Note: Descriptions are shown in the official language in which they were submitted.
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ACCEPTOR FRAMEWORK FOR CDR GRAFTING
Background of the Invention
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;
U55,693,761). 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
unkown
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. US patent No.
7429487).
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This technology, however, is not widly 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.
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.
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 W008/144757 and
W005/016950 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.
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.
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
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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.
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.
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.
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Summary of the Invention
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, "a72" 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.
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.
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
Figure 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.
Figure 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.
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Detailed Description of the Invention
Definitions
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.
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 VH) 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 VL) and a light chain constant region. The light chain
constant
region is comprised of one domain, CL. The VH and VL 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 VH and VL 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.
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.
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 VI, VH, CL and CH1 domains; (ii) a F(abt)2 fragment, a bivalent
fragment
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comprising two Fab fragments linked by a disulfide bridge at the hinge region;
(iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment
consisting of
the VL and VH 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 VH
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, VL and VH, 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 VL
and VH
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 IgGl,
IgG2,
IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.
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 VL, VH, CL and CH1
domains;
(ii) a F(abt)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.,
3rd ed. 1993); (iv) a Fd fragment consisting of the VH and CH1 domains;
(v) a Fv
fragment consisting of the VL and VH 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 VH or VL 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 ; and (vii) a nanobody, a
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heavy chain variable region containing a single variable domain and two
constant
domains.
The term "single chain antibody", "single chain Fv" or "scFv" refers to a
molecule comprising an antibody heavy chain variable domain (or region; VH)
and an
antibody light chain variable domain (or region; VL) connected by a linker.
Such
scFv molecules can have the general structures: NH2-VL-linker-VH-COOH or NH2-
VH-linker-VL-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)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.
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.
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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 Ll, CDR L2, CDR L3, or Ll, 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 Figure 1 for
illustration).
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.
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).
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 (KD) of approximately
less
than 10-7 M, such as approximately less than 10 -8 M, 10-9 M or 10-10 M or
even lower.
The term "KD" 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 (KD) of less than
approximately
10-7 M, such as less than approximately 10-8 M, 10-9 M or 10-10 M or even
lower, for
example, as determined using surface plasmon resonance (SPR) technology in a
BIACORE instrument.
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-
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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.
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).
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.
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.
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
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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.
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.
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.
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.
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 a72 (SEQ ID NO: 1) was surprisingly found
to be in essence highly compatible with the antigen-binding sites of rabbit
antibodies.
Therefore, the a72 VH represents a suitable scaffold to construct stable
humanized
scFv antibody fragments derived from grafting of rabbit loops.
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.
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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
W003/097697 and designated KI27, or any other VL sequence as disclosed in
W003/097697.
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)4 peptide (SEQ ID NO: 4), or a linker as disclosed in
Alfthan et
al. (1995) Protein Eng. 8:725-731.
Accordingly, the present invention provides an immunobinder acceptor
framework comprising
(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
(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.
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.
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.
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.
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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.
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.
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
(i) variable light chain CDRs of a lagomorph; and
(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.
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.
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.
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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
(i) identifying the respective germ line progenitor sequence or,
alternatively,
by using the consensus sequences in case of highly homologous framework
sequences;
(ii) generating a sequence alignment of donor variable domain sequences with
germ line progenitor sequence or consensus sequence of step (i); and
(iii) identifying differing residues.
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.
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.
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.
The immunobinder as disclosed herein may e.g. be used in diagnostic
applications, therapeutic application, target validation or gene therapy.
The invention further provides an isolated nucleic acid encoding the
immunobinder acceptor framework disclosed herein or the immunobinder(s) as
disclosed herein.
In another embodiment, a vector is provided which comprises the nucleic acid
disclosed herein.
The nucleic acid or the vector as disclosed herein can e.g. be used in gene
therapy.
The invention further encompasses a host cell comprising the vector and/or the
nucleic acid disclosed herein.
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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.
