U.S. patent application number 15/195411 was filed with the patent office on 2016-10-20 for acceptor framework for cdr grafting.
The applicant listed for this patent is ESBATech - a Novartis Company LLC. Invention is credited to Dominik Escher.
Application Number | 20160304627 15/195411 |
Document ID | / |
Family ID | 47844686 |
Filed Date | 2016-10-20 |
United States Patent
Application |
20160304627 |
Kind Code |
A1 |
Escher; Dominik |
October 20, 2016 |
ACCEPTOR FRAMEWORK FOR CDR GRAFTING
Abstract
The present invention relates to an antibody acceptor framework
and to methods for grafting non-human antibodies, e.g., rabbit
antibodies, using a particularly well suited antibody acceptor
framework. Antibodies generated by the methods of the invention are
useful in a variety of diagnostic and therapeutic applications.
Inventors: |
Escher; Dominik;
(Huenenberg, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ESBATech - a Novartis Company LLC |
Schlieren |
|
CH |
|
|
Family ID: |
47844686 |
Appl. No.: |
15/195411 |
Filed: |
June 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14644441 |
Mar 11, 2015 |
9409979 |
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15195411 |
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13765302 |
Feb 12, 2013 |
9005924 |
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14644441 |
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12823551 |
Jun 25, 2010 |
8399625 |
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13765302 |
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61220503 |
Jun 25, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/94 20130101;
C07K 2317/92 20130101; C07K 16/00 20130101; C07K 16/18 20130101;
C07K 16/464 20130101; C07K 2317/622 20130101; C07K 2317/55
20130101; C07K 2317/24 20130101; C07H 21/00 20130101; C07K 2317/565
20130101; C07K 16/241 20130101; C07K 2317/567 20130101; C07K
2317/14 20130101 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/24 20060101 C07K016/24 |
Claims
1. A human heavy chain acceptor framework comprising SEQ ID NO:
1.
2. The human heavy chain acceptor framework of claim 1, comprising
an amino acid substitution at position 12, 103, and/or 144 (Aho
numbering).
3. The human heavy chain acceptor framework of claim 2, wherein the
substitution is (a) Serine (S) at position 12; (b) Serine (S) or
Threonine (T) at position 103; and/or (c) Serine (S) or Threonine
(T) at position 144.
4. An isolated nucleic acid encoding the acceptor framework of
claim 1.
5. A vector comprising the nucleic acid of claim 4.
6. A host cell comprising the vector of claim 5.
7. An immunobinder specific to a desired antigen comprising: (a) a
light chain acceptor framework comprising variable light chain CDRs
of a lagomorph immunobinder; and (b) human heavy chain acceptor
framework of claim 1 comprising variable heavy chain CDRs of a
lagomorph immunobinder.
8. The immunobinder of claim 7, wherein the light chain acceptor
framework has at least 85% identity to SEQ ID NO: 2.
9. The immunobinder of claim 7, further comprising a linker
sequence that links the variable light chain framework and the
heavy chain acceptor framework, wherein the linker sequence is SEQ
ID NO: 4.
10. The immunobinder of claim 7, further comprising donor framework
residues involved in antigen binding.
11. The immunobinder of claim 7, wherein the immunobinder is a scFv
antibody, a full-length immunoglobulin or a Fab fragment.
12. A method of humanizing a rabbit immunobinder, the method
comprising: (a) grafting at least one heavy chain CDR of the group
consisting of CDR H1, CDR H2 and CDR H3 sequences from a donor
rabbit immunobinder into the human heavy chain acceptor framework
of claim 1; and (b) grafting at least one light chain CDR of the
group consisting of CDR L1, CDR L2 and CDR L3 sequences from a
donor rabbit immunobinder into a human light chain acceptor
framework into a light chain acceptor framework has at least 85%
identity to SEQ ID NO: 2.
13. The method of claim 12, further comprising substituting
framework residues in one or both of the human heavy chain acceptor
framework and the human light chain framework with framework
residues of the donor rabbit immunobinder.
14. The method of claim 12, wherein the heavy chain acceptor
framework has a substitution at one or more of heavy chain amino
positions 12, 103 and 144 (AHo numbering).
15. The method of claim 14, wherein the substitution at one or more
of positions 12, 103 and 144 are selected from the group consisting
of: (a) Serine (S) at position 12; (b) Threonine (T) at position
103; and (c) Threonine (T) at position 144.
16. An immunobinder humanized according to the method of claim 12.
Description
[0001] The present application is a divisional of U.S. application
Ser. No. 14/644,441 filed Mar. 11, 2015 (now Allowed), which is a
divisional of U.S. Pat. No. 9,005,924 filed Feb. 12, 2013, which is
a divisional of U.S. Pat. No. 8,399,625 filed Jun. 25, 2010
(Granted), which claims the benefit of U.S. Provisional Patent
Application Ser. No. 61/220,503, filed on Jun. 25, 2009, the
disclosure of which is specifically incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] Monoclonal antibodies, their conjugates and derivatives are
hugely commercially important as therapeutic and diagnostic agents.
Non-human antibodies elicit a strong immune response in patients,
usually following a single low dose injection (Schroff, 1985 Cancer
Res 45:879-85, Shawler. J Immunol 1985 135:1530-5; Dillman, Cancer
Biother 1994 9:17-28). Accordingly, several methods for reducing
the immunogenicity of murine and other rodent antibodies as well as
technologies to make fully human antibodies using e.g. transgenic
mice or phage display were developed. Chimeric antibodies were
engineered, which combine rodent variable regions with human
constant regions (e.g., Boulianne Nature 1984 312:643-6) reduced
immunogenicity problems considerably (e.g., LoBuglio, Proc Natl
Acad Sci 1989 86:4220-4; Clark, Immunol Today 2000 21:397-402).
Humanized antibodies were also engineered, in which the rodent
sequence of the variable region itself is engineered to be as close
to a human sequence as possible while preserving at least the
original CDRs, or where the CDRs from the rodent antibody were
grafted into framework of a human antibody (e.g., Riechmann, Nature
1988 332:323-7; U.S. Pat. No. 5,693,761). Rabbit polyclonal
antibodies are widely used for biological assays such as ELISAs or
Western blots. Polyclonal rabbit antibodies are oftentimes favored
over polyclonal rodent antibodies because of their usually much
higher affinity. Furthermore, rabbit oftentimes are able to elicit
good antibody responses to antigens that are poorly immunogenic in
mice and/or which give not rise to good binders when used in phage
display. Due to these well-known advantages of rabbit antibodies,
they would be ideal to be used in the discovery and development of
therapeutic antibodies. The reason that this is not commonly done
is mainly due to technical challenges in the generation of
monoclonal rabbit antibodies. Since myeloma-like tumors are unknown
in rabbits, the conventional hybridoma technology to generate
monoclonal antibodies is not applicable to rabbit antibodies.
Pioneering work in providing fusion cell line partners for rabbit
antibody-expressing cells has been done by Knight and colleagues
(Spieker-Polet et al., PNAS 1995, 92:9348-52) and an improved
fusion partner cell line has been described by Pytela et al. in
2005 (see e.g. U.S. Pat. No. 7,429,487). This technology, however,
is not widely spread since the corresponding know-how is basically
controlled by a single research group. Alternative methods for the
generation of monoclonal antibodies that involve the cloning of
antibodies from selected antibody-expressing cells via RT-PCR are
described in the literature, but have never been successfully
reported for rabbit antibodies.
