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CA 02552999 2006-07-10
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Identification of Novel IgE Epitopes
CROSS RELATED APPLICATIONS .
[0001] The present application claims priority to PCT Application No.
PCT/US04/02892
and PCT/US04/02894 filed Feb. 2, 2004, all of which are incorporated by
reference herein.
BACKGROUND OF THE INVENTION
[0002] Allergy is a hypersensitive state induced by an exaggerated immune
response to
a foreign agent, such as an allergen. Immediate (type I) hypersensitivity,
characterized by allergic reactions immediately following contact with the
allergen,
is mediated via B cells and is based on antigen-antibody reactions. Delayed
hypersensitivity is mediated via T cells and based on mechanisms of cellular
immunity. In recent years, the term "allergy" has become more and more
synonymous with type I hypersensitivity.
[0003] Immediate hypersensitivity is a response based on the production of
antibodies of
the immunoglobulin class E (IgE antibodies) by B cells which upon exposure to
an
allergen differentiate into antibody secreting plasma cells. The IgE induced
reaction is a local event occurring at the site of the allergen's entry into
the body,
i.e. at mucosal surfaces and/or at local lymph nodes. Locally produced IgE
will
first sensitize local mast cells, i.e. IgE antibodies bind with their~constant
regions
to FcE receptors on the surface of the mast cells, and then "spill-over" IgE
enters
the circulation and binds to receptors on both circulating basophils and
tissue-
fixed mast cells throughout the body. When the bound IgE is subsequently
contacted with the allergen, the FcE receptors are crosslinked by binding of
the
allergen causing the cells to degranulate and release a number of anaphylactic
mediators such as histamine, prostaglandins, leukotrienes, etc. It is the
release of
these substances which is responsible for the clinical symptoms typical of
immediate hypersensitivity, namely contraction of smooth muscle in the
respiratory tract or the intestine, the dilation of small blood vessels and
the
increase in their permeability to water and plasma proteins, the secretion of
mucus resulting, e.g in allergic rhinitis, atopic excema and asthma, and the
stimulation of nerve endings in the skin resulting in itching and pain. In
addition,
the reaction upon second contact with the allergen is intensified because some
B
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cells form a "memory pool" of surface IgE positive B cells (slgE+ B cells)
after the
first contact with the allergen by expressing IgE on the cell surface.
[0004] There are two major receptors for IgE, the high affinity receptor FceRl
and the low-
affinity receptor FcsRll. FceRl is predominantly expressed on the surface of
mast
cells and basophils, but low levels of FceRl can also be found on human
Langerhan's cells, dendritic cells, and monocytes, where it functions in IgE-
mediated allergen presentation. In addition, FcERI has been reported on human
eosinophils and platelets (Hasegawa, S. et. al., Hematopoiesis, 1999, 93:2543-
2551 ). FcERI is not found on the surface of B cells, T cells, or neutrophils.
The
expression of FceRl on Langerhan's cells and dermal dendritic cells is
functionally
and biologically important for IgE-bound antigen presentation in allergic
individuals
(Klubal R. et al., J. Invest. Dermatol. 1997, 108 (3):336-42).
[0005] The low-affinity receptor, Fc~Rll (CD23) is a lectin-like molecule
comprising three
identical subunits with head structures extending from a long a-helical coiled
stalk
from the cellular plasma membrane (Dierks, A.E. et al., J. Immunol. 1993,
150:2372-2382). Upon binding to IgE, FceRll associates with CD21 on B cells
involved in the regulation of synthesis of IgE (Sanon, A. et al., J. Allergy
Clin.
lmmunol. 1990, 86:333-344, Bonnefoy, J. et al., Eur. Resp. J. 1996, 9:63s-
66s).
FcERII has long been recognized for allergen presentation (Sutton and Gould
,1993, Nature, 366:421-428). IgE bound to Fc~Rll on epithelial cells is
responsible for specific and rapid allergen presentation (Yang, P.P., J. Clin.
Invest., 2000, 106:879-886). FcERII is present on several cell types,
including B-
cells, eosinophils, platelets, natural killer cells, T-cells, follicular
dendritic cells, and
Langerhan's cells.
[0006] The structural entities on the IgE molecule that interact with FceRl
and FcERII
have also been identified. Mutagenesis studies have indicated that the CH3
domain mediates IgE interaction with both FceRl (Presta et al., J. Biol. Chem.
1994, 269:26368-26373; Henry A.J. et al., Biochemistry, 1997, 36:15568-15578)
and FceRll (Sutton and Gould, Nature, 1993, 366: 421-428; Shi, J. et al.,
Biochemistry, 1997, 36:2112-2122). The binding sites for both high- and low-
affinity receptors are located symmetrically along a central rotational axis
through
the two CH3 domains. The FceRl-binding site is located in a CH3 domain on the
2
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outward side near the junction of the CH2 domain, whereas the FcERII-binding
site is on the carboxyl-terminus of CH3.
[0007] A promising concept for the treatment of allergy involves the
application of
monoclonal antibodies, which are IgE isotype-specific and are thus capable of
binding IgE. This approach is based on the inhibition of allergic reactions by
downregulating the IgE immune response, which is the earliest event in the
induction of allergy and provides for the maintenance of the allergic state.
As the
response of other antibody classes is not affected, both an immediate and a
long
lasting effect on allergic symptoms is achieved. Early studies of human
basophil
density showed a correlation between the level of IgE in the plasma of a
patient
and the number of FceRl receptors per basophil (Malveaux et al., J. Clin.
Invest.,
1978, 62:176). They noted that the FceRl densities in allergic and non-
allergic
persons range from 104 to 106 receptors per basophil. Later it was shown that
treatment of allergic diseases with anti-IgE decreased the amount of
circulating
IgE to 1 % of pretreatment levels (MacGlashan et al., J. Immunol., 1997,
158:1438-1445). MacGlashan analyzed serum obtained from patients treated
with whole anti-IgE antibody, which binds free IgE circulating in the serum of
the
patient. They reported that lowering the level of circulating IgE in a patient
resulted in a lower number of receptors present on the surface of basophils.
Thus, they hypothesized that FceRl density on the surface of basophils and
mast
cells is directly or indirectly regulated by the level of circulating IgE
antibody.
[0008] More recently, WO 99/62550 disclosed the use of IgE molecules and
fragments,
which bind to FcERI and FcERII IgE binding sites to block IgE binding to
receptors.
However, effective therapies that lack deleterious side effects for the
management
of these allergic diseases are limited. One therapeutic approach to treating
allergic diseases involved using humanized anti-IgE antibody to treat allergic
rhinitis and asthma (Corne, J. et al., J. Clin. Invest.1997, 99:879-887;
Racine-
Poon, A. et al., Clin. Pharmcol. Ther. 1997, 62:675-690; Fahy, J.V. et al.,
Am. J.
Resp. Crit. Care Med. 1997, 155:1824-1834; Boulet, L. P. et al., Am. J. Resp.
Crit.
Care Med., 1997, 155:1835-1840; Milgrom, E. et al., N. Engl. J. Med., 1999,
341:1966-1973). These clinical data demonstrate that inhibition of IgE binding
to
its receptors is an effective approach to treating allergic diseases.
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[0009] Antibodies suitable as anti-allergic agents should react with surface
IgE positive B
cells which differentiate into IgE producing plasma cells, so that they can be
used
to functionally eliminate those B cells. However, antibodies to IgE in
principle may
also induce mediator release from IgE sensitized mast cells by crosslinking
the
Fce receptors, thus antagonizing the beneficial effect exerted on the serum
IgE
and slgE+ B cell level. One of the potentially dangerous problems with
developing
anti-IgE therapies is the possibility of IgE-crosslinking caused by the
therapetic
antibody binding to IgE already bound to the high affinity receptor and
triggering
histamine release resulting in a potentially anaphylactic reaction.
[0010] Therefore, antibodies applicable for therapy of allergy must not be
capable of
reacting with IgE bound on sensitized mast cells and basophils, but should
retain
the capability to recognize slgE+ B cells. Such IgE isotype-specific
antibodies
have been described e.g. by Chang et al. (Biotechnology 8, 122-126 (1990)), in
European Patent No. EP0407392, and several U.S. Patents, e.g., U.S. Patent No.
5,449,760.
[0011] Peptides used to generate anti-IgE antibodies also suffer from the
dangerous
potential to induce anaphylactic antibodies. Generation of anti-IgE antibodies
during active vaccination may be capable of triggering histamine release in
the
same way passively administered anti-IgE antibodies, if the antibodies
generated
during immunization bind to IgE bound to the high affinity IgE receptor or by
other
mechanisms.
[0012] Thus, there is a need for higher affinity non-anaphylactic antibodies
that bind
specifically to IgE but do not bind IgE already bound to its high affinity
receptor, as
well as peptides for active immunization that do not induce anaphylactic
antibodies. The inventors have identified the specific epitope of IgE that
provides
high affinity binding of antibodies without binding to IgE on mast cells or
basophils.
These specific epitopes can in turn be used to gnerate specific peptides for
active
immunization to generate antibodies to IgE that only bind to the region of IgE
that
binds to the receptor, ensuring that the antibodies do not crosslink IgE
already
bound to the receptor and thus are non-anaphylactic.
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SUMMARY OF THE INVENTION
[0013] The present invention relates to novel peptide epitopes derived from
the CH3
domain of IgE. These peptide epitopes are recognized by high affinity
antibodies
that specifically bind IgE. These novel peptides may be used for both the
active
immunization of a mammal by administering these peptides to generate high
affinity antibodies in the mammal. The peptide epitopes may also be used in
generating high affinity anti-IgE antibodies in a non-human host that
specifically
bind to these regions of IgE and use the resulting antibodies for the passive
immunization of a of a mammal.
[0014] One immunogen (epitope A, Fig. 11 ) of the present invention comprises
the amino
acid sequence:
Asn Pro Arg Gly Val Ser Xaa Tyr Xaa Xaa Arg Xaa (SEQ ID NO. 72).
One example of epitope A is:
Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro (SEQ ID NO. 73)
Another immunogen (epitope B, Fig. 11 ) comprises the amino acid sequence:
Leu Pro Arg Ala Leu Xaa Arg Ser Xaa (SEQ ID NO. 74).
Examples of Epitope B include:
Leu Pro Arg Ala Leu Met Arg Ser Thr (SEQ ID NO. 75)
His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr (SEQ ID NO 76)
Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr (SEQ ID NO 77).
In either SEQ ID NO: 72 or SEQ ID NO: 74, Xaa may be any amino acid.
[0015] These peptides may be included in a composition comprising at least one
of the
peptides and a physiologically acceptable carrier, diluent, stabilizer or
excipient,
as well as an immunogenic carrier. The immunogenic carrier may be, e.g., BSA,
KLH, tetanus toxoid, and diphtheria toxoid. The present invention also relates
to
polynucleotides encoding SEQ ID NOS. 72-77, vectors comprising said
polynucleotides, and cells harboring said vectors.
[0016] The present invention also relates to antibodies that specifically bind
to epitope A
and/or epitope B. The present invention is also directed to a method of making
antibodies that specifically bind to epitope A and/or epitope B.
[0017] The present invention relates to the administration of peptides
comprising SEQ ID
NO: 72 and/or SEQ ID NO 74 to a subject suffering from an IgE-mediated disease
or condition.
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WO 2005/075504 PCT/US2004/024360
[0018] The present invention relates to the administration of high affinity
antibodies
generated using peptides comprising SEQ ID NO: 72 and/or SEQ ID NO 74 to a
mammal suffering from an IgE-mediated disease or condition. The high affinity
antibody may be human, humanized, or chimeric. The antibody may be polyclonal
or monoclonal. Such IgE mediated diseases or conditions include, e.g., asthma,
atopic dermatitis, urticaria, allergic rhinitis and eczema.
BRIEF DESCRIPTION OF THE FIGURES
[0019] Figure 1 is a schematic representation of the phage vector used in
antibody
cloning and screening.
(0020] Figure 2 is a schematic representation of oligonucleotides useful in
generating
antibody variants.
[0021] Figure 3A depicts the comparison of the light chains of the murine anti-
IgE
antibody TES-C21 and the combined human template of L16 and JK4.
[0022] Figure 3B depicts the comparison of the heavy chains of TES-C21 and the
combined human template DP88 and JH4b.
[0023] Figure 4 presents a table of the framework residue variants having high
affinity as
compared to the parent TES-C21.
[0024] Figure 5A and B depict the ELISA titration curves for clones 4, 49, 72,
78, and
136, as compared to the parent Fab of TES-C21 and negative control (5D12).
[0025] Figure 6 depicts an inhibition assay of clones 2C, 5A, and 51, as
compared to the
parent TES-C21 and a negative control antibody.
[0026] Figure 7A depicts the sequences of clones having a combination of
beneficial
mutations which resulted in even greater affinity for IgE.
[0027] Figure 8A & 8B depict the framework sequences of the entire light chain
variable
region for clones 136, 1, 2, 4, 8, 13, 15, 21, 30, 31, 35, 43, 44, 53, 81, 90,
and
113.
[0028] Figure 9A & 9B depict the framework sequences of the entire heavy chain
variable
region for 35 clones.
[0029] Figures 10 A-F depict the complete heavy and light chain sequences for
clones
136, 2C, 51, 5A, 2B, and 1136-2C.
[0030] Figure11 depicts the CH3 region amino acid sequence of human IgE and
highlights Epitope "A" and Epitope "B".
[0031] Figure 12 depicts the overlapping peptides used to identify Epitope B.
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[0032] Figure 13 depicts the identification of important residues in the
binding region of
Epitope A.
[0033] Figure 14 depicts the identification of important residues in the
binding region of
Epitope B.
[0034] Figure 15 depicts a western blot analysis of MAb binding to mutant
peptides.
[0035] Figure 16 depicts the generation of anti-IgE antibodies in a transgenic
animal
expressing human IgE.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0036] Terms used throughout this application are to be construed with
ordinary and
typical meaning to those of ordinary skill in the art. However, Applicants
desire
that the following terms be given the particular definition as defined below.
