U.S. patent application number 13/758858 was filed with the patent office on 2013-08-15 for antibodies that bind il-4 and/or il-13 and their uses.
This patent application is currently assigned to SANOFI. The applicant listed for this patent is Matthew DAVISON, Jochen KRUIP, Danxi LI, Vincent MIKOL, Ercole RAO. Invention is credited to Matthew DAVISON, Jochen KRUIP, Danxi LI, Vincent MIKOL, Ercole RAO.
Application Number | 20130209469 13/758858 |
Document ID | / |
Family ID | 39313041 |
Filed Date | 2013-08-15 |
United States Patent
Application |
20130209469 |
Kind Code |
A1 |
RAO; Ercole ; et
al. |
August 15, 2013 |
ANTIBODIES THAT BIND IL-4 AND/OR IL-13 AND THEIR USES
Abstract
The present invention relates to novel humanized anti-IL-4 and
IL-13 antibodies and fragments thereof and novel bispecific
antibodies and fragments thereof that specifically bind to IL-4 and
IL-13. The invention also includes uses of the antibodies to treat
or prevent IL-4 and/or IL-13 mediated diseases or disorders,
including allergic asthma and dermatitis.
Inventors: |
RAO; Ercole;
(Morfelden-Waldorf, DE) ; MIKOL; Vincent;
(Charenton-le-Pont, FR) ; LI; Danxi; (Skillman,
NJ) ; KRUIP; Jochen; (Erzhausen, DE) ;
DAVISON; Matthew; (Long Valley, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RAO; Ercole
MIKOL; Vincent
LI; Danxi
KRUIP; Jochen
DAVISON; Matthew |
Morfelden-Waldorf
Charenton-le-Pont
Skillman
Erzhausen
Long Valley |
NJ
NJ |
DE
FR
US
DE
US |
|
|
Assignee: |
SANOFI
Paris
FR
|
Family ID: |
39313041 |
Appl. No.: |
13/758858 |
Filed: |
February 4, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12682864 |
Apr 13, 2010 |
8388965 |
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PCT/US08/79787 |
Oct 14, 2008 |
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13758858 |
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61037128 |
Mar 17, 2008 |
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Current U.S.
Class: |
424/136.1 ;
435/320.1; 435/328; 435/69.6; 530/387.3; 530/391.3; 530/391.7;
536/23.53 |
Current CPC
Class: |
C07K 2317/64 20130101;
C07K 16/244 20130101; C07K 16/247 20130101; A61P 37/02 20180101;
C07K 2317/76 20130101; A61P 37/00 20180101; A61P 35/00 20180101;
A61P 37/08 20180101; C07K 16/468 20130101; C07K 2317/31 20130101;
A61P 29/00 20180101; A61P 43/00 20180101; A61P 11/06 20180101; A61P
11/00 20180101; A61K 39/00 20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 530/391.7; 536/23.53; 435/320.1; 435/328; 435/69.6;
530/391.3 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 15, 2007 |
EP |
07291259.5 |
Claims
1-49. (canceled)
50. A bispecific antibody or bispecific antibody fragment thereof
that specifically binds to IL-13 and IL-4, wherein the bispecific
antibody or bispecific antibody fragment thereof comprises two
light chains and two heavy chains; wherein the light chains
comprise an outer variable light chain domain linked to an inner
variable light chain domain which is linked to a constant light
chain domain (CL), and the heavy chains comprise an outer variable
heavy chain domain linked to an inner variable heavy chain domain
which is linked to a constant heavy chain domain (CH1); and
wherein: (a) the outer variable light chain domain comprises the
amino acid sequence of SEQ ID NO: 1, the inner variable light chain
domain comprises the amino acid sequence of SEQ ID NO: 3, the outer
variable heavy chain domain comprises the amino acid sequence of
SEQ ID NO: 2 and the inner variable heavy chain domain comprises
the amino acid sequence of SEQ ID NO: 4; (b) the outer variable
light chain domain comprises the amino acid sequence of SEQ ID NO:
1 the inner variable light chain domain comprises the amino acid
sequence of SEQ ID NO: 3, the outer variable heavy chain domain
comprises the amino acid sequence of SEQ ID NO: 2, and the inner
variable heavy chain domain comprises the amino acid sequence of
SEQ ID NO: 5; (c) the outer variable light chain domain comprises
the amino acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES
(SEQ ID NO: 9), and QQNAEDSRT SEQ ID NO: 10 the inner variable
light chain domain comprises the amino acid sequences HASQNIDVWLS
(SEQ ID NO: 14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID
NO: 16), the outer variable heavy chain domain comprises the amino
acid sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12),
and DGYFPYAMDF (SEQ ID NO: 13), and the inner variable heavy chain
domain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO:
17), IDPSDGETR (SEQ ID NO: 18), and LKEYGNYDSFYFDV (SEQ ID NO: 19);
or (d) the outer variable light chain domain comprises the amino
acid sequences RASESVDSYGQSYMH (SEQ ID NO: 8), LASNLES (SEQ ID NO:
9), and QQNAEDSRT (SEQ ID NO: 10) the inner variable light chain
domain comprises the amino acid sequences HASQNIDVWLS (SEQ ID NO:
14), KASNLHTG (SEQ ID NO: 15), and QQAHSYPFT (SEQ ID NO: 16), the
outer variable heavy chain domain comprises the amino acid
sequences GFSLTDSSIN (SEQ ID NO: 11), DGRID (SEQ ID NO: 12), and
DGYFPYAMDF (SEQ ID NO: 13), and the inner variable heavy chain
domain comprises the amino acid sequences GYSFTSYWIH (SEQ ID NO:
20), IDASDGETR (SEQ ID NO: 21), and LKEYGNYDSFYFDV (SEQ ID NO:
22).
51-54. (canceled)
55. The bispecific antibody or bispecific antibody fragment thereof
of claim 50 that further comprises additional constant region
domains.
56. The bispecific antibody or bispecific antibody fragment thereof
of claim 55, wherein the additional constant region domains are
C.sub.H2 and C.sub.H3.
57. A pharmaceutical composition comprising the bispecific antibody
or bispecific antibody fragment thereof of claim 50 and a
pharmaceutically acceptable carrier.
58. The bispecific antibody or bispecific antibody fragment thereof
of claim 50 that is further conjugated to an effector molecule.
59. The bispecific antibody or bispecific antibody fragment thereof
of claim 58, wherein the effector molecule is selected from the
group consisting of heterologous polypeptides, drugs,
radionucleotides, and toxins.
60-67. (canceled)
68. The bispecific antibody or bispecific antibody fragment thereof
of claim 50 comprising a label, wherein the label is a radiolabel,
a fluorophore, a chromophore, an imaging agent, or a metal ion.
69. (canceled)
70. The bispecific antibody or bispecific antibody fragment thereof
of claim 50, wherein a peptide linker links the outer variable
light chain domain to the inner variable light chain domain, and a
peptide linker links the outer variable heavy chain domain to the
inner variable heavy chain domain.
71. The bispecific antibody or bispecific antibody fragment thereof
of claim 70, wherein the peptide linker consists of the amino acid
sequence of SEQ ID NO: 6.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel anti-IL-4 antibodies,
anti-IL-13 antibodies and bispecific anti-IL-4/anti-IL-13
antibodies and their use in the amelioration, treatment or
prevention of diseases or disorders in mammals, including humans,
resulting from improper IL-4 and/or IL-13 activity or metabolism.
An antibody of interest may block engagement and/or signaling of a
ligand, such as IL-4 or IL-13, with a receptor or receptor complex,
such as IL-4R.alpha., IL-13R.alpha.1 and IL-13R.alpha.2.
Prophylactic, immunotherapeutic and diagnostic compositions
comprising the antibodies of interest and their use in methods for
preventing or treating diseases in mammals, including humans,
caused by inappropriate metabolism and/or activity of lymphoid and
non-lymphoid cells, including monocytes, fibroblasts and
endothelial cells, are disclosed. Such diseases include autoimmune
deficiencies and diseases caused by or characterized by
inflammation, such as allergic asthma and dermatitis.
BACKGROUND OF THE INVENTION
[0002] Interleukin-4 (IL-4) is a pleiotropic cytokine that has a
broad spectrum of biological effects on lymphoid B and T cells, and
many non-lymphoid cells including monocytes, endothelial cells and
fibroblasts. For example, IL-4 stimulates the proliferation of
several IL-2- and IL-3-dependent cell lines, induces the expression
of class II major histocompatability complex molecules on resting B
cells, and enhances the secretion of IgG4 and IgE by human B cells.
IL-4 is associated with a Th2-type immune response, and is produced
by and promotes differentiation of Th2 cells. IL-4 has been
implicated in a number of disorders, such as allergy and
asthma.
[0003] IL-13 is a recently identified (Minty, A. et al., Nature,
1993, 362, 248-250, and McKenzie, A. N. et al., Proc. Natl. Acad.
Sci. U.S.A, 1993, 90, 3735-3739) cytokine of 112 amino acids
secreted by the activated T lymphocytes, the B lymphocytes and the
mastocytes after activation.
[0004] By virtue of its numerous biological properties shared with
IL-4, IL-13 has been described as an IL-4-like cytokine. Its
activities are indeed similar to those of IL-4 on the B cells
(Defrance, T. et al., J. Exp. Med., 1994, 179, 135-143, Punnonen,
J. et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 3730-3734, Fior,
R. et al., Eur. Cytokine Network, 1994, 5, 593-600), the monocytes
(Muzio, M. R. F. et al., Blood, 1994, 83, 1738-1743, De Waal
Malefyt, R. et al., J. Immunol, 1993, 151, 6370-6381, Doyle, A. et
al., Eur. J. Immunol. 1994, 24, 1441-1445, Montaner, L. J. et al.,
J. Exp. Med., 1993, 178, 743-747, Sozzani, P. et al., J. Biol.
Chem., 1995, 270, 5084-5088) and other non-haematopoietic cells
(Herbert, J. M. et al., Febs Lett., 1993, 328, 268-270, and Derocq,
J. M. et al., Febs Lett. 1994, 343, 32-36). On the other hand,
contrary to IL-4, it does not exert a specific effect on resting or
activated T cells (Zurawuki, G. et al., Immunol. Today, 1994, 15,
19-26).
[0005] Various biological activities of IL-13 on the
monocytes/macrophages,
[0006] the B lymphocytes and certain haematopoietic precursors have
been described in detail by A. J. Minty as well as in review
articles on IL-13. Several data indicate, in addition, that this
cytokine has a pleiotropic effect on other cell types. These
non-haematopoietic cells which are directly affected by IL-13 are
endothelial and microglial cells, keratinocytes and kidney and
colon carcinomas.
[0007] One of the stages in the analysis of the signal transmitted
by a biological molecule within a cell consists in identifying its
membrane receptor. The research studies carried out to this end on
the IL-13 receptor have shown that IL-13 and IL-4 have a common
receptor, or at the very least some of the components of a common
receptor complex, as well as common signal transduction elements
(Zurawski S. M. et al., Embo Journal, 1993, 12, 2663-2670, Aversa,
G. et al., J. Exp. Med., 1993, 178, 2213-2218, Vita, N. et al.,
Biol. Chem., 1995, 270, 3512-3517, Lefort, S. et al., Febs Lett.,
1995, 366, 122-126). This receptor is present at the surface of
various cell types, in a variable number according to the cell type
considered. The comparative distribution of the IL-13 and IL-4
receptors has been indicated by A. J. Minty (Interleukin-13 for
Cytokines in Health and Disease. Eds D. G. Remick and J. S. Frie,
Marcel Decker, N.Y. 1996).
[0008] The cell surface receptors and receptor complexes bind IL-4
and/or IL-13 with different affinities. The principle components of
receptors and receptor complexes that bind IL-4 and/or IL-13 are
IL-4R.alpha., IL-13R.alpha.1 and IL-13R.alpha.2. These chains are
expressed on the surface of cells as monomers or heterodimers of
IL-4R.alpha./IL-13R.alpha.1 (Type II IL-4R) or
IL-4R.alpha./.gamma.c (Type I IL-4R). IL-4R.alpha. monomer and
IL-4R.alpha./.gamma.c heterodimer bind IL-4, but not IL-13.
IL-13R.alpha.1 and IL-13R.alpha.2 monomers bind IL-13, but do not
bind IL-4. IL-4R.alpha./IL-13R.alpha.1 heterodimer binds both IL-4
and IL-13 (Murata et al., Int. J. Hematol., 1999, 69, 13-20).
[0009] Th2-type immune responses promote antibody production and
humoral immunity, and are elaborated to fight off extracellular
pathogens. Th2 cells are mediators of Ig production (humoral
immunity) and produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13
(Tanaka, et, al., Cytokine Regulation of Humoral Immunity, 251-272,
Snapper, ed., John Wiley and Sons, New York (1996)). Th2-type
immune responses are characterized by the generation of certain
cytokines (e.g., IL-4, IL-13) and specific types of antibodies
(IgE, IgG4) and are typical of allergic reactions, which may result
in watery eyes and asthmatic symptoms, such as airway inflammation
and contraction of airway muscle cells in the lungs.
[0010] Both IL-4 and IL-13 are therapeutically important cytokines
based on their biological functions and play critical roles in many
diseases, including asthma (Curr Opin Allergy Clin Immunol 2005,
Vo. 5, 161-166). IL-4 has been shown to be able to inhibit
autoimmune disease and IL-4 and IL-13 have both shown the potential
to enhance anti-tumor immune responses. Since both cytokines are
involved in the pathogenesis of allergic diseases, inhibitors of
these cytokines could provide therapeutic benefits.
[0011] Accordingly, a need exists for improved agents that inhibit
IL-4, inhibit IL-13, and single agents that inhibit both IL-4 and
IL-13.
SUMMARY OF THE INVENTION
[0012] The present invention provides novel humanized monoclonal
and bispecific antibodies, and fragments and derivatives thereof,
which specifically bind to IL-4 and/or IL-13. Some of the anti-IL-4
and/or IL-13 mono- or bispecific antibodies, and fragments thereof,
can be altered to prevent intrachain disulfide bond formation
resulting in a molecule that is stable through manufacturing and
use in vivo. The antibodies of the present invention neutralize
IL-4 and/or IL-13 activity in the biological assays described
herein.
[0013] The invention includes the amino acid sequences of the
variable heavy and light chain of the antibodies and their
corresponding nucleic acid sequences.
[0014] Another embodiment of the present invention includes the
cell lines and vectors harboring the antibody sequences of the
present invention.
[0015] Another embodiment of the present invention is the use of
the antibodies for the preparation of a pharmaceutical composition
for the treatment of diseases and disorders associated with IL-4
and/or IL-13 function and metabolism. In particular, the present
invention relates to the treatment of cancer, autoimmune
deficiencies and diseases caused by or characterized by
inflammation, such as allergic asthma and dermatitis.
[0016] Additional features and advantages are described herein, and
will be apparent from, the following Detailed Description and the
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is a schematic drawing of a bispecific
anti-IL-4/IL-13 antibody molecule containing four polypeptide
chains. Two lighter chains consist of
N-VL.sub.hB-B13-linker-VL.sub.h8D4-8-CL-C(CL, light chain constant
region), two heavier chains consist of
N-VH.sub.B-B13-linker-VH.sub.h8D4-8-CH1-CH2-CH3-C. The linker
sequence (G4S).sub.2 is GGGGSGGGGS (SEQ ID NO: 6).
[0018] FIG. 2 illustrates the amino acid sequences of humanized
variable domains of B-B13 anti-IL-13 antibody (SEQ ID NOS: 1 and 2)
and humanized variable domains of 8D4-8 anti-IL-4 antibody (SEQ ID
NOS: 3, 4 and 5). Underline indicates amino acid changes made. Bold
indicates the CDR.
DETAILED DESCRIPTION OF THE INVENTION
[0019] This invention is not limited to the particular methodology,
protocols, cell lines, vectors, or reagents described herein
because they may vary without departing from the spirit and scope
of the invention. Further, the terminology used herein is for the
purpose of exemplifying particular embodiments only and is not
intended to limit the scope of the present invention. Unless
defined otherwise, all technical and scientific terms and any
acronyms used herein have the same meanings as commonly understood
by one of ordinary skill in the art in the field of the invention.
Any method and material similar or equivalent to those described
herein can be used in the practice of the present invention and
only exemplary methods, devices, and materials are described
herein.
[0020] All patents and publications mentioned herein are
incorporated herein in entirety by reference for the purpose of
describing and disclosing the proteins, enzymes, vectors, host
cells and methodologies reported therein that might be used with
and in the present invention. However, nothing herein is to be
construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
[0021] Prior to teaching the making and using of the IL-4 and/or
IL-13 related methods and products of interest, the following
non-limiting definitions of some terms and phrases are provided to
guide the artisan.
[0022] "Interleukin-4" (IL-4) relates to the naturally occurring,
or endogenous mammalian IL-4 proteins and to proteins having an
amino acid sequence which is the same as that of a naturally
occurring or endogenous corresponding mammalian IL-4 protein {e.g.,
recombinant proteins, synthetic proteins (i.e., produced using the
methods of synthetic organic chemistry)). Accordingly, as defined
herein, the term includes mature IL-4 protein, polymorphic or
allelic variants, and other isoforms of an IL-4 and modified or
unmodified forms of the foregoing (e.g., lipidated, glycosylated).
Naturally occurring or endogenous IL-4 includes wild type proteins
such as mature IL-4, polymorphic or allelic variants and other
isoforms and mutant forms which occur naturally in mammals (e.g.,
humans, non-human primates). Such proteins can be recovered or
isolated from a source which naturally produces IL-4, for example.
These proteins and proteins having the same amino acid sequence as
a naturally occurring or endogenous corresponding IL-4, are
referred to by the name of the corresponding mammal. For example,
where the corresponding mammal is a human, the protein is
designated as a human IL-4. Several mutant IL-4 proteins are known
in the art, such as those disclosed in WO 03/038041.
[0023] "Interleukin-13" (IL-13) refers to naturally occurring or
endogenous mammalian IL-13 proteins and to proteins having an amino
acid sequence which is the same as that of a naturally occurring or
endogenous corresponding mammalian IL-13 protein (e.g., recombinant
proteins, synthetic proteins (i.e., produced using the methods of
synthetic organic chemistry)). Accordingly, as defined herein, the
term includes mature IL-13 protein, polymorphic or allelic
variants, and other isoforms of IL-13 (e.g., produced by
alternative splicing or other cellular processes), and modified or
unmodified forms of the foregoing (e.g., Hpidated, glycosylated).
Naturally occurring or endogenous IL-13 include wild type proteins
such as mature IL-13, polymorphic or allelic variants and other
isoforms and mutant forms which occur naturally in mammals (e.g.,
humans, non-human primates). For example, as used herein IL-13
encompasses the human IL-13 variant in which Arg at position 110 of
mature human IL-13 is replaced with Gin (position 110 of mature
IL-13 corresponds to position 130 of the precursor protein) which
is associed with asthma (atopic and nonatopic asthma) and other
variants of IL-13. (Heinzmann et al, Hum Mol. Genet. 9:549-559
(2000).) Such proteins can be recovered or isolated from a source
which naturally produces IL-13, for example.
[0024] These proteins and proteins having the same amino acid
sequence as a naturally occurring or endogenous corresponding IL-13
are referred to by the name of the corresponding mammal. For
example, where the corresponding mammal is a human, the protein is
designated as a human IL-13. Several mutant IL-13 proteins are
known in the art, such as those disclosed in WO 03/035847.
[0025] The phrase "substantially identical" with respect to an
antibody chain polypeptide sequence may be construed as an antibody
chain exhibiting at least 70%, 80%, 90%, 95% or more 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%, 90%, 95%, or 97% or
more sequence identity to the reference nucleic acid sequence.
[0026] The terms, "identity" or "homology" may mean the percentage
of nucleotide bases or 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-terminal or C-terminal extensions nor insertions shall be
construed as reducing identity or homology. Methods and computer
programs for the alignment are available and well known in the art.
Sequence identity may be measured using sequence analysis
software.
[0027] The phrases and terms "functional fragment, variant,
derivative or analog" and the like, as well as forms thereof, of an
antibody or antigen is a compound or molecule having qualitative
biological activity in common with a full-length antibody or
antigen of interest. For example, a functional fragment or analog
of an anti-IL-4 antibody is one which can bind to an IL-4 molecule
or one which can prevent or substantially reduce the ability of a
ligand, or an agonistic or antagonistic antibody, to bind to
IL-4.
[0028] "Substitutional" variants are those that have at least one
amino acid residue in a native sequence removed and replaced with a
different amino acid inserted in its place at the same position.
The substitutions may be single, where only one amino acid in the
molecule is substituted, or may be multiple, where two or more
amino acids are substituted in the same molecule. The plural
substitutions may be at consecutive sites. Also, one amino acid can
be replaced with plural residues, in which case such a variant
comprises both a substitution and an insertion. "Insertional"
variants are those with one or more amino acids inserted
immediately adjacent to an amino acid at a particular position in a
native sequence.