[0003] Rabbit antibodies, like mouse antibodies are expected to
elicit strong immune responses if used for human therapy, thus,
rabbit antibodies need to be humanized before they can be used
clinically. However, the methods that are used to make humanized
rodent antibodies cannot easily be extrapolated for rabbit
antibodies due to structural differences between rabbit and mouse
and, respectively, between rabbit and human antibodies. For
example, the light chain CDR3 (CDRL3) is often much longer than
previously known CDRL3s from human or mouse antibodies.
[0004] There are few rabbit antibody humanization approaches
described in the prior art, which are, however, no classical
grafting approach in which the CDRs of a non-human donor are
transplanted on a human acceptor antibody. WO 04/016740 describes a
so-called "resurfacing" strategy. The goal of a "resurfacing"
strategy is to remodel the solvent-accessible residues of the
non-human framework such that they become more human-like. Similar
humanization techniques for rabbit antibodies as described in WO
04/016740 are known in the art. Both WO08/144757 and WO05/016950
disclose methods for humanizing a rabbit monoclonal antibody which
involve the comparison of amino acid sequences of a parent rabbit
antibody to the amino acid sequences of a similar human antibody.
Subsequently, the amino acid sequence of the parent rabbit antibody
is altered such that its framework regions are more similar in
sequence to the equivalent framework regions of the similar human
antibody. In order to gain good binding capacities, laborious
development efforts need to be made for each immunobinder
individually.
[0005] A potential problem of the above-described approaches is
that not a human framework is used, but the rabbit framework is
engineered such that it looks more human-like. Such approach
carries the risk that amino acid stretches that are buried in the
core of the protein still might comprise immunogenic T cell
epitopes.
[0006] To date, the applicants have not identified a rabbit
antibody, which was humanized by applying state-of-the-art grafting
approaches. This might be explained by fact that rabbit CDRs may be
quite different from human or rodent CDRs. As known in the art,
many rabbit VH chains have extra paired cysteines relative to the
murine and human counterparts. In addition to the conserved
disulfide bridge formed between cys22 and cys92, there is also a
cys21-cys79 bridge as well as an interCDR S-S bridge formed between
the last residue of CDRH1 and the first residue of CDR H2 in some
rabbit chains. Besides, pairs of cysteine residues are often found
in the CDR-L3. Moreover, many rabbit antibody CDRs do not belong to
any previously known canonical structure. In particular the CDR-L3
is often much longer than the CDR-L3 of a human or murine
counterpart.
[0007] Hence, the grafting of non-human CDRs antibodies into a
human framework is a major protein engineering task. The transfer
of antigen binding loops from a naturally evolved framework to a
different artificially selected human framework must be performed
so that native loop conformations are retained for antigen binding.
Often antigen binding affinity is greatly reduced or abolished
after loop grafting. The use of carefully selected human frameworks
in grafting the antigen binding loops maximizes the probability of
retaining binding affinity in the humanized molecule (Roguzka et al
1996). Although the many grafting experiments available in the
literature provide a rough guide for CDR grafting, it is not
possible to generalize a pattern. Typical problems consist in
loosing the specificity, stability or producibility after grafting
the CDR loops.
[0008] Accordingly, there is an urgent need for improved methods
for reliably and rapidly humanizing rabbit antibodies for use as
therapeutic and diagnostic agents. Furthermore, there is a need for
human acceptor frameworks for reliably humanizing rabbit
antibodies, providing functional antibodies and/or antibody
fragments with drug-like biophysical properties.
SUMMARY OF THE INVENTION
[0009] It has surprisingly been found that a highly soluble and
stable human antibody framework identified by a Quality Control
(QC) assay (as disclosed in WO 0148017 and in Auf der Maur et al
(2001), FEBS Lett 508, p. 407-412) is particularly suitable for
accommodating CDRs from other non-human animal species, for
example, rabbit CDRs. Accordingly, in a first aspect, the invention
provides the heavy chain variable regions of a particular human
antibody (the so called, "a58" VH framework sequence) which is
especially suitable as acceptor for CDRs from a variety of
antibodies, in particular from rabbit antibodies, of different
binding specificities, independent of whether a disulfide bridge is
present in a CDR or not.
[0010] Humanized immunobinders generated by the grafting of rabbit
CDRs into this highly compatible variable chain framework
consistently and reliably retain the spatial orientation of the
rabbit antibodies from which the donor CDRs are derived. Therefore,
no structurally relevant positions of the donor immunobinder need
to be introduced into the acceptor framework. Due to these
advantages, high-throughput humanization of rabbit antibodies with
no or little optimization of the binding capacities can be
achieved.
[0011] Accordingly, in another aspect, the invention provides
methods for grafting rabbit and other non-human CDRs, into the
soluble and stable light chain and/or heavy chain human antibody
framework sequences disclosed herein, thereby generating humanized
antibodies with superior biophysical properties. In particular,
immunobinders generated by the methods of the invention exhibit
superior functional properties such as solubility and
stability.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts the CDR H1 definition used herein for
grafting antigen binding sites from rabbit monoclonal antibodies
into the highly soluble and stable human antibody frameworks.
[0013] FIG. 2: An analysis of rabbit antibody sequences extracted
from the Kabat database confirms that CDR3 of the variable heavy
chain is typically by three amino acids longer than its murine
counterpart.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0014] In order that the present invention may be more readily
understood, certain terms will be defined as follows. Additional
definitions are set forth throughout the detailed description.
[0015] The term "antibody" refers to whole antibodies and any
antigen binding fragment. The term "antigen binding polypeptide"
and "immunobinder" are used simultaneously herein. An "antibody"
refers to a protein, optionally glycosylated, comprising at least
two heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, or an antigen binding portion thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as V.sub.H) and a heavy chain constant region. The heavy
chain constant region is comprised of three domains, CH1, CH2 and
CH3. Each light chain is comprised of a light chain variable region
(abbreviated herein as V.sub.L) and a light chain constant region.
The light chain constant region is comprised of one domain, CL. The
V.sub.H and V.sub.L regions can be further subdivided into regions
of hypervariability, termed complementarity determining regions
(CDR), interspersed with regions that are more conserved, termed
framework regions (FR). Each V.sub.H and V.sub.L is composed of
three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system.
[0016] The term "antigen-binding portion" of an antibody (or simply
"antibody portion") refers to one or more fragments of an antibody
that retain the ability to specifically bind to an antigen (e.g.,
TNF). It has been shown that the antigen-binding function of an
antibody can be performed by fragments of a full-length
antibody.
[0017] Examples of binding fragments encompassed within the term
"antigen-binding portion" of an antibody include (i) a Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
CL and CH1 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 Fd fragment consisting of the V.sub.H
and CH1 domains; (iv) a Fv fragment consisting of the V.sub.L and
V.sub.H domains of a single arm of an antibody, (v) a single domain
or dAb fragment (Ward et al., (1989) Nature 341:544-546), which
consists of a V.sub.H domain; and (vi) an isolated complementarity
determining region (CDR) or (vii) a combination of two or more
isolated CDRs which may optionally be joined by a synthetic linker.
Furthermore, although the two domains of the Fv fragment, V.sub.L
and V.sub.H, are coded for by separate genes, they can be joined,
using recombinant methods, by a synthetic linker that enables them
to be made as a single protein chain in which the V.sub.L and
V.sub.H regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. These
antibody fragments are obtained using conventional techniques known
to those with skill in the art, and the fragments are screened for
utility in the same manner as are intact antibodies.