[0037] The phrase "substantially identical" with respect to an antibody chain
polypeptide
sequence may be construed as an antibody chain exhibiting at least 70%, or
80%,
or 90% or 95% sequence identity to the reference polypeptide sequence. The
term with respect to a nucleic acid sequence may be construed as a sequence of
nucleotides exhibiting at least about 85%, or 90%, or 95% or 97% sequence
identity to the reference nucleic acid sequence.
[0038] The term "identity" or "homology" shall be construed to mean the
percentage of
amino acid residues in the candidate sequence that are identical with the
residue
of a corresponding sequence to which it is compared, after aligning the
sequences
and introducing gaps, if necessary to achieve the maximum percent identity for
the entire sequence, and not considering any conservative substitutions as
part of
the sequence identity. Neither N- or C-terminal extensions nor insertions
shall be
construed as reducing identity or homology. Methods and computer programs for
the alignment are well known in the art. Sequence identity may be measured
using sequence analysis software.
[0039] The term "antibody" is used in the broadest sense, and specifically
covers
monoclonal antibodies (including full length monoclonal antibodies),
polyclonal
antibodies, and multispecific antibodies (e.g., bispecific antibodies).
Antibodies
(Abs) and immunoglobulins (Igs) are glycoproteins having the same structural
characteristics. While antibodies exhibit binding specificity to a specific
target,
immunoglobulins include both antibodies and other antibody-like molecules
which
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lack target specificity. Native antibodies and immunoglobulins are usually
heterotetrameric glycoproteins of about 150,000 daltons, composed of two
identical light (L) chains and two identical heavy (H) chains. Each heavy
chain has
at one end a variable domain (VH) followed by a number of constant domains.
Each light chain has a variable domain at one end (V~) and a constant domain
at
its other end. "High affinity" antibodies refers to those antibodies having a
binding
affinity of at least 10'~°, preferably 10'~Z.
[0040] As used herein, "anti-human IgE antibody" means an antibody which binds
to
human IgE in such a manner so as to inhibit or substantially reduce the
binding of
such IgE to the high affinity receptor, FceRl.
[0041] The term "variable" in the context of variable domain of antibodies,
refers to the
fact that certain portions of the variable domains differ extensively in
sequence
among antibodies and are used in the binding and specificity of each
particular
antibody for its particular target. However, the variability is not evenly
distributed
through the variable domains of antibodies. It is concentrated in three
segments
called complementarity determining regions (CDRs) also known as hypervariable
regions both in the light chain and the heavy chain variable domains. The more
highly conserved portions of variable domains are called the framework (FR).
The
variable domains of native heavy and light chains each comprise four FR
regions,
largely a adopting a a-sheet configuration, connected by three CDRs, which
form
loops connecting, and in some cases forming part of, the a-sheet structure.
The
CDRs in each chain are held together in close proximity by the FR regions and,
with the CDRs from the other chain, contribute to the formation of the target
binding site of antibodies (see Kabat et al.) As used herein, numbering of
immunoglobulin amino acid residues is done according to the immunoglobulin
amino acid residue numbering system of Kabat et al., (Sequences of Proteins of
Immunological Interest, National Institute of Health, Bethesda, Md. 1987),
unless
otherwise indicated.
[0042] The term "antibody fragment" refers to a portion of a full-length
antibody, generally
the target binding or variable region. Examples of antibody fragments include
Fab,
Fab', F(ab')2 and Fv fragments. The phrase "functional fragment or analog" of
an
antibody is a compound having qualitative biological activity in common with a
full-
length antibody. For example, a functional fragment or analog of an anti-IgE
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antibody is one which can bind to an IgE immunoglobulin in such a manner so as
to prevent or substantially reduce the ability of such molecule from having
the
ability to bind to the high affinity receptor, FceRl. As used herein,
"functional
fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')2
fragments. An
"Fv" fragment is the minimum antibody fragment which contains a complete
target
recognition and binding site. This region consists of a dimer of one heavy and
one
light chain variable domain in a tight, non-covalent association (VH -V~
dimer). It is
in this configuration that the three CDRs of each variable domain interact to
define
an target binding site on the surface of the VH -V~ dimer. Collectively, the
six
CDRs confer target binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs specific for an
target) has the ability to recognize and bind target, although at a lower
affinity than
the entire binding site. "Single-chain Fv" or "sFv" antibody fragments
comprise the
VH and V~ domains of an antibody, wherein these domains are present in a
single
polypeptide chain. Generally, the Fv polypeptide further comprises a
polypeptide
linker between the VH and V~ domains which enables the sFv to form the desired
structure for target binding.
[0043] The Fab fragment contains the constant domain of the light chain and
the first
constant domain (CH 1 ) of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxyl terminus of the
heavy
chain CH1 domain including one or more cysteines from the antibody hinge
region. F(ab') fragments are produced by cleavage of the disulfide bond at the
hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical
couplings of antibody fragments are known to those of ordinary skill in the
art.
[0044] The term "monoclonal antibody" as used herein refers to an antibody
obtained
from a population of substantially homogeneous antibodies, i.e., the
individual
antibodies comprising the population are identical except for possible
naturally
occurring mutations that may be present in minor amounts. Monoclonal
antibodies
are highly specific, being directed against a single targetic site.
Furthermore, in
contrast to conventional (polyclonal) antibody preparations which typically
include
different antibodies directed against different determinants (epitopes), each
monoclonal antibody is directed against a single determinant on the target. In
addition to their specificity, monoclonal antibodies are advantageous in that
they
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may be synthesized by the hybridoma culture, uncontaminated by other
immunoglobulins. The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous population of
antibodies, and is not to be construed as requiring production of the antibody
by
any particular method. For example, the monoclonal antibodies for use with the
present invention may be isolated from phage antibody libraries using the well
known techniques. The parent monoclonal antibodies to be used in accordance
with the present invention may be made by the hybridoma method first described
by Kohler and Milstein, Nature 256, 495 (1975), or may be made by recombinant
methods.
[0045] "Humanized" forms of non-human (e.g. murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab', F(ab')2 or other target-binding subsequences of antibodies) which
contain
minimal sequence derived from non-human immunoglobulin. In general, the
humanized antibody will comprise substantially all of at least one, and
typically
two, variable domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or substantially all
of
the FR regions are those of a human immunoglobulin consensus sequence. The
humanized antibody may also comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin template
chosen.
[0046] The terms "cell", "cell line" and "cell culture" include progeny. It is
also understood
that all progeny may not be precisely identical in DNA content, due to
deliberate or
inadvertent mutations. Variant progeny that have the same function or
biological
property, as screened for in the originally transformed cell, are included.
The "host
cells" used in the present invention generally are prokaryotic or eukaryotic
hosts.
[0047] "Transformation" of a cellular organism with DNA means introducing DNA
into an
organism so that the DNA is replicable, either as an extrachromosomal element
or
by chromosomal integration. "Transfection" of a cellular organism with DNA
refers
to the taking up of DNA, e.g., an expression vector, by the cell or organism
whether or not any coding sequences are in fact expressed. The terms
"transfected host cell" and "transformed" refer to a cell in which DNA was
introduced. The cell is termed "host cell" and it may be either prokaryotic or
eukaryotic. Typical prokaryotic host cells include various strains of E. coli.
Typical
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eukaryotic host cells are mammalian, such as Chinese hamster ovary or cells of
human origin. The introduced DNA sequence may be from the same species as
the host cell of a different species from the host cell, or it may be a hybrid
DNA
sequence, containing some foreign and some homologous DNA.
[004] The term "vector" means a DNA construct containing a DNA sequence which
is
operably linked to a suitable control sequence capable of effecting the
expression
of the DNA in a suitable host. Such control sequences include a promoter to
effect
transcription, an optional operator sequence to control such transcription, a
sequence encoding suitable mRNA ribosome binding sites, and sequences which
control the termination of transcription and translation. The vector may be a
plasmid, a phage particle, or simply a potential genomic insert. Once
transformed
into a suitable host, the vector may replicate and function independently of
the
host genome, or may in some instances, integrate into the genome itself. In
the
present specification, "plasmid" and "vector" are sometimes used
interchangeably,
as the plasmid is the most commonly used form of vector. However, the
invention
is intended to include such other forms of vectors which serve equivalent
function
as and which are, or become, known in the art.
[0049] The expression "control sequences" refers to DNA sequences necessary
for the
expression of an operably linked coding sequence in a particular host
organism.
The control sequences that are suitable for prokaryotes, for example, include
a
promoter, optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation signals, and
enhancers. DNA for a presequence or secretory leader may be operably linked to
DNA for a polypeptide if it is expressed as a preprotein that participates in
the
secretion of the polypeptide; a promoter or enhancer is operably linked to a
coding
sequence if it affects the transcription of the sequence; or a ribosome
binding site
is operably linked to a coding sequence if it affects the transcription of the
sequence; or a ribosome binding site is operably linked to a coding sequence
if it
is positioned so as to facilitate translation. Generally, "operably linked"
means that
the DNA sequences being linked are contiguous, and, in the case of a secretory
leader, contiguous and in reading phase. However, enhancers do not have to be
contiguous.
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[0050] "Mammal" for purposes of treatment refers to any animal classified as a
mammal,
including human, domestic and farm animals, nonhuman primates, and zoo,
sports, or pet animals, such as dogs, horses, cats, cows, etc.
[0051] The term "epitope tagged" when used herein in the context of a
polypeptide refers
to a polypeptide fused to an "epitope tag". The epitope tag polypeptide has
enough residues to provide an epitope against which an antibody can be made,
yet is short enough such that it does not interfere with activity of the
polypeptide.
The epitope tag preferably also is fairly unique so that the antibody does not
substantially cross-react with other epitopes. Suitable tag polypeptides
generally
have at least 6 amino acid residues and usually between about 8-50 amino acid
residues (preferably between about 9-30 residues). Examples include the flu HA
tag polypeptide and its antibody 12CA5 (Field et al, Mol Cell. Biol. 8: 2159-
2165
(1988))); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies
thereagainst (Evan et al., Mol Cell. Biol. 5(12): 3610-3616 (1985)); and the
Herpes
Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al.,
Protein
Engineering 3(6): 547-553 (1990)). In certain embodiments, the epitope tag may
be an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3 or
IgG4)
that is responsible for increasing the in vivo serum half-life of the IgG
molecule.
[0052] The word "label" when used herein refers to a detectable compound or
composition which can be conjugated directly or indirectly to a molecule or
protein, e.g., an antibody. The label may itself be detectable (e.g.,
radioisotope
labels or fluorescent labels) or, in the case of an enzymatic label, may
catalyze
chemical alteration of a substrate compound or composition which is
detectable.
[0053] As used herein, "solid phase" means a non-aqueous matrix to which the
antibody
of the present invention can adhere. Example of solid phases encompassed
herein include those formed partially or entirely of glass (e.g. controlled
pore
glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene,
polyvinyl
alcohol and silicones. In certain embodiments, depending on the context, the
solid
phase can comprise the well of an assay plate; in others it is a purification
column
(e.g. an affinity chromatography column).
[0054] As used herein, the term "IgE-mediated disorder" means a condition or
disease
which is characterized by the overproduction andlor hypersensitivity to the
immunoglobulin IgE. Specifically it would be construed to include conditions
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associated with anaphylactic hypersensitivity and atopic allergies, including
for
example: asthma, allergic rhinitis & conjunctivitis (hay fever), eczema,
urticaria,
atopic dermatitis, and food allergies. The serious physiological condition of
anaphylactic shock caused by, e.g., bee stings, snake bites, food or
medication, is
also encompassed under the scope of this term.
Generation of Antibodies
[0055] The starting or "parent" antibody may be prepared using techniques
available in
the art for generating such antibodies. These techniques are well known.
Exemplary methods for generating the starting antibody are described in more
detail in the following sections. These descriptions are possible alternatives
for
making or selecting a parent antibody and in no way limit the methods by which
such a molecule may be generated.
[0056] The antibody's binding affinity is determined prior to generating a
high affinity
antibody of the present invention. Also, the antibody may be subjected to
other
biological activity assays, e.g., in order to evaluate effectiveness as a
therapeutic.
Such assays are known in the art and depend on the target and intended use for
the antibody.
[0057] To screen for antibodies which bind to a particular epitope (e.g.,
those which block
binding of IgE to its high affinity receptor), a routine cross-blocking assay
such as
that described in "Antibodies: A Laboratory Manual" (Cold Spring Harbor
Laboratory, Ed Harlow and David Lane (1988)) can be performed. Alternatively,
epitope mapping can be performed to determine where the antibody binds an
epitope of interest. Optionally, the binding affinity of the antibody for a
homolog of
the target used to generate the antibody (where the homolog is from a
different
species) may be assessed using techniques known in the art. In one embodiment,
the other species is a nonhuman mammal to which the antibody will be
administered in preclinical studies. Accordingly, the species may be a
nonhuman
primate, such as rhesus, cynomolgus, baboon, chimpanzee and macaque. In
other embodiments, the species may be a rodent, cat or dog, for example.
[0058] The parent antibody is altered according to the present invention so as
to
generate an antibody which has a higher or stronger binding affinity for the
target
than the parent antibody. Antibody specificity results from the unique
interface that
is formed between the antibody and its target; the surfaces complement each
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other to produce a unique fit (Jones, S. & Thornton, J. M. (1996) Proc. Natl.
Acad.
Sci. USA 93: 13-20). By further improving the contacts along this interface,
the
overall affinity can increase as a result of the lower energy cost needed to
favor
the association of the binding partners.
[0059] The binding surface of the antibody is generally composed of six
complemetarity
determining regions (CDRs) which are loops that extend out from the core. The
CDRs are composed of amino acids having a sequence that is unique for binding
to the specific target. To increase the affinity of an antibody for its
antigen, the
environment around these amino acids must become more favorable by
introducing or improving various noncovalent forces, which ultimately lowers
the
energetics of the interaction, resulting in higher affinity.
[0060] Van der Waals forces are noncovalent interactions which occur between
two
electrically neutral molecules (Voet, D. & Voet, J. G. (1990) Biochemistry
John
Wiley and Sons, NY, NY). Associations can occur between two surfaces from
electrostatic interactions that arise from permanent or induced dipoles. These
dipoles can exist along the ends of a-helices or near polar amino acids. By
increasing the number of van der Waals forces along a binding interface, a
more
favorable association will. result.