[0029] Immediately adjacent to an amino acid means connected to
either the .alpha.-carboxyl or .alpha.-amino functional group of
the amino acid. "Deletional" variants are those with one or more
amino acids in the native amino acid sequence removed. Ordinarily,
deletional variants will have one or two amino acids deleted in a
particular region of the molecule.
[0030] The term "antibody" is used in the broadest sense, and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), antibody fragments or
synthetic polypeptides carrying one or more CDR or CDR-derived
sequences so long as the polypeptides exhibit the desired
biological activity. Antibodies (Abs) and immunoglobulins (Igs) are
glycoproteins having the same structural characteristics.
Generally, antibodies are considered Igs with a defined or
recognized specificity. Thus, while antibodies exhibit binding
specificity to a specific target, immunoglobulins include both
antibodies and other antibody-like molecules which lack target
specificity. The antibodies of the invention can be of any class
(e.g., IgG, IgE, IgM, IgD, IgA and so on), or subclass (e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.2a, IgG.sub.3, IgG.sub.4, IgA.sub.1,
IgA.sub.2 and so on) ("type" and "class", and "subtype" and
""subclass", are used interchangeably herein). Native or wildtype,
that is, obtained from a non-artificially manipulated member of a
population, 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 (V.sub.H)
followed by a number of constant domains. Each light chain has a
variable domain at one end (V.sub.L) and a constant domain at the
other end. By "non-artificially manipulated" is meant not treated
to contain or express a foreign antigen binding molecule. Wildtype
can refer to the most prevalent allele or species found in a
population or to the antibody obtained from a non-manipulated
animal, as compared to an allele or polymorphism, or a variant or
derivative obtained by a form of manipulation, such as mutagenesis,
use of recombinant methods and so on to change an amino acid of the
antigen-binding molecule.
[0031] As used herein, "anti-IL-4 antibody" means an antibody or
polypeptide derived therefrom (a derivative) which binds
specifically to IL-4 as defined herein, including, but not limited
to, molecules which inhibit or substantially reduce the binding of
IL-4 to its receptor or inhibit IL-4 activity.
[0032] As used herein, "anti-IL-13 antibody" means an antibody or
polypeptide derived therefrom (a derivative) which binds
specifically to IL-13 as defined herein, including, but not limited
to, molecules which inhibit or substantially reduce the binding of
IL-13 to its receptor or inhibit IL-13 activity.
[0033] The term "variable" in the context of a variable domain of
antibodies refers to certain portions of the pertinent molecule
which differ extensively in sequence between and among antibodies
and are used in the specific recognition and binding of a
particular antibody for its particular target. However, the
variability is not evenly distributed through the variable domains
of antibodies. The variability is concentrated in three segments
called complementarity determining regions (CDRs; i.e., CDR1, CDR2,
and CDR3) 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) regions or sequences. The variable domains of native heavy and
light chains each comprise four FR regions, largely adopting a
.beta.-sheet configuration, connected by three CDRs, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The CDRs in each chain are held together
often in proximity by the FR regions and, with the CDRs from the
other chain, contribute to the formation of the target (epitope or
determinant) binding site of antibodies (see Kabat et al. Sequences
of Proteins of Immunological Interest, National Institute of
Health, Bethesda, Md. (1987)). As used herein, numbering of
immunoglobulin amino acid residues is done according to the
immunoglobulin amino acid residue numbering system of Kabat et al.,
unless otherwise indicated. One CDR can carry the ability to bind
specifically to the cognate epitope.
[0034] The term "hinge" or "hinge region" as used in the present
invention refers to the flexible polypeptide comprising the amino
acids between the first and second constant domains of an
antibody.
[0035] The term "antibody fragment" refers to a portion of an
intact or a full-length chain or an antibody, generally the target
binding or variable region. Examples of antibody fragments include,
but are not limited to, F.sub.ab, F.sub.ab', F.sub.(ab')2 and
F.sub.v fragments. A "functional fragment" or "analog of an
anti-IL-4 and/or IL-13 antibody" is one which can prevent or
substantially reduce the ability of the receptor to bind to a
ligand or to initiate signaling. As used herein, functional
fragment generally is synonymous with, "antibody fragment" and with
respect to antibodies, can refer to fragments, such as F.sub.v,
F.sub.ab, F.sub.(ab').sub.2 and so on which can prevent or
substantially reduce the ability of the receptor to bind to a
ligand or to initiate signaling. An "F.sub.v" fragment consists of
a dimer of one heavy and one light chain variable domain in a
non-covalent association (V.sub.H-V.sub.L dimer). In that
configuration, the three CDRs of each variable domain interact to
define a target binding site on the surface of the V.sub.H-V.sub.L
dimer, as in an intact antibody. Collectively, the six CDRs confer
target binding specificity on the intact antibody. However, even a
single variable domain (or half of an F.sub.v comprising only three
CDRs specific for a target) can have the ability to recognize and
to bind target.
[0036] "Single-chain F.sub.v," "sF.sub.v" or "scAb" antibody
fragments comprise the V.sub.H and V.sub.L domains of an antibody,
wherein these domains are present in a single polypeptide chain.
Generally, the F.sub.v polypeptide further comprises a polypeptide
linker, often a flexible molecule, between the V.sub.H and V.sub.L
domains, which enables the sFv to form the desired structure for
target binding.
[0037] The term "diabodies" refers to antibody fragments with two
antigen-binding sites, which fragments can comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain. By using a linker
that is too short to allow pairing between the two variable domains
on the same chain, the diabody domains are forced to pair with the
binding domains of another chain to create two antigen-binding
sites.
[0038] The F.sub.ab fragment contains the variable and constant
domains of the light chain and the variable and first constant
domain (C.sub.H1) of the heavy chain. F.sub.ab' fragments differ
from F.sub.ab fragments by the addition of a few residues at the
carboxyl terminus of the C.sub.H1 domain to include one or more
cysteines from the antibody hinge region. F.sub.ab' fragments can
be produced by cleavage of the disulfide bond at the hinge
cysteines of the F.sub.(ab')2 pepsin digestion product. Additional
enzymatic and chemical treatments of antibodies can yield other
functional fragments of interest.
[0039] The term "linear Fab" refers to a tetravalent antibody as
described by Miller et al. (2003), J. Immunol. 170: 4854-4861. The
"linear Fab" is composed of a tandem of the same CH1-VH domain,
paired with the identical light chain at each CH1-VH position.
These molecules have been developed in order to increase the
valency of an antibody to enhance its functional affinity through
the avidity effect, but they are monospecific.
[0040] The term "bispecific antibodies (BsAbs)" refers to molecules
which combine the antigen-binding sites of two antibodies within a
single molecule. Thus, a bispecific antibody is able to bind two
different antigens simultaneously. Besides applications for
diagnostic purposes, BsAbs pave the way for new therapeutic
applications by redirecting potent effector systems to diseased
areas or by increasing neutralizing or stimulating activities of
antibodies.
[0041] Initial attempts to couple the binding specificities of two
whole antibodies against different target antigens for therapeutic
purposes utilized chemically fused heteroconjugate molecules
(Staerz et al. (1985), Nature 314: 628-631).
[0042] Bispecific antibodies have been produced from hybrid
hybridomas by heterohybridoma techniques and have demonstrated in
vitro properties similar to those observed for heteroconjugates
(Milstein & Cuello (1983) Nature 305:537-540).
[0043] Despite the promising results obtained using
heteroconjugates or bispecific antibodies produced from cell
fusions as cited above, several factors made them impractical for
large scale therapeutic applications. Such factors include: rapid
clearance of large heteroconjugates in vivo, the labor intensive
techniques required for generating either type of molecule, the
need for extensive purification of heteroconjugates away from
homoconjugates or mono-specific antibodies and generally low
yields.
[0044] Genetic engineering has been used with increasing frequency
to design, modify, and produce antibodies or antibody derivatives
with a desired set of binding properties and effector
functions.
[0045] A variety of recombinant methods have been developed for
efficient production of BsAbs, both as antibody fragments (Carter
et al. (1995), J. Hematotherapy 4: 463-470; Pluckthun et al. (1997)
Immunotechology 3: 83-105; Todorovska et al. (2001) J. Immunol.
Methods 248: 47-66) and full length IgG formats (Carter (2001) J.
Immunol. Methods 248: 7-15).
[0046] Combining two different scFvs results in BsAb formats with
minimal molecular mass, termed sc-BsAbs or Ta-scFvs (Mack et al.
(1995), Proc. Acad. Sci. USA. 92: 7021-7025; Mallender et al.
(1994) J. Biol. Chem. 269: 199-206). BsAbs have been constructed by
genetically fusing two scFvs via dimerization functionality such as
a leucine zipper (Kostelny et al. (1992) J. Immunol. 148: 1547-53;
de Kruif et al. (1996) J. Biol. Chem. 271: 7630-4).
[0047] As mentioned above, diabodies are small bivalent and
bispecific antibody fragments. The fragments comprise a VH
connected to a VL on the same polypeptide chain, by using a linker
that is too short (less than 12 amino acids) to allow pairing
between the two domains on the same chain. The domains are forced
to pair intermolecularly with the complementary domains of another
chain and create two antigen-binding sites. These dimeric antibody
fragments, or "diabodies," are bivalent and bispecific. (Holliger
et al. (1993), Proc. Natl. Acad. Sci. USA. 90: 6444-6448).
Diabodies are similar in size to a Fab fragment. Polypeptide chains
of VH and VL domains joined with linker between 3 and 12 amino
acids form predominantly dimers (diabodies), whereas with linker
between 0 and 2 amino acid residues, trimers (triabodies) and
tetramers (tetrabodies) find favor. In addition to the linker
length, the exact pattern of oligomerization seems to depend on the
composition as well as the orientation of the V-domains (Hudson et
al. (1999), J Immunol Methods 231: 177-189). The predictability of
the final structure of diabody molecules is very poor.
[0048] Although sc-BsAbs and diabodies based constructs display
interesting clinical potential, it was shown that such
non-covalently associated molecules are not sufficient stable under
physiological conditions. The overall stability of a scFv fragment
depends on the intrinsic stability of the VL and VH domains as well
as on the stability of the domain interface. Insufficient stability
of the VH-VL interface of scFv fragments has often been suggested
as a main cause of irreversible scFv inactivation, since transient
opening of the interface, which would be allowed by the peptide
linker, exposes hydrophobic patches that favor aggregation and
therefore instability and poor production yield (Worn and Pluckthun
(2001), J. Mol. Biol. 305: 989-1010).
[0049] An alternative method of manufacturing bispecific bivalent
antigen-binding proteins from VH and VL domains is disclosed in
U.S. Pat. No. 5,989,830. Such double head antibody fragments are
obtained by expressing a dicistronic vector which encodes two
polypeptide chains, whereby one polypeptide chain has two times a
VH in series by a peptide linker (VH1-linker-VH2) and the other
polypeptide chain consisting of complementary VL domains connected
in series by a peptide linker (VL1-linker-VL2). It was described in
U.S. Pat. No. 5,989,830 that each linker should comprise at least
10 amino acid residues.
[0050] Polyvalent protein complexes (PPC) with an increased valency
are described in US 2005/0003403 A1. PPCs comprise two polypeptide
chains generally arranged laterally to one another. Each
polypeptide chain typically comprises 3 or 4 "v-regions", which
comprise amino acid sequences capable of forming an antigen binding
site when matched with a corresponding v-region on the opposite
polypeptide chain. Up to about 6 "v-regions" can be used on each
polypeptide chain. The v-regions of each polypeptide chain are
connected linearly to one another and may be connected by
interspersed linking regions. When arranged in the form of the PPC,
the v-regions on each polypeptide chain form individual antigen
binding sites. The complex may contain one or several binding
specificities.
[0051] However, the use of such molecules showed aggregation,
unstability and poor expression yield (Wu et al. (2001) Prot. Eng.
14: 1025-1033). These are typical stability problems that may occur
expressing single chain based antibodies. (Worn and Pluckthun
(2001), J. Mol. Biol. 305: 989-1010).
[0052] Thus, it is the object of the present invention to provide a
bispecific polyvalent antibody by means of which the formation of
aggregates can be avoided. Furthermore, it shall have a stability
which makes it usable for therapeutic uses.
[0053] 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.
[0054] Monoclonal antibodies herein specifically include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass (type or subtype), with the
remainder of the chain(s) identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity of binding to IL-4 and/or IL-13 or impacting
IL-4 and/or IL-13 activity or metabolism (U.S. Pat. No. 4,816,567;
and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984)). Thus,
CDRs from one class of antibody can be grafted into the FR of an
antibody of different class or subclass.
[0055] Monoclonal antibodies are highly specific, being directed
against a single target site, epitope or determinant. Furthermore,
in contrast to conventional (polyclonal) antibody preparations
which typically include different antibodies directed against
different determinants (epitopes) of an antigen, each monoclonal
antibody is directed against a single determinant on the target. In
addition to their specificity, monoclonal antibodies are
advantageous being synthesized by a host cell, uncontaminated by
other immunoglobulins, and provides for cloning the relevant gene
and mRNA encoding the antibody of chains thereof. 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 well known techniques or can be purified
from a polyclonal prep. The parent monoclonal antibodies to be used
in accordance with the present invention may be made by the
hybridoma method described by Kohler et al., Nature 256:495 (1975),
or may be made by recombinant methods well known in the art.
[0056] The term "polyvalent antibody" as used in the present
invention refers to an antibody comprising two or more antigen
binding sites, thus being able to bind two or more antigens, which
may have the same or a different structure, simultaneously. The
term "bivalent" means that the antibody comprises two antigen
binding sites. The term "tetravalent" means that the antibody
comprises four antigen binding sites.
[0057] The term "antigen binding site" as used in the present
invention refers to the part of the antibody which comprises the
area which specifically binds to and is complementary to part or
all of an antigen. Where an antigen is large, an antibody may only
bind to a particular part of the antigen, which part is termed on
epitope. An antigen binding domain may be provided by one or more
antibody variable domains. Preferably, an antigen binding domain is
made of the association of an antibody light chain variable domain
(VL) and an antibody heavy chain variable domain (VH).
[0058] The term "antigen" as used in the present invention refers
to a molecule or a portion of a molecule capable of being bound by
the antibodies of the present invention. An antigen can have one or
more than one epitope. Examples of antigens recognized by the
antibodies of the present invention include, but are not limited
to, serum proteins, e.g. cytokines such as IL-4, IL5, IL9 and
IL-13, bioactive peptides, cell surface molecules, e.g. receptors,
transporters, ion-channels, viral and bacterial proteins.
[0059] The term "monospecific" as used in the present invention
means that the polyvalent antibody of the present invention
recognizes only one antigen, all the antigen binding sites being
identical.
[0060] The tem "bispecific" as used in the present invention means
that the polyvalent antibody of the present invention recognizes
two different epitopes on the same or on two different
antigens.
[0061] The term "multispecific" as used in the present invention
means that the polyvalent antibody of the present invention
recognizes multiple different epitopes on the same or on multiple
different antigens.
[0062] The term "linker" as used in the present invention refers to
a peptide adapted to connect the variable domains of the antibody
constructs of the present invention. The peptide linker may contain
any amino acids, the amino acids glycine (G) and serine (S) being
preferred. The linkers may be equal or differ from each other
between and within the heavy chain polypeptide and the light chain
polypeptide. Furthermore, the linker may have a length of 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
amino acids. A preferred peptide linker unit for the heavy chain
domains as for the light chain domains is GGGGS. The numbers of
linker units of the heavy chain and of the light chain may be equal
(symmetrical order) or differ from each other (asymmetrical
order).
[0063] A peptide linker is preferably long enough to provide an
adequate degree of flexibility to prevent the antibody moieties
from interfering with each others activity, for example by steric
hindrance, to allow for proper protein folding and, if necessary,
to allow the antibody molecules to interact with two or more,
possibly widely spaced, receptors on the same cell; yet it is
preferably short enough to allow the antibody moieties to remain
stable in the cell.
[0064] Therefore, the length, composition and/or conformation of
the peptide linkers can readily be selected by one skilled in the
art in order to optimize the desired properties of the polyvalent
antibody.
[0065] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as F.sub.v, F.sub.ab, F.sub.ab', F.sub.(ab')2 or
other target-binding subsequences of antibodies) which contain
sequences derived from non-human immunoglobulin, as compared to a
human antibody. In general, the humanized antibody will comprise
substantially all of 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 template
sequence. The humanized antibody may also comprise at least a
portion of an immunoglobulin constant region (F.sub.c), typically
that of the human immunoglobulin template chosen. In general, the
goal is to have an antibody molecule that is minimally immunogenic
in a human. Thus, it is possible that one or more amino acids in
one or more CDRs also can be changed to one that is less
immunogenic to a human host, without substantially minimizing the
specific binding function of the one or more CDRs to IL-4 and/or
IL-13. Alternatively, the FR can be non-human but those amino acids
most immunogenic are replaced with ones less immunogenic.
Nevertheless, CDR grafting, as discussed above, is not the only way
to obtain a humanized antibody. For example, modifying just the CDR
regions may be insufficient as it is not uncommon for framework
residues to have a role in determining the three-dimensional
structure of the CDR loops and the overall affinity of the antibody
for its ligand. Hence, any means can be practiced so that the
non-human parent antibody molecule is modified to be one that is
less immunogenic to a human, and global sequence identity with a
human antibody is not always a necessity. So, humanization also can
be achieved, for example, by the mere substitution of just a few
residues, particularly those which are exposed on the antibody
molecule and not buried within the molecule, and hence, not readily
accessible to the host immune system. Such a method is taught
herein with respect to substituting "mobile" or "flexible" residues
on the antibody molecule, the goal being to reduce or dampen the
immunogenicity of the resultant molecule without comprising the
specificity of the antibody for its epitope or determinant. See,
for example, Studnicka et al., Prot Eng 7(6)805-814, 1994; Mol 1 mm
44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia
et al., J Mol Biol 196:901 (1987); Carter et al., Proc Nail Acad
Sci USA 89:4285 (1992); Presta et al., J Immunol 151:2623 (1993),
WO 2006/042333 and U.S. Pat. No. 5,869,619.
[0066] A humanization method of interest is based on the impact of
the molecular flexibility of the antibody during and at immune
recognition. Protein flexibility is related to the molecular motion
of the protein molecule. Protein flexibility is the ability of a
whole protein, a part of a protein or a single amino acid residue
to adopt an ensemble of conformations which differ significantly
from each other. Information about protein flexibility can be
obtained by performing protein X-ray crystallography experiments
(see, for example, Kundu et al. 2002, Biophys J 83:723-732.),
nuclear magnetic resonance experiments (see, for example, Freedberg
et al., J Am Chem Soc 1998, 120(30:7916-7923) or by running
molecular dynamics (MD) simulations. An MD simulation of a protein
is done on a computer and allows one to determine the motion of all
protein atoms over a period of time by calculating the physical
interactions of the atoms with each other. The output of a MD
simulation is the trajectory of the studied protein over the period
of time of the simulation. The trajectory is an ensemble of protein
conformations, also called snapshots, which are periodically
sampled over the period of the simulation, e.g. every 1 picosecond
(ps). It is by analyzing the ensemble of snapshots that one can
quantify the flexibility of the protein amino acid residues. Thus,
a flexible residue is one which adopts an ensemble of different
conformations in the context of the polypeptide within which that
residue resides. MD methods are known in the art, see, e.g., Brooks
et al. "Proteins: A Theoretical Perspective of Dynamics, Structure
and Thermodynamics" (Wiley, New York, 1988). Several software
enable MD simulations, such as Amber (see Case et al. (2005) J Comp
Chem 26:1668-1688), Charmm (see Brooks et al. (1983) J Comp Chem
4:187-217; and MacKerell et al. (1998) in "The Encyclopedia of
Computational Chemistry" vol. 1:271-177, Schleyer et al., eds.
Chichester: John Wiley & Sons) or Impact (see Rizzo et al. J Am
Chem Soc; 2000; 122(51):12898-12900.)
[0067] Most protein complexes share a relatively large and planar
buried surface and it has been shown that flexibility of binding
partners provides the origin for their plasticity, enabling them to
conformationally adapt to each other (Structure (2000)8,
R137-R142). As such, examples of "induced fit" have been shown to
play a dominant role in protein-protein interfaces. In addition,
there is a steadily increasing body of data showing that proteins
actually bind ligands of diverse shapes sizes and composition
(Protein Science (2002) 11:184-187) and that the conformational
diversity appears to be an essential component of the ability to
recognize different partners (Science (2003) 299, 1362-1367).
Flexible residues are involved in the binding of protein-protein
partners (Structure (2006) 14, 683-693).