Antigen-binding portions can be produced by recombinant DNA
techniques, or by enzymatic or chemical cleavage of intact
immunoglobulins. Antibodies can be of different isotype, for
example, an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1,
IgA2, IgD, IgE, or IgM antibody.
[0018] 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, (vi) a
single domain antibody such as a Dab fragment (Ward et al., (1989)
Nature 341:544-546), which consists of a V.sub.H or V.sub.L domain,
a Camelid (see Hamers-Casterman, et al., Nature 363:446-448 (1993),
and Dumoulin, et al., Protein Science 11:500-515 (2002)) or a Shark
antibody (e.g., shark Ig-NARs Nanobodies.RTM.; and (vii) a
nanobody, a heavy chain variable region containing a single
variable domain and two constant domains.
[0019] The term "single chain antibody", "single chain Fv" or
"scFv" refers to a molecule comprising an antibody heavy chain
variable domain (or region; V.sub.H) and an antibody light chain
variable domain (or 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. A suitable state of the art
linker consists of repeated GGGGS amino acid sequences or variants
thereof. In a preferred embodiment of the present invention a
(GGGGS).sub.4 linker of the amino acid sequence set forth in SEQ ID
NO: 8 is used, but variants of 1-3 repeats are also possible
(Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
Other linkers that can be used for the present invention are
described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et
al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer
Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol.
293:41-56 and Roovers et al. (2001), Cancer Immunol.
[0020] As used herein, the term "functional property" is a property
of a polypeptide (e.g., an immunobinder) for which an improvement
(e.g., relative to a conventional polypeptide) is desirable and/or
advantageous to one of skill in the art, e.g., in order to improve
the manufacturing properties or therapeutic efficacy of the
polypeptide. In one embodiment, the functional property is
stability (e.g., thermal stability). In another embodiment, the
functional property is solubility (e.g., under cellular
conditions). In yet another embodiment, the functional property is
aggregation behavior. In still another embodiment, the functional
property is protein expression (e.g., in a prokaryotic cell). In
yet another embodiment the functional property is refolding
behavior following inclusion body solubilization in a manufacturing
process. In certain embodiments, the functional property is not an
improvement in antigen binding affinity. In another preferred
embodiment, the improvement of one or more functional properties
has no substantial effect on the binding affinity of the
immunobinder.
[0021] The term "CDR" refers to one of the six hypervariable
regions within the variable domains of an antibody that mainly
contribute to antigen binding. One of the most commonly used
definitions for the six CDRs was provided by Kabat E. A. et al.,
(1991) Sequences of proteins of immunological interest. NIH
Publication 91-3242). As used herein, Kabat's definition of CDRs
only apply for CDR1, CDR2 and CDR3 of the light chain variable
domain (CDR L1, CDR L2, CDR L3, or L1, L2, L3), as well as for CDR2
and CDR3 of the heavy chain variable domain (CDR H2, CDR H3, or H2,
H3). CDR1 of the heavy chain variable domain (CDR H1 or H1),
however, as used herein is defined by the residue positions (Kabat
numbering) starting with position 26 and ending prior to position
36. This definition is basically a fusion of CDR H1 as differently
defined by Kabat and Chotia (see also FIG. 1 for illustration).
[0022] The term "antibody framework" as used herein refers to the
part of the variable domain, either VL or VH, which serves as a
scaffold for the antigen binding loops (CDRs) of this variable
domain. In essence it is the variable domain without the CDRs.
[0023] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds (e.g., a specific site on the TNF molecule). An
epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14 or 15 consecutive or non-consecutive amino acids in a
unique spatial conformation. See, e.g., Epitope Mapping Protocols
in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.
(1996).
[0024] 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 10.sup.-7 M, such as approximately less than 10.sup.-8 M,
10.sup.-9 M or 10.sup.-19 M or even lower. The term "K.sub.D" or
"Kd" refers to the dissociation equilibrium constant of a
particular antibody-antigen interaction. Typically, the antibodies
of the invention bind to TNF with a dissociation equilibrium
constant (K.sub.D) of less than approximately 10.sup.-7 M, such as
less than approximately 10.sup.-8 M, 10.sup.-9 M or 10.sup.-19 M or
even lower, for example, as determined using surface plasmon
resonance (SPR) technology in a BIACORE instrument.
[0025] The term "nucleic acid molecule," as used herein 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.
[0026] The term "vector," refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
In one embodiment, the vector is a "plasmid," which refers to a
circular double stranded DNA loop into which additional DNA
segments may be ligated. In another embodiment, the vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. The vectors disclosed herein can be 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) or can be 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 (e.g., non-episomal mammalian vectors).
[0027] The term "host cell" refers to a cell into which an
expression vector has been introduced. Host cells include
bacterial, microbial, plant or animal cells, preferably,
Escherichia coli, Bacillus subtilis; Saccharomyces cerevisiae,
Pichia pastoris, CHO (Chinese Hamster Ovary lines) or NSO
cells.
[0028] The term "lagomorphs" refers to members of the taxonomic
order Lagomorpha, comprising the families Leporidae (e.g. hares and
rabbits), and the Ochotonidae (pikas). In a most preferred
embodiment, the lagomorphs is a rabbit. The term "rabbit" as used
herein refers to an animal belonging to the family of the
leporidae.
[0029] As used herein, "identity" refers to the sequence matching
between two polypeptides, molecules or between two nucleic acids.
When a position in both of the two compared sequences is occupied
by the same base or amino acid monomer subunit (for instance, if a
position in each of two polypeptides is occupied by a lysine), then
the respective molecules are identical at that position. The
"percentage identity" between two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
Generally, a comparison is made when two sequences are aligned to
give maximum identity. Such alignment can be provided using, for
instance, the method of the Needleman and Wunsch (J. MoI. Biol.
(48):444-453 (1970)) algorithm which has been incorporated into the
GAP program in the GCG software package, using either a Blossum 62
matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8,
6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
[0030] 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.
[0031] Various aspects of the invention are described in further
detail in the following subsections. It is understood that the
various embodiments, preferences and ranges may be combined at
will. Further, depending of the specific embodiment, selected
definitions, embodiments or ranges may not apply.
[0032] If not otherwise stated, the amino acid positions are
indicated according to the AHo numbering scheme. The AHo numbering
system is described further in Honegger, A. and Pluckthun, A.
(2001) J. Mol. Biol. 309:657-670). Alternatively, the Kabat
numbering system as described further in Kabat et al. (Kabat, E.
A., et al. (1991) Sequences of Proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242) may be used. Conversion tables for the two
different numbering systems used to identify amino acid residue
positions in antibody heavy and light chain variable regions are
provided in A. Honegger, J. Mol. Biol. 309 (2001) 657-670.
[0033] In a first aspect, the present invention provides a human
acceptor framework sequence for the grafting of CDRs from lagomorph
species, for example, from rabbit. The human single-chain VH
framework a58 (SEQ ID NO: 1) was surprisingly found to be in
essence highly compatible with the antigen-binding sites of rabbit
antibodies. Therefore, the a58 VH represents a suitable scaffold to
construct stable humanized scFv antibody fragments derived from
grafting of rabbit loops.
[0034] Thus, in one aspect, the invention provides an immunobinder
acceptor framework, comprising a VH sequence having at least 70%
identity to SEQ ID No. 1.
[0035] Said sequence may be combined with any other suitable
variable light chain. A preferred variable light chain is SEQ ID
NO: 2 which was also disclosed in WO03/097697 and designated KI27,
or any other VL sequence as disclosed in WO03/097697.