[0061] Introducing hydrogen bonds will also increase the specificity of an
interaction
between an antibody and its antigen. Common donors and acceptors involved in
hydrogen bonding are nitrogen, oxygen and sulfer atoms, of which amino acids
are predominantly composed (See Voet, et al., supra). Hydrogen bonds tend
cross only short distances (usually 2.7 to 3.1 ,~), hence the binding partners
must
come within close proximity for these interactions to occur. Thus, one manner
by
which affinity can be improved is to bring potential donor and acceptor
molecules
into close contact to establish hydrogen bonds.
[0062] Finally, improving the hydrophobic interactions will also increase~the
favorable
energetics between two binding partners. The nonpolar residues that lie close
to
the binding surface should be surrounded by other nonpolar residues, and thus
will exist in a favorable environment. By satisfying the burial of nonpolar
side
chains, the energetics of the interaction are favorable for a strong binding
interface.
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[0063] Interactions that stabilize the protein-protein interface lower the
energetic cost of
maintaining these contacts and thus will increase the overall affinity. By
improving
the environment around individual amino acids that are near the binding
interface,
a more favorable environment is produced resulting in higher binding affinity.
Therefore, by introducing favorable contacts and improving the interface
through
further complementation, the overall binding interaction between antibody and
antigen will be greatly improved.
[0064] The resulting high affinity antibody preferably has a binding affinity
for the target
which is at feast about 10 fold higher, or at least about 20 fold higher, or
at least
about 500 fold higher or may be 1000 to 5000 fold higher, than the binding
affinity
of the parent antibody for the target. The degree of enhancement in binding
affinity necessary or desired will depend on the initial binding affinity of
the parent
antibody.
[0065] In general, the method for making high affinity antibodies from a
parent antibody
involves the following steps:
[0066] 1. Obtaining or selecting a parent antibody which binds the target of
interest,
which comprises heavy and light chain variable domains. This may be done by
traditional hybridoma techniques, phage-display techniques, or any other
method
of generating a target specific antibody.
[0067] 2. Selecting a framework sequence which is close in sequence to the
parent
framework, preferably a human template sequence. This template may be
chosen based on, e.g., its comparative overall length, the size of the CDRs,
the
amino acid residues located at the junction between the framework and the
CDRs,
overall homology, etc. The template chosen can be a mixture of more than one
sequence or may be a consensus template.
[0068] 3. Generating a library of clones by making random amino acid
substitutions
at each and every possible CDR position. One may also randomly substitute the
amino acids in the human framework template that are, e..g., adjacent to the
CDRs
or that may affect binding or folding, with all possible amino acids,
generating a
library of framework substitutions. These framework substitutions can be
assessed for their potential effect on target binding and antibody folding.
The
substitution of amino acids in the framework may be done either simultaneously
or
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sequentially with the substitution of the amino acids in the CDRs. One method
for
generating the library of variants by oligonucleotide synthesis.
[0069] 4. Constructing an expression vector comprising the heavy and/or light
chain
variants generated in step (3) which may comprise the formulas: FRH1-CDRH1-
FRH2-CDRH2-FRH3-CDRH3-FRH4(I) and FRL1-CDRL1-FRL2-CDRL2-FRL3-
CDRL3-FRL4 (II), wherein FRL1, FRL2, FRL3, FRL4, FRH1, FRH2, FRH3 and
FRH4 represent the variants of the framework template light chain and heavy
chain sequences chosen in step 3 and the CDRs represent the variant CDRs of
the parent antibody CDRs. An example of a vector containing such light and
heavy chain sequences is depicted in Figure 1.
[0070] 5. Screening the library of clones against the specific target and
those clones
that bind the target are screened for improved binding affinity. Those clones
that
bind with greater affinity than the parent molecule may be selected. The
optimal
high affinity candidate will have the greatest binding affinity possible
compared to
the parent antibody, preferably greater then 20 fold, 100 fold, 1000 fold or
5000
fold. If the chosen variant contains certain amino acids that are undesirable,
such
as a glycosylation site that has been introduced or a potentially immunogenic
site,
those amino acids may be replaced with more beneficial amino acid residues and
the binding affinity reassessed.
[0071] One may also use this method to generate high affinity antibodies from
a fully
human parent antibody by randomly substituting only the CDR regions, leaving
the human framework intact.
[0072] Due to improved high throughput screening techniques and vectors such
as the
one depicted in Figure 1, an artisan can rapidly and efficiently screen a
comprehensive library of substitutions at all sites in a given CDR and/or
framework region. By randomly substituting all amino acids at all positions
simultaneously, one is able to screen possible combinations that significantly
increase affinity that would not have been anticipated or identified by
individual
substitution due to, e.g., synergy.
PARENT ANTIBODY PREPARATION
Target Preparation
[0073] Soluble targets or fragments thereof can be used as immunogens for
generating
antibodies. The antibody is directed against the target of interest.
Preferably, the
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target is a biologically important polypeptide and administration of the
antibody to
a mammal suffering from a disease or disorder can result in a therapeutic
benefit
in that mammal. However, antibodies may be directed against non polypeptide
targets. Where the target is a polypeptide, it may be a transmembrane molecule
(e.g. receptor) or ligand such as a growth factor. One target of the present
invention is IgE. Whole cells may be used as the immunogen for making
antibodies. The target may be produced recombinantly or made using synthetic
methods. The target may also be isolated from a natural source.
[0074] Antigens used in producing antibodies of the invention may include
polypeptides
and polypeptide fragments of the invention, including epitope A and/or B. A
polypeptide used to immunize an animal can be obtained by standard
recombinant, chemical synthetic, or purification methods. As is well known in
the
art, in order to increase immunogenicity, an antigen can be conjugated to a
carrier
protein. Commonly used carriers include, but are not limited to, keyhole
limpet
hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus
toxoid. The coupled peptide is then used to immunize an animal (e.g., a mouse,
a
rat, or a rabbit). In addition to such carriers, well known adjuvants can be
administered with the antigen to facilitate induction of a strong immune
response.
Polyclonal Antibodies
[0075] Polyclonal antibodies are usually generated in non-human mammals by
multiple
subcutaneous (sc) or intraperitoneal (ip) injections of the relevant target in
combination with an adjuvant. Numerous agents capable of eliciting an
immunological response are well known in the art.
[0076] Animals are immunized against the target, immunogenic conjugates, or
derivatives by combining the protein or conjugate (for rabbits or mice,
respectively) with Freund's complete adjuvant and injecting the sol ution
intradermally. One month later the animals are boosted with 1/5 to 1/10 the
original amount of peptide or conjugate in Freund's incomplete adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later the animals
are
bled and the serum is assayed for antibody titer. Animals are boosted until
the titer
plateaus.
[0077] The mammalian antibody selected will normally have a sufficiently
strong binding
affinity for the target. For example, the antibody may bind the human anti-IgE
17
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target with a binding affinity (Kd) value of about 1 x 10-$ M. Antibody
affinities may
be determined by saturation binding; enzyme-linked immunoabsorbant assay
(ELISA); and competition assays (e.g., radioimmunoassays).
[0078] To screen for antibodies that bind the target of interest, a routine
cross-linking
assay such as that described in Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, Ed Harlow and David Lane (1988) can be performed.
Alternatively, epitope mapping, e.g., as described in Champe, et al. J. Biol.
Chem.
270: 1388-1394 (1995), can be performed to determine binding.
Monoclonal Antibodies
[0079] Monoclonal antibodies are antibodies which recognize a single antigenic
site.
Their uniform specificity makes monoclonal antibodies much more useful than
polyclonal antibodies, which usually contain antibodies that recognize a
variety of
different antigenic sites. Monoclonal antibodies may be made using the
hybridoma
method first described by Kohler et al., Nature, 256: 495 (1975), or may be
made
by recombinant DNA methods.
[0080] In the hybridoma method, a mouse or other appropriate host animal, such
as a
rodent, is immunized as hereinabove described to elicit lymphocytes that
produce
or are capable of producing antibodies that will specifically bind to the
protein
used for immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes then are fused with myeloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (coding, Monoclonal
Antibodies: Principals and Practice, pp. 590-103 (Academic Press,1986)).
[0081] The hybridoma cells thus prepared are seeded and grown in a suitable
culture
medium that preferably contains one or more substances that inhibit the growth
or
survival of the unfused, parental myeloma cells. For example, if the parental
myeloma cells lack the enzyme hypoxanthine guanine phophoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), substances which
prevent the growth of HGPRT-deficient cells. Preferred myeloma cells are those
that fuse efficiently, support stable high-level production of antibody by the
selected antibody-producing cells, and are sensitive to a medium such as HAT
medium. Human myeloma and mouse-human heteromyeloma cell lines have
been described for the production of human monoclonal antibodies (Kozbar, J.
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Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
[0082] After hybridoma cells are identified that produce antibodies of the
desired
specificity, affinity, andlor activity, the clones may be subcloned by
limiting dilution
procedures and grown by standard methods (coding, Monoclonal Antibodies:
Principals and Practice, pp. 59-103, Academic Press, 1986)). Suitable culture
media for this purpose include. The monoclonal antibodies secreted by the
subclones are suitably separated from the culture medium by conventional
immunoglobulin purification procedures such as, for example, protein A-
Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
[0083] DNA encoding the monoclonal antibodies is readily isolated and
sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of
binding specifically to genes encoding the heavy and light chains of the
monoclonal antibodies). The hybridoma cells serve as a source of such DNA.
Once isolated, the DNA may be placed into expression vectors, which are then
transferred into host cells such as E. coli cells, NSO cells, Chinese hamster
ovary
(CHO) cells, or myeloma cells to obtain the synthesis of monoclonal antibodies
in
the recombinant host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567;
Morrison et al., Proc. Natl Acad. Sci. USA 81: 6851 (1984)), or by covalently
joining to the immunoglobulin pofypeptide.
Humanized Antibodies
[0084] Humanization is a technique for making a chimeric antibody wherein
substantially
less than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. A humanized antibody has
one or more amino acid residues introduced into it from a source which is non-
human. These non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable domain.
Humanization can be essentially performed following the method of Winter and
co-workers (Jones et al, Nature 321: 522-525 (1986); Riechman et al., Nature
332: 323-327 (1988); Verhoeyens et al., Science 239: 1534-1536 (1988)), by
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substituting non-human CDR's or CDR sequences for the corresponding
sequences in a human antibody (See, e.g., U.S. Pat. No. 4,816,567). As
practiced in the present invention, the humanized antibody may have some CDR
residues and some FR residues substituted by residues from analogous sites in
murine antibodies.
[0085] The choice of human variable domains, both light and heavy, to be used
in
making the humanized antibodies is very important to reduce antigenicity.
According to the so-called "best fit" method, the sequence of the variable
domain
of a non-human antibody is compared with the library of known human variable-
domain sequences. The human sequence which is closest to that of the non-
human parent antibody is then accepted as the human framework for the
humanized antibody (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al.,
J.
Mol. Biol. 196: 901 (1987)). Another method uses a particular framework
derived
from the consensus sequence of all human antibodies of a particular subgroup
of
light or heavy chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285
(1992);
Presta et al., J. Immunol. 151: 2623 (1993)).
Antibody Fragments
[0086] Various techniques have been developed for the production of antibody
fragments. Traditionally, these fragments were derived via proteolytic
digestion of
intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and
Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81
(1985)). However, these fragments can now be produced directly by recombinant
host cells. For example, the antibody fragments can be isolated from an
antibody
phage library. Alternatively, F(ab')2 -SH fragments can be directly recovered
from
E. coli and chemically coupled to form F(ab')2 fragments (Carter et al.,
Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')~
fragments can be isolated directly from recombinant host cell culture. Other
techniques for the production of antibody fragments will be apparent to the
skilled
practitioner. In other embodiments, the antibody of choice is a single chain
Fv
fragment (scFv). (PCT patent application WO 93/16185).
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PREPARATION OF HIGH AFFINITY ANTIBODIES
[0087] Once the parent antibody has been identified and isolated, one or more
amino
acid residues may be altered in one or more of the variable regions of the
parent
antibody. Alternatively, or in addition, one or more substitutions of
framework
residues may be introduced in the parent antibody where these result in an
improvement in the binding affinity of the antibody, for example, for human
IgE.
Examples of framework region residues to modify include those which non-
covalently bind target directly (Amit et al. Science 233: 747-753 (1986));
interact
with/effect the conformation of CDR (Chothia et al. J. Mol. Biol. 196: 901-917
(1987)); and/or participate in the VL-VH interface (EP 239 400 B1 ). In
certain
embodiments, modification of one or more of such framework region residues
results in an enhancement of the binding affinity of the antibody for the
target of
interest.
[0088] Modifications in the antibodies' biological properties may be
accomplished by
selecting substitutions that differ significantly in their effect on
maintaining, e.g.,
(a) the structure of the polypeptide backbone in the area of the substitution,
for
example, as a sheet or helical conformation; (b) the charge or hydrophobicity
of
the molecule at the target site, or (c) the bulk of the side chain. Non-
conservative
substitutions will entail exchanging a member of one of these classes for
another
class.
[0089] Nucleic acid molecules encoding amino acid sequence variants are
prepared by a
variety of methods known in the art. These methods include, but are not
limited to,
oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and
cassette mutagenesis of an earlier prepared variant or a non-variant version
of the
species-dependent antibody. The preferred method for generating variants is an
oligonucleotide-mediated synthesis. In certain embodiments, the antibody
variant
will only have a single hypervariable region residue substituted, e.g. from
about
two to about fifteen hypervariable region substitutions.