[0068] The flexible residues can adopt a variety of conformations
that provide an ensemble of interaction areas that are likely to be
recognized by memory B cells and to trigger an immunogenic
response. Thus, antibody can be humanized by modifying a number of
residues from the framework so that the ensemble of conformations
and of recognition areas displayed by the modified antibody
resemble as much as possible those adopted by a human antibody.
[0069] That can be achieved by modifying a limited number of
residues by: (1) building a homology model of the parent mAb and
running an MD simulation; (2) analyzing the flexible residues and
identification of the most flexible residues of a non-human
antibody molecule, as well as identifying residues or motifs likely
to be a source of heterogeneity or of degradation reaction; (3)
identifying a human antibody which displays the most similar
ensemble of recognition areas as the parent antibody; (4)
determining the flexible residues to be mutated, residues or motifs
likely to be a source of heterogeneity and degradation are also
mutated; and (5) checking for the presence of known T cell or B
cell epitopes. The flexible residues can be found using an MD
calculation as taught herein using an implicit solvent model, which
accounts for the interaction of the water solvent with the protein
atoms over the period of time of the simulation. Once the set of
flexible residues has been identified within the variable light and
heavy chains, a set of human heavy and light chain variable region
frameworks that closely resemble that of the antibody of interest
are identified. That can be done, for example, using a blast search
on the set of flexible residues against a database of antibody
human germline sequence. It can also be done by comparing the
dynamics of the parent mAb with the dynamics of a library of
germline canonical structures. The CDR residues and neighboring
residues are excluded from the search to ensure high affinity for
the antigen is preserved.
[0070] Flexible residues then are replaced. When several human
residues show similar homologies, the selection is driven also by
the nature of the residues that are likely to affect the solution
behavior of the humanized antibody. For instance, polar residues
will be preferred in exposed flexible loops over hydrophobic
residues. Residues which are a potential source of instability and
heterogeneity are also mutated even if there are found in the CDRs.
That will include exposed methionines as sulfoxide formation can
result from oxygen radicals, proteolytic cleavage of acid labile
bonds such as those of the Asp-Pro dipeptide (Drug Dev Res (2004)
61:137-154), deamidation sites found with an exposed asparagine
residue followed by a small amino acid, such as Gly, Ser, Ala, His,
Asn or Cys (J Chromatog (2006) 837:35-43) and N-glycosylation
sites, such as the Asn-X-Ser/Thr site. Typically, exposed
methionines will be substituted by a Leu, exposed asparagines will
be replaced by a glutamine or by an aspartate, or the subsequent
residue will be changed. For the glycosylation site
(Asn-X-Ser/Thr), either the Asn or the Ser/Thr residue will be
changed.
[0071] The resulting composite sequence is checked for the presence
of known B cell or linear T-cell epitopes. A search is performed,
for example, with the publicly available IEDB. If a known epitope
is found within the composite sequence, another set of human
sequences is retrieved and substituted
[0072] Unlike the resurfacing method of U.S. Pat. No. 5,639,641,
both B-cell-mediated and T-cell-mediated immunogenic responses are
addressed by the method. The method also avoids the issue of loss
of activity that is sometimes observed with CDR grafting (U.S. Pat.
No. 5,530,101). In addition, stability and solubility issues also
are considered in the engineering and selection process, resulting
in an antibody that is optimized for low immunogenicity, high
antigen affinity and improved biophysical properties.
[0073] Strategies and methods for resurfacing antibodies, and other
methods for reducing immunogenicity of antibodies within a
different host, are disclosed, for example, in U.S. Pat. No.
5,639,641. Briefly, in a preferred method, (1) position alignments
of a pool of antibody heavy and light chain variable regions are
generated to yield heavy and light chain variable region framework
surface exposed positions, wherein the alignment positions for all
variable regions are at least about 98% identical; (2) a set of
heavy and light chain variable region framework surface exposed
amino acid residues is defined for a non-human, such as a rodent
antibody (or fragment thereof); (3) a set of heavy and light chain
variable region framework surface exposed amino acid residues that
is most closely identical to the set of rodent surface exposed
amino acid residues is identified; and (4) the set of heavy and
light chain variable region framework surface exposed amino acid
residues defined in step (2) is substituted with the set of heavy
and light chain variable region framework surface exposed amino
acid residues identified in step (3), except for those amino acid
residues that are within 5A of any atom of any residue of a CDR of
the rodent antibody, to yield a humanized, such as a rodent
antibody retaining binding specificity.
[0074] Antibodies can be humanized by a variety of other techniques
including CDR grafting (EPO 0 239 400; WO 91/09967; and U.S. Pat.
Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EPO 0 592
106; EPO 0 519 596; Padlan, 1991, Molec 1 mm 28(4/5):489-498;
Studnicka et al., 1994, Prot Eng 7(6):805-814; and Roguska et al.,
1994, PNAS 91:969-973) and chain shuffling (U.S. Pat. No.
5,565,332). Human antibodies can be made by a variety of methods
known in the art including, but not limited to, phage display
methods, see U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806 and
5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654,
WO 96/34096, WO 96/33735 and WO 91/10741, using transgenic animals,
such as rodents, using chimeric cells and so on.
[0075] "Antibody homolog" or "homolog" refers to any molecule which
specifically binds IL-4 and/or IL-13 as taught herein. Thus, an
antibody homolog includes native or recombinant antibody, whether
modified or not, portions of antibodies that retain the biological
properties of interest, such as binding IL-4 or IL-13, such as an
F.sub.ab or F.sub.v molecule, a single chain antibody, a
polypeptide carrying one or more CDR regions and so on. The amino
acid sequence of the homolog need not be identical to that of the
naturally occurring antibody but can be altered or modified to
carry substitute amino acids, inserted amino acids, deleted amino
acids, amino acids other than the twenty normally found in proteins
and so on to obtain a polypeptide with enhanced or other beneficial
properties.
[0076] Antibodies with homologous sequences are those antibodies
with amino acid sequences that have sequence homology with the
amino acid sequence of a IL-4, IL-13 or bispecific IL-4/IL-13
antibody of the present invention. Preferably, homology is with the
amino acid sequence of the variable regions of an antibody of the
present invention. "Sequence homology" as applied to an amino acid
sequence herein is defined as a sequence with at least about 90%,
91%, 92%, 93%, 94% or more sequence homology, and more preferably
at least about 95%, 96%, 97%, 98% or 99% sequence homology to
another amino acid sequence, as determined, for example, by the
FASTA search method in accordance with Pearson & Lipman, Proc
Natl Acad Sci USA 85, 2444-2448 (1988).
[0077] A chimeric antibody is one with different portions of an
antibody derived from different sources, such as different
antibodies, different classes of antibody, different animal
species, for example, an antibody having a variable region derived
from a murine monoclonal antibody paired with a human
immunoglobulin constant region and so on. Thus, a humanized
antibody is a species of chimeric antibody. Methods for producing
chimeric antibodies are known in the art, see, e.g., Morrison,
1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214;
Gillies et al., 1989, J Immunol Methods 125:191-202; and U.S. Pat.
Nos. 5,807,715, 4,816,567, and 4,816,397.
[0078] Artificial antibodies include scFv fragments, chimeric
antibodies, diabodies, triabodies, tetrabodies and mru (see reviews
by Winter & Milstein, 1991, Nature 349:293-299; and Hudson,
1999, Curr Opin Imm 11:548-557), each with antigen-binding or
epitope-binding ability. In the single chain F.sub.v fragment
(scF.sub.v), the V.sub.H and V.sub.L domains of an antibody are
linked by a flexible peptide. Typically, the linker is a peptide of
about 15 amino acids. If the linker is much smaller, for example, 5
amino acids, diabodies are formed. The smallest binding unit of an
antibody is a CDR, typically the CDR2 of the heavy chain which has
sufficient specific recognition and binding capacity. Such a
fragment is called a molecular recognition unit or mru. Several
such mrus can be linked together with short linker peptides,
therefore forming an artificial binding protein with higher avidity
than a single mru.
[0079] Also included within the scope of the invention are
functional equivalents of an antibody of interest. The term
"functional equivalents" includes antibodies with homologous
sequences, antibody homologs, chimeric antibodies, artificial
antibodies and modified antibodies, for example, wherein each
functional equivalent is defined by the ability to bind to IL-4
and/or IL-13, inhibiting IL-4 and/or IL-13 signaling ability or
function, or inhibiting binding of IL-4 and/or IL-13 to its
receptor. The skilled artisan will understand that there is an
overlap in the group of molecules termed "antibody fragments" and
the group termed "functional equivalents." Methods of producing
functional equivalents which retain IL-4 and/or IL-13 binding
ability are known to the person skilled in the art and are
disclosed, for example, in WO 93/21319, EPO Ser. No. 239,400, WO
89/09622, EPO Ser. No. 338,745 and EPO Ser. No. 332,424.
[0080] The functional equivalents of the present application also
include modified antibodies, e.g., antibodies modified by the
covalent attachment of any type of molecule to the antibody. For
example, modified antibodies include antibodies that have been
modified, e.g., by glycosylation, acetylation, pegylation,
deamidation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand, linkage to a toxin or cytotoxic moiety or other
protein etc. The covalent attachment need not yield an antibody
that is immune from generating an anti-idiotypic response. The
modifications may be achieved by known techniques, including, but
not limited to, specific chemical cleavage, acetylation,
formylation, metabolic synthesis etc. Additionally, the modified
antibodies may contain one or more non-classical amino acids.
[0081] Many techniques are available to one of ordinary skill in
the art which permit the optimization of binding affinity.
Typically, the techniques involve substitution of various amino
acid residues at the site of interest, followed by a screening
analysis of binding affinity of the mutant polypeptide for the
cognate antigen or epitope.
[0082] Once the antibody is identified and isolated, it is often
useful to generate a variant antibody or mutant, or mutein, wherein
one or more amino acid residues are altered, for example, in one or
more of the hypervariable regions of the antibody. Alternatively,
or in addition, one or more alterations (e.g., substitutions) of
framework residues may be introduced in the antibody where these
result in an improvement in the binding affinity of the antibody
mutant for IL-4 and/or IL-13. Examples of framework region residues
that can be modified include those which non-covalently bind
antigen directly (Amit et al., Science 233:747-753 (1986));
interact with/affect the conformation of a CDR (Chothia et al., J
Mol Biol 196:901-917 (1987)); and/or participate in the
V.sub.L-V.sub.H interface (EP 239 400). 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 cognate antigen. For example, from about one to about five
framework residues may be altered in this embodiment of the
invention. Sometimes, this may be sufficient to yield an antibody
mutant suitable for use in preclinical trials, even where none of
the hypervariable region residues have been altered. Normally,
however, the antibody mutant can comprise one or more hypervariable
region alteration(s). The constant regions also can be altered to
obtain desirable or more desirable effector properties.
[0083] The hypervariable region residues which are altered may be
changed randomly, especially where the starting binding affinity of
the parent antibody is such that randomly-produced antibody mutants
can be readily screened for altered binding in an assay as taught
herein.
[0084] One procedure for obtaining antibody mutants, such as CDR
mutants, is "alanine scanning mutagenesis" (Cunningham & Wells,
Science 244:1081-1085 (1989); and Cunningham & Wells, Proc Nat
Acad Sci USA 84:6434-6437 (1991)). One or more of the hypervariable
region residue(s) are replaced by alanine or polyalanine
residue(s). Those hypervariable region residue(s) demonstrating
functional sensitivity to the substitutions then are refined by
introducing further or other mutations at or for the sites of
substitution. Thus, while the site for introducing an amino acid
sequence variation is predetermined, the nature of the mutation per
se need not be predetermined. Similar substitutions can be
attempted with other amino acids, depending on the desired property
of the scanned residues.
[0085] A more systematic method for identifying amino acid residues
to modify comprises identifying hypervariable region residues
involved in binding IL-4 and/or IL-13 and those hypervariable
region residues with little or no involvement with IL-4 and/or
IL-13 binding. An alanine scan of the non-binding hypervariable
region residues is performed, with each ala mutant tested for
enhancing binding to IL-4 and/or IL-13. In another embodiment,
those residue(s) significantly involved in binding IL-4 and/or
IL-13 are selected to be modified. Modification can involve
deletion of a residue or insertion of one or more residues adjacent
to a residue of interest. However, normally the modification
involves substitution of the residue by another amino acid. A
conservative substitution can be a first substitution. If such a
substitution results in a change in biological activity (e.g.,
binding affinity), then another conservative substitution can be
made to determine if more substantial changes are obtained.
[0086] Even more substantial modification in an antibody range and
presentation of biological properties can be accomplished by
selecting an amino acid that differs more substantially in
properties from that normally resident at a site. Thus, such a
substitution can be made while maintaining: (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.
[0087] For example, the naturally occurring amino acids can be
divided into groups based on common side chain properties:
[0088] (1) hydrophobic: methionine (M or met), alanine (A or ala),
valine (V or val), leucine (L or leu) and isoleucine (I or
ile);
[0089] (2) neutral, hydrophilic: cysteine (C or cys), serine (S or
ser), threonine (T or thr), asparagine (N or asn) and glutamine (Q
or gln);
[0090] (3) acidic: aspartic acid (D or asp) and glutamic acid (E or
glu);
[0091] (4) basic: histidine (H or his), lysine (K or lys) and
arginine (R or arg);
[0092] (5) residues that influence chain orientation: glycine (G or
gly) and proline (P or pro), and
[0093] (6) aromatic: tryptophan (W or trp), tyrosine (Y or tyr) and
phenylalanine (F or phe).
[0094] Non-conservative substitutions can entail exchanging an
amino acid with an amino acid from another group. Conservative
substitutions can entail exchange of one amino acid for another
within a group.
[0095] Preferred amino acid substitutions include those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity and (4) confer or modify
other physico-chemical or functional properties of such analogs.
Analogs can include various muteins of a sequence other than the
naturally occurring peptide sequence. For example, single or
multiple amino acid substitutions (preferably conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(preferably in the portion of the polypeptide outside the domain
(s) forming intermolecular contacts. A conservative amino acid
substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence) unless of a change in the bulk
or conformation of the R group or side chain, Proteins, Structures
and Molecular Principles (Creighton, ed., W. H. Freeman and
Company, New York (1984)); Introduction to Protein Structure
(Branden & Tooze, eds., Garland Publishing, New York, N.Y.
(1991)); and Thornton et al. Nature 354:105 (1991).
[0096] Ordinarily, the antibody mutant with improved biological
properties will have an amino acid sequence having at least 75%
amino acid sequence identity or similarity with the amino acid
sequence of either the heavy or light chain variable domain of the
parent anti-human IL-4 and/or IL-13 antibody, at least 80%, at
least 85%, at least 90% and often at least 95% identity. Identity
or similarity with respect to parent antibody sequence is defined
herein as the percentage of amino acid residues in the candidate
sequence that are identical (i.e., same residue) or similar (i.e.,
amino acid residue from the same group based on common side-chain
properties, supra) with the parent antibody residues, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity.
[0097] Alternatively, antibody mutants can be generated by
systematic mutation of the FR and CDR regions of the heavy and
light chains, or the F.sub.e region of the anti-IL-4, anti-IL-13 or
bispecific IL-4/IL-13 antibody. Another procedure for generating
antibody mutants involves the use of affinity maturation using
phage display (Hawkins et al., J Mol Biol 254:889-896 (1992) and
Lowman et al., Biochemistry 30(45):10832-10838 (1991)).
Bacteriophage coat-protein fusions (Smith, Science 228:1315 (1985);
Scott & Smith, Science 249:386 (1990); Cwirla et al. Proc Natl
Acad Sci USA 8:309 (1990); Devlin et al. Science 249:404 (1990);
Wells & Lowman, Curr Opin Struct Biol 2:597 (1992); and U.S.
Pat. No. 5,223,409) are known to be useful for linking the
phenotype of displayed proteins or peptides to the genotype of
bacteriophage particles which encode them. The F.sub.ab domains of
antibodies have also been displayed on phage (McCafferty et al.,
Nature 348: 552 (1990); Barbas et al. Proc Natl Acad Sci USA
88:7978 (1991); and Garrard et al. Biotechnol 9:1373 (1991)).
[0098] Monovalent phage display consists of displaying a set of
protein variants as fusions of a bacteriophage coat protein on
phage particles (Bass et al., Proteins 8:309 (1990). Affinity
maturation, or improvement of equilibrium binding affinities of
various proteins, has previously been achieved through successive
application of mutagenesis, monovalent phage display and functional
analysis (Lowman & Wells, J Mol Biol 234:564 578 (1993); and
U.S. Pat. No. 5,534,617), for example, by focusing on the CDR
regions of antibodies (Barbas et al., Proc Natl Acad Sci USA
91:3809 (1994); and Yang et al., J Mol Biol 254:392 (1995)).
[0099] Libraries of many (for example, 10.sup.6 or more) protein
variants, differing at defined positions in the sequence, can be
constructed on bacteriophage particles, each of which contains DNA
encoding the particular protein variant. After cycles of affinity
purification, using an immobilized antigen, individual
bacteriophage clones are isolated, and the amino acid sequence of
the displayed protein is deduced from the DNA.
[0100] Following production of the antibody mutant, the biological
activity of that molecule relative to the parent antibody can be
determined as taught herein. As noted above, that may involve
determining the binding affinity and/or other biological activities
or physical properties of the antibody. In a preferred embodiment
of the invention, a panel of antibody mutants is prepared and
screened for binding affinity for the antigen. One or more of the
antibody mutants selected from the screen are optionally subjected
to one or more further biological activity assays to confirm that
the antibody mutant(s) have new or improved properties. In
preferred embodiments, the antibody mutant retains the ability to
bind IL-4 and/or IL-13 with a binding affinity similar to or
better/higher than that of the parent antibody.
[0101] The antibody mutant(s) so selected may be subjected to
further modifications, often depending on 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. For example, a cysteine residue not
involved in maintaining the proper conformation of the antibody
mutant may be substituted, generally with serine, to improve the
oxidative stability of the molecule and to prevent aberrant
cross-linking. Conversely, a cysteine may be added to the antibody
to improve stability (particularly where the antibody is an
antibody fragment such as an F.sub.v fragment).
[0102] Another type of antibody mutant has an altered glycosylation
pattern. That may be achieved by deleting one or more carbohydrate
moieties found in the antibody and/or by adding one or more
glycosylation sites that are not present in the antibody.
Glycosylation of antibodies is typically either N-linked to Asn or
O-linked to Ser or Thr. The tripeptide sequences,
asparaginc-X-serine and asparaginc-X-threonine, where X is any
amino acid except proline, are common recognition sequences for
enzymatic attachment of a carbohydrate moiety to the asparagine
side chain. N-acetylgalactosamine, galactose, fucose or xylose, for
example, are bonded to a hydroxyamino acid, most commonly serine or
threonine, although 5-hydroxyproline or 5-hydroxylysine also may be
used. Addition or substitution of one or more serine or threonine
residues to the sequence of the original antibody can enhance the
likelihood of O-linked glycosylation.
[0103] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance the
effectiveness of the antibody. For example, cysteine residue(s) may
be introduced in the F.sub.c region, thereby allowing interchain
disulfide bond formation in that region. The homodimeric antibody
thus generated may have improved internalization capability and/or
increased complement-mediated cell killing and antibody-dependent
cellular cytotoxicity (ADCC), see Caron et al., J Exp Med
176:1191-1195 (1992) and Shopes, Immunol 148:2918-2922 (1993).
Alternatively, an antibody can be engineered which has dual F.sub.c
regions and may thereby have enhanced complement lysis and ADCC
capabilities, see Stevenson et al., Anti-Cancer Drug Design 3: 219
230 (1989).
[0104] Covalent modifications of the antibody are included within
the scope of the invention. Such may be made by chemical synthesis
or by enzymatic or chemical cleavage of the antibody, if
applicable. Other types of covalent modifications of the antibody
are introduced into the molecule by reacting targeted amino acid
residues of the antibody with an organic derivatizing agent that is
capable of reacting with selected side chains or with the
N-terminal or C-terminal residue.
[0105] Cysteinyl residues can be reacted with .alpha.-haloacetates
(and corresponding amines), such as chloroacetic acid or
chloroacetamide, to yield carboxylmethyl or carboxyamidomethyl
derivatives. Cysteinyl residues also can be derivatized by reaction
with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercura-4-nitrophenol or
chloro-7-nitrobenzo-2-oxa-1,3-diazole, for example.
[0106] Histidyl residues can be derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0. p-bromophenacyl bromide also
can be used, the reaction is preferably performed in 0.1 M sodium
cacodylate at pH 6.0.
[0107] Lysinyl and a terminal residues can be reacted with succinic
or other carboxylic acid anhydrides to reverse the charge of the
residues. Other suitable reagents for derivatizing
.alpha.-amino-containing residues include imidoesters, such as
methyl picolinimidate, pyridoxal phosphate, pyridoxal,
chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea
and 2,4-pentanedione, and the amino acid can be
transaminase-catalyzed with glyoxylate.