[0036] In a preferred embodiment, the variable heavy chain
framework is linked to a variable light chain framework via a
linker. The linker may be any suitable linker, for example a linker
comprising 1 to 4 repeats of the sequence GGGGS (SEQ ID NO: 5),
preferably a (GGGGS).sub.4 peptide (SEQ ID NO: 4), or a linker as
disclosed in Alfthan et al. (1995) Protein Eng. 8:725-731.
[0037] Accordingly, the present invention provides an immunobinder
acceptor framework comprising
[0038] (i) a variable heavy chain framework having at least 70%
identity, preferably at least 75%, 80%, 85%, 90%, more preferably
at least 95% identity, to SEQ ID No. 1; and/or
[0039] (ii) a variable light chain framework having at least 70%
identity, preferably at least 75%, 80%, 85%, 90%, more preferably
at least 95% identity, to SEQ ID No. 2.
[0040] In a much preferred embodiment, the invention provides an
immunobinder, having a sequence with at least 60%, more preferably
at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, identity to SEQ ID NO:
3.
[0041] The framework is compatible with virtually any rabbit CDRs.
Containing different rabbit CDRs, it is well expressed and good
produced contrary to the rabbit wild type single chains and still
almost fully retains the affinity of the original donor rabbit
antibodies.
[0042] The immunobinder acceptor frameworks as described herein may
comprise solubility enhancing substitution in the heavy chain
framework, preferably at positions 12, 103 and 144 (AHo numbering).
Preferably, a hydrophobic amino acid is substituted by a more
hydrophilic amino acid. Hydrophilic amino acids are e.g. Arginine
(R), Asparagine (N), Aspartic acid (D), Glutamine (Q), Glycine (G),
Histidine (H), Lysine (K), Serine (S) and Threonine (T). More
preferably, the heavy chain framework comprises (a) Serine (S) at
position 12; (b) Serine (S) or Threonine (T) at position 103 and/or
(c) Serine (S) or Threonine (T) at position 144.
[0043] Moreover, stability enhancing amino acids may be present at
one or more positions 1, 3, 4, 10, 47, 57, 91 and 103 of the
variable light chain framework (AHo numbering). More preferably,
the variable light chain framework comprises glutamic acid (E) at
position 1, valine (V) at position 3, leucine (L) at position 4,
Serine (S) at position 10; Arginine (R) at position 47, Serine (S)
at position 57, phenylalanine (F) at position 91 and/or Valine (V)
at position 103.
[0044] As glutamine (Q) is prone to desamination, in another
preferred embodiment, the VH comprises at position 141 a glycine
(G). This substitution may improve long-term storage of the
protein.
[0045] For example, the acceptor frameworks disclosed herein can be
used to generate a human or humanized antibody which retains the
binding properties of the non-human antibody from which the
non-human CDRs are derived. Accordingly, in a preferred embodiment
the invention encompasses an immunobinder acceptor framework as
disclosed herein, further comprising heavy chain CDR1, CDR2 and
CDR3 and/or light chain CDR1, CDR2 and CDR3 from a donor
immunobinder, preferably from a mammalian immunobinder, more
preferably from a lagomorph immunobinder and most preferably from a
rabbit. Thus, in one embodiment, the invention provides an
immunobinder specific to a desired antigen comprising
[0046] (i) variable light chain CDRs of a lagomorph; and
[0047] (ii) a human variable heavy chain framework having at least
70%, preferably at least 75%, 80%, 85%, 90%, 95%, and most
preferably 100% identity to SEQ ID NO. 1.
[0048] Preferably, the lagomorph is a rabbit. More preferably, the
immunobinder comprises heavy chain CDR1, CDR2 and CDR3 and light
chain CDR1, CDR2 and CDR3 from the donor immunobinder.
[0049] As known in the art, many rabbit VH chains have extra paired
cysteines relative to the murine and human counterparts. In
addition to the conserved disulfide bridge formed between cys22 and
cys92, there is also a cys21-cys79 bridge as well as an interCDR
S-S bridge formed between the last residue of CDRH1 and the first
residue of CDR H2 in some rabbit chains. Besides, pairs of cysteine
residues in the CDR-L3 are often found. Besides, many rabbit
antibody CDRs do not belong to any previously known canonical
structure. In particular the CDR-L3 is often much longer than the
CDR-L3 of a human or murine counterpart.
[0050] As stated before, the grafting of the non-human CDRs onto
the frameworks disclosed herein yields a molecule wherein the CDRs
are displayed in a proper conformation. If required, the affinity
of the immunobinder may be improved by grafting antigen interacting
framework residues of the non-human donor immunobinder. These
positions may e.g. be identified by
[0051] (i) identifying the respective germ line progenitor sequence
or, alternatively, by using the consensus sequences in case of
highly homologous framework sequences;
[0052] (ii) generating a sequence alignment of donor variable
domain sequences with germ line progenitor sequence or consensus
sequence of step (i); and
[0053] (iii) identifying differing residues.
[0054] Differing residues on the surface of the molecule were in
many cases mutated during the affinity generation process in vivo,
presumably to generate affinity to the antigen.
[0055] In another aspect, the present invention provides an
immunobinder which comprises the immunobinder acceptor framework
described herein. Said immunobinder may e.g. be a scFv antibody, a
full-length immunoglobulin, a Fab fragment, a Dab or a
Nanobody.
[0056] In a preferred embodiment, the immunobinder is attached to
one or more molecules, for example a therapeutic agent such as a
cytotoxic agent, a cytokine, a chemokine, a growth factor or other
signaling molecule, an imaging agent or a second protein such as a
transcriptional activator or a DNA-binding domain.
[0057] The immunobinder as disclosed herein may e.g. be used in
diagnostic applications, therapeutic application, target validation
or gene therapy.
[0058] The invention further provides an isolated nucleic acid
encoding the immunobinder acceptor framework disclosed herein or
the immunobinder(s) as disclosed herein.
[0059] In another embodiment, a vector is provided which comprises
the nucleic acid disclosed herein.
[0060] The nucleic acid or the vector as disclosed herein can e.g.
be used in gene therapy.
[0061] The invention further encompasses a host cell comprising the
vector and/or the nucleic acid disclosed herein.
[0062] Moreover, a composition is provided, comprising the
immunobinder acceptor framework as disclosed herein, the
immunobinder as disclosed herein, the isolated nucleic acid as
disclosed herein or the vector as disclosed herein.
[0063] The sequences disclosed herein are the following (X residues
are CDR insertion sites and contain at least 3 and up to 50 amino
acids):
TABLE-US-00001 SEQ ID NO: 1: variable heavy chain framework a58
EVQLVESGGGLVQPGGSLRLSCAAS(X).sub.n=3-50WVRQAPGKGLEWVS
(X).sub.n=3-50RFSVSRDNSKNTVYLQINSLRAEDTAVYYCAM(X).sub.n=3-50
WGQGTLVTVSS SEQ ID NO: 2: variable light chain framework KI27
EIVMTQSPSTLSASVGDRVIITC(X).sub.n=3-50 WVQQKPGKAPKLLIY
(X).sub.n=3-50 GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC
(X).sub.n=3-50FGQGTKLTVLG SEQ ID NO: 3: framework sequence
EIVMTQSPSTLSASVGDRVIITC(X).sub.n=3-50 WYQQKPGKAPKLLIY
(X).sub.n=3-50 GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X).sub.n=3-50
FGQGTKLT VLGGGGGSGGGGSGGGGSGGGGS
EVQLVESGGGLVQPGGSLRLSCAAS(X).sub.n=3-50WVRQAPGKGLEWVS
(X).sub.n=3-50 RFSVSRDNSKNTVYLQINSLRAEDTAVYYCAM(X).sub.n=3-50
WGQGTLVTVSS SEQ ID NO: 4: linker GGGGSGGGGSGGGGSGGGGS
[0064] In another aspect, the invention provides methods for the
humanization of non-human antibodies by grafting CDRs of non-human
donor antibodies onto stable and soluble antibody frameworks. In a
particularly preferred embodiment, the CDRs stem from rabbit
antibodies and the frameworks are those described above.