[0090] One method for generating the library of variants is by oligonucleotide
mediated
synthesis according to the scheme depicted in Figure 2. Three oligonucleotides
of
approximately 100 nucleotides each may be synthesized spanning the entire
light
chain or heavy chain variable region. Each oligonucleotide may comprise: (1 )
a
60 amino acid stretch generated by the triplet (NNK)2o where N is any
nucleotide
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and K is G or T, and (2) an approximately 15-30 nucleotide overlap with either
the
next oligo or with the vector sequence at each end. Upon annealing of these
three oligonucleotides in a PCR reaction, the polymerase will fill in the
opposite
strand generating a complete double stranded heavy chain or light chain
variable
region sequence. The number of triplets may be adjusted to any length of
repeats
and their position within the oligonucleotide may be chosen so as to only
substitute amino acds in a given CDR or framework region., By using (NNK), all
twenty amino acids are possible at each position in the encoded variants. The
overlapping sequence of 5-10 amino acids (15-30 nucloetides) will not be
subtituted, but this may be chosen to fall within the stacking regions of the
framework, or may substituted by a separate or subsequent round of synthesis.
Methods for synthesizing oligonucleotides are well known in the art and are
also
commercially available. Methods for generating the antibody variants from
these
oligonucleotides are also well known in the art, e.g., PCR.
[0091] The library of heavy and light chain variants, differing at random
positions in their
sequence, can be constructed in any expression vector, such as a
bacteriophage,
specifically the vector of Fig.1, each of which contains DNA encoding a
particular
heavy and light chain variant.
[0092] Following production of the antibody variants, the biological activity
of variant
relative to the parent antibody is determined. As noted above, this involves
determining the binding affinity of the variant for the target. Numerous high-
throughput methods exist for rapidly screen antibody variants for their
ability to
bind the target of interest.
[0093] One or more of the antibody variants selected from this initial screen
may then be
screened for enhanced binding affinity relative to the parent antibody. One
common method for determining binding affinity is by assessing the association
and dissociation rate constants using a BIAcoreT"~ surface plasmon resonance
system (BIAcore, Inc.). A biosensor chip is activated for covalent coupling of
the
target according to the manufacturer's (BIAcore) instructions. The target is
then
diluted and injected over the chip to obtain a signal in response units (RU)
of
immobilized material. Since the signal in RU is proportional to the mass of
immobilized material, this represents a range of immobilized target densities
on
the matrix. Dissociation data are fit to a one-site model to obtain k~ff +/-
s.d.
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(standard deviation of measurements). Pseudo-first order rate constant (ks)
are
calculated for each association curve, and plotted as a function of protein
concentration to obtain ko~ +/- s.e. (standard error of fit). Equilibrium
dissociation
constants for binding, Kp s, are calculated from SPR measurements as koff/kon~
Since the equilibrium dissociation constant, Kp, is inversely proportional to
koff, an
estimate of affinity improvement can be made assuming the association rate
(kon)
is a constant for all variants.
[0094] The resulting candidates) with high affinity may optionally be
subjected to one or
more further biological activity assays to confirm that the antibody variants)
with
enhanced binding affinity still retain the desired therapeutic attributes. For
example, in the case of an anti-IgE antibody, one may screen for those that
block
binding of IgE to its receptor and inhibit the release of histamine. The
optimal
antibody variant retains the ability to bind the target with a binding
affinity
significantly higher than the parent antibody.
[0095] The antibody variants) so selected may be subjected to further
modifications
oftentimes depending upon the intended use of the antibody. Such modifications
may involve further alteration of the amino acid sequence, fusion to
heterologous
polypeptide(s) and/or covalent modifications such as those elaborated below.
For
example, any cysteine residues not involved in maintaining the proper
conformation of the antibody variant may be substituted, generally with
serine, to
improve the oxidative stability of the molecule and prevent aberrant cross
linking.
Conversely, (a) cysteine bonds) may be added to the antibody to improve its
stability (particularly where the antibody is an antibody fragment such as an
Fv
fragment).
VECTORS
[0096] The invention also provides isolated nucleic acid encoding an antibody
variant as
disclosed herein, vectors and host cells comprising the nucleic acid, and
recombinant techniques for the production of the antibody variant. For
recombinant production of the antibody variant, the nucleic acid encoding it
is
isolated and inserted into a replicable vector for further cloning
(amplification of
the DNA) or for expression. DNA encoding the antibody variant is readily
isolated
and sequenced using conventional procedures (e.g., by using oligonucleotide
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probes that are capable of binding specifically to genes encoding the heavy
and
light chains of the antibody variant).
[0097] Many vectors are available. The vector components generally include,
but are not
limited to, one or more of the following: a signal sequence, an origin of
replication,
one or more marker genes, an enhancer element, a promoter, and a transcription
termination sequence.
[0098] The phage expression vector depicted in Figure 1 is comprised of a
commonly
used M13 vector and M13's own gene III viral secretion signal for rapid
secretion
and screening variant Fabs for proper binding specificity and minimal affinity
criteria. This vector does not use the entire gene III sequence, so there is
no
display on the surface of the bacterial cell, but rather the Fabs are secreted
into
the periplasmic space. Alternatively, the Fabs could be expressed in the
cytoplasm and isolated. The heavy and light chains each have their own viral
secretion signal, but are dependently expressed from a single strong inducible
promoter.
[0099] The vector in Figure 1 also provides a His tag and a myc tag for easy
purification,
as well as detection. A skilled artisan would recognize that the Fabs could be
independently expressed from separate promoters or that the secretion signal
need not be the viral sequence chosen, but could be a prokaryotic or
eukaryotic
signal sequence suitable for the secretion of the antibody fragments from the
chosen host cell. It should also be recognized that the heavy and light chains
may
reside on different vectors.
A. Signal Sequence Component
[0100] The antibody variant of this invention may be produced recombinantly.
The
variant may also be expressed as a fusion polypeptide fused with a
heterologous
polypeptide, which is preferably a signal sequence or other polypeptide having
a
specific cleavage site at the N-terminus of the mature protein or polypeptide.
The
heterologous signal sequence selected preferably is one that is recognized and
processed (i.e., cleaved by signal peptidase) by the host cell. For
prokaryotic host
cells that do not recognize and process the native antibody signal sequence,
the
signal sequence may be substituted by a prokaryotic signal sequence selected,
for example, from the group of the alkaline phosphatase, penicillinase, Ipp,
or
heat-stable enterotoxin II leaders. Or in the case of the vector of Figure 1,
the
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signal sequence chosen was a viral signal sequence from gene III. For yeast
secretion the native signal sequence may be substituted by, a g., the yeast
invertase leader, a-factor leader (including Saccharomyces and Kluyveromyces a-
factor leaders), or acid phosphatase leader, the C. albicans glucoamylase
leader,
or a signal described in e.g., WO 90/13646. In mammalian cell expression,
mammalian signal sequences as well as viral secretory leaders, for example,
the
herpes simplex gD signal, are available. The DNA for such precursor region is
ligated in reading frame to DNA encoding the antibody variant.
B. Origin of Replication Component
[0101] Vectors usually contain a nucleic acid sequence that enables the vector
to
replicate in one or more selected host cells. Generally, this sequence is one
that
enables the vector to replicate independently of the host chromosomal DNA, and
includes origins of replication or autonomously replicating sequences. Such
sequences are well known for a variety of bacteria, yeast, and viruses. The
origin
of replication from the plasmid pBR322 is suitable for most Gram-negative
bacteria, the 2,u plasmid origin is suitable for yeast, and various viral
origins
(SV40, polyoma, adenovirus, VSV or BPV) are useful for vectors in mammalian
cells. Generally, the origin of replication component is not needed for
mammalian
expression vectors (the SV40 origin may typically be used only because it
contains the early promoter).
C. Selection Gene Component
[0102] Vectors may contain a selection gene, also termed a selectable marker.
Typical
selection genes encode proteins that (a) confer resistance to antibiotics or
other
toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement
auxotrophic deficiencies, or (c) supply critical nutrients not available from
complex
media, e.g., the gene encoding D-alanine racemase for Bacilli.
[0103] One example of a selection scheme utilizes a drug to arrest growth of a
host cell.
Those cells that are successfully transformed with a heterologous gene produce
a
protein conferring drug resistance and thus survive the selection regimen.
Examples of such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
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[0104] Another example of suitable selectable markers for mammalian cells are
those
that enable the identification of cells competent to take up the antibody
nucleic
acid, such as DHFR, thymidine kinase, metallothionein-I and -II, preferably
primate metallothionein genes, adenosine deaminase, ornithine decarboxylase,
etc.
[0105] For example, cells transformed with the DHFR selection gene are first
identified
by culturing all of the transformants in a culture medium that contain
methotrexate
(Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-
type
DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in
DHFR activity.
[0106] Alternatively, host cells (particularly wild-type hosts that contain
endogenous
DHFR) transformed or co-transformed with DNA sequences encoding antibody,
wild-type DHFR protein, and another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell growth in medium
containing
a selection agent for the selectable marker such as an aminoglycosidic
antibiotic,
e.g., kanamycin, neomycin, or 6418. (U.S. Pat. No. 4,965,199).
[0107] A suitable selection gene for use in yeast is the trp1 gene present in
the yeast
plasmid Yrp7 (Stinchcomb et al., Nature 282: 39 (1979)). The trp1 gene
provides
a selection marker for a variant strain of yeast lacking the ability to grow
in
typtophan, for example, ATCC No. 44076 or PEP4-1. Jones, Genetics 85: 12
(1977). The presence of the trp1 lesion in the yeast host cell genome then
provides an effective environment for detecting transformation by growth in
the
absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or
38,626) are complemented by known plasmids bearing the Leu2 gene.
D. Promoter Component
[0108] Expression and cloning vectors usually contain a promoter that is
recognized by
the host organism and is operably linked to the antibody nucleic acid.
Promoters
suitable for use with prokaryotic hosts include the phoA promoter, a-lactamase
and lactose promoter systems, alkaline phosphatase, a tryptophan (trp)
promoter
system, and hybrid promoters such as the tac promoter. However, other known
bacterial promoters are suitable. Promoters for use in bacterial systems may
also
contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding
the antibody.
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[0109] Promoter sequences are known for eukaryotes. Virtually all eukaryotic
genes have
an AT-rich region located approximately 25 to 30 bases upstream from the site
where transcription is initiated. Another sequence found 70 to 80 bases
upstream
from the start of transcription of many genes is a CNCAAT region where N may
be
any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence
that may be the signal for addition of the poly A tail to the 3' end of the
coding
sequence. All of these sequences are suitably inserted into eukaryotic
expression
vectors.
[0110] Examples of suitable promotor sequences for use with yeast hosts
include the
promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as
enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-
phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
phosphoglucose isomerase, and glucokinase.
[0111] Other yeast promoters, which are inducible promoters having the
additional
advantage of transcription controlled by growth conditions, are the promoter
regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for
maltose and galactose utilization. Suitable vectors and promoters for use in
yeast
expression are further described in EP 73,657. Yeast enhancers also are
advantageously used with yeast promoters.
[0112] Antibody transcription from vectors in mammalian host cells is
controlled, for
example, by promoters obtained from the genomes of viruses such as polyoma
virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma
virus,
avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most
preferably Simian virus 40 (SV40), from heterologous mammalian promoters,
e.g.,
the actin promoter or an immunoglobulin promoter, from heat-shock promoters--
provided such promoters are compatible with the host cell systems.
[0113] The early and late promoters of the SV40 virus are conveniently
obtained as an
SV40 restriction fragment that also contains the SV40 viral origin of
replication.
The immediate early promoter of the human cytomegalovirus is conveniently
obtained as a Hindlll E restriction fragment. A system for expressing DNA in
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mammalian hosts using the bovine papilloma virus as a vector is disclosed in
U.S
Pat. No. 4,419,446. A modification of this system is described in U.S. Pat.
No.
4,601,978. Alternatively, human /3-interferon cDNA has been expressed in mouse
cells under the control of a thymidine kinase promoter from herpes simplex
virus.
Alternatively, the rous sarcoma virus long terminal repeat can be used as the
promoter.
E. Enhancer Element Component
[0114] Transcription of a DNA encoding the antibody of this invention by
higher
eukaryotes is often increased by inserting an enhancer sequence into the
vector.
Many enhancer sequences are now known from mammalian genes (globin,
elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will
use an
enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on
the late side of the replication origin (bp 100-270), the cytomegalovirus
early
promoter enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) on
enhancing elements for activation of eukaryotic promoters. The enhancer may be
spliced into the vector at a position 5' or 3' to the antibody-encoding
sequence, but
is preferably located at a site 5' from the promoter.
F. Transcription Termination Component
[0115] Expression vectors used in eukaryotic host cells (yeast, fungi, insect,
plant,
animal, human, or nucleated cells from other multicellular organisms) may also
contain sequences necessary for the termination of transcription and for
stabilizing
the mRNA. Such sequences are commonly available from the 5' and, occasionally
3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions
contain nucleotide segments transcribed as polyadenylated fragments in the
untranslated portion of the mRNA encoding the antibody. One useful
transcription
termination component is the bovine growth hormone polyadenylation region. See
e.g.,W094111026.
SELECTION AND TRANSFORMATION OF HOST CELLS
[0116] Suitable host cells for cloning or expressing the DNA in the vectors
herein are
prokaryotic, yeast, or higher eukaryotic cells. Suitable prokaryotes for this
purpose
include both Gram-negative and Gram-positive organisms, for example,
Enterobacteria such as E, coli, Enterobacter, Enwinia, Klebsiella, Proteus,
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Salmonella, Serratia, and Shigella, as well as Bacilli, Pseudomonas, and
Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446),
although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E.
coli
W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than
limiting.
[0117] In addition to prokaryotes, eukaryotic microbes such as filamentous
fungi or yeast
are suitable cloning or expression hosts for antibody-encoding vectors.
Saccharomyces cerevisiae is the most commonly used among cower eukaryotic
host microorganisms. However, a number of other genera, species, and strains
are commonly available and useful herein, such as Schizosaccharomyces pombe;
Kluyveromyces; Candida; Trichoderma; Neurospora crassa; and filamentous fungi
such as e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts,
such
as A. nidulans and A. niger.