[0108] Arginyl residues can be modified by reaction with one or
several conventional reagents, such as phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. Derivatization
of arginine residues often requires alkaline reaction conditions.
Furthermore, the reagents may react with lysine as well as the
arginine .epsilon.-amino group.
[0109] The specific modification of tyrosyl residues can be made
with aromatic diazonium compounds or tetranitromethane. For
example, N-acetylimidizole and tetranitromethane are used to form
O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
Tyrosyl residues can be iodinated using .sup.125I or .sup.131I to
prepare labeled proteins for use in a radioimmunoassay.
[0110] Carboxyl side groups (aspartyl or glutamyl) can be modified
by reaction with carbodiimides (R--N.dbd.C.dbd.C--R'), where R and
R' can be different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues can be converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0111] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues,
respectively, under neutral or basic conditions. The deamidated
form of those residues falls within the scope of this
invention.
[0112] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of serinyl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains (Creighton, Proteins: Structure
and Molecular Properties, W.H. Freeman & Co., San Francisco,
pp. 79-86 (1983)), acetylation of the N-terminal amine and
amidation of any C-terminal carboxyl group.
[0113] Another type of covalent modification involves chemically or
enzymatically coupling glycosides to the antibody. Those procedures
do not require production of the antibody in a host cell that has
glycosylation capabilities for N-linked or O-linked glycosylation.
Depending on the coupling mode used, the sugar(s) may be attached
to: (a) arginine and histidine; (b) free carboxyl groups; (c) free
sulthydryl groups, such as those of cysteine; (d) free hydroxyl
groups, such as those of serine, threonine or hydroxyproline; (c)
aromatic residues such as those of phenylalanine, tyrosine or
tryptophan; or (f) the amide group of glutamine. Such methods are
described in WO 87/05330 and in Aplin & Wriston, CRC Crit. Rev
Biochem, pp. 259-306 (1981).
[0114] Removal of any carbohydrate moieties present on the antibody
may be accomplished chemically or enzymatically. Chemical
deglycosylation, for example, can require exposure of the antibody
to the compound, trifluoromethanesulfonic acid, or an equivalent
compound, resulting in cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the antibody intact. Chemical deglycosylation is described,
for example, in Hakimuddin et al. Arch Biochem Biophys 259:52
(1987) and in Edge et al., Anal Biochem 118:131 (1981). Enzymatic
cleavage of carbohydrate moieties on antibodies can be achieved by
any of a variety of endoglycosidases and exoglycosidases as
described, for example, in Thotakura et al., Meth Enzymol 138:350
(1987).
[0115] Another type of covalent modification of the antibody
comprises linking the antibody to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene
glycol or polyoxylalkylenes, in the manner set forth in U.S. Pat.
No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0116] Another technique preferred for obtaining mutants or muteins
is affinity maturation by phage display (Hawkins et al., J Mol Biol
254:889-896 (1992); and Lowman et al., Biochemistry
30(45):10832-10838 (1991)). Briefly, several hypervariable region
sites (e.g., 6-7 sites) are mutated to generate all possible amino
acid substitutions at each site. The antibody mutants thus
generated are displayed in monovalent fashion on phage particles as
fusions to a protein found on the particles. The phage expressing
the various mutants can be cycled through rounds of binding
selection, followed by isolation and sequencing of those mutants
which display high affinity.
[0117] The method of selecting novel binding polypeptides can
utilize a library of structurally related polypeptides. The library
of structurally related polypeptides, for example, fused to a phage
coat protein, is produced by mutagenesis, and is displayed on the
surface of the particle. The particles then are contacted with a
target molecule and those particles having the highest affinity for
the target are separated from those of lower affinity. The high
affinity binders then are amplified by infection of a suitable
bacterial host and the competitive binding step is repeated. The
process is repeated until polypeptides of the desired affinity are
obtained.
[0118] Alternatively, multivalent phage (McCafferty et al. (1990)
Nature 348:552-554; and Clackson et al. (1991) Nature 352:624-628)
also can be used to express random point mutations (for example,
generated by use of an error-prone DNA polymerase) to generate a
library of phage antibody fragments which then could be screened
for affinity to IL-4 and/or IL-13, Hawkins et al., (1992) J Mol
Biol 254:889-896.
[0119] Preferably, during the affinity maturation process, the
replicable expression vector is under tight control of a
transcription regulatory element, and the culturing conditions are
adjusted so the amount or number of particles displaying more than
one copy of the fusion protein is less than about 1%. Also
preferably, the amount of particles displaying more than one copy
of the fusion protein is less than 10% of the amount of particles
displaying a single copy of the fusion protein. Preferably the
amount is less than 20%.
[0120] Functional equivalents may be produced by interchanging
different CDRs of different antibody chains within a framework or a
composite FR derived from plural antibodies. Thus, for example,
different classes of antibody are possible for a given set of CDRs
by substitution of different heavy chains, for example,
IgG.sub.1-4, IgM, IgA.sub.1-2 or IgD, to yield differing IL-4
and/or IL-13 antibody types and isotypes. Similarly, artificial
antibodies within the scope of the invention may be produced by
embedding a given set of CDRs within an entirely synthetic
framework.
[0121] The antibody fragments and functional equivalents of the
present invention encompass those molecules with a detectable
degree of specific binding to IL-4 and/or IL-13. A detectable
degree of binding includes all values in the range of at least
10-100%, preferably at least 50%, 60% or 70%, more preferably at
least 75%, 80%, 85%, 90%, 95% or 99% of the binding ability of an
antibody of interest. Also included are equivalents with an
affinity greater than 100% that of an antibody of interest.
[0122] The CDRs generally are of importance for epitope recognition
and antibody binding. However, changes may be made to residues that
comprise the CDRs without interfering with the ability of the
antibody to recognize and to bind the cognate epitope. For example,
changes that do not impact epitope recognition, yet increase the
binding affinity of the antibody for the epitope, may be made.
Several studies have surveyed the effects of introducing one or
more amino acid changes at various positions in the sequence of an
antibody, based on the knowledge of the primary antibody sequence,
on the properties thereof, such as binding and level of expression
(Yang et al., 1995, J Mol Biol 254:392-403; Rader et al., 1998,
Proc Natl Acad Sci USA 95:8910-8915; and Vaughan et al., 1998,
Nature Biotechnology 16, 535-539).
[0123] Thus, equivalents of an antibody of interest can be
generated by changing the sequences of the heavy and light chain
genes in the CDR1, CDR2 or CDR3, or framework regions, using
methods such as oligonucleotide-mediated site-directed mutagenesis,
cassette mutagenesis, error-prone PCR, DNA shuffling or
mutator-strains of E. coli (Vaughan et al., 1998, Nat Biotech
16:535-539; and Adey et al., 1996, Chap. 16, pp. 277-291, in Phage
Display of Peptides and Proteins, eds. Kay et al., Academic Press).
The methods of changing the nucleic acid sequence of the primary
antibody can result in antibodies with improved affinity (Gram et
al., 1992, Proc Natl Acad Sci USA 89:3576-3580; Boder et al., 2000,
Proc Natl Acad Sci USA 97:10701-10705; Davies & Riechmann,
1996, Immunotech 2:169-179; Thompson et al., 1996, J Mol Biol
256:77-88; Short et al., 2002, J Biol Chem 277:16365-16370; and
Furukawa et al., 2001, J Biol Chem 276:27622-27628).
[0124] Repeated cycles of "polypeptide selection" can be used to
select for higher and higher affinity binding by, for example, the
selection of multiple amino acid changes which are selected by
multiple selections of cycles. Following a first round of
selection, involving a first region of selection of amino acids in
the ligand or antibody polypeptide, additional rounds of selection
in other regions or amino acids of the ligand are conducted. The
cycles of selection are repeated until the desired affinity
properties are achieved.
[0125] Improved antibodies also include those antibodies having
improved characteristics that are prepared by the standard
techniques of animal immunization, hybridoma formation and
selection for antibodies with specific characteristics.
[0126] "Antagonist" refers to a molecule capable of inhibiting one
or more biological activities of a target molecule, such as
signaling by IL-4 and/or IL-13. Antagonists may interfere with the
binding of a receptor to a ligand and vice versa, by incapacitating
or killing cells activated by a ligand, and/or by interfering with
receptor or ligand activation (e.g., tyrosine kinase activation) or
signal transduction after ligand binding to a receptor. The
antagonist may completely block receptor-ligand interactions or may
substantially reduce such interactions.
[0127] "Agonist" refers to a compound, including a protein, a
polypeptide, a peptide, an antibody, an antibody fragment, a
conjugate, a large molecule, a small molecule, which activates one
or more biological activities of IL-4 and/or IL-13. Agonists may
interact with the binding of a receptor to a ligand and vice versa,
by acting as a mitogen of cells activated by a ligand, and/or by
interfering with cell inactivation or signal transduction
inhibition after ligand binding to a receptor. All such points of
intervention by an agonist shall be considered equivalent for
purposes of the instant invention.
[0128] The terms "cell," "cell line," and "cell culture" include
progeny thereof. It is also understood that all progeny may not be
precisely identical, such as in DNA content, due to deliberate or
inadvertent mutation. Variant progeny that have the same function
or biological property of interest, as screened for in the original
cell, are included.
[0129] The term "vector" means a nucleic acid construct, a carrier,
containing a nucleic acid, the transgene, the foreign gene or the
gene of interest, which can be operably linked to suitable control
sequences for expression of the transgene in a suitable host. Such
control sequences include, for example, 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
just 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 host cell
genome. In the present specification, "plasmid" and "vector" are
used interchangeably, as the plasmid is a commonly used form of
vector. However, the invention is intended to include such other
forms of vectors which serve equivalent carrier function as and
which are, or become, known in the art, such as viruses, synthetics
molecules that carry nucleic acids, liposomes and the like.
[0130] "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.
[0131] The antibodies of interest can be screened or can be used in
an assay as described herein or as known in the art. Often, such
assays require a reagent to be detectable, that is, for example,
labeled. 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, particles or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0132] As used herein, "solid phase" means a non-aqueous matrix to
which an entity or molecule, such as the antibody of the instant
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 can be used in a
purification column (e.g., an affinity chromatography column).
Thus, the solid phase can be a paper, a bead, a plastic, a chip and
so on, can be made from a variety of materials, such as
nitrocellulose, agarose, polystyrene, polypropylene, silicon and so
on, and can be in a variety of configurations.
[0133] The gene or a cDNA encoding TL-4 and IL-13 are known in the
art, may be cloned in a plasmid or other expression vector and
expressed in any of a number of expression systems according to
methods well known to those of skill in the art, and see below, for
example.
[0134] Nucleic acid molecules encoding amino acid sequence mutants
can be prepared by a variety of methods known in the art. The
methods include, but are not limited to, oligonucleotide-mediated
(or site-directed) mutagenesis, PCR mutagenesis and cassette
mutagenesis of an earlier prepared mutant or a non-mutant version
of the molecule of interest, (see, for example, Kunkel, Proc Natl
Acad Sci USA 82:488 (1985)).
[0135] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention, a single chain antibody of
the invention or an antibody mutein of the invention) includes
construction of an expression vector containing a polynucleotide
that encodes the antibody or a fragment of the antibody as
described herein. Once a polynucleotide encoding an antibody
molecule has been obtained, the vector for the production of the
antibody may be produced by recombinant DNA technology as known in
the art. An expression vector is constructed containing antibody
coding sequences and appropriate transcriptional and translational
control signals. The methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques and in vivo
genetic recombination.
[0136] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells then are cultured
by conventional techniques to produce an antibody or fragment of
the invention. In one aspect of the invention, vectors encoding
both the heavy and light chains may be co-expressed in the host
cell for expression of the entire immunoglobulin molecule, as
detailed herein.
[0137] A variety of host/expression vector systems may be utilized
to express the antibody molecules of the invention. Such expression
systems represent vehicles by which the coding sequences of
interest may be produced and subsequently purified, but also
represent cells which may, when transformed or transfected with the
appropriate nucleotide coding sequences, express an antibody
molecule of the invention in situ. Bacterial cells, such as E.
coli, and eukaryotic cells are commonly used for the expression of
a recombinant antibody molecule, especially for the expression of
whole recombinant antibody molecule. For example, mammal cells such
as CHO cells, in conjunction with a vector, such as one carrying
the major intermediate early gene promoter element from human
cytomegalovirus, are an effective expression system for antibodies
(Foecking et al., Gene 45:101 (1986); and Cockett et al.,
Bio/Technology 8:2 (1990)). Plants and plant cell culture, insect
cells and so on also can be used to make the proteins of interest,
as known in the art.
[0138] In addition, a host cell is chosen which modulates the
expression of the inserted sequences, or modifies and processes the
gene product in the specific fashion desired. Such modifications
(e.g., glycosylation) and processing (e.g., cleavage) of protein
products may be important for the function of the protein.
Different host cells have characteristic and specific mechanisms
for the post-translational processing and modification of proteins
and gene products. Appropriate cell lines or host systems can be
chosen to ensure the correct modification and processing of the
expressed antibody of interest. Hence, eukaryotic host cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation and phosphorylation of the gene product
may be used. Such mammalian host cells include, but are not limited
to, CHO, COS, 293, 3T3 or myeloma cells.
[0139] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites etc.)
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for one to two days in
an enriched media, and then are moved to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
a chromosome and be expanded into a cell line. Such engineered cell
lines not only are useful for antibody production but are useful in
screening and evaluation of compounds that interact directly or
indirectly with the antibody molecule.
[0140] A number of selection systems may be used, including but not
limited to the Herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA
48:202 (1992)), glutamate synthase selection in the presence of
methionine sulfoximide (Adv Drug Del Rev 58, 671, 2006 and see the
website or literature of Lonza Group Ltd.) and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
in tk, hgprt or aprt cells, respectively. Also, antimetabolite
resistance can be used as the basis of selection for the following
genes: dhfr, which confers resistance to methotrexate (Wigler et
al., Proc Natl Acad Sci USA 77:357 (1980); O'Hare et al., Proc Natl
Acad Sci USA 78:1527 (1981)); gpt, which confers resistance to
mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78:2072
(1981)); neo, which confers resistance to the aminoglycoside, G-418
(Wu et al., Biotherapy 3:87 (1991)); and hygro, which confers
resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al., eds.,
Current Protocols in Molecular Biology, John Wiley & Sons
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press (1990); Dracopoli et al., eds., Current
Protocols in Human Genetics, John Wiley & Sons (1994); and
Colberre-Garapin et al., J Mol Biol 150:1 (1981).
[0141] The expression levels of an antibody molecule can be
increased by vector amplification (for example, see Bebbington et
al., in DNA Cloning, Vol. 3. Academic Press (1987)). When a marker
in the vector system expressing antibody is amplifiable, an
increase in the level of inhibitor present in the culture will
increase the number of copies of the marker gene. Since the
amplified region is associated with the antibody gene, production
of the antibody will also increase (Crouse et al., Mol Cell Biol
3:257 (1983)).
[0142] The host cell may be co-transfected with two or more
expression vectors of the invention, for example, the first vector
encoding a heavy chain-derived polypeptide and the second vector
encoding a light chain-derived polypeptide. The two vectors may
contain identical selectable markers which enable equal expression
of heavy and light chain polypeptides. Alternatively, a single
vector may be used which encodes, and is capable of expressing,
both heavy and light chain polypeptides. In such situations, the
light chain should be placed before the heavy chain to avoid an
excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986);
and Kohler, Proc Natl Acad Sci USA 77:2197 (1980)). The coding
sequences for the heavy and light chains may comprise cDNA or
genomic DNA.
[0143] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized or recombinantly expressed, it
may be purified by any method known in the art for purification of
an immunoglobulin molecule, for example, by chromatography (e.g.,
ion exchange, affinity, particularly by affinity for IL-4 and/or
IL-13 after Protein A and size-exclusion chromatography and so on),
centrifugation, differential solubility or by any other standard
technique for the purification of proteins. In addition, the
antibodies of the instant invention or fragments thereof can be
fused to heterologous polypeptide sequences described herein or
otherwise known in the art, to facilitate purification.
[0144] The antibodies of the present invention may be generated by
any suitable method known in the art. The antibodies of the present
invention may comprise polyclonal antibodies, although because of
the modification of antibodies to optimize use in human, as well as
to optimize the use of the antibody per se, monoclonal antibodies
are preferred because of ease of production and manipulation of
particular proteins. Methods of preparing polyclonal antibodies are
known to the skilled artisan (Harlow et al., Antibodies: a
Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.
(1988)).
[0145] The antibodies of the present invention preferably comprise
monoclonal antibodies. Monoclonal antibodies may be prepared using
hybridoma technology, such as described by Kohler et al., Nature
256:495 (1975); U.S. Pat. No. 4,376,110; Harlow et al., Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.
(1988) and Hammerling et al., Monoclonal Antibodies and T-Cell
Hybridomas, Elsevier (1981), recombinant DNA methods, for example,
making and using transfectomas, or other methods known to the
artisan. Other examples of methods which may be employed for
producing monoclonal antibodies include, but are not limited to,
the human B-cell hybridoma technique (Kosbor et al., Immunology
Today 4:72 (1983); and Cole et al., Proc Natl Acad Sci USA 80:2026
(1983)), and the EBV-hybridoma technique (Cole et al., Monoclonal
Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss (1985)).
Such antibodies may be of any immunoglobulin class including IgG,
IgM, IgE, IgA and IgD, and any subclass thereof. The hybridoma
producing the mAb of the invention may be cultivated in vitro or in
vivo.
[0146] In the hybridoma model, a host such as a mouse, a humanized
mouse, a transgenic mouse with human immune system genes, hamster,
rabbit, rat, camel or any other appropriate host animal, is
immunized to elicit lymphocytes that produce or are capable of
producing antibodies that specifically bind to IL-4 or IL-13.
Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with mycloma cells using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell (Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press, pp.
59-103 (1986)).
[0147] Generally, in making antibody-producing hybridomas, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. Immortalized cell lines
are usually transformed mammalian cells, particularly myeloma cells
of rodent, bovine or human origin. Typically, a rat or mouse
myeloma cell line is employed. The hybridoma cells may be cultured
in a suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or
HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin and thymidine ("HAT medium"), substances
that prevent the growth of HGPRT-deficient cells.
[0148] Preferred immortalized cell lines 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. Among these myeloma cell lines are
murine myeloma lines, such as those derived from the MOPC-21 and
MPC-11 mouse tumors available from the Salk Institute Cell
Distribution Center, San Diego, Calif. and SP2/0, FO or X63-Ag8-653
cells available from the American Type Culture Collection,
Manassas, Va.
[0149] Human myeloma and mouse-human heteromyeloma cell lines also
have been described for the production of human monoclonal
antibodies (Kozbor, J Immunol 133:3001 (1984); and Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, Marcel
Dekker, Inc, pp. 51-63 (1987)). The mouse myeloma cell line NSO may
also be used (European Collection of Cell Cultures, Salisbury,
Wilshire, UK).
[0150] Another alternative is to use electrical fusion rather than
chemical fusion to form hybridomas. Instead of fusion, a B cell can
be immortalized using, for example, Epstein Barr Virus or another
transforming gene, see, e.g., Zurawaki et al., in Monoclonal
Antibodies, ed., Kennett et al., Plenum Press, pp. 19-33. (1980).
Transgenic mice expressing immunoglobulins and severe combined
immunodeficient (SCID) mice transplanted with human B lymphocytes
also can be used.
[0151] The culture medium in which hybridoma cells are grown is
assayed for production of monoclonal antibodies directed against
IL-4 and/or IL-13. The binding specificity of monoclonal antibodies
produced by hybridoma cells may be determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA), fluorocytometric analysis (FACS) or
enzyme-linked immunosorbent assay (ELISA). Such techniques are
known in the art and are within the skill of the artisan. The
binding affinity of the monoclonal antibody to IL-4 and/or IL-13
can, for example, be determined by a Scatchard analysis (Munson et
al., Anal Biochem 107:220 (1980)).
[0152] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, Academic Press, pp. 59-103 (1986)). Suitable culture
media include, for example, Dulbecco's Modified Eagle's Medium
(D-MEM) or RPMI-1640 medium. In addition, the hybridoma cells may
be grown in vivo as ascites tumors in an animal.
[0153] The monoclonal antibodies secreted by the subclones are
suitably separated or isolated from the culture medium, ascites
fluid or serum by conventional immunoglobulin purification
procedures such as, for example, protein A-Sepharose, protein
G-Sepharose, hydroxylapatite chromatography, gel exclusion
chromatography, gel electrophoresis, dialysis or affinity
chromatography.
