors.
Modified Polypeptides
[0053] Additionally or alternatively, the polypeptides of the
invention can be covalently linked to a functional moiety (e.g.,
PEG) at one or more amino acid residues outside of the linker
region. Any amino acid residue that does not substantially impair
one or more functional properties of the polypeptide can be
employed, for example, surfaced exposed lysine and cysteine
residues. If desired, a polypeptide can be modified (by addition or
substitution) to introduce additional reactive amino acids,
suitable for linkage to a functional moiety. In a particular
embodiment, a polypeptide is modified to contain at least one pair
of cysteine residues, and wherein at least one pair of the cysteine
residues form an intrachain disulphide bond in the mature
polypeptide. A functional moiety can be linked to a single reactive
residue (e.g., a cysteine) using a monofunctional reactive group,
or linked to two disulphide bonded cysteines using a bifunctional
disulphide bond-specific reactive group (e.g., a bis-sulfone).
[0054] Accordingly, in one embodiment, the invention provides a
polypeptide comprising the amino acid sequence set forth in SEQ ID
No. 5, wherein at least one amino acid residue selected from the
group consisting of Lys40, Lys43, Lys46, Lys107, Lys176, Lys197 and
Lys208 is covalently linked to a functional moiety (e.g., PEG).
[0055] In another embodiment, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No. 5,
wherein at least one pair of cysteine residues selected from the
group consisting of C24-C89 and C154-C228 is covalently linked to
the same functional moiety (e.g., PEG). In another embodiment, the
invention provides a polypeptide comprising the amino acid sequence
set forth in SEQ ID No. 6, wherein at least one amino acid residue
selected from the group consisting of Cys123 and Cys136 is
covalently linked to a functional moiety (e.g., PEG).
[0056] In another embodiment, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No. 6,
wherein the pair of cysteine residues Cys123-Cys136 is covalently
linked to the same functional moiety (e.g., PEG).
[0057] In another embodiment, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No. 7,
wherein at least one amino acid residue selected from the group
consisting of Lys44, Lys47, Lys105, Lys109, Lys142, Lys143, Lys149,
Lys173, Lys193 and Lys195 is covalently linked to a functional
moiety (e.g., PEG).
[0058] In another embodiment, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No. 7,
wherein at least one pair of cysteine residues selected from the
group consisting of Cys25-Cys90 and Cys152-Cys226 is covalently
linked to the same functional moiety (e.g., PEG).
[0059] In another embodiment, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No. 8,
wherein at least one amino acid residue selected from the group
consisting of Cys121 and Cys129 is covalently linked to a
functional moiety (e.g., PEG).
[0060] In another embodiment, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No. 8,
wherein the pair of cysteine residues Cys121-Cys129 is covalently
linked to the same functional moiety (e.g., PEG).
[0061] In another embodiment, the invention provides a polypeptide,
comprising the amino acid sequence set forth in SEQ ID No. 5, 6, 7,
or 8, wherein the polypeptide is modified to contain at least one
reactive amino acid (e.g., cysteine or lysine). Such reactive amino
acids can be covalently linked to a functional moiety (e.g.,
PEG).
[0062] In another embodiment, the invention provides a polypeptide
comprising the amino acid sequence set forth in SEQ ID No. 5, 6, 7,
or 8, wherein the polypeptide is modified to contain at least one
pair of cysteine residues, and wherein at least one pair of
cysteine residues are capable of forming an intrachain disulphide
bond in the mature polypeptide. In a particular embodiment, at
least one pair of cysteine residues are covalently linked to the
same functional moiety (e.g., PEG) bifunctional reactive group
(e.g., a bis-sulphone).
[0063] As known in the art, the attachment of PEG improves certain
characteristics of biopharmaceuticals without altering their
function, thereby enhancing their therapeutic effect. Exemplary
effects are (i) improved pharmacokinetics through enhanced
solubility, improved stability, sustained absorption and/or
continuous biopharmaceutical action; (ii) increased circulation
time which decreases the therapeutically effective amount and/or
the dosing frequency; and/or (iii) decreased toxicity, e.g. due to
an improved safety profile, a reduced immunogenicity, a reduced
antigenicity and/or reduced proteolysis.
Polypeptide Compositions and Formulations
[0064] Another aspect of the invention pertains to pharmaceutical
formulations of the polypeptide (e.g. ScFv) compositions of the
invention. Such formulations typically comprise the polypeptide
(e.g. ScFv) composition and a pharmaceutically acceptable carrier.