[0118] Suitable host cells for the expression of glycosylated antibodies are
derived from
multicellular organisms. In principal, any higher eukaryotic cell culture is
workable,
whether from vertebrate or invertebrate culture. Examples of invertebrate
cells
include plant and insect cells, Luckow et al., Bio/Technology 6, 47-55 (1988);
Miller et al., Genetic Engineering, Setlow et al. eds. Vol. 8, pp. 277-279
(Plenam
publishing 1986); Mseda et al., Nature 315, 592-594 (1985). Numerous
baculoviral strains and variants and corresponding permissive insect host
cells
from hosts such as Spodoptera frugiperda (caterpillar), Aedes (mosquito),
Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A
variety of viral strains for transfection are publicly available, e.g., the L-
1 variant of
Autographs californica NPV and the Bm-5 strain of Bombyx mori NPV, and such
viruses may be used as the virus herein according to the present invention,
particularly for transfection of Spodoptera frugiperda cells. Moreover, plant
cells
cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco and
also
be utilized as hosts.
[0119] Vertebrate cells and propagation of vertebrate cells, in culture
(tissue culture) has
become routine. See Tissue Culture, Academic Press, Kruse and Patterson, eds.
(1973). Examples of useful mammalian host cell lines are monkey kidney; human
embryonic kidney line; baby hamster kidney cells; Chinese hamster ovary cells/-
DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse
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sertoli cells; human cervical carcinoma cells (HELA); canine kidney cells;
human
lung cells; human liver cells; mouse mammary tumor; and NSO cells.
[0120] Host cells are transformed with the above-described vectors for
antibody
production and cultured in conventional nutrient media modified as appropriate
for
inducing promoters, selecting transformants, or amplifying the genes encoding
the
desired sequences.
[0121] The host cells used to produce the antibody variant of this invention
may be
cultured in a variety of media. Commercially available media such as Ham's F10
(Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing
host cells. In addition, any of the media described in Ham et al., Meth.
Enzymol.
58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos.
4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; or 6,048,728 may be
used as culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors (such as
insulin, transferrin, or epidermal growth factor), salts (such as X-chlorides,
where
X is sodium, calcium, magnesium; and phosphates), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics (such as
GENTAMYCIN.TM. drug), trace elements (defined as inorganic compounds
usually present at final concentrations in the micromolar range), and glucose
or an
equivalent energy source. Any other necessary supplements may also be
included at appropriate concentrations that would be known to those skilled in
the
art. The culture conditions, such as temperature, pH, and the like, are those
previously used with the host cell selected for expression, and will be
apparent to
the ordinarily skilled artisan.
ANTIBODY PURIFICATION
[0122] When using recombinant techniques, the antibody variant can be produced
intracellularly, in the periplasmic space, or directly secreted into the
medium. If the
antibody variant is produced intracellularly, as a first step, the particulate
debris,
either host cells or lysed fragments, may be removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167
(1992)
describe a procedure for isolating antibodies which are secreted to the
periplasmic
space of E. coli. Briefly, cell paste is thawed in the presence of sodium
acetate
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(pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 minutes.
Cell debris can be removed by centrifugation. Where the antibody variant is
secreted into the medium, supernatants from such expression systems are
generally first concentrated using a commercially available protein
concentration
filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A
protease
inhibitor such as PMSF may be included in any of the foregoing steps to
inhibit
proteolysis and antibiotics may be included to prevent the growth of
adventitious
contaminants.
[0123] The antibody composition prepared from the cells can be purified using,
for
example, hydroxylapatite chromatography, gel elecrophoresis, dialysis, and
affinity chromatography, with affinity chromatography being the preferred
purification technique. The suitability of protein A as an affinity ligand
depends on
the species and isotype of any immunoglobulin Fc domain that is present in the
antibody variant. Protein A can be used to purify antibodies that are based on
human IgG1, IgG2 or IgG4 heavy chains (Lindmark et al., J. Immunol Meth. 62: 1-
13 (1983)). Protein G is recommended for all mouse isotypes and for human IgG3
(Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which, the affinity
ligand
is attached is most often agarose, but other matrices are available.
Mechanically
stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene
allow
for faster flow rates and shorter processing times than can be achieved with
agarose. Where the antibody variant comprises a CH3 domain, the Bakerbond
ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other
techniques for protein purification such as fractionation on an ion-exchange
column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica,
chromatography on heparin SEPHAROSETM chromatography on an anion or
cation exchange resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available depending on
the antibody variant to be recovered.
[0124] Following any preliminary purification step(s), the mixture comprising
the antibody
variant of interest and contaminants may be subjected to low pH hydrophobic
interaction chromatography using an elution buffer at a pH between about 2.5-
4.5,
preferably performed at low salt concentrations (e.g., from about 0-0.25M
salt).
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PHARMACEUTICAL FORMULATIONS
[0125] Therapeutic formulations of the polypeptide or antibody may be prepared
for
storage as lyophilized formulations or aqueous solutions by mixing the
polypeptide
having the desired degree of purity with optional "pharmaceutically-
acceptable"
carriers, excipients or stabilizers typically employed in the art (all of
which are
termed "excipients"). For example, buffering agents, stabilizing agents,
preservatives, isotonifiers, non-ionic detergents, antioxidants and other
miscellaneous additives. (See Remington's Pharmaceutical Sciences, 16th
edition, A. Osol, Ed. (1980)). Such additives must be nontoxic to the
recipients at
the dosages and concentrations employed.
[0126] Buffering agents help to maintain the pH in the range which
approximates
physiological conditions. They are preferably present at concentration ranging
from about 2 mM to about 50 mM. Suitable buffering agents for use with the
present invention include both organic and inorganic acids and salts thereof
such
as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric
acid-
trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.),
succinate
buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-
sodium
hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate
buffers
(e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate
mixture,
tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric
acid-
monosodium fumarate mixture, etc.), fumarate buffers (e.g., fumaric acid-
monosodium fumarate mixture, fumaric acid-disodium fumarate mixture,
monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g.,
gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide
mixture,
gluconic acid-potassium glyuconate mixture, etc.), oxalate buffer (e.g.,
oxalic acid-
sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-
potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium
lactate
mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate
mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture,
acetic
acid-sodium hydroxide mixture, etc.). Additionally, there may be mentioned
phosphate buffers, histidine buffers and trimethylamine salts such as Tris.
[0127] Preservatives may be added to retard microbial growth, and may be added
in
amounts ranging from 0.2%-1 % (w/v). Suitable preservatives for use with the
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present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben,
propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium
halides (e.g., chloride, bromide, iodide), hexamethonium chloride, alkyl
parabens
such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-
pentanol.
[0128] Isotonicifiers sometimes known as "stabilizers" may be added to ensure
isotonicity
of liquid compositions of the present invention and include polhydric sugar
alcohols, preferably trihydric or higher sugar alcohols, such as glycerin,
erythritol,
arabitol, xylitol, sorbitol and mannitol.
[0129] Stabilizers refer to a broad category of excipients which can range in
function from
a bulking agent to an additive which solubilizes the therapeutic agent or
helps to
prevent denaturation or adherence to the container wall. Typical stabilizers
can be
polyhydric sugar alcohols (enumerated above); amino acids such as arginine,
lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-
leucine, 2-
phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar
alcohols,
such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol,
myoinisitol,
galactitol, glycerol and the like, including cyclitols such as inositol;
polyethylene
glycol; amino acid polymers; sulfur containing reducing agents, such as urea,
glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-
monothioglycerol
and sodium thio sulfate; low molecular weight polypeptides (i.e. <10
residues);
proteins such as human serum albumin, bovine serum albumin, gelatin or
immunoglobulins; hydrophylic polymers, such as polyvinylpyrrolidone
monosaccharides, such as xylose, mannose, fructose, glucose; disaccharides
such as lactose, maltose, sucrose and trisaccacharides such as raffinose;
polysaccharides such as dextran. Stabilizers may be present in the range from
0.1
to 10,000 weights per part of weight active protein.
[0130] Non-ionic surfactants or detergents (also known as "wetting agents")
may be
added to help solubilize the therapeutic agent as well as to protect the
therapeutic
protein against agitation-induced aggregation, which also permits the
formulation
to be exposed to shear surface stressed without causing denaturation of the
protein. Suitable non-ionic surfactants include polysorbates (20, 80, etc.),
polyoxamers (184, 188 etc.), Pluronic~ polyols, polyoxyethylene sorbitan
monoethers (TWEEN~-20, TWEEN~-80, etc.). Non-ionic surfactants may be
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present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about
0.07
mg/ml to about 0.2 mglml.
[0131] Additional miscellaneous excipients include bulking agents, (e.g.
starch), chelating
agents (e.g. EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E),
and
co-solvents. The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated, preferably
those with complementary activities that do not adversely affect each other.
For
example, it may be desirable to further provide an immunosuppressive agent.
Such molecules are suitably present in combination in amounts that are
effective
for the purpose intended. The active ingredients may also be entrapped in
microcapsule prepared, for example, by coascervation techniques or by
interfacial
polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule
and
poly-(methylmethacylate) microcapsule, respectively, in colloidal drug
delivery
systems (for example, fiposomes, albumin micropheres, microemulsions, nano-
particles and nanocapsules) or in macroemulsions. Such techniques are
disclosed
in Remington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).
[0132] The formulations to be used for in vivo administration must be sterile.
This is
readily accomplished, for example, by filtration through sterile filtration
membranes. Sustained-release preparations may be prepared. Suitable examples
of sustained-release preparations include semi-permeable matrices of solid
hydrophobic polymers containing the antibody variant, which matrices are in
the
form of shaped articles, e.g., films, or microcapsules. Examples of sustained-
release matrices include polyesters, hydrogels (for example, poly(2-
hydroxyethyl-
methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),
copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-
vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the
LUPRON DEPOTT"~ (injectable microspheres composed of lactic acid-glycolic acid
copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While
polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable
release of molecules for over 100 days, certain hydrogels release proteins for
shorter time periods. When encapsulated antibodies remain in the body for a
long
time, they may denature or aggregate as a result of exposure to moisture at
37° C
resulting in a loss of biological activity and possible changes in
immunogenicity.
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Rational strategies can be devised for stabilization depending on the
mechanism
involved. For example, if the aggregation mechanism is discovered to be
intermolecular S--S bond formation through thin-disulfide interchange,
stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from acidic
solutions, controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0133] The amount of therapeutic polypeptide, antibody or fragment thereof
which will be
effective in the treatment of a particular disorder or condition will depend
on the
nature of the disorder or condition, and can be determined by standard
clinical
techniques. Where possible, it is desirable to determine the dose-response
curve
and the pharmaceutical compositions of the invention first in vitro, and then
in
useful animal model systems prior to testing in humans.
[0134] In a preferred embodiment, an aqueous solution of therapeutic
polypeptide,
antibody or fragment thereof is administered by subcutaneous injection. Each
dose may range from about 0.5,~g to about 50,ug per kilogram of body weight,
or
more preferably, from about 3 ~ug to about 30,ug per kilogram body weight.
(0135] The dosing schedule for subcutaneous administration may vary form once
a
month to daily depending on a number of clinical factors, including the type
of
disease, severity of disease, and the subject's sensitivity to the therapeutic
agent.
USES FOR THE ANTIBODY VARIANT
[0I36] The antibody variants of the invention may be used as affinity
purification agents.
In this process, the antibodies are immobilized on a solid phase such as
SEPHADEXT"~ resin or filter paper, using methods well known in the art. The
immobilized antibody variant is contacted with a sample containing the target
to
be purified, and thereafter the support is washed with a suitable solvent that
will
remove substantially all the material in the sample except the target to be
purified,
which is bound to the immobilized antibody variant. Finally, the support is
washed
with another suitable solvent, such as glycine buffer, that will release the
target
from the antibody variant.
[0137] The variant antibodies may also be useful in diagnostic assays, e.g.,
for detecting
expression of a target of interest in specific cells, tissues, or serum. For
diagnostic
applications, the antibody variant typically will be labeled with a detectable
moiety.
Numerous labels are available Techniques for quantifying a change in
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fluorescence are described above. The cflemiluminescent substrate becomes
electronically excited by a chemical reaction and may then emit light which
can be
measured (using a chemiluminometer, for example) or donates energy to a
fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g.,
firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),
luciferin, 2,3-
dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as
horseradish peroxidase (HRPO), alkaline phosphatase, .beta.-galactosidase,
glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose
oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such
as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the
like.
Techniques for conjugating enzymes to antibodies are described in O'Sullivan
et
al., Methods for the Preparation of Enzyme-Antibody Conjugates for Use in
Enzyme Immunoassay, in Methods in Enzym. (Ed. J. Langone & H. Van Vunakis),
Academic press, New York, 73: 147-166 ('I 981 ).
[0138] Sometimes, the label is indirectly conjugated with the antibody
variant. The skilled
artisan The skilled artisan will be aware of various techniques for achieving
this.
For example, the antibody variant can be conjugated with biotin and any of the
three broad categories of labels mentioned above can be conjugated with
avidin,
or vice versa. Biotin binds selectively to avidin and thus, the label can be
conjugated with the antibody variant in this indirect manner. Alternatively,
to
achieve indirect conjugation of the label with the antibody variant, the
antibody
variant is conjugated with a small hapten (e.g. digloxin) and one of the
different
types of labels mentioned above is conjugated with an anti-hapten antibody
variant (e.g. anti-digloxin antibody). Thus, i ndirect conjugation of the
label with the
antibody variant can be achieved.
[0139] In another embodiment of the invention, the antibody variant need not
be labeled,
and the presence thereof can be detected using a labeled antibody which binds
to
the antibody variant.
[0140] The antibodies of the present invention may be employed in any known
assay
method, such as competitive binding assays, direct and indirect sandwich
assays,
and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of
Techniques, pp. 147-158 (CRC Press, Inc. 1987).
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[0141] Competitive binding assays rely on the ability of a labeled standard to
compete
with the test sample for binding with a limited amount of antibody variant.
The
amount of target in the test sample is inversely proportional to the amount of
standard that becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies generally are
insolubilized before or after the competition. As a result, the standard and
test
sample that are bound to the antibodies may conveniently be separated from the
standard and test sample which remain unbound.
[0142] Sandwich assays involve the use of two antibodies, each capable of
binding to a
different immunogenic portion, or epitope, or the protein to be detected. In a
sandwich assay, the test sample to be analyzed is bound by a first antibody
which
is immobilized on a solid support, and thereafter a second antibody binds to
the
test sample, thus forming an insoluble three-part complex. See e.g., U.S. Pat.