[0065] A general method for grafting CDRs into human acceptor
frameworks has been disclosed by Winter in U.S. Pat. No. 5,225,539
and by Queen et al. in WO09007861A1, which are hereby incorporated
by reference in their entirety. The general strategy for grafting
CDRs from rabbit monoclonal antibodies onto selected frameworks is
related to that of Winter et al. and Queen et al., but diverges in
certain key respects. In particular, the methods of the invention
diverge from the typical Winter and Queen methodology known in the
art in that the human antibody frameworks as disclosed herein are
particularly suitable as acceptors for human or non-human donor
antibodies. Thus, unlike the general method of Winter and Queen,
the framework sequence used for the humanization methods of the
invention is not necessarily the framework sequence which exhibits
the greatest sequence similarity to the sequence of the non-human
(e.g., rabbit) antibody from which the donor CDRs are derived. In
addition, framework residue grafting from the donor sequence to
support CDR conformation is not required. At most, antigen binding
amino acids located in the framework or other mutations that
occurred during somatic hypermutation may be introduced.
[0066] Particular details of the grafting methods to generate
humanized rabbit-derived antibodies with high solubility and
stability are described below.
[0067] In exemplary embodiments of the methods of the invention,
the amino acid sequence of the CDR donor antibody is first
identified and the sequences aligned using conventional sequence
alignment tools (e.g., Needleman-Wunsch algorithm and Blossum
matrices). The introduction of gaps and nomenclature of residue
positions may be done using a conventional antibody numbering
system. For example, the AHo numbering system for immunoglobulin
variable domains may be used. The Kabat numbering scheme may also
be applied since it is the most widely adopted standard for
numbering the residues in an antibody. Kabat numbering may e.g. be
assigned using the SUBIM program. This program analyses variable
regions of an antibody sequence and numbers the sequence according
to the system established by Kabat and co-workers (Deret et al
1995). The definition of framework and CDR regions is generally
done following the Kabat definition which is based on sequence
variability and is the most commonly used. However, for CDR-H1, the
designation is preferably a combination of the definitions of
Kabat's, mean contact data generated by analysis of contacts
between antibody and antigen of a subset of 3D complex structures
(MacCallum et al., 1996) and Chotia's which is based on the
location of the structural loop regions (see also FIG. 1).
Conversion tables for the two different numbering systems used to
identify amino acid residue positions in antibody heavy and light
chain variable regions are provided in A. Honegger, J. Mol. Biol.
309 (2001) 657-670. The Kabat numbering system is described further
in Kabat et al. (Kabat, E. A., et al. (1991) Sequences of Proteins
of Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242). The AHo numbering
system is described further in Honegger, A. and Pluckthun, A.
(2001) J. Mol. Biol. 309:657-670).
[0068] The variable domains of the rabbit monoclonal antibodies may
e.g. be classified into corresponding human sub-groups using e.g.
an EXCEL implementation of sequence analysis algorithms and
classification methods based on analysis of the human antibody
repertoire (Knappik et al., 2000, J. Mol Biol. February 11;
296(1):57-86).
[0069] CDR conformations may be assigned to the donor antigen
binding regions, subsequently residue positions required to
maintain the different canonical structures can also be identified.
The CDR canonical structures for five of the six antibody
hypervariable regions of rabbit antibodies (L1, L2, L3, H1 and H2)
are determined using Chothia's (1989) definition.
[0070] The antibodies of the invention may be further optimized to
show enhanced functional properties, e.g., enhanced solubility
and/or stability. In certain embodiments, the antibodies of the
invention are optimized according to the "functional consensus"
methodology disclosed in PCT Application Serial No.
PCT/EP2008/001958, entitled "Sequence Based Engineering and
Optimization of Single Chain Antibodies", filed on Mar. 12, 2008,
which is incorporated herein by reference.
[0071] Exemplary framework residue positions for substitution and
exemplary framework substitutions are described in PCT Application
No. PCT/CH2008/000285, entitled "Methods of Modifying Antibodies,
and Modified Antibodies with Improved Functional Properties", filed
on Jun. 25, 2008, and PCT Application No. PCT/CH2008/000284,
entitled "Sequence Based Engineering and Optimization of Single
Chain Antibodies", filed on Jun. 25, 2008.
[0072] In other embodiments, the immunobinders of the invention
comprise one or more of the stability enhancing mutations described
in U.S. Provisional Application Ser. No. 61/075,692, entitled
"Solubility Optimization of Immunobinders", filed on Jun. 25, 2008.
In certain preferred embodiments, the immunobinder comprises a
solubility enhancing mutation at an amino acid position selected
from the group of heavy chain amino acid positions consisting of
12, 103 and 144 (AHo Numbering convention). In one preferred
embodiment, the immunobinder comprises one or more substitutions
selected from the group consisting of: (a) Serine (S) at heavy
chain amino acid position 12; (b) Serine (S) or Threonine (T) at
heavy chain amino acid position 103; and (c) Serine (S) or
Threonine (T) at heavy chain amino acid position 144. In another
embodiment, the immunobinder comprises the following substitutions:
(a) Serine (S) at heavy chain amino acid position 12; (b) Serine
(S) or Threonine (T) at heavy chain amino acid position 103; and
(c) Serine (S) or Threonine (T) at heavy chain amino acid position
144.
[0073] In certain preferred embodiments, the immunobinder comprises
stability enhancing mutations at a framework residue of the light
chain acceptor framework in at least one of positions 1, 3, 4, 10,
47, 57, 91 and 103 of the light chain variable region according to
the AHo numbering system. In a preferred embodiment, the light
chain acceptor framework comprises one or more substitutions
selected from the group consisting of (a) glutamic acid (E) at
position 1, (b) valine (V) at position 3, (c) leucine (L) at
position 4; (d) Serine (S) at position 10; (e) Arginine (R) at
position 47; (e) Serine (S) at position 57; (0 phenylalanine (F) at
position 91; and (g) Valine (V) at position 103.
[0074] One can use any of a variety of available methods to produce
a humanized antibody comprising a mutation as described above.
[0075] Accordingly, the present invention provides an immunobinder
humanized according to the method described herein.
[0076] In certain preferred embodiments, the target antigen of said
immunobinder is VEGF or TNF.alpha..
[0077] The polypeptides described in the present invention or
generated by a method of the present invention can, for example, be
synthesized using techniques known in the art. Alternatively
nucleic acid molecules encoding the desired variable regions can be
synthesized and the polypeptides produced by recombinant
methods.
[0078] For example, once the sequence of a humanized variable
region has been decided upon, that variable region or a polypeptide
comprising it can be made by techniques well known in the art of
molecular biology. More specifically, recombinant DNA techniques
can be used to produce a wide range of polypeptides by transforming
a host cell with a nucleic acid sequence (e.g., a DNA sequence that
encodes the desired variable region (e.g., a modified heavy or
light chain; the variable domains thereof, or other antigen-binding
fragments thereof)).