As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Preferably, the carrier
is suitable for, for example, intravenous, intramuscular,
subcutaneous, topical (e.g., to eye, skin, or epidermal layer),
inhalation, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the polypeptides (e.g. ScFv) may be coated in a
material to protect the compound from the action of acids and other
natural conditions that may inactivate the compound.
[0065] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al.
(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic
acids, hydroxy alkanoic acids, aromatic acids, aliphatic and
aromatic sulfonic acids and the like. Base addition salts include
those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0066] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0067] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0068] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents that delay absorption such
as aluminum monostearate and gelatin.
[0069] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0070] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0071] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying (lyophilization) that yield a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0072] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, preferably from about 0.1 percent to
about 70 percent, most preferably from about 1 percent to about 30
percent of active ingredient in combination with a pharmaceutically
acceptable carrier.
[0073] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
TABLE-US-00001 TABLE 1 Linker and scFv sequences SEQ ID NO: Linker
Sequence 1 SS (X).sub.3-15C(X).sub.3-50C(X).sub.3-15 linker 2 SS
GGGGSGGGGSC(X).sub.3-50CGGGGSGGGGS linker 3 SS
GGGGSGGGGSCGGGSGGGCGGGGSGGGGS linker 4 SS Pep-1
GGGGSGGGGSCGAHWQFNALTVRCGGGGS linker GGGGS 5 ESBA903
MEIVMTQSPSTLSASVGDRVIITCQASEI Normal IHSWLAWYQQKPGKAPKLLIYLASTLASG
linker VPSRFSGSGSGAEFTLTISSLQPDDFATY YCQNVYLASTNGANFGQGTKLTVLGGGGG
SGGGGSGGGGSGGGGSEVQLVESGGGLVQ PGGSLRLSCTASGFSLTDYYYMTWVRQAP
GKGLEWVGFIDPDDDPYYATWAKGRFTIS RDISKNIVYLQMNSLRAEDTAVYYCAGGD
HNSGWGLDIWGQGTLVTVSS 6 ESBA903 MEIVMTQSPSTLSASVGDRVIITCQASEI SS
Pep-1 IHSWLAWYQQKPGKAPKLLIYLASTLASG linker
VPSRFSGSGSGAEFTLTISSLQPDDFATY YCQNVYLASTNGANFGQGTKLTVLGGGGG
SGGGGSCGAHWQFNALTVRCGGGGSGGGG SEVQLVESGGGLVQPGGSLRLSCTASGFS
LTDYYYMTWVRQAPGKGLEWVGFIDPDDD PYYATWAKGRFTISRDTSKNIVYLQMNSL
RAEDTAVYYCAGGDHNSGWGLDIWGQGTL VTVSS 7 ESBA105
MADIVMTQSPSSLSASVGDRVTLTCTASQ Normal SVSNDVVWYQQRPGKAPKLLIYSAFNRYT
linker GVPSRFSGRGYGTDFTLTISSLQPEDVAV YYCQQDYNSPRTFGQGTKLEVKRGGGGSG
GGGSGGGGSSGGGSQVQLVQSGAEVKKPG ASVKVSCTASGYTFTHYGMNWVRQAPGKG
LEWMGWINTYTGEPTYADKFKDRFTFSLE TSASTVYMELTSLTSDDTAVYYCARERGD
AMDYWGQGTLVTVSS 8 ESBA105 MADIVMTQSPSSLSASVGDRVTLTCTASQ SS
SVSNDVVWYQQRPGKAPKLLIYSAFNRYT linker GVPSRFSGRGYGTDFTLTISSLQPEDVAV
YYCQQDYNSPRTFGQGTKLEVKRGGGGSG GGGSCGGGSGGGCGGGGSGGGGSQVQLVQ
SGAEVKKPGASVKVSCTASGYTFTHYGMN WVRQAPGRGLEWMGWINTYTGEPTYADKF
KDRFTESLETSASTVYMELTSLTSDDTAV YYCARERGDAMDYWGQGTLVTVSS
Exemplification
[0074] The present disclosure is further illustrated by the
following examples, which should not be construed as further
limiting. The contents of all figures, references, patents and
published patent applications cited throughout this application are
expressly incorporated herein by reference in their entireties.