No.
4,376,110. The second antibody may itself be labeled with a detectable moiety
(direct sandwich assays) or may be measured using an anti-immunoglobulin
antibody that is labeled with a detectable moiety (indirect sandwich assay).
For
example, one type of sandwich assay is an ELISA assay, in which case the
detectable moiety is an enzyme.
[0143] For immunohistochemistry, the tumor sample may be fresh or frozen or
may be
embedded in paraffin and fixed with a preservative such as formalin, for
example.
[0144j The antibodies may also be used fvr in vivo diagnostic assays,
Generally, the
antibody variant is labeled with a radionucleotide (such as <sup>111</sup> In,
<sup>99</sup> Tc,
<sup>14</sup> C, <sup>131</sup> I, <sup>3</sup> H, <sup>32</sup> P or <sup>35</sup> S) so that the tumor can
be
localized using immunoscintiography. For example, a high affinity anti-IgE
antibody of the present invention may be used to detect the amount of IgE
present
in, e.g., the lungs of an asthmatic patient.
[0145] The antibody of the present invention can be provided in a kit, i.e.,
packaged
combination of reagents in predetermined amounts with instructions for
performing the diagnostic assay. Where the antibody variant is labeled with an
enzyme, the kit may include substrates and cofactors required by the enzyme ,
(e,g., a substrate precursor which provides the detectable chromophore or
fluorophore). in addition, other additives may be included such as
stabilizers,
buffers (e.g., a block buffer or lysis buffer) and the like. The relative
amounts of
37
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WO 2005/075504 PCT/US2004/024360
various reagents may be varied widely to provide for concentrations in
solution
the reagents which substantially optimize the sensitivity of the assay.
~rticularly, the reagents may be provided as dry powders, usually lyophilized,
eluding excipients which on dissolution will provide a reagent solution having
the
ppropriate concentration.
N VIVO USES FOR THE ANTIBODY
It is contemplated that the antibodies of the present invention may be used to
treat
a mammal. In one embodiment, the antibody is administered to a nonhuman
mammal for the purposes of obtaining preclinical data, for examp~ 1e.
Exemplary
nonhuman mammals to be treated include nonhuman primates, clogs, cats,
rodents and other mammals in which preclinical studies are perFormed. Such
mammals may be established animal models for a disease to b~e treated with the
antibody or may be used to study toxicity of the antibody of interest. !n each
of
these embodiments, dose escalation studies may be performe d on the mammal.
The antibody or polypeptide is administered by any suitable rr~eans, including
parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal,
and, if
desired for local immunosuppressive treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous administration. In addition, the antibody
variant is
suitably administered by pulse infusion, particularly with declining doses of
the
antibody variant. Preferably the dosing is given by inject'sor~s, most
preferably
intravenous or subcutaneous injections, depending in part an whether the
administration is brief or chronic.
For the prevention or treatment of disease, the apprapria~e dosage of the
antibody
or polypeptide will depend on the type of disease to be trweated, the severity
and
course of the disease, whether the antibody variant is administered for
preventive
or therapeutic purposes, previous therapy, the patient's clinical history and
response to the antibody variant, and the discretion of'~he attending
physician.
The very high affinity anti-human fgE antibodies of the invention may be
suitably
administered to the patient at one time or over a series of treatments.
Depending on the type and severity of the disease, about 0.1 mg/kg to 150
mgJkg
{e.g., 0.1-20 mglkg) of antibody is an initial candidate: dosage for
administration to
the patient, whether, for example, by one or more se: parate administrations,
o~- by
38
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continuous infusion. A typical daily dosage might range from about 1 mglkg to
100
mg/kg or more, depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the condition, the
treatment is sustained until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful. The progress of this therapy is
easily monitored by conventional techniques and assays. An exemplary dosing
regimen for an anti-LFA-1 or anti-ICAM-1 antibody is disclosed in WO 94/04188.
(0150] The antibody variant composition will be formulated, dosed and
administered in a
manner consistent with good medical practice. Factors for consideration in
this
context include the particular disorder being treated, the particular mammal
being
treated, the clinical condition of the individual patient, the cause of the
disorder,
the site of delivery of the agent, the method of administration, the
scheduling of
administration, and other factors known to medical practitioners. The
"therapeutically effective amount" of the antibody variant to be administered
will
be governed by such considerations, and is the minimum amount necessary to
prevent, ameliorate, or treat a disease or disorder. The antibody variant need
not
be, but is optionally formulated with one or more agents currently used to
prevent
or treat the disorder in question. The effective amount of such other agents
depends on the amount of antibody present in the formulation, the type of
disorder
or treatment, and other factors discussed above. These are generally used in
the
same dosages and with administration routes as used hereinbefore or about from
1 to 99°I° of the heretofore employed dosages.
[0151] The antibodies of the present invention which recognize 1gE as their
target may be
used to treat "IgE-mediated disorders". These include diseases such as asthma,
allergic rhinitis & conjunctivitis (hay fever), eczema, urticaria, atopic
dermatitis,
and food allergies. The serious physiological condition of anaphylactic shock
caused by, e.g., bee stings, snake bites, food or medication, is also
encompassed
under the scope of this invention.
ANTIBODY EPITOPE MAPPING
[0152] The term "epitope" refers to a site on an antigen to which B and/or T
cells
respond. B-calf epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of a protein.
Epitopes
formed from contiguous amino acids are typically retained on exposure to
39
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denaturing solvents, whereas epitopes formed by tertiary folding are typically
lost
on treatment with denaturing solvents. An epitope typically includes at least
3, and
more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
Antibodies that recognize the same epitope can be identified in a simple
immunoassay showing the ability of one antibody to block the binding of
another
antibody to a target antigen.
[0153] Epitope mapping of the binding site for high affinity antibodies IgE of
the present
invention involved Western blot analysis for binding, a peptide scan of the
CH3
domain of IgE, an alanine scan of the regions that showed binding, amino acid
replacement from corresponding regions of IgG1 and site directed mutagenesis.
[0154] The peptide scan of the entire CH3 domain of IgE required seventy-three
overlapping peptides. Each peptide was subjected to binding by labeled anti-
IgE
antibodies of the present invention to determine the specific epitope(s) of
IgE that
block binding of IgE to its high affinity receptor. The peptide scan
identified two
peptides as potential anti-IgE MAb contact sites on IgE, designated Epitope
'A'
and Epitope 'B' (See Figure 11 ). Although the Epitope 'A' and Epitope 'B'
sequences are about 80 amino acids apart in the linear sequence, they are
positioned close to each other in the three dimensional structure of IgE. Both
are
surface exposed, they overlap the FceRl binding site of IgE, and in both
peptides,
there are positively charged residues of Arg and hydrophobic residues of Pro.
Figure 12 illustrates the binding region of Epitope B as determined by ELISA
using
peptide scan.
[0155] A determiriation of which amino acid residues are critical for binding
of high affinity
antibodies within these epitopes was performed by alanine scanning
mutagenesis.
(Cunningham et al., "High-Resolution Epitope Mapping of hGH-Receptor
Interactions by Alanine-Scanning Mutagenesis" Science 244:1081-1085). Alanine
was substituted for each residue of Epitope A and Epitope B and binding of
high
affinity monoclonal antibodies was determined. (See Example 12 below and
Figures 13 and 14).
ACTIVE AND PASSIVE IMMUNIZATION
[0156] The invention also relates to pharmaceutical compositions, e.g.,
vaccines,
comprising the peptide immunogen molecules of the present invention, including
SEQ ID NO 72 and/or SEQ ID NO 74, and diluents, excipients, adjuvants, or
CA 02552999 2006-07-10
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carriers. It further concerns a process for the preparation of an immunogen of
the
invention, comprising covalently coupling at least one peptide of the
invention with
a moiety capable of eliciting an immune response against that peptide.
[0157] It also relates to immunogenic peptides as defined above, for use as a
pharmaceutical, e.g. in the treatment of IgE-mediated diseases or conditions,
such
as allergy and atopic dermatitis.
[0158] It further relates to a method of immunizing a mammal against IgE-
mediated
diseases or conditions, such as allergies and atopic dermatitis, comprising
the
administration of a therapeutically effective amount of the immunogenic
peptides
as defined above to a patient in need of such treatment.
[0159] The immunogenic peptides of the present invention, while being
substantially
incapable of, mediating non-cytolytic histamine release, are capable of
eliciting
antibodies with strong serological cross-reactivity with the target amino acid
sequences of Epitope A and/or Epitope B.
[0160] The initial dose of peptide (e.g. from about 0.2 mg to about 5 mg) may
be
administered, for example, intramuscularly, followed by repeat (booster) doses
of
the same at 14 to 28 days later. Doses will of course depend to some extent on
the age, weight and general health of the patient. Immunization may be
"active" or
"passive". In "active" immunization the subject receives an immunogenic
peptide
of the present invention and an anti-IgE response is actively induced by the
subject's immune system.
(0161] "Active" immunization, this is preferred for human use, but other
mammalian
species may be treated similarly, as e.g. in the dog. The term "immunogenic
carrier" herein includes those materials which have the property of
independently
eliciting an immunogenic response in a host animal and which can be covalently
coupled to polypeptide either directly via formation of peptide or ester bonds
between free carboxyl, amino or hydroxyl groups in the polypeptide and
corresponding groups on the immunogenic carrier material, or alternatively by
bonding through a conventional bifunctional linking group, or as a fusion
protein.
[0162] Examples of such immunogenic carriers include: albumins, such as BSA;
globulins; thyroglobulins; hemoglobins; hemocyanins (particularly Keyhole
Limpet
Hemocyanin [KLH]); proteins extracted from ascaris, e.g. ascaris extracts such
as
those described in J. Immunol. 111 [1973] 260-268, J. Immunol. 122 [1979] 302-
41
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WO 2005/075504 PCT/US2004/024360
308, J. Immun. 98 [1967] 893-900, a Am. J. Physiol. 199 [1960] 575-578 or
purified products thereof; polylysine; polyglutamic acid; lysine-glutamic acid
copolymers; copolymers containing lysine or ornithine; etc. Vaccines have
been.
produced using diphteria toxoid or tetanus toxoid as immunogenic carrier
material
(Lepow M. L. et al., J. of Infectious Diseases 150 [1984] 402-406; Coen
Beuvery,
E. et al., Infection and Immunity 40 [1983] 39-45) and these toxoid materials
can
also be used in the present invention. The purified protein derivative of
tuberculin
(PPD) is particularly preferred for utilization in the "active" immunization
scheme
since (I) it does not induce a T-cell response itself (i.e. it is in effect a
"T-cell
hapten"), and yet behaves as a fully processed antigen and is recognized by T-
cells as such; (2) it is known to be one of the most powerful hapten
"carriers" in
the linked recognition mode; and (3) it can be used in humans without further
testing.
[0163] The present invention also relates to polynucleotides encoding the
peptides of the
present invention, vectors comprising said polynucleotides, and cells
harboring
said vectors. In addition, active immunization may be achieved by
administering
the polynucelotides encoding the peptides of the present invention. Vectors
suitable for such herapy are known in the art and include, e.g., adenovirus
vectors.
[0164] "Passive" immunization is achieved by administering anti-IgE antibodies
of the
present invention, to a patient suffering from IgE-mediated disease or
condition.
[0165] These antibodies can be prepared by administering an immunogenic
peptide of
the present invention to a non-human mammal and collecting the resultant
antiserum. Improved titres can be obtained by repeated injections over a
period of
time. There is no particular limitation to the species of mammals which may be
used for eliciting antibodies; it is generally preferred to use rabbits or
guinea pigs,
but horses, cats, dogs, goats, pigs, rats, cows, sheep, etc., can also be
used.
Antibody is recovered by collecting blood from the immunized animal after the
passage of 1 to 2 weeks subsequently to the last administration, centrifuging
the
blood and isolating serum from the blood. Monoclonal antibodies may e.g. be
human or murine.
[0166] When immunizing a subject, an antibody of the present invention can be
introduced into the mammal by, e.g., intramuscular injection. However, any
form
42
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of antibody administration may be used. Any conventional liquid or solid
vehicle
may be employed which is acceptable to the subject and does not have adverse
side effects. Phosphate-buffered saline (PBS), at a physiological pH, e.g.
about
pH 6.8 to 7.2, preferably about pH 7.0, may be used as a vehicle, alone or
with a
suitable adjuvant, such as an aluminium hydroxide-based adjuvant.
[0167] The following examples are offered by way of illustration and not by
way of
limitation.
EXAMPLES
Example 1
Humanization of Anti-IgE Murine MAb TES-C21
[0168] The sequences of the heavy chain variable region (VH) and the light
chain variable
region (V~) of murine mAb TES-C21 were compared with human antibody
germline sequences available in the public databases. Several criteria were
used
when deciding on a template as described in step 1 above, including overall
length, similar CDR position within the framework, overall homology, size of
the
CDR, etc. All of these criteria taken together provided a result for choosing
the
optimal human template as shown in the sequence alignment between TES-C21
MAb heavy and light chain sequences and the respective human template
sequences depicted in Figure 3A and 3B.
[0169] In this case, more than one human framework template was used to design
this
antibody. The human template chosen for the VH chain was a combination of
DP88 (aa residues 1-95) and JH4b (aa residues 103-113) (See Figure 3B). The
human template chosen for the V~ chain was a combination of L16 (VK subgroup
III, as residues 1-87) combined with JK4 (aa residues 98-107) (See Figure 3A).
The framework homology between the murine sequence and the human template
was about 70% for VH and about 74% for V~.
[0170] Once the template was chosen, a Fab library was constructed by DNA
synthesis
and overlapping PCR as described above and depicted in Fig.2. The library was
composed of synthesized TES-C21 CDRs synthesized with the respective chosen
human templates, DP88lJH4b and L16/JK4. The complexity of the library was
4096 (= 2~2). The overlapping nucleotides encoding partial VH and V~ sequences
43
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WO 2005/075504 PCT/US2004/024360
were synthesized in the range of about 63 to about 76 nucleotides with 18 to
21
nucleotide overlaps.