[0154] A variety of methods exist in the art for the production of
monoclonal antibodies and thus, the invention is not limited to
their sole production in hybridomas. For example, the monoclonal
antibodies may be made by recombinant DNA methods, such as those
described in U.S. Pat. No. 4,816,567. In this context, the term
"monoclonal antibody" refers to an antibody derived from a single
eukaryotic, phage or prokaryotic clone.
[0155] DNA encoding the monoclonal antibodies of the invention 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 murine
antibodies, or such chains from human, humanized or other sources)
(Innis et al. in PCR Protocols. A Guide to Methods and
Applications, Academic (1990), and Sanger et al., Proc Natl Acad
Sci 74:5463 (1977)). The hybridoma cells serve as a source of such
DNA. Once isolated, the DNA may be placed into expression vectors,
which are then transfected into host cells such as E. coli cells,
NSO cells, COS cells, Chinese hamster ovary (CHO) cells or myeloma
cells that do not otherwise produce immunoglobulin protein, 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; and Morrison et al., Proc Natl Acad Sci USA
81:6851 (1984)) or by covalently joining to the immunoglobulin
coding sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an
antibody of the invention, or can be substituted for the variable
domains of one IL-4 or IL-13 combining site of an antibody of the
invention to create a chimeric bivalent antibody.
[0156] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the F.sub.c region so
as to prevent heavy chain cross-linking Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent cross-linking
[0157] Antibody fragments which recognize specific epitopes may be
generated by known techniques. Traditionally, these fragments were
derived via proteolytic digestion of intact antibodies (see, e.g.,
Morimoto et al., J Biochem Biophys Methods 24:107 (1992); and
Brennan et al., Science 229:81 (1985)). For example, F.sub.ab and
F.sub.(ab')2 fragments of the invention may be produced by
proteolytic cleavage of immunoglobulin molecules, using enzymes
such as papain (to produce F.sub.ab fragments) or pepsin (to
produce F.sub.(ab')2 fragments). F.sub.(ab')2 fragments contain the
variable region, the light chain constant region and the C.sub.H1
domain of the heavy chain. However, those fragments can be produced
directly by recombinant host cells. For example, the antibody
fragments can be isolated from an antibody phage library.
Alternatively, F.sub.(ab')2-SH fragments can be directly recovered
from E. coli and chemically coupled to form F(ab').sub.2 fragments
(Carter et al., Bio/Technology 10:163 (1992). According to another
approach, F.sub.(ab')2 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
F.sub.v fragment (F.sub.v) (WO 93/16185).
[0158] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use
chimeric, humanized or human antibodies. Methods for producing
chimeric antibodies are known in the art, see e.g., Morrison,
Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986);
Gillies et al., J Immunol Methods 125:191 (1989); and U.S. Pat.
Nos. 5,807,715; 4,816,567; and 4,816397.
[0159] Humanized antibodies are derived from antibody molecules
generated in a non-human species that bind IL-4 and/or IL-13
wherein one or more CDRs therefrom are inserted into the FR regions
from a human immunoglobulin molecule. Antibodies can be humanized
using a variety of techniques known in the art including, for
example, CDR grafting (EPO 239,400; WO 91/09967; and U.S. Pat. Nos.
5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EPO
592,106; EPO 519,596; Padlan, Molecular Immunology 28:489 (1991);
Studnicka et al., Protein Engineering 7:805 (1994); and Roguska et
al., Proc Natl Acad Sci USA 91:969 (1994)), and chain shuffling
(U.S. Pat. No. 5,565,332).
[0160] A humanized antibody has one or more amino acid residues
from a source that is non-human. The 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 methods of Winter and
co-workers (Jones et al., Nature 321:522 (1986); Riechmann et al.,
Nature 332:323 (1988); and Verhoeyen et al., Science 239:1534
(1988)), by substituting non-human CDRs or portions of CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possible some FR residues are substituted from
analogous sites in rodent antibodies. The heavy chain constant
region and hinge region can be from any class or subclass to obtain
a desired effect, such as a particular effector function.
[0161] Often, framework residues in the human framework regions can
be substituted with the corresponding residue from the CDR donor
antibody to alter, and possibly improve, antigen binding. The
framework substitutions are identified by methods known in the art,
e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions, see, e.g., U.S. Pat. No.
5,585,089; and Riechmann et al., Nature 332:323 (1988).
[0162] It is further preferable that humanized antibodies retain
high affinity for IL-4 and/or IL-13, and retain or acquire other
favorable biological properties. Thus, humanized antibodies are
prepared by a process of analysis of the parental sequences and
various conceptual humanized products using three-dimensional
models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of the displays permits analysis of the likely role of
certain residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind IL-4 and/or IL-13. In that
way, FR residues can be selected and combined from the recipient
and import sequences so that the desired antibody characteristic,
such as increased affinity for the target antigen, is maximized,
although it is the CDR residues that directly and most
substantially influence IL-4 or IL-13 binding. The CDR regions also
can be modified to contain one or more amino acids that vary from
that obtained from the parent antibody from which the CDR was
obtained, to provide enhanced or different properties of interest,
such as binding of greater affinity or greater avidity, for
example.
[0163] Certain portions of the constant regions of antibody can be
manipulated and changed to provide antibody homologs, derivatives,
fragments and the like with properties different from or better
than that observed in the parent antibody. Thus, for example, many
IgG4 antibodies form intrachain disulfide bonds near the hinge
region. The intrachain bond can destabilize the parent bivalent
molecule forming monovalent molecules comprising a heavy chain with
the associated light chain. Such molecules can reassociate, but on
a random basis.
[0164] It was observed that modifying amino acids in the hinge
region of IgG4 molecules can reduce the likelihood of intrachain
bond formation, thereby stabilizing the IgG4 molecule, which will
minimize the likelihood of forming bispecific molecules. That
modification can be beneficial if a therapeutic antibody is an IgG4
molecule as the enhanced stability will minimize the likelihood of
having the molecule dissociate during production and manufacture,
as well as in vivo. A monovalent antibody may not have the same
effectiveness as the bivalent parent molecule. For example, when
bivalent IgG4 is administered to a patient, the percentage of
bivalent IgG4 decays to about 30% over a two-week period. An amino
acid substitution at position 228 enhances IgG4 stability. The
serine that resides at 228 can be replaced with another amino acid,
such as one of the remaining 19 amino acids. Such a change can be
made particularly with recombinant antibodies wherein the nucleic
acid coding sequence can be mutated to yield a replacement amino
acid at position 228. For example, the S can be replaced with a
proline.
[0165] Another set of amino acids suitable for modification include
amino acids in the area of the hinge which impact binding of a
molecule containing a heavy chain with binding to the F.sub.c
receptor and internalization of bound antibody. Such amino acids
include, in IgG1 molecules, residues from about 233 to about 237
(Glu-Leu-Leu-Gly-Gly); (SEQ ID NO:49) from about 252 to about 256
(Met-11e-Ser-Arg-Thr) (SEQ ID NO:50) and from about 318 (Glu) to
about 331 (Pro), including, for example, Lys.sub.320, Lys.sub.322
and Pr.sub.329.
[0166] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences, see, U.S. Pat. Nos. 4,444,887
and 4,716,111; and WO 98/46645, WO 98/50433, WO 98/24893, WO
98/16654, WO 96/34096, WO 96/33735 and WO 91/10741. The techniques
of Cole et al. and Boerder et al. are also available for the
preparation of human monoclonal antibodies (Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss (1985); and Boerder et
al., J Immunol 147:86 (1991)).
[0167] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which also express certain human
immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene complexes may be introduced randomly or by
homologous recombination into mouse embryonic stem cells.
Alternatively, the human variable region, constant region and
diversity region may be introduced into mouse embryonic stem cells,
in addition to the human heavy and light chain genes. The mouse
heavy and light chain immunoglobulin genes may be rendered
non-functional separately or simultaneously with the introduction
of the human immunoglobulin loci by homologous recombination. In
particular, homozygous deletion of the JH region prevents
endogenous antibody production. The modified embryonic stem cells
are expanded and microinjected into blastocysts to produce chimeric
mice. The chimeric mice are then bred to produce homozygous
offspring which express human antibodies, see, e.g., Jakobovitis et
al., Proc Natl Acad Sci USA 90:2551 (1993); Jakobovitis et al.,
Nature 362:255 (1993); Bruggermann et al., Year in Immunol 7:33
(1993); and Duchosal et al., Nature 355:258 (1992)).
[0168] The transgenic mice are immunized in the normal fashion with
IL-4 or IL-13 cytokine, e.g., all or a portion of IL-4 or IL-13
Monoclonal antibodies directed against IL-4 and IL-13 can be
obtained from the immunized, transgenic mice using conventional
hybridoma technology. The human immunoglobulin transgenes harbored
by the transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview, see
Lonberg et al., Int Rev Immunol 13:65-93 (1995). For a discussion
of producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., WO 98/24893; WO
92/01047; WO 96/34096; and WO 96/33735; EPO No. 0 598 877; and U.S.
Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;
5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598. In
addition, companies such as Amgen (Fremont, Calif.), Genpharm (San
Jose, Calif.) and Medarex, Inc. (Princeton, N.J.) can be engaged to
provide human antibodies directed against IL-4 and/or IL-13 using
technology similar to that described above.
[0169] Also, human mAbs could be made by immunizing mice
transplanted with human peripheral blood leukocytes, splenocytes or
bone marrow (e.g., trioma technique of XTL Biopharmaceuticals,
Israel). Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In that approach, a selected non-human monoclonal
antibody, e.g., a mouse antibody is used to guide the selection of
a completely human antibody recognizing the same epitope (Jespers
et al., Bio/technology 12:899 (1988)).
[0170] 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 (1992) describe a procedure for isolating antibodies which
are secreted to the periplasmic space of E. coli. Briefly, cell
paste is exposed to sodium acetate (pH 3.5) and EDTA. Cell debris
can be removed by centrifugation. Where the antibody variant is
secreted into the medium, supernatant 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 to inhibit proteolysis, and antibiotics may be
included to prevent growth of adventitious contaminants.
[0171] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis and affinity chromatography. The
suitability of protein A or protein G as an affinity ligand depends
on the species and isotype of any immunoglobulin F.sub.c 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 (1983)). Protein G can
be used for mouse isotypes and for human IgG3 (Guss et al., EMBO J.
5:1567 (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 C.sub.H3 domain, the Bakerbond
ABX.TM. resin (JT 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 agarose 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 or variant to be recovered.
[0172] Following any preliminary purification step(s), the mixture
comprising the antibody or variant of interest and contaminants may
be subjected to low pH hydrophobic interaction chromatography using
an elution buffer at a pH of between about 2.5-4.5, preferably
performed at low salt concentrations (e.g., from about 0-0.25 M
salt).
[0173] The antibodies of the present invention may be bispecific
antibodies. Bispecific antibodies can be monoclonal, preferably
human or humanized, antibodies that have binding specificities for
at least two different antigens. In a preferred embodiment, the
bispecific antibody, fragment thereof and so on has binding
specificities directed towards IL-4 and IL-13.
[0174] Methods for making bispecific antibodies are well known.
Traditionally, the recombinant production of bispecific antibodies
is based on the co-expression of two immunoglobulin heavy
chain/light chain pairs, where the two heavy chains have different
specificities (Milstein et al., Nature 305:537 (1983)). Because of
the random assortment of immunoglobulin heavy and light chains, the
hybridomas (quadromas) produce a potential mixture of ten different
antibody molecules, of which only one has the correct bispecific
structure. The purification of the correct molecule is usually
accomplished by affinity chromatography steps. Similar procedures
are disclosed in WO 93/08829 and in Traunecker et al., EMBO J.
10:3655 (1991). Other methods for making bispecific antibodies are
provided in, for example, Kufer et al., Trends Biotech 22:238-244,
2004.
[0175] Antibody variable domains with the desired binding
specificities can be fused to immunoglobulin constant domain
sequences. The fusion preferably is with an immunoglobulin heavy
chain constant domain, comprising at least part of the hinge,
C.sub.H2, and C.sub.H3 regions. It may have the first heavy chain
constant region (C.sub.H1) containing the site necessary for light
chain binding present in at least one of the fusions. DNAs encoding
the immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transformed into a suitable host organism. For
further details of generating bispecific antibodies see, for
example Suresh et al., Meth Enzym 121:210 (1986).
[0176] Heteroconjugate antibodies are also contemplated by the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells (U.S.
Pat. No. 4,676,980). It is contemplated that the antibodies may be
prepared in vitro using known methods in synthetic protein
chemistry, including those involving cross-linking agents. For
example, immunotoxins may be constructed using a disulfide exchange
reaction or by forming a thioester bond. Examples of suitable
reagents for that purpose include iminothiolate and
methyl-4-mercaptobutyrimidate, and those disclosed, for example, in
U.S. Pat. No. 4,676,980.
[0177] In addition, one can generate single-domain antibodies to
IL-4 and/or IL-13. Examples of that technology have been described
in WO9425591 for antibodies derived from Camelidae heavy chain Ig,
as well as in US20030130496 describing the isolation of single
domain fully human antibodies from phage libraries.
[0178] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423 (1988); Huston et al., Proc Natl Acad Sci USA 85:5879
(1988); and Ward, et al., Nature 334:544 (1989)) can be adapted to
produce single chain antibodies. Single chain antibodies are formed
by linking the heavy and light chain fragments of the F.sub.v
region via an amino acid bridge, resulting in a single chain
polypeptide. Techniques for the assembly of functional F.sub.v
fragments in E. coli may also be used (Skerra et al., Science
242:1038 (1988)).
[0179] The instant invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide. Fused or conjugated
antibodies of the present invention may be used for ease in
purification, sec e.g., WO 93/21232; EP 439,095; Naramura et al.,
Immunol Lett 39:91 (1994); U.S. Pat. No. 5,474,981; Gillies et al.,
Proc Natl Acad Sci USA 89:1428 (1992); and Fell et al., J Immunol
146:2446 (1991). The marker amino acid sequence can be a
hexa-histidine peptide, such as the tag provided in a pQE vector
(QIAGEN, Inc., Chatsworth, Calif.), among others, many of which are
commercially available, Gentz et al., Proc Natl Acad Sci USA 86:821
(1989). Other peptide tags useful for purification include, but are
not limited to, the "HA" tag, which corresponds to an epitope
derived from the influenza hemagglutinin protein (Wilson et al.,
Cell 37:767 (1984)) and the "flag" tag.
[0180] One can also create a single peptide chain binding molecules
in which the heavy and light chain F.sup.v regions are connected.
Single chain antibodies ("scF.sub.v") and the method of their
construction are described in, for example, U.S. Pat. No.
4,946,778. Alternatively, F.sub.ab can be constructed and expressed
by similar means. All of the wholly and partially human antibodies
can be less immunogenic than wholly murine monoclonal antibodies,
and the fragments and single chain antibodies also can be less
immunogenic.
[0181] Antibodies or antibody fragments can be isolated from
antibody phage libraries generated using the techniques described
in McCafferty et al., Nature 348:552 (1990). Clarkson et al.,
Nature 352:624 (1991) and Marks et al., J Mol Biol 222:581 (1991)
describe the isolation of murine and human antibodies,
respectively, using phage libraries. Subsequent publications
describe the production of high affinity (nM range) human
antibodies by chain shuffling (Marks et al., Bio/Technology 10:779
(1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nucl Acids Res 21:2265 (1993)). Thus,
the techniques are viable alternatives to traditional monoclonal
antibody hybridoma techniques for isolation of monoclonal
antibodies.
[0182] Candidate anti-IL-4 and/or IL-13 antibodies are tested by
enzyme-linked immunosorbent assay (ELISA), FACS, Western
immunoblotting or other immunochemical techniques as known in the
art.
[0183] To determine whether a particular antibody homolog binds to
human IL-4 and/or IL-13, any conventional binding assay may be
used. Useful IL-4 and IL-13 binding assays include FACS analysis,
ELISA assays, Surface Plasmon Resonance (Biacore),
radioimmunoassays and the like, which detect binding of antibody,
and functions resulting therefrom, to human IL-4 and/or IL-13.
Full-length and soluble forms of human IL-4 and IL-13 taught herein
are useful in such assays. The binding of an antibody or homolog to
IL-4 and/or IL-13, or to soluble fragments thereof, may
conveniently be detected through the use of a second antibody
specific for immunoglobulins of the species from which the antibody
or homolog is derived.
[0184] To determine whether a particular antibody or homolog does
or does not significantly block binding to IL-4 and/or IL-13, any
suitable competition assay may be used. Useful assays include, for
example, ELISA assays, FACS assays, radioimmunoassays and the like
that quantify the ability of the antibody or homolog to compete
with IL-4 and/or IL-13. Preferably, the ability of ligand to block
binding of labeled human IL-4 and/or IL-13 to immobilized antibody
or homolog is measured.
[0185] Antibodies of the instant invention may be described or
specified in terms of the epitope(s) or portion(s) of IL-4 and/or
IL-13 to which the antibody recognizes or specifically binds. The
epitope(s) or polypeptide portion(s) may be specified as described
herein, e.g., by N-terminal and C-terminal positions, by size in
contiguous amino acid residues, conformational epitopes and so
on.
[0186] Antibodies of the instant invention may also be described or
specified in terms of cross-reactivity. Antibodies that bind IL-4
and/or IL-13 polypeptides, which have at least 95%, at least 90%,
at least 85%, at least 80%, at least 75%, at least 70%, at least
65%, at least 60%, at least 55%, and at least 50% identity (as
calculated using methods known in the art and described herein) to
IL-4 and/or IL-13 are also included in the instant invention.
[0187] Antibodies of the instant invention also may be described or
specified in terms of binding affinity to IL-4 and/or IL-13.
Anti-IL-4 and/or anti-IL-13 antibodies may bind with a K.sub.D of
less than about 10.sup.-7 M, less than about 10.sup.-6 M, or less
than about 10.sup.-5 M. Higher binding affinities in an antibody of
interest can be beneficial, such as those with an equilibrium
dissociation constant or K.sub.D of from about 10.sup.-8 to about
10.sup.-15 M, from about 10.sup.-8 to about 10.sup.-12 M, from
about 10.sup.-9 to about 10.sup.-11 M, or from about 10.sup.-8 to
about 10.sup.-10 M. The invention also provides antibodies that
competitively inhibit binding of an antibody to an epitope of the
invention as determined by any method known in the art for
determining competitive binding, for example, the immunoassays
described herein. In preferred embodiments, the antibody
competitively inhibits binding to the epitope by at least 95%, at
least 90%, at least 85%, at least 80%, at least 75%, at least 70%,
at least 60%, or at least 50%.
[0188] The instant invention also includes conjugates comprising an
antibody of interest. The conjugates comprise two primary
components, an antibody of interest and a second component, which
may be a cell-binding agent, a cytotoxic agent and so on.
[0189] As used herein, the term "cell-binding agent" refers to an
agent that specifically recognizes and binds to a molecule on the
cell surface. Thus, the cell-binding agent can be a CD antigen, a
pathogen antigen, such as a virus antigen, a differentiation
antigen, a cancer antigen, a cell-specific antigen, a
tissue-specific antigen, an Ig or Ig-like molecule and so on.
[0190] Cell-binding agents may be of any type as presently known,
or that become known, and includes peptides, non-peptides,
saccharides, nucleic acids, ligands, receptors and so on, or
combinations thereof. The cell-binding agent may be any compound
that can bind a cell, either in a specific or non-specific manner.
Generally, the agent can be an antibody (especially monoclonal
antibodies), lymphokines, hormones, growth factors, vitamins,
nutrient-transport molecules (such as transferrin), or any other
cell-binding molecule or substance.
[0191] Other examples of cell-binding agents that can be used
include: polyclonal antibodies; monoclonal antibodies; and
fragments of antibodies such as F.sub.ab, F.sub.ab', F.sub.(ab')2
and F.sub.v (Parham, J. Immunol. 131:2895-2902 (1983); Spring et
al., J. Immunol. 113:470-478 (1974); and Nisonoff et al., Arch.
Biochem. Biophys. 89: 230-244 (1960)).
[0192] The second component also can be a cytotoxic agent. The term
"cytotoxic agent" as used herein refers to a substance that reduces
or blocks the function, or growth, of cells and/or causes
destruction of cells. Thus, the cytotoxic agent can be a taxol, a
maytansinoid, such as DM1 or DM4, CC-1065 or a CC-1065 analog, a
ricin, mitomycin C and so on. In some embodiments, the cytotoxic
agent, as with any binding agent of a conjugate of the instant
invention is covalently attached, directly or via a cleavable or
non-cleavable linker, to an antibody of interest.
[0193] Examples of suitable maytansinoids include maytansinol and
maytansinol analogs. Maytansinoids inhibit microtubule formation
and are highly toxic to mammalian cells.
[0194] Examples of suitable maytansinol analogues include those
having a modified aromatic ring and those having modifications at
other positions. Such suitable maytansinoids are disclosed in U.S.
Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946;
4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254;
4,322,348; 4,371,533; 6,333,410; 5,475,092; 5,585,499; and
5,846,545.
[0195] Examples of suitable analogues of maytansinol having a
modified aromatic ring include: (1) C-19-dechloro (U.S. Pat. No.
4,256,746) (prepared, for example, by LAH reduction of ansamytocin
P2); (2) C-20-hydroxy (or C-20-demethyl)+/-C-19-dechloro (U.S. Pat.
Nos. 4,361,650 and 4,307,016) (prepared, for example, by
demethylation using Streptomyces or Actinomyces or dechlorination
using lithium aluminum hydride (LAH)); and (3) C-20-demethoxy,
C-20-acyloxy (--OCOR), +/-dechloro (U.S. Pat. No. 4,294,757)
(prepared by acylation using acyl chlorides).
[0196] Examples of suitable analogues of maytansinol having
modifications of other positions include: (1) C-9-SH (U.S. Pat. No.
4,424,219) (prepared by the reaction of maytansinol with H.sub.2S
or P.sub.2S.sub.5); (2) C-14-alkoxymethyl (demethoxy/CH.sub.2OR)
(U.S. Pat. No. 4,331,598); (3) C-14-hydroxymethyl or acyloxymethyl
(CH.sub.2OH or CH.sub.2OAc) (U.S. Pat. No. 4,450,254) (prepared
from Nocardia); (4) C-15-hydroxy/acyloxy (U.S. Pat. No. 4,364,866)
(prepared by the conversion of maytansinol by Streptomyces); (5)
C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated
from Trewia nudiflora); (6) C-18-N-demethyl (U.S. Pat. Nos.
4,362,663 and 4,322,348) (prepared by the demethylation of
maytansinol by Streptomyces); and (7) 4,5-deoxy (U.S. Pat. No.
4,371,533) (prepared by the titanium trichloride/LAH reduction of
maytansinol).
[0197] The cytotoxic conjugates may be prepared by in vitro
methods. To link a cytotoxic agent, drug or prodrug to the
antibody, commonly, a linking group is used. Suitable linking
groups are known in the art and include disulfide groups, thioether
groups, acid labile groups, photolabile groups, peptidase labile
groups and esterase labile groups. For example, conjugates can be
constructed using a disulfide exchange reaction or by forming a
thioether bond between an antibody of interest and the drug or
prodrug.
[0198] As discussed above, the instant invention provides isolated
nucleic acid sequences encoding an antibody or functional fragment
or variant thereof as disclosed herein, vector constructs
comprising a nucleotide sequence encoding the IL-4 and/or
IL-13-binding portion of the antibody or functional fragment
thereof of the present invention, host cells comprising such a
vector, and recombinant techniques for the production of the
polypeptide.
[0199] The vector normally contains components known in the art and
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, one or more
marker or selection genes, sequences facilitating and/or enhancing
translation, an enhancer element and so on. Thus, the expression
vectors include a nucleotide sequence operably linked to such
suitable transcriptional or translational regulatory nucleotide
sequences such as those derived from mammalian, microbial, viral or
insect genes. Examples of additional regulatory sequences include
operators, mRNA ribosomal binding sites, and/or other appropriate
sequences which control transcription and translation, such as
initiation and termination thereof. Nucleotide sequences are
"operably linked" when the regulatory sequence functionally relates
to the nucleotide sequence for the appropriate polypeptide. Thus, a
promoter nucleotide sequence is operably linked to, e.g., the
antibody heavy chain sequence if the promoter nucleotide sequence
controls the transcription of that nucleotide sequence.
[0200] In addition, sequences encoding appropriate signal peptides
that are not naturally associated with antibody heavy and/or light
chain sequences can be incorporated into expression vectors. For
example, a nucleotide sequence for a signal peptide (secretory
leader) may be fused in-frame to the polypeptide sequence so that
the antibody is secreted to the periplasmic space or into the
medium. A signal peptide that is functional in the intended host
cells enhances extracellular secretion of the appropriate antibody
or portion thereof. The signal peptide may be cleaved from the
polypeptide on secretion of antibody from the cell. Examples of
such secretory signals are well known and include, e.g., those
described in U.S. Pat. Nos. 5,698,435; 5,698,417; and
6,204,023.
[0201] The vector may be a plasmid, a single-stranded or
double-stranded viral vector, a single-stranded or double-stranded
RNA or DNA phage vector, a phagemid, a cosmid or any other carrier
of a transgene of interest. Such vectors may be introduced into
cells as polynucleotides by well known techniques for introducing
DNA and RNA into cells. The vectors, in the case of phage and viral
vectors also may be introduced into cells as packaged or
encapsulated virus by well known techniques for infection and
transduction. Viral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells and using
plural vectors carrying the various virus components necessary to
produce a particle. Cell-free translation systems may also be
employed to produce the protein using RNAs derived from the present
DNA constructs (see, e.g., WO 86/05807 and WO 89/01036; and U.S.
Pat. No. 5,122,464).
[0202] The antibodies of the present invention can be expressed
from any suitable host cell. Examples of host cells useful in the
instant invention include prokaryotic, yeast or higher eukaryotic
cells and include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis, Enterobacter, Erwinia,
Klebsiella, Proteus, Salmonella, Serratia, and Shigella, as well as
Bacilli, Pseudomonas and Streptomyces) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing the antibody coding sequences of interest; yeast (e.g.,
Saccharomyces, Pichia, Actinomycetes, Kluyveromyces,
Schizosaccharomyces, Candida, Trichoderma, Neurospora, and
filamentous fungi, such as Neurospora, Penicillium, Tolypocladium
and Aspergillus) transformed with recombinant yeast expression
vectors containing antibody coding sequences; insect cell systems
infected with recombinant virus expression vectors (e.g.,
Baculovirus) containing antibody coding sequences; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; or tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing antibody coding sequences; or mammalian cell
systems (e.g., COS, CHO, BHK, 293 or 3T3 cells) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; or the
vaccinia virus 7.5K promoter).
[0203] Expression vectors for use in prokaryotic host cells
generally comprise one or more phenotypic selectable marker genes.
A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids, such as pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, Wis.),
pET (Novagen, Madison, Wis.) and the pRSET (Invitrogen, Carlsbad,
Calif.) series of vectors (Studier, J Mol Biol 219:37 (1991); and
Schoepfer, Gene 124:83 (1993)). Promoter sequences commonly used
for recombinant prokaryotic host cell expression vectors include
T7, (Rosenberg et al., Gene 56:125 (1987)), .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al., Nature
275:615 (1978); and Goeddel et al., Nature 281:544 (1979)),
tryptophan (tip) promoter system (Goeddel et al., Nucl Acids Res
8:4057 (1980)), and tac promoter (Sambrook et al., Molecular
Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory (1990)).
[0204] Yeast vectors will often contain an origin of replication
sequence, such as from a 2.mu. yeast plasmid, an autonomously
replicating sequence (ARS), a promoter region, sequences for
polyadenylation, sequences for transcription termination and a
selectable marker gene. Suitable promoter sequences for yeast
vectors include, among others, promoters for metallothionein,
3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem 255:2073
(1980)) or other glycolytic enzymes (Holland et al., Biochem
17:4900 (1978)) 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. Other suitable
vectors and promoters for use in yeast expression are further
described in Fleer et al., Gene 107:285 (1991). Other suitable
promoters and vectors for yeast and yeast transformation protocols
are well known in the art. Yeast transformation protocols are well
known. One such protocol is described by Hinnen et al., Proc Natl
Acad Sci 75:1929 (1978), which selects for Trp.sup.+ transformants
in a selective medium.
[0205] Any 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
(1988); Miller et al., Genetic Engineering, Setlow et al., eds.,
vol. 8, pp. 277-9, Plenum Publishing (1986); and Maeda et al.,
Nature 315:592 (1985)). For example, Baculovirus systems may be
used for production of heterologous proteins. In an insect system,
Autographa californica nuclear polyhedrosis virus (AcNPV) may be
used as a vector to express foreign genes. The virus grows in
Spodoptera frugiperda cells. The antibody coding sequence may be
cloned under control of an AcNPV promoter (for example the
polyhedrin promoter). Other hosts that have been identified include
Aedes, Drosophila melanogaster and Bombyx mori. A variety of viral
strains for transfection are publicly available, e.g., the L-1
variant of AcNPV and the Bm-5 strain of Bombyx mori NPV. Moreover,
plant cell cultures of cotton, corn, potato, soybean, petunia,
tomato, and tobacco and also be utilized as hosts as known in the
art.
[0206] Vertebrate cells and propagation of vertebrate cells in
culture (tissue culture) can be a routine procedure, although
fastidious cell lines do exist which require, for example, a
specialized medium with unique factors, feeder cells and so on, see
Tissue Culture, Kruse et al., eds., Academic Press (1973). Examples
of useful mammal 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 sertoli cells; human cervical carcinoma cells (for
example, HeLa); canine kidney cells; human lung cells; human liver
cells; mouse mammary tumor; and NS0 cells.
[0207] Host cells are transformed with vectors for antibody
production and cultured in conventional nutrient medium containing
growth factors, vitamins, minerals and so on, as well as inducers
appropriate for the cells and vectors used. Commonly used promoter
sequences and enhancer sequences are derived from polyoma virus,
Adenovirus 2, Simian virus 40 (SV40) and human cytomegalovirus
(CMV). DNA sequences derived from the SV40 viral genome may be used
to provide other genetic elements for expression of a structural
gene sequence in a mammalian host cell, e.g., SV40 origin, early
and late promoter, enhancer, splice and polyadenylation sites.
Viral early and late promoters are particularly useful because both
are easily obtained from a viral genome as a fragment which may
also contain a viral origin of replication. Exemplary expression
vectors for use in mammalian host cells are commercially
available.
[0208] Commercially available medium such as Ham's F10, Minimal
Essential Medium (MEM), RPMI-1640 and Dulbecco's Modified Eagle's
Medium (DMEM) are suitable for culturing host cells. In addition,
any of the media described in Ham et al., Meth Enzymol 58:44 (1979)
and Barnes et al., Anal Biochem 102:255 (1980), and in 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 a culture medium for the host cells. Any
of those media may be supplemented as necessary with hormones
and/or other growth factors (such as insulin, transferrin or
epidermal growth factor), salts (such as chlorides, such as sodium,
calcium or magnesium chloride; and phosphates), buffers (such as
HEPES), nucleotides (such as adenosine and thymidine), antibiotics,
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 be
included at appropriate concentrations, as a design choice. The
culture conditions, such as temperature, pH and the like, are as
known in the art appropriate for the cell and to enable the desired
expression of the transgene.
[0209] The polynucleotides of interest may be obtained, and the
nucleotide sequence of the polynucleotides determined, by any
method known in the art. For example, if the nucleotide sequence of
the antibody is known, a polynucleotide encoding the antibody may
be assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., Bio/Techniques 17:242 (1994)) and
then amplifying the ligated oligonucleotides, for example, by
PCR.
[0210] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid of a cell expressing same. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be obtained from a
suitable source, such as a library, which may be one specific for
antibody-producing cells, such as hybridoma cells selected to
express an antibody of the invention. Suitable primers can be
configured for PCR amplification. Amplified nucleic acids generated
by PCR may then be cloned into replicable cloning vectors using any
method well known in the art.
[0211] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody are determined, the nucleotide sequence of
the antibody may be manipulated to obtain the equivalents of
interest described herein using methods known in the art for
manipulating nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR etc. (see, for example,
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory (1990); and Ausubel et al., eds.,
Current Protocols in Molecular Biology, John Wiley & Sons
(1998) to generate antibodies having a different amino acid
sequence, for example, to create amino acid substitutions,
deletions and/or insertions.
[0212] The amino acid sequence of the heavy and/or light chain
variable domain may be inspected to identify the sequences of the
CDRs by well known methods, e.g., by comparison to known amino acid
sequences of other heavy and light chain variable regions to
determine the regions of sequence hypervariability. Using routine
recombinant DNA techniques, one or more of the CDRs may be inserted
within framework regions, e.g., into human framework regions to
humanize a non-human antibody, as described supra. The
polynucleotide of interest generated by the combination of the
framework regions and one or more CDRs encodes an antibody that
specifically binds IL-4 and/or IL-13, or at least the ED domain
thereof. For example, such methods may be used to make amino acid
substitutions or deletions of one or more variable region cysteine
residues participating in an intrachain disulfide bond to generate
antibody molecules lacking one or more intrachain disulfide
bonds.
[0213] The antibodies or antibody fragments of the invention can be
used to detect IL-4 and/or IL-13, and hence cells expressing IL-4
and/or IL-13, in a biological sample in vitro or in vivo. In one
embodiment, the anti-IL-4 and/or IL-13 antibody of the invention is
used to determine the presence and the level of IL-4 and/or IL-13
in a tissue or in cells derived from the tissue. The levels of IL-4
and/or IL-13 in the tissue or biopsy can be determined, for
example, in an immunoassay with the antibodies or antibody
fragments of the invention. The tissue or biopsy thereof can be
frozen or fixed. The same or other methods can be used to determine
other properties of IL-4 and/or IL-13, such as the level thereof,
cellular localization, mRNA levels, mutations thereof and so
on.
[0214] The above-described method can be used, for example, to
diagnose a cancer in a subject known to be or suspected to have a
cancer, wherein the level of IL-4 and/or IL-13 measured in said
patient is compared with that of a normal reference subject or
standard. The assay of interest also can be used to diagnose
arthritis or other autoimmune diseases characterized by B cell
infiltration and concentration, along with development of
differentiated lymphoid tissue.
[0215] The instant invention further provides for monoclonal
antibodies, humanized antibodies and epitope-binding fragments
thereof that are further labeled for use in research or diagnostic
applications. In some embodiments, the label is a radiolabel, a
fluorophore, a chromophore, an imaging agent or a metal ion.
[0216] A method for diagnosis is also provided in which said
labeled antibodies or epitope-binding fragments thereof are
administered to a subject suspected of having a cancer, arthritis,
autoimmune diseases or other IL-4 and/or IL-13 mediated disease,
and the distribution of the label within the body of the subject is
measured or monitored.
[0217] The antibody and fragments thereof of the instant invention
may be used as affinity purification agents. In that process, the
antibodies are immobilized on a solid phase, such as a dextran or
agarose resin or filter paper, using methods known in the art. The
immobilized antibody is contacted with a sample containing IL-4
and/or IL-13 or cells carrying same 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 IL-4 and/or
IL-13 or cell to be purified, which is bound to the immobilized
antibody of interest. Finally, the support is washed with another
suitable solvent, such as glycine buffer, pH 5.0 that will release
the IL-4 and/or IL-13 or cell from the antibody of interest.
[0218] For diagnostic applications, the antibody of interest
typically will be labeled with a detectable moiety. Numerous labels
are available which can be generally grouped into the following
categories: (a) radioisotopes, such as .sup.36S, .sup.14C,
.sup.125I, .sup.3H and .sup.131I (The antibody can be labeled with
the radioisotope using a techniques described in Current Protocols
in Immunology, vol. 12, Coligen et al., ed., Wiley-Interscience,
New York (1991), for example, and radioactivity can be measured
using scintillation counting); (b) fluorescent labels, such as rare
earth chelates (europium chelates), fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, lissamine,
phycoerythrin and Texas Red, the fluorescent labels can be
conjugated to the antibody using a technique disclosed in Current
Protocols in Immunology, supra, for example, where fluorescence can
be quantified using a fluorimeter; and (c) various enzyme substrate
labels are available (U.S. Pat. No. 4,275,149 provides a review),
the enzyme generally catalyzes a chemical alteration of the
chromogenic substrate which can be measured using various
techniques, for example, the enzyme may catalyze a color change in
a substrate, which can be measured spectrophotometrically, or the
enzyme may alter the fluorescence or chemiluminescence of the
substrate. Techniques for quantifying a change in fluorescence are
known, for example, using a luminometer, or the label 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., Meth Enz, ed. Langone & Van
Vunakis, Academic Press, New York, 73 (1981).
[0219] When such labels are used, suitable substrates are
available, such as: (i) for horseradish peroxidase with hydrogen
peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes
a dye precursor (e.g., orthophenylene diamine (OPD) or
3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)); (ii) for
alkaline phosphatase (AP) with p-nitrophenyl phosphate as the
chromogenic substrate; and (iii) .beta.-D-galactosidase
(.beta.-D-Gal) with a chromogenic substrate (e.g.,
p-nitrophenyl-.beta.-D-galactosidase) or a fluorogenic substrate
such as 4-methylumbelliferyl-.beta.-D-galactosidase.
[0220] Other enzyme-substrate combinations are available to those
skilled in the art. For a general review, see U.S. Pat. Nos.
4,275,149 and 4,318,980.
[0221] Sometimes, the label is indirectly conjugated with the
antibody. For example, the antibody can be conjugated with biotin
and any of the reporters 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 in that indirect
manner. Alternatively, to achieve indirect conjugation of the
label, the antibody is conjugated with a small hapten (e.g.,
digoxin) and one of the different types of labels or reporters
mentioned above is conjugated with an anti-digoxin antibody. Thus,
indirect conjugation of the label with the antibody or mutein can
be achieved using a second antibody.
[0222] In another embodiment of the invention, the antibody need
not be labeled, and the presence thereof can be detected using a
labeled antibody which binds to the antibody, another form of a
second antibody.
[0223] 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 (CRC Press, Inc.
1987).
[0224] 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. The amount of antigen 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.
[0225] Sandwich assays involve the use of two antibodies, each
capable of binding to a different immunogenic portion, determinant
or epitope, of the target to be detected. In a sandwich assay, the
test sample to be analyzed is bound by a first antibody which is
immobilized directly or indirectly on a solid support, and
thereafter a second antibody directly or indirectly labeled binds
to the bound 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 or other
suitable member of the binding pair (antibody/antigen,
receptor/ligand, enzyme/substrate, for example) 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.
[0226] The instant invention also includes kits, e.g., comprising
an antibody, fragment thereof, homolog, derivative thereof and so
on, such as a labeled or cytotoxic conjugate, and instructions for
the use of the antibody, conjugate for killing particular cell
types and so on. The instructions may include directions for using
the antibody, conjugate and so on in vitro, in vivo or ex vivo. The
antibody can be in liquid form or as a solid, generally
lyophilized. The kit can contain suitable other reagents, such as a
buffer, a reconstituting solution and other necessary ingredients
for the intended use. A packaged combination of reagents in
predetermined amounts with instructions for use thereof, such as
for a therapeutic use of for performing a diagnostic assay is
contemplated. Where the antibody is labeled, such as with an
enzyme, the kit can 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
the various reagents may be varied to provide for concentrates of a
solution of a reagent, which provides user flexibility, economy of
space, economy of reagents and so on. The reagents may be provided
as dry powders, usually lyophilized, including excipients, which on
dissolution provide a reagent solution having the appropriate
concentration.
[0227] The antibodies of the present invention may be used to treat
a mammal. In one embodiment, the antibody or equivalent of interest
is administered to a nonhuman mammal for the purposes of obtaining
preclinical data, for example. Exemplary nonhuman mammals to be
treated include nonhuman primates, dogs, cats, rodents and other
mammals in which preclinical studies are performed. Such mammals
may be established animal models for a disease to be treated with
the antibody, or may be used to study toxicity of the antibody of
interest. In each of those embodiments, dose escalation studies may
be performed in the mammal.
[0228] An antibody, with or without a second component, such as a
therapeutic moiety conjugated to same, administered alone or in
combination with cytotoxic factor(s) can be used as a therapeutic.
The present invention is directed to antibody-based therapies which
involve administering antibodies of the invention to an animal, a
mammal, or a human, for treating a IL-4 and/or IL-13 mediated
disease, disorder or condition.
[0229] The term "treatment" as used in the present invention refers
to both therapeutic treatment and prophylactic or preventative
measures. It refers to preventing, curing, reversing, attenuating,
alleviating, minimizing, suppressing or halting the deleterious
effects of a disease state, disease progression, disease causative
agent (e.g., bacteria or viruses) or other abnormal condition.
[0230] Thus the invention also includes polyvalent antibodies,
including bispecific anti-IL-4/IL-13 antibodies, having attached
thereto diagnostically or therapeutically functional effector
molecules, atoms or other species. For example, the antibody may
have a radioactive diagnostic label or radioactive cytotoxic atom
or metal or cytotoxic species, e.g. ricin chain, attached thereto
for in vivo diagnosis or therapy of cancer.
[0231] Moreover, the antibodies according to the invention may be
used in immunoassays, in purification methods and in other methods
in which immunoglobulins or fragments thereof are used. Such uses
are well-known in the art.
[0232] Accordingly, the invention also provides compositions
comprising the anti-IL-13 and/or anti-IL-4 antibodies or fragments
thereof according to the invention, conveniently in combination
with a pharmaceutically acceptable carrier, diluent or excipient
which are conventional in the art.