[0079] In one embodiment, one can prepare an expression vector
including a promoter that is operably linked to a DNA sequence that
encodes at least V.sub.H or V.sub.L. If necessary, or desired, one
can prepare a second expression vector including a promoter that is
operably linked to a DNA sequence that encodes the complementary
variable domain (i.e., where the parent expression vector encodes
V.sub.H, the second expression vector encodes V.sub.L and vice
versa). A cell line (e.g., an immortalized mammalian cell line) can
then be transformed with one or both of the expression vectors and
cultured under conditions that permit expression of the chimeric
variable domain or chimeric antibody (see, e.g., International
Patent Application No. PCT/GB85/00392 to Neuberger et. al.).
[0080] In one embodiment, variable regions comprising donor CDRs
and acceptor FR amino acid sequences can be made and then changes
introduced into the nucleic acid molecules to effect the CDR amino
acid substitution.
[0081] Exemplary art recognized methods for making a nucleic acid
molecule encoding an amino acid sequence variant of a polypeptide
include, but are not limited to, preparation by site-directed (or
oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and
cassette mutagenesis of an earlier prepared DNA encoding the
polypeptide.
[0082] Site-directed mutagenesis is a preferred method for
preparing substitution variants. This technique is well known in
the art (see, e.g., Carter et al. Nucleic Acids Res. 13:4431-4443
(1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA 82:488
(1987)). Briefly, in carrying out site-directed mutagenesis of DNA,
the parent DNA is altered by first hybridizing an oligonucleotide
encoding the desired mutation to a single strand of such parent
DNA. After hybridization, a DNA polymerase is used to synthesize an
entire second strand, using the hybridized oligonucleotide as a
primer, and using the single strand of the parent DNA as a
template. Thus, the oligonucleotide encoding the desired mutation
is incorporated in the resulting double-stranded DNA.
[0083] PCR mutagenesis is also suitable for making amino acid
sequence variants of polypeptides. See Higuchi, in PCR Protocols,
pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids
Res. 17:723-733 (1989). Briefly, when small amounts of template DNA
are used as starting material in a PCR, primers that differ
slightly in sequence from the corresponding region in a template
DNA can be used to generate relatively large quantities of a
specific DNA fragment that differs from the template sequence only
at the positions where the primers differ from the template.
[0084] Another method for preparing variants, cassette mutagenesis,
is based on the technique described by Wells et al., Gene
34:315-323 (1985). The starting material is the plasmid (or other
vector) comprising the DNA to be mutated. The codon(s) in the
parent DNA to be mutated are identified. There must be a unique
restriction endonuclease site on each side of the identified
mutation site(s). If no such restriction sites exist, they may be
generated using the above-described oligonucleotide-mediated
mutagenesis method to introduce them at appropriate locations in
the DNA encoding the polypeptide. The plasmid DNA is cut at these
sites to linearize it. A double-stranded oligonucleotide encoding
the sequence of the DNA between the restriction sites but
containing the desired mutation(s) is synthesized using standard
procedures, wherein the two strands of the oligonucleotide are
synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as
the cassette. This cassette is designed to have 5' and 3' ends that
are compatible with the ends of the linearized plasmid, such that
it can be directly ligated to the plasmid. This plasmid now
contains the mutated DNA sequence.
[0085] A variable region generated by the methods of the invention
can be re-modeled and further altered to further increase antigen
binding. Thus, the steps described above can be preceded or
followed by additional steps, including, e.g. affinity maturation.
In addition, empirical binding data can be used for further
optimization.
[0086] Aside from amino acid substitutions, the present invention
contemplates other modifications, e.g., to Fc region amino acid
sequences in order to generate an Fc region variant with altered
effector function. One may, for example, delete one or more amino
acid residues of the Fc region in order to reduce or enhance
binding to an FcR. In one embodiment, one or more of the Fc region
residues can be modified in order to generate such an Fc region
variant. Generally, no more than one to about ten Fc region
residues will be deleted according to this embodiment of the
invention. The Fc region herein comprising one or more amino acid
deletions will preferably retain at least about 80%, and preferably
at least about 90%, and most preferably at least about 95%, of the
starting Fc region or of a native sequence human Fc region.
[0087] In one embodiment, the polypeptides described in the present
invention or generated by a method of the present invention, e.g.,
humanized Ig variable regions and/or polypeptides comprising
humanized Ig variable regions may be produced by recombinant
methods. For example, a polynucleotide sequence encoding a
polypeptide can be inserted in a suitable expression vector for
recombinant expression. Where the polypeptide is an antibody,
polynucleotides encoding additional light and heavy chain variable
regions, optionally linked to constant regions, may be inserted
into the same or different expression vector. An affinity tag
sequence (e.g. a His(6) tag) may optionally be attached or included
within the polypeptide sequence to facilitate downstream
purification. The DNA segments encoding immunoglobulin chains are
the operably linked to control sequences in the expression
vector(s) that ensure the expression of immunoglobulin
polypeptides. Expression control sequences include, but are not
limited to, promoters (e.g., naturally-associated or heterologous
promoters), signal sequences, enhancer elements, and transcription
termination sequences. Preferably, the expression control sequences
are eukaryotic promoter systems in vectors capable of transforming
or transfecting eukaryotic host cells. Once the vector has been
incorporated into the appropriate host, the host is maintained
under conditions suitable for high level expression of the
nucleotide sequences, and the collection and purification of the
polypeptide.
[0088] These expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers (e.g., ampicillin-resistance,
hygromycin-resistance, tetracycline resistance or neomycin
resistance) to permit detection of those cells transformed with the
desired DNA sequences (see, e.g., U.S. Pat. No. 4,704,362).
[0089] E. coli is one prokaryotic host particularly useful for
cloning the polynucleotides (e.g., DNA sequences) of the present
invention. Other microbial hosts suitable for use include bacilli,
such as Bacillus subtilus, and other enterobacteriaceae, such as
Salmonella, Serratia, and various Pseudomonas species. Other
microbes, such as yeast, are also useful for expression.
Saccharomyces and Pichia are exemplary yeast hosts, with suitable
vectors having expression control sequences (e.g., promoters), an
origin of replication, termination sequences and the like as
desired. Typical promoters include 3-phosphoglycerate kinase and
other glycolytic enzymes. Inducible yeast promoters include, among
others, promoters from alcohol dehydrogenase, isocytochrome C, and
enzymes responsible for methanol, maltose, and galactose
utilization.
[0090] Within the scope of the present invention, E. coli and S.
cerevisiae are preferred host cells.
[0091] In addition to microorganisms, mammalian tissue culture may
also be used to express and produce the polypeptides of the present
invention (e.g., polynucleotides encoding immunoglobulins or
fragments thereof). See Winnacker, From Genes to Clones, VCH
Publishers, N.Y., N.Y. (1987). Eukaryotic cells are actually
preferred, because a number of suitable host cell lines capable of
secreting heterologous proteins (e.g., intact immunoglobulins) have
been developed in the art, and include CHO cell lines, various Cos
cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed
B-cells, and hybridomas. Expression vectors for these cells can
include expression control sequences, such as an origin of
replication, a promoter, and an enhancer (Queen et al., Immunol.
Rev. 89:49 (1986)), and necessary processing information sites,
such as ribosome binding sites, RNA splice sites, polyadenylation
sites, and transcriptional terminator sequences. Preferred
expression control sequences are promoters derived from
immunoglobulin genes, SV40, adenovirus, bovine papilloma virus,
cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149
(1992).