[0171] PCR amplification of V~ and VH gene was performed using a biotinylated
forward
primer containing the specific sequence to the framework region FR1 and an
overhanging sequence annealed to the end of leader sequence (Genelll) and a
reverse primer from the conserved constant region (Co or CH 1 ) under standard
PCR conditions. The PCR product was purified by agarose gel electrophoresis,
or
by commercial PCR purification kit to remove unincorporated biotinylated
primers
and non-specific PCR.
[0172] 5'-Phosphorylation of PCR product was performed using 2,ug PCR product,
1,uL of
T4 polynucleotide kinase (10 units/,uL), 2~L of 10x PNK buffer, 1,uL of 10mM
ATP
in a total volume of 20,~L adjusted by ddH20. After incubating at 37°C
for 45
minutes, and heat denaturation at 65°C for 10 min, the reaction volume
was
adjusted to 200~L by adding ddH~O for the next step.
[0173] The 100,uL of streptavidin-coated magnetic beads were washed twice with
200,uL
2x B&W buffer and resuspended in 200~L 2x B&W buffer. The phosphorylated
PCR product was mixed with beads, and incubated at room temperature (RT) for
16 min with mild shaking.
[0174] The beads were sedimented and washed twice with 200,uL 2x B&W buffer.
The
non-biotinylated ssDNA (minus strand) was eluted with 300,uL freshly prepared
0.15M NaOH at RT for 10 min with mild shaking. A second NaOH elution can
increase the yield slightly (optional). The eluant was centrifuged to remove
any
trace beads.
[0175] The ssDNA was precipitated from the supernatant by adding l,uL glycogen
(10mg/mL), 1/10 volume of 3M NaOAc (pH 5.2), and 2.5 volumes of EtOH. The
precipitated ssDNA was then washed with 70% EtOH followed by lyophilizing for
3
min and dissolving in 20,uL ddH20. The ssDNA was quantitated by spotting on an
ethidium bromide (EtBr) agarose plate with DNA standards, or by measuring
OD2so.
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Example 2
Cloning of VH and V~ into Phage-Expression Vector
[0176] VH and V~ were cloned into a phage-expression vector by hybridization
mutagenesis. Uridinylated templates were prepared by infecting CJ236 E. coli
strain (duf ung'} with M13-based phage (phage-expression vector TN003).
[0177] The following components [200 ng of uridinylated phage vector (8.49
kb); 92 ng
phosphorylated single-stranded H chain (489 bases}; 100 ng phosphorylated
single-stranded L chain (525 bases); l,uL 10X annealing buffer; adjust volume
with ddH20 to 10,1] were annealed (at about 8-fold molar ratio of insert to
vector)
by PCR holding the temperature at 85°C for 5 min (denaturation) and
then
tamping to 55°C over 1 hour. The samples were chilled on ice.
[0178] To the annealed product the following components were added: 1.4,uL 10
X
synthesis buffer; 0.5,~L T4 DNA ligase (1 unit/,uL); 1 ,uL T4 DNA polymerase
(1
unit/~rL} followed by incubating on ice for 5 min, and 37°C for 1.5
hours. The
product was then ethanol precipitated, and dissolved in 10,uL of ddH20 or TE.
[0179] DNA was digested with 1 ,~L Xbal (1 Ounit/,uL} for 2 h, and heat
inactivated at 65°C
for 20 min. Digested DNA was transfected into 50 ~L of electro-competent
DH10B cells by electroporation. The resulting phage were titered by growing on
XL-1 Blue bacterial lawn at 37°C overnight. Clones were sequenced to
confirm
composition.
Example 3
Deep Well Culture for Library Screening
A. Plating Phage Library
[0180] The phage library was diluted in LB media to achieve the desired number
of
plaques per plate. High titer phage was mixed with 200 ~uL XL-1 B cell
culture.
3mL LB top agar was mixed, poured onto an LB plate, and allowed to sit at room
temperature for 10 minutes. The plate was incubated overnight at 37°C.
B. Phage Elution
[0181] 100,uL of phage elution buffer (10mM Tris-CI, pH 7.5, 10mM EDTA, 100mM
NaCI) was added to each well of a sterile U-bottom 96 well plate. A single
phage
plaque from the overnight library plate was transferred with a filtered
pipette tip to
a well. The phage elution plate was incubated at 37°C for 1 hour. The
plate may
be stored at 4°C following incubation.
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C. Culture for Deep Well Plates
[0182] XL1 B cells from 50mL culture were added to 2xYT media at a 1:100
dilution. The
cells were grown at 37°C in a shaker until the A6oo was between 0.9 to
1.2.
C. Infection with Phage in Deep Well Plates
[0183] When the cells reached the appropriate OD, 1 M IPTG (1:2000) was added
to the
XL1 B culture. The final concentration of IPTG was 0.5mM. 750,uL of cell
culture
was transferred to each well of a 96 well - deep well plate (Fisher
Scientific).
Each well was inoculated with 25pL of eluted phage. The deep well plate was
placed in the shaker (250rpm) and incubated overnight at 37°C.
D. Preparing Supernatant for ELISA Screening
[0184] Following incubation, the deep well plates were centrifuged at 3,250
rpm for 20
minutes using the Beckman JA-5.3 plate rotor. 50,uL of supernatant was
withdrawn from each well for ELISA.
E. Innoculation of 15mL Liquid Cultures of XL-1 cells
[0185] XL-1s were grown at 37°C in the shaker (250rpm) in 2xYT
containing 10,~g/mL of
tetracycline until A6oo = 0.9 to 1.2. IPTG was added at a final concentration
of
0.5mM and 15mL of the culture was tranferred to a 50mL conical tube for each
clone to be characterized. The cells were inoculated with 10,~L of phage from
the
high titer stock (titer = -10~~ pfu/mL) and incubated for 1 hour at
37°C. The cells
were grown overnight at room temperature with shaking.
F. Isolation of Soluble Fab from Periplasm
[0186] The cells were pelleted in an IEC centrifuge at 4,500 rpm for 20
minutes. Culture
medium was removed the pellet was resuspend in 650,~L of resuspension buffer
(50mM Tris, pH 8.0 containing 1 mM EDTA and 500mM sucrose), vortexed, and
placed on ice for 1 hour with gentle shaking. Cellular debris was removed by
centrifugation at 9,000 rpm for 10 minutes at 4°C. The supernatant
containing the
soluble Fabs was collected and stored at 4°C.
Example 4
Framework Modification
[0187] There were twelve murine/human wobble residues within the framework at
the
potential key positions described above. Position 73 in VH was kept as the
murine
residue threonine in the humanization library because this position was
46
CA 02552999 2006-07-10
WO 2005/075504 PCT/US2004/024360
determined to affect binding. It was noted, however, that threonine at VH 73
is a
common human residue in the human germline VH subgroup 1 and 2.
[0188] The framework residues that differed between the TES-C21 sequence and
the
human template were randomly substituted as described above and then
assessed for their potential affect on target binding, and antibody folding.
Potential framework residues that may have affected the binding were
identified.
In this case, they were residues 12, 27, 43, 48, 67, 69 in VH, and 1, 3, 4,
49, 60,
85 in V~ (Kabat number system). (See Figure 4) It was later demonstrated that
only positions 27 and 69 significantly affected binding in the VH region
(clone
number 1136-2C).
[0189] The primary screen used was a single point ELISA (SPE) using culture
media
(See description below). The primary screen selected clones that that bind to
the
antibody's target molecule. Clones that gave equal or better signal than the
parent molecule were selected for the next round of screening.
[0190] In the second round of screening, individual phage were grown in a 15
ml
bacterial culture and periplasmic preparations were used for SPE and ELISA
titration assays. The clones that retained higher binding in this assay were
further
characterized. Once all the selected primary clones were processed, the top 10-
15% clones were sequenced and the clones arranged according to sequence.
Representatives from each sequence group were compared against each other
and the best clones selected. Sequences from these chosen clones were
combined and the effects of various combinations were evaluated.
[0191] The constructed library was subjected to an ELISA screen for improved
binding to
the recombinant human IgE, SE44.. Clones with binding affinity greater than
murine TES-C21 Fab were isolated and sequenced. Clone ID #4, 49, 72, 76, and
136 were further characterized. ELISA titration curves for clone 4, 49, 72,
78, and
136 ire shown in Figure 5A and 5B indicating that their affinity is similar to
the
parent, TES-C21. These clones compete with murine TES-C21 for binding to
human IgE indicating that the binding epitope was not changed during the
humanization process. The humanized Fabs did not bind to FcsRl-bound IgE
suggesting that it is less likely that the humanized antibodies will crosslink
the
receptor to cause histamine release when they were constructed into divalent
IgG.
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[0192] Humanized clone 136 retained 5 murine heavy chain framework residues (=
94.3
human VH framework homology), with a 100% human light chain framework
selected for by affinity maturation. The inhibition of IgE binding to FcERI by
the
humanized Fab was demonstrated (Figure 6).
Example 5
Single Point ELISA Protocol for Screening anti IgE
[0193] Plates were coated with 2ug/mL sheep anti-human Fd in carbonate coating
buffer
overnight at 4°C. The coating solution was removedand the plates were
blocked
with 200uL/well 3% BSA/PBS for 1 hour at 37°C. After washing the plates
4x with
PBS/0.1 % TWEEN~ (PBST), 50uL/well Fab sample (i.e., supernatant containing
high titer phage and secreted Fab or periplasmic prep from DMB block, or 15mL
prep) was added. Plates were incubated for 1 hour at room temperature followed
by washing 4x with PBST. 50uL/well of biotinylated SE44 at 0.015ug/mL diluted
in 0.5% BSA/PBS and 0.05% TWEEN~was then added. Plates were then
incubated for 2 hours at room temperature and washed 4X PBST. 50uL/well
StreptAvidin HRP 1:2000 dilution in 0.5% BSA/PBS and 0.05% TWEEN~ was
added and the plates incubated 1 hour at room temperature. Plates were washed
6x with PBST. 50uLlwell TMB substrate (sigma) was added to develop and then
stopped by adding 50uL/well 0.2M H2S04.
Example 6
ELISA Titration: anti IgE
[0194] Plates were coated with 0.25uglmL (for purified Fab 0.1 ug/ml) SE44 in
carbonate
coating buffer overnight at 4°C. Coating solution was removed and the
plates
were blocked with 200uL/well 3 % BSA/PBS for 1 hour at 37°C.
[0195] The plates were washed 4x with PBS/0.1% TWEEN~ (PBST). 50uL/well Fab
(from 15mL periplasmic prep) was added starting with a dilution of 1:2 and
diluting
3 fold serially in 0.5% BSA/PBS and .05% TWEEN~20. Plates were incubated for
2 hours at room temperature.
[0196] The plates were washed 4x with PBST and 50uL/well 1:1000 (0.3ug/ml)
dilution of
biotin-sheep anti human Fd in 0.5% BSA/PBS and 0.05% TWEEN~20 was added.
The plates were incubated again for 2 hours at room temperature.
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[0197] Following a wash 4x with PBST, 50uL/well Neutra-avidin-AP 1: 2000(0.9
ug/ml) in
0.5% BSAIPBS and 0.05% TWEEN~ 20 was added and the plates were incubated
1 hour at room temperature.
[0198] The plates were washed 4x with PBST. And developed by adding 50uLlwell
pNPP substrate. Development was stopped by adding 50uL/well 3M NaOH. The
absorbance of each well was read at 405nm or 41 Onm.
Example 7
Protocol for Affinity Purification of M13 phage Expressed Soluble Fab
A. DAY 1
[0199] Two 500 mL cultures (2xYT) containing 10 mg/mL tetracycline were
innoculated
with 5 mL overnight stock XL1 B and grown at 37°C to A600 = 0.9 to 1.2.
IPTG
was added to a concentration of 0.5mM. The cell culture was then infected with
200 pL phage per culture and incubated for 1 hour at 37° C with
shaking.
Following infection, the cells were grown at 25°C overnight with
shaking.
B. DAY 2
[0200] Cells were pelleted at 3500 x g for 30 minutes at 4 °C in 250mL
centrifuge tubes.
Culture medium was aspirated and the pellets were resuspended in a total of 12-
15 mL (ysis buffer (Buffer A + protease inhibitor cocktail).
Buffer A: (1 liter)
50mM NaH2P04 6.9 g NaH2PO4H20 (or 6 g NaH2P04)
300mM NaCI 17.54 g NaCI
10mM imidazole 0.68 g imidazole (MW 68.08)
adjust pH to 8.0 using NaOH
Lysis buffer:
Mix 25 mL of Buffer A with one tablet of Complete Protease Inhibitor
Cocktail (Roche, Basel, Switzerland).
[0201] Resuspended cells were transferred into a 50mL conical tube and lysed
with
100,~L 100 mg/mL Vysozyme by inverting the tube several times until the
mixture
moves together as a blob (due to the lysis). Cells were sonicated on ice
followed
by the addition of 10 pL DNase f (about 1000 units) and gently rocked at
4°C for
30 minutes. Debri was pelleted by centrifugation at 12000 x g for 30 minutes
at 4
°C, using 50 mL centrifuge tubes. Supernatants were transferred to a
new conical
tube and stored at 4 °C.
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[0202) Ni-NT agarose (Qiagen, Valencis, CA) was used to purify the soluble
Fabs
according to the manufacturer's protocol. The lysate was mixed with Ni-NTA and
loaded into a column. The flow through was collected for SDS-PAGE analysis.
The column was washed with 20 mL buffer (50rriM NaH2P04, 300mM NaCI,
15mM imidazole, adjust pH to 8.0 with NaOH) followed by a 20 mL wash with
50mM NaH2P04, 300mM NaCI, 20mM imidazole. Fabs were eluted with 6 x 500
~L elution buffer (50mM NaH2P04, 300mM NaCI, 450mM imidazole, adjust pH to
8.0 with NaOH) and analyzed by SDS PAGE. Column fractions were stored at 4
°C. Column fractions were analyzed by SDS-PAGE and the fraction with
the
greatest amount of Fab was selected and dialyzed in PBS at 4 °C.