[0075] In general, the practice of the present invention employs,
unless otherwise indicated, conventional techniques of chemistry,
molecular biology, recombinant DNA technology, and immunology
(especially, e.g., immunoglobulin technology). See, e.g., Sambrook,
Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor
Laboratory Press (1989); Antibody Engineering Protocols (Methods in
Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody
Engineering: A Practical Approach (Practical Approach Series, 169),
McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual,
Harlow et al., C.S.H.L. Press, Pub. (1999); Current Protocols in
Molecular Biology, eds. Ausubel et al., John Wiley & Sons
(1992). See also, e.g., Polytherics U.S. Pat. No .6,803,438;
EP1701741A2; EP1648518A2; WO05065712A2; WO05007197A2; EP1496941A1;
EP1222217B1; EP1210093A4; EP1461369A2; WO03089010A1; WO03059973A2;
and EP1210093A1); Genentech US20070092940A1 and EP1240337B1; and
ESBATech U.S. Ser. No. 60/899,907 and WO03097697A2.
PEGylation of ESBA105
[0076] Methyl-PEO.sub.12-Malemide (Pierce), a sulfhydryl-reactive
PEGylation reagent was used for modification of sulfhydryl groups
in ESBA105 (SEQ ID No:7) and ESBA105-SS-LINKER (SEQ ID No:8). Since
ESBA105 contains cysteine residues whose side-chain sulfur atoms
occur in pairs as disulfide bonds, reduction of these disulfide
bonds is required to expose the sulfhydryl groups that serve as a
target for PEGylation with Methyl-PEO.sub.12-Malemide. Pegylation
of ESBA105 and ESBA105-SS-LINKER was performed as recommended by
the supplier of PEG (Thermo Scientific: Pierce Protein Research
Products). Shortly, disulfide bonds were reduced by incubation of
approximately 2 mg of ESBA105 in the presence of 20 mM DTT for 30
minutes at 4.degree. C. For removal of DTT the reduced ESBA105 was
dialysed against PBS (pH 6.5) using Slide-A-Lyzer Dialysis
cassettes (Pierce; cut-off: 7000 Da). After dialysis and
up-concentration, 2 mg/ml of protein was pegylated by incubation of
a 20-fold molar excess of Methyl-PEO.sub.12-Malemide at 4.degree.
C. overnight. The labeled proteins were purified from nonreacted
Methyl-PEO.sub.12-Malemide by dialysis using Slide-A-Lyzer Dialysis
cassettes (Pierce; cut-off: 7000 Da).
SDS-PAGE
[0077] SDS-PAGE was performed under non-reducing conditions using
the commercially available Bis-Tris electrophoresis system from
Invitrogen according to the recommendations of the provider. 5
.mu.g of protein samples were loaded on precast 12% Bis-Tris gels.
The gels were stained using Coomassie reagent (0.1% (w/v) Coomassie
G250, 10% glacial acetic acid, 50% ethanol) for 15 minutes.
Destaining was done using 10% (v/v) acetic acid.
Western Blot Analysis to Confirm Pegylation
[0078] 1, 10 and 100 ng of PEGylated ESBA105-SS-LINKER was loaded
on a precast 12%
[0079] Bis-Tris gel. Samples were blotted on nitrocellulose
membranes and the PEG moiety was detected with a rabbit monoclonal
anti-PEG antibody (Epitomics). After incubation with the primary
antibody, membranes were incubated with horseradish peroxidase
(HRP)-conjugated goat anti-rabbit polyclonal antibody (Santa Cruz).
Specific binding of antibodies to the membranes was detected by a
chemiluminescence detection system (Pierce).
Direct ELISA to Confirm Binding of Pegylated ESBA105 to Human TNF
Alpha
[0080] Binding of ESBA105 and its derivatives was assessed by a
direct ELISA. Human TNF alpha (PeproTech EC Ltd) was coated on a
96-well microtitre plate and then blocked using BSA (bovine serum
albumin). ESBA105 and ESBA105 derivates were tested at
concentrations of 50 nM, 12.5 nM, 3.13 nM, 1.56 nM, 0.78 nM, 0.39
nM, 0.20 nM, 0.10 nM and 0.05 nM. Binding of ESBA105 and its
derivatives to human TNF alpha was visualized by the subsequent
addition of a biotinylated rabbit polyclonal anti-ESBA105 antibody
(AK3A, generated at ESBATech), streptavidin Poly HRP and a
chromogenic substrate (POD). The product of this reaction was
detected by measurement of the optical densitiy (OD) at 450 nM
using a spectrophotometer. Data were analyzed using a 4-parameter
logistic curve fit, and EC.sub.50 values were calculated from the
dose-response curves of the scFvs.