[0233] The term "pharmaceutical composition" as used in the present
invention refers to formulations of various preparations. The
formulations containing therapeutically effective amounts of the
polyvalent antibodies are sterile liquid solutions, liquid
suspensions or lyophilized versions and optionally contain
stabilizers or excipients.
[0234] The term "disorder" as used in the present invention refers
to any condition that would benefit from treatment with the
antibody of the present invention. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal, and in particular humans, to the disorder in
question. Non-limiting examples of disorders to be treated herein
include cancers, inflammation, autoimmune diseases, infections,
cardiovascular diseases, respiratory diseases, neurological
diseases and metabolic diseases.
[0235] The antibodies of the present invention may be used to
treat, suppress or prevent disease, such as an allergic disease, a
Th2-mediated disease, IL-13-mediated disease, IL-4-mediated
disease, and/or IL-4/IL-13-mediated disease. Examples of such
diseases include, Hodgkin's disease, asthma, allergic asthma,
atopic dermatitis, atopic allergy, ulcerative colitis, scleroderma,
allergic rhinitis, COPD3 idiopathic pulmonary fibrosis, chronic
graft rejection, bleomycin-induced pulmonary fibrosis,
radiation-induced pulmonary fibrosis, pulmonary granuloma,
progressive systemic sclerosis, schistosomiasis, hepatic fibrosis,
renal cancer, Burkitt lymphoma, Hodgkins disease, non-Hodgkins
disease, Sezary syndrome, asthma, septic arthritis, dermatitis
herpetiformis, chronic idiopathic urticaria, ulcerative colitis,
scleroderma, hypertrophic scarring, Whipple's Disease, benign
prostate hyperplasia, a lung disorder in which IL-4 receptor plays
a role, condition in which IL-4 receptor-mediated epithelial
barrier disruption plays a role, a disorder of the digestive system
in which IL-4 receptor plays a role, an allergic reaction to a
medication, Kawasaki disease, sickle cell disease, Churg-Strauss
syndrome, Grave's disease, pre-eclampsia, Sjogren's syndrome,
autoimmune lymphoproliferative syndrome, autoimmune hemolytic
anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis,
cystic fibrosis, allergic bronchopulmonary mycosis, chronic
obstructive pulmonary disease, bleornycin-induced pneumopathy and
fibrosis, pulmonary alveolar proteinosis, adull respiratory
distress syndrome, sarcoidosis, hyper IgE syndrome, idiopathic
hypercosinophil syndrome, an autoimmune blistering disease,
pemphigus vulgaris, bullous pemphigoid, myasthenia gravis, chronic
fatigue syndrome, nephrosis).
[0236] The term "allergic disease" refers to a pathological
condition in which a patient is hypersensitized to and mounts an
immunologic reaction against a substance that is normally
nonimmunogenic. Allergic disease is generally characterized by
activation of mast cells by IgE resulting in an inflammatory
response (e.g. local response, systemic response) that can result
in symptoms as benign as a runny nose, to life-threatening
anaphylactic shock and death. Examples of allergic disease include,
but are not limited to, allergic rhinitis (e.g., hay fever), asthma
(e.g., allergic asthma), allergic dermatitis (e.g., eczema),
contact dermatitis, food allergy and urticaria (hives).
[0237] As used herein "Th2-mediated disease" refers to a disease in
which pathology is produced (in whole or in part) by an immune
response (Th2-type immune response) that is regulated by CD4.sup.+
Th2 T lymphocytes, which characteristically produce IL-4, IL-5,
IL-9 and IL-13. A Th2-type immune response is associated with the
production of certain cytokines (e.g., IL-4, IL-13) and of certain
classes of antibodies (e.g., IgE), and is associate with humoral
immunity. Th2-mediated diseases are characterized by the presence
of elevated levels of Th2 cytokines (e.g., IL-4, IL-13) and/or
certain classes of antibodies (e.g., IgE) and include, for example,
allergic disease (e.g., allergic rhinitis, atopic dermatitis,
asthma (e.g., atopic asthma), allergic airways disease (AAD),
anaphylactic shock, conjunctivitis), autoimmune disorders
associated with elevated levels of IL-4 and/or IL-13 (e.g.,
rheumatoid arthritis, host-versus-graft disease, renal disease
(e.g., nephritic syndrome, lupus nephritis)), and infections
associated with elevated levels of IL-4 and/or IL-13 (e.g., viral,
parasitic, fungal (e.g., C. albicans) infection). Certain cancers
are associated with elevated levels of IL-4 and/or IL-13 or
associated with IL-4-induced and/or IL-13-induced cancer cell
proliferation (e.g., B cell lymphoma, T cell lymphoma, multiple
myeloma, head and neck cancer, breast cancer and ovarian cancer).
These cancers can be treated, suppressed or prevented using the Ii
gaud of the invention.
[0238] The term "cancer" as used in the present invention refers to
or describes the physiological condition in mammals, in particular
humans, which is typically characterized by unregulated cell
growth. Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
[0239] The term "autoimmune disease" as used in the present
invention refers to a non-malignant disease or disorder arising
from and directed against an individual's own tissues. Examples of
autoimmune diseases or disorders include, but are not limited to,
inflammatory responses such as inflammatory skin diseases including
psoriasis and dermatitis; allergic conditions such as eczema and
asthma; other conditions involving infiltration of T cells and
chronic inflammatory responses; atherosclerosis; diabetes mellitus
(e.g. Type I diabetes mellitus or insulin dependent diabetes
mellitis); multiple sclerosis and central nervous system (CNS)
inflammatory disorder.
[0240] The antibodies of the present invention may be used as
separately administered compositions or in conjunction with other
agents. The antibodies can be used in combination therapy with
existing IL-13 therapeutics (e.g. existing IL-13 agents such as
anti-IL-13R.alpha.1, IL-4/13 Trap, anti-IL-13) plus anti-IL-4
antibody and existing IL-4 agents (for example, anti-IL-4R, IL-4
Mutein, IL-4/13 Trap) plus anti-IL-13 antibody and IL-4 antibodies
(for example, WO05/0076990 (CAT), WO03/092610 (Regeneron),
WO00/64944 (Genetic Inst.) and WO2005/062967 (Tanox)).
[0241] The antibodies of the present invention may be administered
and/or formulated together with one or more additional therapeutic
or active agents. When a ligand is administered with an additional
therapeutic agent, the ligand can be administered before,
simultaneously with or subsequent to administration of the
additional agent. Generally, the ligand and additional agent are
administered in a manner that provides an overlap of therapeutic
effect. Additional agents that can be administered or formulated
with the ligand of the invention include, for example, various
immunotherapeutic dings?, such as cylcosporine, methotrexate,
adriamycin or cisplatimun, antibiotics, antimycotics, anti-viral
agents and immunotoxins. For example, when the antagonist is
administered to prevent, suppress or treat lung inflammation or a
respiratory disease (e.g., asthma), it can be administered in
conjuction with phosphodiesterase inhibitors (e.g., inhibitors of
phosphodiesterase 4), bronchodilators (e.g., .beta.2-agonists,
anticholinergerics, theophylline), short-acting beta-agonists
(e.g., albuterol, salbuiamol, bambuterol, fenoter[sigma]l,
isoetherine, isoproterenol, leva[iota]buterol, metaproterenol,
pirbuterol, terbutaline and tornlate), long-acting beta-agonists
(e.g., formoterol and salmeterol), short acting anticholinergics
(e.g., ipratropium bromide and oxitropium bromide), long-acting
anticholinergics (e.g., tiotropium), theophylline (e.g. short
acting formulation, long acting formulation), inhaled steroids
(e.g., beclomethasone, beclometasone, budesonide, flunisolide,
fluticasone propionate and triamcinolone), oral steroids (e.g.,
methylprednisolone, prednisolone, prednisolon and prednisone),
combined short-acting beta-agonists with anticholinergics (e.g.,
albuterol/salbutamol/ipratopium, and fenoterol/ipratopium),
combined long-acting beta-agonists with inhaled steroids (e.g.,
salmeterol/fluticasone, and formolerol/budesonide) and mucolytic
agents (e.g., erdosteine, acetylcysteine, bromheksin,
carbocyslcine, guiafencsin and iodinated glycerol
[0242] Other suitable co-therapeutic agents that can be
administered with antibody of the present invention to prevent,
suppress or treat asthma (e.g., allergic asthma), include a
corticosteroid (e.g., beclomethasone, budesonide, fluticasone),
cromoglycate, nedocromil, beta-agonist (e.g., salbutamol,
terbutaline, bambuterol, fenoterol, reproterol, tolubuterol,
salmeterol, fomtero), zafirlukast, salmeterol, prednisone,
prednisolone, theophylline, zileutron, montelukast, and leukotriene
modifiers. The ligands of the invention can be coadministered with
a variety of co-therapeutic agents suitable for treating diseases
(e.g., a Th-2 mediated disease, YL-A-mediated disease, IL-13
mediated disease, IL-4 mediated disease and cancer), including
cytokines, analgesics/antipyretics, antiemetics, and
chemotherapeutics.
[0243] Antibodies of the invention may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein. The term "physiologically acceptable,"
"pharmacologically acceptable" and so on mean approved by a
regulatory agency of the Federal or a state government or listed in
the U.S. Pharmacopeia or other generally recognized pharmacopeia
for use in animals and more particularly in humans.
[0244] The anti-IL-4, anti-IL-13 and bispecific anti-IL-4/IL-13
antibodies may be administered to a mammal and in particular
humans, in any acceptable manner. Methods of introduction include,
but are not limited to, parenteral, subcutaneous, intraperitoneal,
intrapulmonary, intranasal, epidural, inhalation and oral routes,
and if desired for immunosuppressive treatment, intralesional
administration. Parenteral infusions include intramuscular,
intradermal, intravenous, intraarterial or intraperitoneal
administration. The antibodies or compositions may be administered
by any convenient route, for example, by infusion or bolus
injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa etc.) and
may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, it may be
desirable to introduce the therapeutic antibodies or compositions
of the invention into the central nervous system by any suitable
route, including intraventricular and intrathecal injection;
intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. In addition, the antibody is suitably
administered by pulse infusion, particularly with declining doses
of the antibody. Preferably the dosing is given by injection,
preferably intravenous or subcutaneous injections, depending, in
part, on whether the administration is brief or chronic.
[0245] Various other delivery systems are known and can be used to
administer an antibody of the present invention, including, e.g.,
encapsulation in liposomes, microparticles, microcapsules (see
Langer, Science 249:1527 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein et al.,
eds., p. 353-365 (1989); and Lopez-Berestein, ibid., p. 317-327)
and recombinant cells capable of expressing the compound;
receptor-mediated endocytosis (see, e.g., Wu et al., J Biol Chem
262:4429 (1987)); construction of a nucleic acid as part of a
retroviral or other vector etc.
[0246] 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,
liposomes, albumin microspheres, microemulsions, nanoparticles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed.
(1980).
[0247] Pulmonary administration can also be employed, e.g., by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent. The antibody may also be administered into the lungs of a
patient in the form of a dry powder composition, see e.g., U.S.
Pat. No. 6,514,496.
[0248] In a specific embodiment, it may be desirable to administer
the therapeutic antibodies or compositions of the invention locally
to the area in need of treatment; that may be achieved by, for
example, and not by way of limitation, local infusion, topical
application, by injection, by means of a catheter, by means of a
suppository or by means of an implant, said implant being of a
porous, non-porous or gelatinous material, including membranes,
such as sialastic membranes or fibers. Preferably, when
administering an antibody of the invention, care is taken to use
materials to which the protein does not absorb or adsorb.
[0249] In yet another embodiment, the antibody can be delivered in
a controlled release system. In one embodiment, a pump may be used
(see Langer, Science 249:1527 (1990); Sefton, CRC Crit. Ref Biomed
Eng 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and
Saudek et al., N Engl J Med 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer et al., eds., CRC Press (1974);
Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen et al., eds., Wiley (1984); Ranger et al., J
Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al.,
Science 228:190 (1985); During et al., Ann Neurol 25:351 (1989);
and Howard et al., J Neurosurg 71:105 (1989)). In yet another
embodiment, a controlled release system can be placed in proximity
of the therapeutic target.
[0250] 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,
diluents, excipients or stabilizers typically employed in the art,
i.e., buffering agents, stabilizing agents, preservatives,
isotonifiers, non-ionic detergents, antioxidants and other
miscellaneous additives, see Remington's Pharmaceutical Sciences,
16th ed., Osol, ed. (1980). Such additives are generally nontoxic
to the recipients at the dosages and concentrations employed,
hence, the excipients, diluents, carriers and so on are
pharmaceutically acceptable.
[0251] An "isolated" or "purified" antibody is substantially free
of cellular material or other contaminating proteins from the cell
or tissue source or medium from which the protein is derived, or
substantially free of chemical precursors or other chemicals when
chemically synthesized. The language "substantially free of
cellular material" includes preparations of an antibody in which
the polypeptide/protein is separated from cellular components of
the cells from which same is isolated or recombinantly produced.
Thus, an antibody that is substantially free of cellular material
includes preparations of the antibody having less than about 30%,
20%, 10%, 5%, 2.5% or 1%, (by dry weight) of contaminating protein.
When the antibody is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, 10%, 5%, 2.5% or 1% of the volume
of the protein preparation. When antibody is produced by chemical
synthesis, it is preferably substantially free of chemical
precursors or other chemicals and reagents, i.e., the antibody of
interest is separated from chemical precursors or other chemicals
which are involved in the synthesis of the protein. Accordingly,
such preparations of the antibody have less than about 30%, 20%,
10%, 5% or 1% (by dry weight) of chemical precursors or compounds
other than antibody of interest. In a preferred embodiment of the
present invention, antibodies are isolated or purified.
[0252] As used herein, the phrase "low to undetectable levels of
aggregation" refers to samples containing no more than 5%, no more
than 4%, no more than 3%, no more than 2%, no more than 1% and
often no more than 0.5% aggregation, by weight protein, as measured
by, for example, high performance size exclusion chromatography
(HPSEC).
[0253] As used herein, the term "low to undetectable levels of
fragmentation" refers to samples containing equal to or more than
80%, 85%, 90%, 95%, 98% or 99%, of the total protein, for example,
in a single peak, as determined by HPSEC, or in two (2) peaks
(heavy chain and light chain) by, for example, reduced capillary
gel electrophoresis (rCGE) and containing no other single peaks
having more than 5%, more than 4%, more than 3%, more than 2%, more
than 1% or more than 0.5% of the total protein, each. The rCGE as
used herein refers to capillary gel electrophoresis under reducing
conditions sufficient to reduce disulfide bonds in an antibody or
antibody-type or derived molecule.
[0254] As used herein, the terms "stability" and "stable" in the
context of a liquid formulation comprising a 11-4 and/or IL-13
antibody or binding fragment thereof refer to the resistance of the
antibody or antigen-binding fragment thereof in the formulation to
thermal and chemical unfolding, aggregation, degradation or
fragmentation under given manufacture, preparation, transportation
and storage conditions. The "stable" formulations of the invention
retain biological activity equal to or more than 80%, 85%, 90%,
95%, 98%, 99% or 99.5% under given manufacture, preparation,
transportation and storage conditions. The stability of said
antibody preparation can be assessed by degrees of aggregation,
degradation or fragmentation by methods known to those skilled in
the art, including, but not limited to, rCGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPSEC,
compared to a reference.
[0255] The term, "carrier," refers to a diluent, adjuvant,
excipient or vehicle with which the therapeutic is administered.
Such physiological carriers can be sterile liquids, such as water
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a suitable carrier when the
pharmaceutical composition is administered intravenously. Saline
solutions and aqueous dextrose and glycerol solutions also can be
employed as liquid carriers, particularly for injectable solutions.
Suitable pharmaceutical excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene glycol, water, ethanol and the
like. The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, capsules, powders, sustained-release formulations,
depots and the like. The composition can be formulated as a
suppository, with traditional binders and carriers such as
triglycerides. Oral formulations can include standard carriers such
as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate etc.
Examples of suitable carriers are described in "Remington's
Pharmaceutical Sciences," Martin. Such compositions will contain an
effective amount of the antibody, preferably in purified form,
together with a suitable amount of carrier so as to provide the
form for proper administration to the patient. As known in the art,
the formulation will be constructed to suit the mode of
administration.
[0256] Buffering agents help to maintain the pH in the range which
approximates physiological conditions. Buffers are preferably
present at a concentration ranging from about 2 mM to about 50 mM.
Suitable buffering agents for use with the instant 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, 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
gluconate mixture etc.), oxalate buffers (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.). Phosphate buffers, carbonate buffers,
histidine buffers, trimethylamine salts such as Tris, HEPES and
other such known buffers can be used.
[0257] 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 present invention include phenol,
benzyl alcohol, m-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzyaconium halides
(e.g., chloride, bromide and iodide), hexamethonium chloride, alkyl
parabens such as methyl or propyl paraben, catechol, resorcinol,
cyclohexanol and 3-pentanol.
[0258] Isotonicifiers are present to ensure physiological
isotonicity of liquid compositions of the instant invention and
include polhydric sugar alcohols, preferably trihydric or higher
sugar alcohols, such as glycerin, erythritol, arabitol, xylitol,
sorbitol and mannitol. Polyhydric alcohols can be present in an
amount of between about 0.1% to about 25%, by weight, preferably 1%
to 5% taking into account the relative amounts of the other
ingredients.
[0259] 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; 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,
arabitol, erythritol, 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, .alpha.-monothioglycerol
and sodium thiosulfate; low molecular weight polypeptides (i.e.,
<10 residues); proteins, such as human serum albumin, bovine
serum albumin, gelatin or immunoglobulins; hydrophilic polymers,
such as polyvinylpyrrolidone, saccharides, monosaccharides, such as
xylose, mannose, fructose, glucose; disaccharides, such as lactose,
maltose and sucrose; trisaccharides such as raffinose;
polysaccharides such as dextran and so on. Stabilizers are present
in the range from 0.1 to 10,000 w/w per part of active protein.
[0260] Additional miscellaneous excipients include bulking agents,
(e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g.,
ascorbic acid, methionine or vitamin E) and cosolvents.
[0261] The formulation herein also may contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely impact each other. For example, it may be desirable to
further provide an immunosuppressive agent. Such molecules suitably
are present in combination in amounts that are effective for the
purpose intended.
[0262] As used herein, the term "surfactant" refers to organic
substances having amphipathic structures, namely, are composed of
groups of opposing solubility tendencies, typically an oil-soluble
hydrocarbon chain and a water-soluble ionic group. Surfactants can
be classified, depending on the charge of the surface-active
moiety, into anionic, cationic and nonionic surfactants.
Surfactants often are used as wetting, emulsifying, solubilizing
and dispersing agents for various pharmaceutical compositions and
preparations of biological materials.
[0263] 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 stresses without causing
denaturation of the protein. Suitable non-ionic surfactants include
polysorbates (20, 80 etc.), polyoxamers (184, 188 etc.),
Pluronic.RTM. polyols and polyoxyethylene sorbitan monoethers
(TWEEN-20.RTM., TWEEN-80.RTM. etc.). Non-ionic surfactants may be
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 mg/ml.
[0264] As used herein, the term, "inorganic salt," refers to any
compound, containing no carbon that result from replacement of part
or all of the acid hydrogen or an acid by a metal or group acting
like a metal, and often are used as a tonicity adjusting compound
in pharmaceutical compositions and preparations of biological
materials. The most common inorganic salts are NaCl, KCl,
NaH.sub.2PO.sub.4 etc.
[0265] The instant invention encompasses liquid formulations having
stability at temperatures found in a commercial refrigerator and
freezer found in the office of a physician or laboratory, such as
from about -20.degree. C. to about 5.degree. C., said stability
assessed, for example, by high performance size exclusion
chromatography (HPSEC), for storage purposes, such as for about 60
days, for about 120 days, for about 180 days, for about a year, for
about 2 years or more. The liquid formulations of the present
invention also exhibit stability, as assessed, for example, by
HSPEC, at room temperatures, for a at least a few hours, such as
one hour, two hours or about three hours prior to use.
[0266] The term "small molecule" and analogous terms include, but
are not limited to, peptides, peptidomimetics, amino acids, amino
acid analogues, polynucleotides, polynucleotide analogues,
nucleotides, nucleotide analogues, organic or inorganic compounds
(i.e., including heterorganic and/or ganometallic compounds) having
a molecular weight less than about 10,000 grams per mole, organic
or inorganic compounds having a molecular weight less than about
5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.
[0267] Thus, in the case of cancer, the antibodies of the invention
may be administered alone or in combination with other types of
cancer treatments, including conventional chemotherapeutic agents
(paclitaxel, carboplatin, cisplatin and doxorubicin), anti-EGFR
agents (gefitinib, erlotinib and cetuximab), anti-angiogenesis
agents (bevacizumab and sunitinib), as well as immunomodulating
agents, such as interferon-.alpha. and thalidomide.