[0092] The vectors containing the polynucleotide sequences of
interest (e.g., the heavy and light chain encoding sequences and
expression control sequences) can be transferred into the host cell
by well-known methods, which vary depending on the type of cellular
host. For example, calcium chloride transfection is commonly
utilized for prokaryotic cells, whereas calcium phosphate
treatment, electroporation, lipofection, biolistics or viral-based
transfection may be used for other cellular hosts. (See generally
Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Press, 2nd ed., 1989). Other methods used to
transform mammalian cells include the use of polybrene, protoplast
fusion, liposomes, electroporation, and microinjection (see
generally, Sambrook et al., supra). For production of transgenic
animals, transgenes can be microinjected into fertilized oocytes,
or can be incorporated into the genome of embryonic stem cells, and
the nuclei of such cells transferred into enucleated oocytes.
[0093] The subject polypeptide can also be incorporated in
transgenes for introduction into the genome of a transgenic animal
and subsequent expression, e.g., in the milk of a transgenic animal
(see, e.g., Deboer et al. U.S. Pat. No. 5,741,957; Rosen U.S. Pat.
No. 5,304,489; and Meade U.S. Pat. No. 5,849,992. Suitable
transgenes include coding sequences for light and/or heavy chains
in operable linkage with a promoter and enhancer from a mammary
gland specific gene, such as casein or beta lactoglobulin.
[0094] Polypeptides can be expressed using a single vector or two
vectors. For example, antibody heavy and light chains may be cloned
on separate expression vectors and co-transfected into cells.
[0095] In one embodiment, signal sequences may be used to
facilitate expression of polypeptides of the invention.
[0096] Once expressed, the polypeptides can be purified according
to standard procedures of the art, including ammonium sulfate
precipitation, affinity columns (e.g., protein A or protein G),
column chromatography, HPLC purification, gel electrophoresis and
the like (see generally Scopes, Protein Purification
(Springer-Verlag, N.Y., (1982)).
[0097] Either the humanized Ig variable regions or polypeptides
comprising them can be expressed by host cells or cell lines in
culture. They can also be expressed in cells in vivo. The cell line
that is transformed (e.g., transfected) to produce the altered
antibody can be an immortalized mammalian cell line, such as those
of lymphoid origin (e.g., a myeloma, hybridoma, trioma or quadroma
cell line). The cell line can also include normal lymphoid cells,
such as B-cells, that have been immortalized by transformation with
a virus (e.g., the Epstein-Barr virus).
[0098] Although typically the cell line used to produce the
polypeptide is a mammalian cell line, cell lines from other sources
(such as bacteria and yeast) can also be used. In particular, E.
coli-derived bacterial strains can be used, especially, e.g., phage
display.
[0099] Some immortalized lymphoid cell lines, such as myeloma cell
lines, in their normal state, secrete isolated Ig light or heavy
chains. If such a cell line is transformed with a vector that
expresses an altered antibody, prepared during the process of the
invention, it will not be necessary to carry out the remaining
steps of the process, provided that the normally secreted chain is
complementary to the variable domain of the Ig chain encoded by the
vector prepared earlier.
[0100] If the immortalized cell line does not secrete or does not
secrete a complementary chain, it will be necessary to introduce
into the cells a vector that encodes the appropriate complementary
chain or fragment thereof.
[0101] In the case where the immortalized cell line secretes a
complementary light or heavy chain, the transformed cell line may
be produced for example by transforming a suitable bacterial cell
with the vector and then fusing the bacterial cell with the
immortalized cell line (e.g., by spheroplast fusion).
Alternatively, the DNA may be directly introduced into the
immortalized cell line by electroporation.
[0102] In one embodiment, a humanized Ig variable region as
described in the present invention or generated by a method of the
present invention can be present in an antigen-binding fragment of
any antibody. The fragments can be recombinantly produced and
engineered, synthesized, or produced by digesting an antibody with
a proteolytic enzyme. For example, the fragment can be a Fab
fragment; digestion with papain breaks the antibody at the region,
before the inter-chain (i.e., V.sub.H-V.sub.H) disulphide bond,
that joins the two heavy chains. This results in the formation of
two identical fragments that contain the light chain and the
V.sub.H and C.sub.H1 domains of the heavy chain. Alternatively, the
fragment can be an F(ab').sub.2 fragment. These fragments can be
created by digesting an antibody with pepsin, which cleaves the
heavy chain after the inter-chain disulfide bond, and results in a
fragment that contains both antigen-binding sites. Yet another
alternative is to use a "single chain" antibody. Single-chain Fv
(scFv) fragments can be constructed in a variety of ways. For
example, the C-terminus of V.sub.H can be linked to the N-terminus
of V.sub.L. Typically, a linker (e.g., (GGGGS).sub.4; SEQ ID NO: 4)
is placed between V.sub.H and V.sub.L. However, the order in which
the chains can be linked can be reversed, and tags that facilitate
detection or purification (e.g., Myc-, His-, or FLAG-tags) can be
included (tags such as these can be appended to any antibody or
antibody fragment of the invention; their use is not restricted to
scFv). Accordingly, and as noted below, tagged antibodies are
within the scope of the present invention. In alternative
embodiments, the antibodies described herein, or generated by the
methods described herein, can be heavy chain dimers or light chain
dimers. Still further, an antibody light or heavy chain, or
portions thereof, for example, a single domain antibody (DAb), can
be used.
[0103] In another embodiment, a humanized Ig variable region as
described in the present invention or generated by a method of the
present invention is present in a single chain antibody (ScFv) or a
minibody (see e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1).
Minibodies are dimeric molecules made up of two polypeptide chains
each comprising an ScFv molecule (a single polypeptide comprising
one or more antigen binding sites, e.g., a V.sub.L domain linked by
a flexible linker to a V.sub.H domain fused to a CH3 domain via a
connecting peptide). ScFv molecules can be constructed in a
V.sub.H-linker-V.sub.L orientation or V.sub.L-linker-V.sub.H
orientation. The flexible hinge that links the V.sub.L and V.sub.H
domains that make up the antigen binding site preferably comprises
from about 10 to about 50 amino acid residues. An exemplary
connecting peptide for this purpose is (Gly4Ser)3 (Huston et al.
(1988). PNAS, 85:5879). Other connecting peptides are known in the
art.
[0104] Methods of making single chain antibodies are well known in
the art, e.g., Ho et al. (1989), Gene, 77:51; Bird et al. (1988),
Science 242:423; Pantoliano et al. (1991), Biochemistry 30:10117;
Milenic et al. (1991), Cancer Research, 51:6363; Takkinen et al.
(1991), Protein Engineering 4:837. Minibodies can be made by
constructing an ScFv component and connecting peptide-CH.sub.3
component using methods described in the art (see, e.g., U.S. Pat.
No. 5,837,821 or WO 94/09817A1). These components can be isolated
from separate plasmids as restriction fragments and then ligated
and recloned into an appropriate vector. Appropriate assembly can
be verified by restriction digestion and DNA sequence analysis. In
one embodiment, a minibody of the invention comprises a connecting
peptide. In one embodiment, the connecting peptide comprises a
Gly/Ser linker, e.g., GGGSSGGGSGG (SEQ ID NO: 6).
[0105] In another embodiment, a tetravalent minibody can be
constructed. Tetravalent minibodies can be constructed in the same
manner as minibodies, except that two ScFv molecules are linked
using a flexible linker, e.g., having an amino acid sequence
(G.sub.4S).sub.4G.sub.3AS (SEQ ID NO: 7).