Surface Plasmon Resonance Analysis of ESBA903 and ESBA903 with Pep1
in the Linker
[0081] For binding affinity measurements, surface Plasmon resonance
measurements with BIAcore.TM.-T100 were employed. In these
experiments, purified Escherichia coli-expressed recombinant human
VEGF.sub.165 (PeproTech EC Ltd) was used. Carboxymethylated dextran
biosensor chips (CM4, GE Healthcare) were activated with
N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and
N-hydroxysuccinimide according to the supplier's instructions.
Human VEGF.sub.165 was coupled to 1 of the 4 different flow cells
on a CM4 sensor chip using a standard amine-coupling procedure. The
range of responses obtained with the immobilized hVEGF.sub.165
molecule after coupling and blocking was approximately 120-140
response units (RU). The 4th flow cell of each chip was treated
similarly except no proteins were immobilized prior to blocking,
and the flow cell was used as in-line reference. Various
concentrations of anti-VEGF scFvs (20 nM, 10 nM, 5 nM, 2.5 nM, 1.25
nM, 0.63 nM, 0.31 nM and 0.16 nM) in HBS-EP buffer (0.01 M HEPES,
pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) were
injected into the flow cells at a flow rate of 30 .mu.l/min for 5
min. Dissociation of the anti-VEGF scFv from the VEGF on the CM4
chip was allowed to proceed for 10 min at 25.degree. C. Sensorgrams
were generated for each anti-VEGF scFv sample after in-line
reference cell correction followed by buffer sample subtraction.
The apparent dissociation rate constant (k.sub.d), the apparent
association rate constant (k.sub.a) and the apparent dissociation
equilibrium constant (K.sub.D) were calculated using one-to-one
Langmuir binding model with BlAcore T100 evaluation Software
version 1.1.
Cloning and Expression of scFvs
[0082] The scFvs described and characterized herein were produced
as follows. In some cases DNA sequences encoding for the various
scFvs were de novo synthesized at the service provider Entelechon
GmbH (www.entelechon.com). The resulting DNA inserts were cloned
into the bacterial expression vector pGMP002 via NcoI and HindIII
restriction sites introduced at the 5' and 3' end of the scFv DNA
sequence, respectively. In some cases the modified linkers were
introduced by state-of-the-art methods as annealed, complementary
oligos that encode the respective amino acid molecules, by cloning
them into suitable restriction sites between the VH and VL domains.
In other cases, point mutations were introduced into the VH and/or
VL domain using state of the art assembling PCR methods.
[0083] The cloning of GMP002 is described in Example 1 of
WO2008006235. The production of the scFvs was performed as
described for ESBA105 in Example 1 of WO08/006235, which is hereby
incorporated by reference.
EXAMPLE 1
Pegylation of ESBA105 and ESBA105-SS-Linker
[0084] ESBA105 (SEQ ID No: 7) is a single chain antibody that
specifically binds and inhibits human TNFalpha (see e.g. WO
06/131013, which is hereby incorporated by reference).
ESBA105-SS-linker (SEQ ID No: 8) is an ESBA105 variant, in which
the linker was replaced by the SS-linker (SEQ ID No:3).
[0085] ESBA105, ESBA105 reduced with DTT, ESBA105 reduced and
subjected to cysteine pegylation and ESBA105 reduced and subjected
to cysteine pegylation followed by dialysis were analyzed by
SDS-PAGE. Upon pegylation of the protein an increase of molecular
weight of approximately 4 kDa is expected. "ESBA105 reduced" and
"ESBA105 reduced cysteine-pegylated" migrate at a slightly higher
position compared to ESBA105. Upon dialysis of cys pegylated
ESBA105, the protein migrates at the same position as ESBA105
suggesting that the protein shift was not due to pegylation but
rather due to reduction of the disulfide bonds that were formed
again upon oxidation of the sulfhydryl groups during dialysis.
These data indicate that the reduced intramolecular cysteines (SH)
of ESBA105 have low accessibility for PEG-NHS. In contrast to the
sulfhydryl grougs of the intramolecular cysteines in ESBA105,
primary amines (Lysine residues at the N-terminus) are accessible
for PEG-NHS, as shown in lane 6 and 7 of FIG. 1.