The sequences disclosed herein are the following (X residues are CDR
insertion sites and contain at least 3 and up to 50 amino acids):
SEQ ID NO.1: variable heavy chain framework a72
EVQLVESGPGLVKPSQTLSLTCGVS (X)11=3-50WIRQHPVKGLEWIG(X)11=3-50
RLTIS VDTS KTQVS LNLRS VTAADTAVYYCAR(X)n=3-50 WGQGTTVTVS S
SEQ ID NO. 2: variable light chain framework KI27
EIVMTQSPSTLSASVGDRVIITC(X)11=3-50 WYQQKPGKAPKLLIY(X)11=3-50
GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)11=3-50 FGQGTKLT VLG
SEQ ID NO. 3: framework sequence
EIVMTQSPSTLSASVGDRVIITC(X)11=3-50 WYQQKPGKAPKLLIY(X)11=3-50
GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC(X)11=3-
50FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGPGLVKPSQTLSL
TCGVS(X)11=3-50WIRQHPVKGLEWIG(X)11=3-50
RLTIS VDTS KTQVS LNLRS VTAADTAVYYCAR(X)n= 3 -50 WGQGTTVTVS S
SEQ ID NO: 4: linker
GGGGSGGGGSGGGGSGGGGS
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.
A general method for grafting CDRs into human acceptor frameworks has
been disclosed by Winter in US Patent No. 5,225,539 and by Queen et al. in
W09007861A1, 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
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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.
Particular details of the grafting methods to generate humanized rabbit-
derived
antibodies with high solubility and stability are described below.
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 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-
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3242). The AHo numbering system is described further in Honegger, A. and
Pluckthun, A. (2001) J. Mol. Biol. 309:657-670).
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. Feb 11;296(1):57-
86).
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.
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 March 12, 2008, which is incorporated
herein by
reference.
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 June 25, 2008, and PCT Application No. PCT/
CH2008/000284, entitled "Sequence Based Engineering and Optimization of Single
Chain Antibodies", filed on June 25, 2008.
In other embodiments, the immunobinders of the invention comprise one or
more of the stability enhancing mutations described in U.S. Provisional
Application
Serial No. 61/075,692, entitled "Solubility Optimization of Immunobinders",
filed on
June 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
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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.
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; (f) phenylalanine (F) at position 91; and (g)
Valine (V) at
position 103.
One can use any of a variety of available methods to produce a humanized
antibody comprising a mutation as described above.
Accordingly, the present invention provides an immunobinder humanized
according to the method described herein.
In certain preferred embodiments, the target antigen of said immunobinder is
VEGF or TNFa.
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.
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)).
In one embodiment, one can prepare an expression vector including a
promoter that is operably linked to a DNA sequence that encodes at least VH or
VL. 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
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variable domain (i.e., where the parent expression vector encodes VH, the
second
expression vector encodes VL 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.).
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.
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.
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.
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.
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
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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.
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.
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.
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
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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.
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. Patent
No. 4,704,362).
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.
Within the scope of the present invention, E. coli and S. cerevisiae are
preferred host cells.
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,
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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).
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.
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. 5,741,957; Rosen 5,304,489; and
Meade
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.
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.
In one embodiment, signal sequences may be used to facilitate expression of
polypeptides of the invention.
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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)).
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).
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. co/i-derived bacterial strains can be used,
especially,
e.g., phage display.
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.
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.
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.
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
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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., VH-VH)
disulphide bond, that joins the two heavy chains. This results in the
formation of two
identical fragments that contain the light chain and the VH and CH1 domains of
the
heavy chain. Alternatively, the fragment can be an F(ab')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 VH can be linked to the N-terminus of VL.
Typically,
a linker (e.g., (GGGGS)4; SEQ ID NO: 4) is placed between VH and VL. 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.
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., US 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 VL domain linked by a flexible linker to a VH
domain
fused to a CH3 domain via a connecting peptide). ScFv molecules can be
constructed
in a VH-linker-VL orientation or VL-linker-VH orientation. The flexible hinge
that
links the VL and VH 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.
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;
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Takkinen et al. (1991), Protein Engineering 4:837. Minibodies can be made by
constructing an ScFv component and connecting peptide-CH3 component using
methods described in the art (see, e.g., US patent 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).
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 (G45)4G3A5 (SEQ ID NO: 7).
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 VL and VH domains on the same polypeptide chain can not
interact.
Instead, the VL and VH domain of one polypeptide chain interact with the VH
and VL
domain (respectively) on a second polypeptide chain (WO 02/02781).
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.
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.
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 105 M-1, 106 N4-1, 107 N4-1, 108 N4-1, 109 N4-
1, 1010N4-1,
1011 N4-1, 12 -
or 10 M1, (including affinities intermediate of these values).
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
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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).
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 KinEXATM 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.)
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.
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".
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
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A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis
factor
or interferon-y; 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.
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).
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
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
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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.
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.
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