Example 8
Soluble Receptor Assay
[0203) A 96 well assay plate suitable for ELISA was coated with 0.05 mL
0.5,ug/mL FceRl
alpha-chain receptor coating buffer (50 mM carbonate/bicarbonate, pH 9.6) for
12
hours at 4-8°C. The wells were aspirated and 250~rL blocking buffer
(PBS, 1
BSA, pH 7.2) was added and incubated for 1 hour at 37°C. In a
separate assay
plate the samples and reference TES-C21 MAbs were titered from 200 to
0.001~g/mL by 1:4 dilutions with assay buffer (0.5% BSA and 0.05% Tween 20,
PBS, pH 7.2) and an equal volume of 100ng/mL biotinylated IgE was added and
the plate incubated for 2-3 hours at 25°C. The FceRl - coated wells
were washed
three times with PBS and 0.05% TWEEN 20 and 50,uL from the sample wells
were transferred and incubated with agitation for 30 minutes at 25°C.
Fifty ~L/well
of 1 mg/mL Streptavidin-HRP, diluted 1:2000 in assay buffer, was incubated for
30
minutes with agitation and then the plate was washed as before. Fifty,uL/well
of
TMB substrate was added and color was developed. The reaction was stopped
by adding an equal volume of 0.2 M H2S04 and the absorbance measured at
450nm.
Example 9
Binding of Antibodies to IgE-loaded FceRl
[0204) Antibody binding to human IgE associated with the alpha-subunit of
FcERI was
determined by preincubating with 10,ug/mL human IgE for 30 min at 4°C.
Plates
were washed three times followed by a one hour incubation with varying
concentrations of either murine anti-human IgE MAb E-10-10 or the humanized
CA 02552999 2006-07-10
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Fab variant. Binding of Fabs was detected with a biotin labeled anti human Fd
antibody followed by SA-HRP. Murine MAb E-10-10 was detected by Goat
anti-murine Ig Fc HRP-conjugated Ab.
Example 10
Clone Characterization
[0205] Each candidate was assayed for binding affinity and positive clones
were
sequenced. Antibody variants having beneficial mutations in CDR regions that
increase binding affinity were further characterized. Assays included Biacore
analysis; inhibition of IgE binding to its receptor; and cross linking of
receptor
bound IgE.
[0206] A library of variants was created. The amino acid sequences for the
various
CDRs which demonstrated improved affinity are depicted in Table 1. Figure 7
presents high affinity candidates having combinations of substitutions.
TABLE 1.
CDRL1: CDRH1:
P RASQSIGTNIHSEQ ID NO P MYWLE SEQ ID NO
5 15
#1 RASRSIGTNIHSEQ ID NO #1 WYWLE SEO ID NO
6 16
#2 RASQRIGTNIHSEQ ID NO #2 YYWLE SEQ ID NO
7 17
CDRL2: CDRH2:
P YASESIS SEQ ID NO P EISPGTFTTNYNEKFKA SEQ ID NO
8 18
#1 YAYESIS SEQ ID NO #1 EIEPGTFTTNYNEKFKASEQ ID NO
9 19
#2 YASESIY SEQ ID NO #2 EIDPGTFTTNYNEKFKASEQ ID NO
10 20
#3 YASESDS SEQ (D NO #3 EISPDTFTTNYNEKFKASEQ ID NO
11 21
#4 YASESES SEQ ID NO #4 EISPETFTTNYNEKFKASEQ ID NO
12 22
CDRL3: #5 EISPGTFETNYNEKFKASEQ ID NO
23
P QQSDSWPTT SEQ ID NO #6 EIEPGTFETNYNEKFKASEQ ID NO
13 24
#1 AASWSWPTT SEQ ID NO #7 EIDPGTFETNYNEKFKASEQ ID NO
14 25
CDRH3:
P FSHFSGSNYDYFDY SEQ ID NO
26
#1 FSHFSGMNYDYFDY SEQ ID NO
27
#2 FSHFSGQNYDYFDY SEQ ID NO
28
#3 FSHFTGSNYDYFDY SEQ ID NO
29
P = Parent
51
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[0207] Nineteen heavy chain variants are presented in Figure 9 and 35 light
chain
variants are presented in Figure 8. Three candidates were further
characterized
for binding affinity and these are presented in Table 2.
TABLE 2 Binding Affinity
Mab ~ KD Foid Increase
in
Binding Affinity
TES-C21 614 200 pM
MAb 1 (CL-5A) 0.158 pM 3886
MAb 2 (CL-2C) 1.47 0.5 pM 417
MAb 3 (CL-51) 3.2 2.2 pM 191
Example 11
Expression and purification of anti-IgE antibodies and HRP-conjugation
(0208] High affinity MAbs candidates were generated. For the generation of
intact anti-
IgE MAbs, the heavy and light chains variable regions were PCR amplified from
phage vectors templates and subcloned separately into H- and L-chain
expression
vectors under the expression of a CMV promoter. Six full antibody clones were
constructed and are represented in Figure 10 A-F. Appropriate heavy and light
chain plasmids were co-transfected into the mouse myeloma cell line NSO using
electroporation by techniques well known in the art. See, e.g., Liou et al. J
Immunol. 143(12):3967-75 (1989). Antibodies were purified from the single
stable
cell line supernatants using protein A-sepharose (Pharmacia). The
concentration
of the antibody was determined using spectrophotometer at 280nm and FCA
assay (IDEXX).
[0209] Purified antibodies were conjugated by horseradish peroxidase (HRP)
using
peroxidase conjugation kit (Zymed Labs, San Francisco, CA) according to the
manufacturer's protocol. The titer of each conjugated anti-IgE MAb was
determined using ELISA with plates coated with a monoclonal human IgE (SE44).
52
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WO 2005/075504 PCT/US2004/024360
[0210] The following cultures have been deposited with the American Type
Culture
Collection, 10801 University Boulevard, Manassas Va. 20110-2209 USA (ATCC):
Hybridoma ATCC NO. Deposit Date
Anti-IgE CL-2C PTA-5678 December 3, 2003
Anti-IgE CL-5A PTA-5679 December 3, 2003
Anti-IgE CL-51 PTA-5680 December 3, 2003
[0211] This deposit was made under the provisions of the Budapest Treaty on
the
International Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure and the Regulations thereunder (Budapest Treaty). This
assures maintenance of a viable culture for 30 years from the date of deposit.
The
organism will be made available by ATCC under the terms of the Budapest
Treaty, which assures permanent and unrestricted availability of the progeny
of
the culture to the public upon issuance of the pertinent U.S. patent.
[0212] The assignee of the present application has agreed that if the culture
on deposit
should die or be Post or destroyed when cultivated under suitable conditions,
it will
be promptly replaced on notification with a viable specimen of the same
culture.
Availability of the deposited strain is not to be construed as a license to
practice
the invention in contravention of the rights granted under the authority of
any
government in accordance with its patent laws.
[0213] The foregoing written specification is considered to be sufficient to
enable one
skilled in the art to practice the invention. The present invention is not to
be limited
in scope by the cultures deposited, since the deposited embodiments are
intended
as illustration of one aspect of the invention and any culture that are
functionally
equivalent are within the scope of this invention. The deposit of material
herein
does not constitute an admission that the written description herein contained
is
inadequate to enable the practice of any aspect of the invention, including
the best
mode thereof, nor is it to be construed as limiting the scope of the claims to
the
specific illustration that it represents. Indeed, various modifications of the
invention
in addition to those shown and described herein will become apparent to those
skilled in the art from the foregoing description and fall within the scope of
the
appended claims.
53
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Example 12
Mapping of the High Affinity Binding Epitope of Human IgE
A. Peptide synthesis and anti-IgE binding assay
[0214] Studies have shown that IgE binds to its receptor through the CH3
domain. Since
the HA Anti-IgE antibodies of the present invention very efficiently block IgE
from
binding to its receptor, we mapped the epitope using peptides that encompassed
the entire CH3 domain. First, we prepared two V5-tagged peptides, one
comprised the entire constant region of human IgE and one comprised just the
CH2-CH3 region of human IgE. These two peptides were expressed by in vitro
transcription-translation and used in a Western blot assay to detect HA Anti-
IgE
MAb binding. Both CL-2C and CL-51 MAbs were able to bind to the intact human
IgE as well as both the peptides.
[0215] To map the epitope more specifically, 73 overlapping peptides were
synthesized
which encompassed amino acid residues 141 to 368 of human IgE, which
included the entire CH3 domain. Each peptide consisted of 12 amino acid
residues having a 3 amino acid overlap with the 3' end of the previous
peptide.
SPQTs membranes were synthesized with fluorenylmethoxycarboyl (Fmoc) amino
acids on cellulose membrane. The membranes were rinsed in methanol and then
washed in TBS (pH 7.5) 3X for 10 min. After an overnight incubation in
blocking
solution (5% milk or 3% BSA in TBS), HRP-labeled anti-IgE MAbs diluted in
blocking solution were incubated with the membrane for 3 hrs. After washing 3X
for 15 min in TBS-TWEEN~, using SuperSignal HRP substrate (Pierce), IgE
reactivity was measured by chemiluminescence exposure of BioMax MS film
(Kodak) for the desired time.
[0216] The results from the experiment indicate that the HA Anti-IgE MAbs bind
to two
regions in the CH3 portion of IgE, which are represented by the following two
peptide sequences: NPRGVSAYLSRP (epitope "A") and HPHLPRALMRST
(Epitope "B"). (See Figure 12.) Binding to epitope A was several times weaker
than to epitope B.
B. Alanine Scan Mutagenesis
[0217] An alanine scan along with substitutions of amino acids in the peptides
with those
that are found in IgG1 was carried out to determine which amino acids are
critically involved in HA Anti-IgE MAb's binding to these peptides. Amino
acids
54
CA 02552999 2006-07-10
WO 2005/075504 PCT/US2004/024360
that were determined to be important for HA Anti-IgE MAb binding were replaced
using an in vitro mutagenesis strategy in the E chain of IgE. Another peptide
covering the CE2 and Ce3 regions as described earlier was also used in this
study.
(See Figures 13 and 14).
[0218] The EU numbering scheme for human IgE amino acid residues has been
used.
Polymerase chain reaction (PCR) was used to amplify the entire Fc region of
IgE,
and a truncated form of IgE Fc containing only the CH2-CH3 domain. The DNA
products were cloned directly into pcDNA3 expression vector (Invitrogene,
Carlsbad,CA) using TOPO cloning (Invitrogene, Carlsbad, CA).
[0219] Mutagenesis in IgE-Fc was performed using overlapping PCR (Ho et al.,
1989).
The DNA products were purified by agarose gel electrophoresis, digested with
an
appropriate restriction enzyme(s), and subcloned into the pcDNA3 expression
vector. For each variant construct, PCR amplified regions were completely
sequenced using dideoxynucleotide method from both strands of DNA.
Recombinant human IgE Fc and its mutants were expressed using reticulocyte
lysate based in-vitro transcription and translation coupled system (Promega,
Madison, WI)
[0220] Lysates from this in-vitro transcription and translation coupled system
(10 u1
reaction mix) were subjected to SDS-PAGE (12 %) and then transferred to
nitrocellulalose membranes. The membranes were blocked with 5% dry milk in
Tris-buffered saline (TBS) and subsequently stained with the primary antibody,
anti-IgE MAbs. Specific reactive bands were detected using a goat anti-human
IgG Fc conjugated to horseradish peroxidase (Jackson Labs, Bar Harbor, Maine)
and the immunoreactive bands were visualized by the SuperSignal Western
blotting detection kit (Pierce). Anti-V5 antibodies were used as a positive
control
that detected the V5 tag introduced at the C-terminus of these peptides. The
Western blot with anti-V5 antibodies demonstrated that all the peptides were
expressed at almost equal level. Interestingly, HA Anti-IgE MAbs were able to
bind to the peptide that carried mutations in epitope 'A', but, they did not
bind to
the peptide that carried mutations in epitope 'B', indicating that this second
site
was more important for binding. (See Figure 15).
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Example 13
Active Immunization of Transgenic Mice Using an Immunogenic Peptide of
Epitope B
[0221] Transgenic mice that constitutively express human IgE were used to
demonstrate
the active production of antibodies to a human immunogenic peptide of Epitope
B.
Two fusion peptides, each comprising an immunogenic peptide of the invention,
a
cysteine residue and KLH, were chemically synthesized. The sequence of
peptide 1 was
(KLH-Cys) - Leu Pro Arg Ala Leu Met Arg Ser Thr
and the sequence of peptide 2 was:
Leu Pro Arg Ala Leu Met Arg Ser Thr - (Cys-KLH)
[0222] The transgenic mice were injected subcutaneously with 20,ug of the
immunogenic
peptide in complete Freund's adjuvant (Difco Laboratories, Detroit, MI) in
200,~L
of PBS pH 7.4. At two-week intervals the mice were twice injected
subcutaneously with 20 ~g of the peptide immunogen in incomplete Freund's
adjuvant. Then, two weeks later and three days prior to sacrifice, the mice
were
again injected intraperitoneally with 20,ug of the same immunogen in PBS.
Serum
was collected and tested for the presence of anti-IgE antibodies specific for
Epitope B. As seen in Figure 16, the peptide elicited anti-IgE antibodies in
these
transgenic mice.
[0223] Those skilled in the art will recognize, or be able to ascertain using
no more than
routine experimentation, many equivalents to the specific embodiments of the
invention described herein. Such equivalents are intended to be encompassed by
the following claims.
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IN THE UNITED STATES RECEIVING OFFICE FOR THE PCT
Applicant: Tanox, Inc. et al.
PCT Application Claiming Priority to PCT/US04/02892 and PCT/US04/02894
International Filing Date: 29 JULY 2004
For: IDENTIFICATION OF NOVEL IgE EPITOPES
Dear Sirs:
STATEMENT REGARDING SEQUENCE LISTING
Applicants hereby state that the Sequence Listing contains 77 amino
acid sequences appearing in the specification, figures and claims. This
Sequence Listing does no introduce any new matter and does not go beyond
the disclosure as originally filed. Applicants also state that the information
recorded on the computer readable disks is identical to the written Sequence
Listing attached herewith.
Respectfully Submitted,
Dated: 29 Jul 2004. BY: '
Cheryl A iljestrand
Reg. N . 5,275
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST L,E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional valumes please contact the Canadian Patent Office.