[0086] The activities of differently treated ESBA105 preparations
as described above were assessed in ELISA experiments. The ELISA
analysis depicted in FIG. 2 show that reduced ESBA105 is as active
as oxidized ESBA105. Cys pegylated ESBA105 shows only a slight loss
of binding activity compared to ESBA105. However, as shown in FIG.
1 the extent of PEGylation of intrachain disulfides in ESBA105 is
low. Lys-pegylation was successful with only minor loss of binding
activity towards human TNF alpha.
[0087] One purpose of the present invention was to provide scFv
antibodies that are susceptible to cys-pegylation. In a first step,
it was examined, whether the un-pegylated ESBA105-SS-linker
molecule is still active as compared to ESBA105. Once this was
confirmed by means of an ELISA assay as depicted in FIG. 4A,
ESBA105-SS-linker was subjected to cys-pegylation. To test for
successful cys-pegylation, ESBA105-SS-linker, reduced
ESBA105-SS-linker, reduced cys pegylated ESBA105-SS-linker and cys
pegylated ESBA105-SS-linker subjected to dialysis were analyzed by
SDS-PAGE (see FIG. 4B). PEGylated ESBA105-SS-linker migrates at a
higher position compared to ESBA105-SS-linker indicating successful
PEGylation of ESBA105-SS-linker. PEGylation of ESBA105-SS-linker
was confirmed by Western blot analysis using a rabbit anti-PEG
monoclonal antibody (see FIG. 4C). A signal at the expected size
was detected with 100 ng/ml pegylated ESBA105-SS-linker.
Furthermore, the ELISA assay as depicted in FIG. 6 confirms that
cys-pegylated ESBA105-SS-linker almost fully active as compared to
naked ESBA105.
[0088] Conventional cys-pegylation of the ESBA105 SS-linker using a
PEG with a monospecific reactive group is depicted schematically on
FIG. 3 (middle). An alternative method of cys-pegylation is also
depicted in FIG. 3 (right). This, latter, method employs a PEG
attached to a bifunctional reactive group (schematically
represented as a horizontal "T" having a PEG molecule on its left
end), which allows connection of a PEG molecule to both cysteine
residues of a disulphide bond in a protein. Such linker technology
is described by Shaunak et al. Nature Chemical Biology 2006, volume
2 page 312; Brocchini et al, Nature Protocols, 2006: 1(5), 241, and
WO05/007197.
EXAMPLE 2
Introduction of a Binding Actvity Into the SS-Linker
[0089] A second application of the present invention is to
introduce an additional binding specificity into polypeptides
(e.g., scFv). In this example, the Pep-1 peptide, which was
identified to bind to hyaluronic acid during a phage display
selection (Mummert et al. J. Exp. Med. 2000, volume 192, page 769),
was introduced into the loop region of the SS linker to give rise
to the SS Pep1 linker (SEQ ID NO:4). The SS Pep1 linker was
introduced into the scFv ESBA903 to produce ESBA903 SS Pep1 linker
(sometimes also referred to as ESBA903-Pep1; SEQ ID NO:6). As
demonstrated by the surface plasmon resonance results shown in FIG.
7, ESBA903-Pep1 displaying the Pep1 12mer peptide in its loop,
binds equally well to its target (VEGF) as unmodified ESBA903 (SEQ
ID No: 5), and therefore is still fully functional (Kd values
measured were 4.436E-11 M and 5.608E-11 M, respectively).
ESBA903-Pep1 was designed to prolong the local half-life as
compared to naked ESBA903 when applied to a site where hyaluronic
acid is present, for example such as the vitreous body or a
joint.