[0106] In another embodiment, a humanized variable region as
described in the present invention or generated by a method of the
present invention can be present in a diabody. Diabodies are
similar to scFv molecules, but usually have a short (less than 10
and preferably 1-5) amino acid residue linker connecting both
variable domains, such that the V.sub.L and V.sub.H domains on the
same polypeptide chain can not interact. Instead, the V.sub.L and
V.sub.H domain of one polypeptide chain interact with the V.sub.H
and V.sub.L domain (respectively) on a second polypeptide chain (WO
02/02781).
[0107] In another embodiment, a humanized variable region of the
invention can be present in an immunoreactive fragment or portion
of an antibody (e.g. an scFv molecule, a minibody, a tetravalent
minibody, or a diabody) operably linked to an FcR binding portion.
In an exemplary embodiment, the FcR binding portion is a complete
Fc region.
[0108] Preferably, the humanization methods described herein result
in Ig variable regions in which the affinity for antigen is not
substantially changed compared to the donor antibody.
[0109] In one embodiment, polypeptides comprising the variable
domains of the instant invention bind to antigens with a binding
affinity greater than (or equal to) an association constant Ka of
about 10.sup.5 M.sup.-1, 10.sup.6 M.sup.-1, 10.sup.7 M.sup.-1,
10.sup.8 M.sup.-1, 10.sup.9 M.sup.-1, 10.sup.10 M.sup.-1,
10.sup.11M.sup.-1, or 10.sup.12 M.sup.-1, (including affinities
intermediate of these values).
[0110] Affinity, avidity, and/or specificity can be measured in a
variety of ways. Generally, and regardless of the precise manner in
which affinity is defined or measured, the methods of the invention
improve antibody affinity when they generate an antibody that is
superior in any aspect of its clinical application to the antibody
(or antibodies) from which it was made (for example, the methods of
the invention are considered effective or successful when a
modified antibody can be administered at a lower dose or less
frequently or by a more convenient route of administration than an
antibody (or antibodies) from which it was made).
[0111] More specifically, the affinity between an antibody and an
antigen to which it binds can be measured by various assays,
including, e.g., an ELISA assay, a BiaCore assay or the KinExA.TM.
3000 assay (available from Sapidyne Instruments (Boise, Id.)).
Briefly, sepharose beads are coated with antigen (the antigen used
in the methods of the invention can be any antigen of interest
(e.g., a cancer antigen; a cell surface protein or secreted
protein; an antigen of a pathogen (e.g., a bacterial or viral
antigen (e.g., an HIV antigen, an influenza antigen, or a hepatitis
antigen)), or an allergen) by covalent attachment. Dilutions of
antibody to be tested are prepared and each dilution is added to
the designated wells on a plate. A detection antibody (e.g. goat
anti-human IgG-HRP conjugate) is then added to each well followed
by a chromagenic substrate (, e.g. HRP). The plate is then read in
ELISA plate reader at 450 nM, and EC50 values are calculated. (It
is understood, however, that the methods described here are
generally applicable; they are not limited to the production of
antibodies that bind any particular antigen or class of
antigens.)
[0112] Those of ordinary skill in the art will recognize that
determining affinity is not always as simple as looking at a single
figure. Since antibodies have two arms, their apparent affinity is
usually much higher than the intrinsic affinity between the
variable region and the antigen (this is believed to be due to
avidity). Intrinsic affinity can be measured using scFv or Fab
fragments.
[0113] In another aspect, the present invention features a
humanized rabbit antibody, or a fragment thereof, conjugated to a
therapeutic moiety, such as a cytotoxin, a drug (e.g., an
immunosuppressant) or a radiotoxin. Such conjugates are referred to
herein as "immunoconjugates".
[0114] The antibody conjugates of the invention can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0115] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0116] In one aspect the invention provides pharmaceutical
formulations comprising humanized rabbit antibodies for the
treatment disease. The term "pharmaceutical formulation" refers to
preparations which are in such form as to permit the biological
activity of the antibody or antibody derivative to be unequivocally
effective, and which contain no additional components which are
toxic to the subjects to which the formulation would be
administered. "Pharmaceutically acceptable" excipients (vehicles,
additives) are those which can reasonably be administered to a
subject mammal to provide an effective dose of the active
ingredient employed.
EQUIVALENTS
[0117] Numerous modifications and alternative embodiments of the
present invention will be apparent to those skilled in the art in
view of the foregoing description. Accordingly, this description is
to be construed as illustrative only and is for the purpose of
teaching those skilled in the art the best mode for carrying out
the present invention. Details of the structure may vary
substantially without departing from the spirit of the invention,
and exclusive use of all modifications that come within the scope
of the appended claims is reserved. It is intended that the present
invention be limited only to the extent required by the appended
claims and the applicable rules of law.
[0118] All literature and similar material cited in this
application, including, patents, patent applications, articles,
books, treatises, dissertations, web pages, figures and/or
appendices, regardless of the format of such literature and similar
materials, are expressly incorporated by reference in their
entirety. In the event that one or more of the incorporated
literature and similar materials differs from or contradicts this
application, including defined terms, term usage, described
techniques, or the like, this application controls.
Sequence CWU 1
1
71232PRTArtificialvariable heavy chain framework a58 1Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser 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 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp
Val Arg Gln Ala 65 70 75 80 Pro Gly Lys Gly Leu Glu Trp Val Ser Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Phe Ser Val Ser 130 135 140 Arg Asp
Asn Ser Lys Asn Thr Val Tyr Leu Gln Ile Asn Ser Leu Arg 145 150 155
160 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Met Xaa Xaa Xaa Xaa Xaa
165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Trp Gly Gln 210 215 220 Gly Thr Leu Val Thr Val Ser Ser
225 230 2231PRTArtificialvariable light chain framework KI27 2Glu
Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Ile Ile Thr Cys 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 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp
Tyr Gln Gln Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140
Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu 145
150 155 160 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa
Xaa Xaa 165 170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu
Gly 225 230 3483PRTArtificialframework sequence 3Glu Ile Val Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Ile Ile Thr Cys 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 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Tyr Gln Gln
Lys Pro Gly 65 70 75 80 Lys Ala Pro Lys Leu Leu Ile Tyr Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Gly Val Pro Ser Arg Phe 130 135 140 Ser Gly Ser
Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser Ser Leu 145 150 155 160
Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa 165
170 175 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 180 185 190 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Phe Gly Gln Gly 210 215 220 Thr Lys Leu Thr Val Leu Gly Gly Gly
Gly Gly Ser Gly Gly Gly Gly 225 230 235 240 Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Val 245 250 255 Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 260 265 270 Cys Ala
Ala Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 275 280 285
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290
295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu 325 330 335 Glu Trp Val Ser Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa
Xaa Xaa Arg Phe Ser Val Ser Arg Asp Asn Ser Lys 385 390 395 400 Asn
Thr Val Tyr Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala 405 410
415 Val Tyr Tyr Cys Ala Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
420 425 430 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 435 440 445 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 450 455 460 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Gly
Gln Gly Thr Leu Val Thr 465 470 475 480 Val Ser Ser
420PRTArtificiallinker sequence 4Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20
55PRTArtificiallinker 5Gly Gly Gly Gly Ser 1 5
611PRTArtificiallinker sequence 6Gly Gly Gly Ser Ser Gly Gly Gly
Ser Gly Gly 1 5 10 725PRTArtificiallinker sequence 7Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly
Gly Ser Gly Gly Gly Ala Ser 20 25
* * * * *