Equivalents
[0090] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
8183PRTArtificialSynthetic peptide 1Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60
Xaa Xaa Cys Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65
70 75 80 Xaa Xaa Xaa272PRTArtificiallinker sequence 2Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25
30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Gly Gly 50 55 60 Gly Gly Ser Gly Gly Gly Gly Ser 65 70
329PRTArtificial Sequencelinker sequence 3Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Cys Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Cys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 434PRTArtificial
Sequencelinker sequence 4Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Cys Gly Ala His Trp Gln 1 5 10 15 Phe Asn Ala Leu Thr Val Arg Cys
Gly Gly Gly Gly Ser Gly Gly Gly 20 25 30 Gly Ser 5252PRTArtificial
SequencescFv ESBA903 5Met Glu Ile Val Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Ile Ile Thr Cys Gln
Ala Ser Glu Ile Ile His Ser 20 25 30 Trp Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Leu Ala Ser
Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro
Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Tyr Leu Ala Ser 85 90
95 Thr Asn Gly Ala Asn Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Thr Ala Ser Gly Phe Ser 145 150 155 160 Leu Thr Asp Tyr Tyr Tyr Met
Thr Trp Val Arg Gln Ala Pro Gly Lys 165 170 175 Gly Leu Glu Trp Val
Gly Phe Ile Asp Pro Asp Asp Asp Pro Tyr Tyr 180 185 190 Ala Thr Trp
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys 195 200 205 Asn
Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 210 215
220 Val Tyr Tyr Cys Ala Gly Gly Asp His Asn Ser Gly Trp Gly Leu Asp
225 230 235 240 Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245
250 6266PRTArtificial SequencescFv ESBA903 with SS linker 6Met Glu
Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val 1 5 10 15
Gly Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Glu Ile Ile His Ser 20
25 30 Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu 35 40 45 Ile Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys
Gln Asn Val Tyr Leu Ala Ser 85 90 95 Thr Asn Gly Ala Asn Phe Gly
Gln Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Cys Gly Ala His Trp Gln 115 120 125 Phe Asn Ala
Leu Thr Val Arg Cys Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Gly
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 145 150
155 160 Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Ser Leu
Thr 165 170 175 Asp Tyr Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu 180 185 190 Glu Trp Val Gly Phe Ile Asp Pro Asp Asp Asp
Pro Tyr Tyr Ala Thr 195 200 205 Trp Ala Lys Gly Arg Phe Thr Ile Ser
Arg Asp Thr Ser Lys Asn Thr 210 215 220 Val Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr 225 230 235 240 Tyr Cys Ala Gly
Gly Asp His Asn Ser Gly Trp Gly Leu Asp Ile Trp 245 250 255 Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 260 265 7247PRTArtificial
SequencescFv ESBA105 7Met Ala Asp Ile Val Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val Thr Leu Thr Cys
Thr Ala Ser Gln Ser Val Ser 20 25 30 Asn Asp Val Val Trp Tyr Gln
Gln Arg Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Ala
Phe Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Arg
Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln
Pro Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr Asn Ser 85 90
95 Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Val Lys Arg Gly Gly
100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser
Gly Gly 115 120 125 Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro 130 135 140 Gly Ala Ser Val Lys Val Ser Cys Thr Ala
Ser Gly Tyr Thr Phe Thr 145 150 155 160 His Tyr Gly Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175 Trp Met Gly Trp Ile
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp 180 185 190 Lys Phe Lys
Asp Arg Phe Thr Phe Ser Leu Glu Thr Ser Ala Ser Thr 195 200 205 Val
Tyr Met Glu Leu Thr Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr 210 215
220 Tyr Cys Ala Arg Glu Arg Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly
225 230 235 240 Thr Leu Val Thr Val Ser Ser 245 8256PRTArtificial
SequencescFv ESBA105 with SS linker 8Met Ala Asp Ile Val Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser 1 5 10 15 Val Gly Asp Arg Val
Thr Leu Thr Cys Thr Ala Ser Gln Ser Val Ser 20 25 30 Asn Asp Val
Val Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu
Ile Tyr Ser Ala Phe Asn Arg Tyr Thr Gly Val Pro Ser Arg Phe 50 55
60 Ser Gly Arg Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
65 70 75 80 Gln Pro Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asp Tyr
Asn Ser 85 90 95 Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Val
Lys Arg Gly Gly 100 105 110 Gly Gly Ser Gly Gly Gly Gly Ser Cys Gly
Gly Gly Ser Gly Gly Gly 115 120 125 Cys Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Val Gln Leu Val 130 135 140 Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala Ser Val Lys Val Ser 145 150 155 160 Cys Thr Ala
Ser Gly Tyr Thr Phe Thr His Tyr Gly Met Asn Trp Val 165 170 175 Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Trp Ile Asn Thr 180 185
190 Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Lys Phe Lys Asp Arg Phe Thr
195 200 205 Phe Ser Leu Glu Thr Ser Ala Ser Thr Val Tyr Met Glu Leu
Thr Ser 210 215 220 Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala
Arg Glu Arg Gly 225 230 235 240 Asp Ala Met Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 245 250 255
* * * * *