[0268] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any agent(s) which can be used in the
treatment, management or amelioration of a disease, disorder,
malady and the like associated with aberrant IL-4 and/or IL-13
metabolism and activity.
[0269] In addition, the antibodies of the instant invention may be
conjugated to various effector molecules such as heterologous
polypeptides, drugs, radionucleotides or toxins, see, e.g., WO
92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and
EPO 396,387. An antibody or fragment thereof may be conjugated to a
therapeutic moiety such as a cytotoxin (e.g., a cytostatic or
cytocidal agent), a therapeutic agent or a radioactive metal ion
(e.g., a emitters, such as, for example, .sup.213Bi). A cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy
anthracindione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol and puromycin and analogs or homologues
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil and decarbazine),
alkylating agents (e.g., mechlorethamine, chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin, daunomycin and doxorubicin), antibiotics
(e.g., dactinomycin, actinomycin, bleomycin, mithramycin and
anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
[0270] Techniques for conjugating such a therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., in Monoclonal
Antibodies and Cancer Therapy, Reisfeld et al. (eds.), p. 243-56
Alan R. Liss (1985); Hellstrom et al., in Controlled Drug Delivery,
2nd ed., Robinson et al., eds., p. 623-53, Marcel Dekker (1987);
Thorpe, in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al., eds., p. 475-506 (1985); Monoclonal
Antibodies For Cancer Detection and Therapy, Baldwin et al., eds.,
p. 303-16, Academic Press (1985); and Thorpe, et al., Immunol Rev
62:119 (1982). Alternatively, an antibody can be conjugated to a
second antibody to form an antibody heteroconjugate, such as a
bifunctional antibody, see, e.g., U.S. Pat. No. 4,676,980.
[0271] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-.alpha., TNF-.beta., AIM I
(WO 97/33899), AIM II (WO 97/34911), Fas ligand (Takahashi et al.,
Int Immunol, 6:1567 (1994)), VEGF (WO 99/23105); a thrombotic
agent; an anti-angiogenic agent, e.g., angiostatin or endostatin;
or biological response modifiers such as, for example, lymphokines,
interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),
granulocyte macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (GCSF) or other growth
factors.
[0272] The formulations to be used for in vivo administration must
be sterile. That can be accomplished, for example, by filtration
through sterile filtration membranes. For example, the liquid
formulations of the present invention may be sterilized by
filtration using a 0.2 .mu.m or a 0.22 .mu.m filter.
[0273] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films or
matrices. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethylmethacrylate), 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 injectable
microspheres composed of lactic acid-glycolic acid copolymer) 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. 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 thio-disulfide interchange, stabilization
may be achieved by modifying sulfhydryl residues, lyophilizing from
acidic solutions, controlling moisture content, using appropriate
additives, amino acid substitution and developing specific polymer
matrix compositions.
[0274] The antibody or 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 or human
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 or variant to be administered
will be governed by such considerations, and can be the minimum
amount necessary to prevent, ameliorate or treat a IL-4 and/or
IL-13 mediated disease, condition or disorder.
[0275] The antibody or variant optionally is 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% of the heretofore employed
dosages.
[0276] As used herein, the term "effective amount" refers to the
amount of a therapy (e.g., a prophylactic or therapeutic agent),
which is sufficient to reduce the severity and/or duration of a
IL-4 and/or IL-13 mediated disease, ameliorate one or more symptoms
thereof, prevent the advancement of a IL-4 and/or IL-13 mediated
disease or cause regression of a disease, or which is sufficient to
result in the prevention of the development, recurrence, onset, or
progression of a IL-4 and/or IL-13 mediated disease or one or more
symptoms thereof, or enhance or improve the prophylactic and/or
therapeutic effect(s) of another therapy (e.g., another therapeutic
agent) useful for treating a IL-4 and/or IL-13 mediated
disease.
[0277] The amount of therapeutic antibody or fragment thereof which
will be effective in the use or 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, a dose-response curve and the pharmaceutical
compositions of the invention can be first derived in vitro. If a
suitable animal model system is available, again a dose-response
curve can be obtained and used to extrapolate a suitable human dose
practicing methods known in the art. However, based on common
knowledge of the art, a pharmaceutical composition effective in
promoting a diminution of an inflammatory effect, for example, may
provide a local therapeutic agent concentration of between about 5
and 20 ng/ml, and, preferably, between about 10 and 20 ng/ml.
[0278] In a preferred embodiment, an aqueous solution of
therapeutic polypeptide, antibody or fragment thereof can be
administered by subcutaneous injection. Each dose may range from
about 0.5 mg to about 50 mg per kilogram of body weight, or more
preferably, from about 3 mg to about 30 mg per kilogram body
weight. The dosage can be ascertained empirically for the
particular disease, patient population, mode of administration and
so on, practicing pharmaceutical methods known in the art.
[0279] The dosing schedule for subcutaneous administration may vary
from once a week to daily depending on a number of clinical
factors, including the type of disease, severity of disease and the
sensitivity of the subject to the therapeutic agent.
[0280] The instant invention provides methods for preparing liquid
formulations of the antibody or IL-4 and/or IL-13 binding fragment
thereof, said methods comprising concentrating a fraction of
purified antibody to a final concentration of about 15 mg/ml, about
20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60
mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100
mg/ml, about 200 mg/ml, about 250 mg/ml, about 300 mg/ml or more
using, for example, a semi-permeable membrane with an appropriate
molecular weight (mw) cutoff (e.g., 30 kD cutoff for F.sub.(ab')2
fragments thereof; and 10 kD cutoff for F.sub.ab fragments).
[0281] In addition, the present invention also encompasses stable
liquid formulations of the products of interest that have improved
half-life in vivo. Thus, the antibody of interest has a half-life
in a subject, preferably a human, of greater than 3 days, greater
than 7 days, greater than 10 days, greater than 15 days, greater
than 25 days, greater than 30 days, greater than 35 days, greater
than 40 days, greater than 45 days, greater than 2 months, greater
than 3 months, greater than 4 months, greater than 5 months or
more.
[0282] To prolong the serum circulation of an antibody in vivo,
various techniques can be used. For example, inert polymer
molecules, such as high molecular weight polyethylene glycol (PEG),
can be attached to an antibody with or without a multifunctional
linker either through site-specific conjugation of the PEG to the
N-terminus or to the C-terminus of the antibody or via .epsilon.
amino groups present on lysine residues. Linear or branched polymer
derivatization that results in minimal loss of biological activity
can be used. The degree of conjugation can be closely monitored by
SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG
molecules to the antibodies. Unreacted PEG can be separated from
antibody-PEG conjugates by size-exclusion or by ion exchange
chromatography. PEG-derivatized antibodies can be tested for
binding activity as well as for in vivo efficacy using methods
known to those of skilled in the art, for example, by immunoassays
described herein.
[0283] An antibody having an increased half-life in vivo can also
be generated by introducing one or more amino acid modifications
(i.e., substitutions, insertions or deletions) into an IgG constant
domain, or F.sub.cR binding fragment thereof (such as an F.sub.e or
hinge F.sub.e domain fragment), see, e.g., WO 98/23289; WO
97/34631; and U.S. Pat. No. 6,277,375.
[0284] Further, an antibody can be conjugated to albumin to make an
antibody more stable in vivo or have a longer half life in vivo.
The techniques are known in the art, see e.g., WO 93/15199, WO
93/15200 and WO 01/77137; and EPO 413, 622. The antibody also can
be modified, for example, by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein and so on.
[0285] In one embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine or other "caine" anesthetic to ease pain at the site
of the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water-free concentrate in a sealed
container, such as an ampule or sachet indicating the quantity of
active agent. Where the composition is to be administered by
infusion, it can be dispensed with an infusion bottle containing
sterile pharmaceutical grade water or saline. Where the composition
is administered by injection, an ampule of sterile water for
injection or saline can be provided, for example, in a kit, so that
the ingredients may be mixed prior to administration.
[0286] The invention also provides that a liquid formulation of the
present invention is packaged in a sealed container such as an
ampule or sachet indicating the quantity of the product of
interest. The liquid formulations of the instant invention can be
in a sealed container indicating the quantity and concentration of
the antibody or antibody fragment. The liquid formulation of the
instant invention can be supplied in a sealed container with at
least 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml,
70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250
mg/ml, or 300 mg/ml of IL-4 and/or IL-13 antibody in a quantity of
1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml
or 20 ml, for example.
[0287] An article of manufacture containing materials useful for
the treatment of the disorders described above is provided. The
article of manufacture comprises a container and a label. Suitable
containers include, for example, bottles, vials, syringes and test
tubes. The containers may be formed from a variety of materials
such as glass or plastic. The container holds a composition which
is effective for diagnosing, preventing or treating a IL-4 and/or
IL-13 mediated condition or disease and may have a sterile access
port (for example, the container may be an intravenous solution bag
or a vial having a stopper pierceable by a hypodermic injection
needle). The label on or associated with the container indicates
that the composition is used for treating the condition of choice.
The article of manufacture may further comprise a second container
comprising a pharmaceutically acceptable buffer, such as
phosphate-buffered saline, Ringer's solution and dextrose solution.
It may further include other materials desirable from a commercial
and user standpoint, including buffers, diluents, filters, needles,
syringes and package inserts with instructions for use.
[0288] The invention now will be exemplified for the benefit of the
artisan by the following non-limiting examples that depict some of
the embodiments by and in which the instant invention can be
practiced.
EXAMPLES
Example 1
Sequencing of the Fv Domain of Mouse Anti-Human IL-13 Monoclonal
Antibody Clone B-B13
[0289] The reagent used in the method below was mouse anti-IL-13
monoclonal antibody clone B-B13 purchased from Cell Sciences, Inc.
(Canton, Mass., USA). Cell Sciences is the US distributor of
Diaclone (Besancon, France), which manufactured the antibody
B-B13.
[0290] The amino acid sequence of anti-IL-13 monoclonal antibody
Clone B-B13 was determined by a combination of Edman N-terminal
sequencing and mass spectrometric analysis. The antibody was
subjected to the following different approaches in order to
generate polypeptide or peptide fragments, and these were
fractionated by different approaches in order to prepare samples
that were subsequently subjected to Edman N-terminal sequencing,
and Liquid Chromatography/Mass Spectrometry/Mass Spectrometry
(LC-MS/MS) analysis with associated protein sequence database
peptide matching
[0291] SDS-Page of the antibody, either untreated or treated with
pyrogluamino peptidase, to separate the heavy and light chains,
followed by blotting to polyvinylidene fluoride (PVDF) membrane and
Edman N-terminal sequencing of the bands.
[0292] Limited partial proteolysis with specific proteases of the
antibody followed by SDS-Page and blotting to PVDF membrane and
Edman N-terminal sequencing of the bands.
[0293] Limited partial chemical cleavage of the whole antibody, or
heavy and light chain SDS-Page gel bands, followed by SDS-Page and
blotting to PVDF membrane and Edman N-terminal sequencing of
bands.
[0294] Proteolysis of the whole antibody or heavy and light chain
SDS-Page gel bands with specific proteases and LC/MS/MS
analysis.
[0295] Proteolysis of heavy and light chain SDS-Page gel bands with
specific proteases followed by reverse phase high pressure
chromatography fractionation (rp-hplc), and subsequent Edman
N-terminal sequencing and LC/MS/MS analysis of fractions.
[0296] Limited proteolysis of the antibody with the protease
papain, fractionation of the Fd (VH-CH1 fragment of the antibody
heavy chain) gel band by SDS-Page, proteolysis with specific
proteases, reverse phase high performance liquid chromatograph
(rp-hplc), and subsequent Edman N-terminal sequencing and LC/MS/MS
analysis of fractions.
Example 2
Sequencing of the Fv Domain of Mouse Anti-Human IL-4 MONOCLONAL
ANTIBODY CLONE 8D4-8
[0297] The reagent mouse anti-IL-4 monoclonal antibody clone 8D4-8
was purchased from Biozol diagnostica Vertrieb GmbH (Eching,
Germany). Biozol is the German distributor of BioLegend (San Diego,
Calif., USA) which manufactured the antibody 8D4-8.
[0298] The amino acid sequence of a mouse monoclonal anti-IL-4
antibody (clone 8D4-8) was determined by a combination of Edman
sequencing and mass spectrometry (Pham et al., 2006, Anal. Biochem.
352: 77-86; Roberts et al., 2005, Anal. Chem. 67: 3613-25).
Briefly, the antibody was first separated into light and heavy
chains and then each chain was cleaved by sequence specific
proteases or chemically. Resulting peptides were separated by
reverse phase chromatography and analyzed by Matrix-assisted laser
desorption/ionization spectrometry (MALDI) and/or LC-MS/MS. Unique
peptides as well as the intact heavy and light chains were than
subjected to Edman sequencing for unambiguous determination of the
protein sequence.
Example 3
Humanization of the Fv Domain of Mouse Anti-Human IL-13 Monoclonal
Antibody Clone B-B13
[0299] The humanization protocol described hereinabove was used to
humanize the B-B 13 clone. Six humanized versions were suggested
which include mutations in the CDRs to address problematic residues
(deamidation site, solvent exposed methionine, acide labile
positions).
[0300] The VL & VH sequences of B-B13 were blasted against the
July 2007 version of the Protein Data Bank (PDB). The most similar
light and heavy chain amino acid sequences were retrieved. The
closest homologue for the variable light chain was found to be
1EGJ. The closest homologue for the heavy chain was found to be
1FNS. The structures 1EGJ & 1FNS were used to build up a
homology model of the variable domains which was subsequently
energy minimized using the standard procedure implemented in
Molecular Operating Environment (MOE). MOE is a comprehensive suite
of softwares for computer assisted drug design distributed by the
Chemical Computing group. A molecular dynamic (MD) calculation of a
3D homology model of B-B13 was subsequently performed for 1.7
nanoseconds in Generalized Born implicit solvent. The resulting
1,700 snapshots from the MD trajectory were then used to calculate,
for each B-B13 amino-acid, the distribution of its root mean square
deviations (rmsd) compared to a reference medoid position. A
statistical test, comparing the rmsd distribution of each
amino-acid to the global rmsd distribution, is finally used to
decide if the amino-acid is flexible enough, as seen during the MD,
to be considered as likely to interact with B-cell receptors and
responsible for activation of the immune response. The flexible
positions of the murine B-B13 variable region were compared to the
corresponding positions in human antibody sequences in the January
2007 version of the ImMunoGeneTics Database that has been
downloaded locally. Only those residues which display flexibility
greater than three times the mean and a few flanking residues that
preserve the 3D structures of these flexible residues were retained
for the search. The human antibody variable region with the most
identical flexible residues, with special considerations given to
positions that come within 5.0 .ANG. of a CDR, was chosen to
replace the murine the B-B13 antibody variable region flexible
residues. A number of mutations in the CDRs were also included in
the proposed versions to avoid problematic residues. The following
motifs of sequences were considered: Asp-Pro (acide labile bond),
Asn-X-Ser/Thr (glycosylation), Asn-Gly/Ser/Thr (deamidation site in
exposed area), Met (oxidation in exposed area). The resulting
humanized sequences were blasted for sequence similarity against
UniProtKB/Swiss-Prot database providing confidence that reasonable
assumption has been made. It was found that all sequences show high
degree of similarity to number of human antibodies. In addition
none of the sequences contains any known B- or T-cell epitope
listed in the Immune Epitope Database and Analysis Resource (IEDB
database).
[0301] Three versions for the heavy chain (H1, H2, H3) and three
versions were suggested for the light chain (L1, L2, L3). The three
versions of the light chain are derived from CAA83271.1 (Genebank
accession number CAA83271). The L1 version has 4 mutations. The L2
version includes an additional mutation to remove a DP site (Pro99)
in CDR3. L3 incorporates two additional mutations located in the
CDRs when compared with L2 which are two presumed deamidation sites
(N34Q, N96A). The H1, H2 and H3 versions of the heavy chain are
derived from CAC39364.1 (Genebank accession number CAC39364). This
template was not the top scoring template but it was the highest
scoring template which did not contain sequence exhibiting high
homology (>70%) with known immunogenic sequence. Version H1
contains 6 mutations and the H2 sequence incorporates two
additional mutations to address three deamidation sites (N60A,
N73T, and N83T). The sequential numbering of amino acidy reflects
their natural order within the protein (N-terminus to C-terminus).
H3 contains two additional mutations (Y100R & D106K) which were
thought to improve potency. Six combinations of VL and VH variants
were recommended for generation of humanized antibodies:
VL1.times.VH1, VL2.times.VH2, VL1.times.VH3, VL3.times.VH1,
VL3.times.VH2 and VL3.times.VH3. As shown in Table 1, the amino
acid changes were made in humanized B-B13 VL and VH variants using
the re-surfacing technology set forth in the detailed description
section of the instant application. The left column indicates the
original amino acids and their positions in the murine B-B 13
mAb.
TABLE-US-00001 TABLE 1 Light Chain (Sequential numbering) (VL1)
(VL2) (VL3) Asn1 Asp Asp Asp Asn34 Asn Asn Gln Pro44 Ala Ala Ala
Glu83 Gln Gln Gln Asp85 Glu Glu Glu Asn96 Asn Asn Ala Pro99 Pro Ser
Ser 4 mutations 5 mutations 7 mutations Heavy Chain (VH1) (VH2)
(VH3) Gln1 Glu Glu Glu Ser15 Gly Gly Gly Gln16 Gly Gly Gly Asn60
Asn Ala Ala Ser61 Asp Asp Asp Asn73 Asn Ser Ser Lys81 Glu Glu Glu
Asn83 Asn Thr Thr Gln86 Arg Arg Arg Tyr100 Tyr Tyr Arg Asp106 Asp
Asp Lys 6 mutations 9 mutations 11 mutations
Example 4
Humanization of the Fv Domain of Mouse Anti-Human IL-4 Monoclonal
Antibody Clone 8D4-8
[0302] The humanization (resurfacing) technology described
hereinabove was used to humanize the 8D4-8 clone. Two humanized
versions were prepared. One version includes one mutation in the
CDRs of the heavy chain which was thought to address a problematic
residue (exposed acide labile positions).
[0303] The VL & VH sequences of 8D4-8 were blasted against the
July 2007 version of the PDB. The most similar light and heavy
chain amino acid sequences were retrieved. The closest homologue
for the variable light chain is 1YDJ. The closest homologue for the
heavy chain was found to be 1IQW. The structures 1YDJ & 1IQW
were used to build up a homology model of the variable domains
which was subsequently energy minimized using the standard
procedure implemented in MOE. A molecular dynamic (MD) calculation
of a 3D homology model of 8D4-8 was subsequently performed for 1.7
nanoseconds in Generalized Born implicit solvent. The resulting
1,700 snapshots from the MD trajectory were then used to calculate,
for each 8D4 amino-acid, the distribution of its root mean square
deviations (rmsd) compared to a reference medoId position. A
statistical test, comparing the rmsd distribution of each
amino-acid to the global rmsd distribution, is finally used to
decide if the amino-acid is flexible enough, as seen during the MD,
to be considered as likely to interact with B-cell receptors and
responsible for activation of the immune response. The flexible
positions of the murine 8D4-8 variable region were compared to the
corresponding positions in human antibody sequences in the January
2007 version of the ImMunoGeneTics Database that has been
downloaded locally. Only those residues which display flexibility
greater than three times the mean and a few flanking residues that
preserve the 3D structures of these flexible residues were retained
for the search. The human antibody variable region with the most
identical flexible residues, with special considerations given to
positions that come within 5.0 .ANG. of a CDR, was chosen to
replace the murine the 8D4-8 antibody variable region flexible
residues. Eventually, some additional mutations were also made to
avoid problematic residues. The following motifs of sequences were
considered: Asp-Pro (acide labile bond), Asn-X-Ser/Thr
(glycosylation), Asn-Gly/Ser/Thr (deamidation site in exposed
area), Met (oxidation in exposed area). The only problematic
residue found was a DP site in the CDR2 of the heavy chain. The
resulting humanized sequences were blasted for sequence
[0304] Two versions for the heavy chain (H1, H2) and one version
for the light chain (L1) were suggested. The L1 version of the
light chain is derived from BAC01676.1 (Genebank accession number
BAC01676). The L1 version has 3 mutations. The H1 and H2 versions
of the heavy chain are derived from BAC02418.1 (Genebank accession
number BAC02418). Version H1 contains 9 mutations and the H2
version includes an additional mutation to remove a DP site (Pro53)
in CDR2. Two combinations, VL1.times.VH1 and VL1.times.VH2, were
prepared.
[0305] Table 2 shows the amino acid changes that were made in
humanized 8D4-8 VL and VH variants using the humanization
(re-surfacing) technology. The left column indicates the original
amino acids and their positions in the murine 8D4-8 mAb.
TABLE-US-00002 TABLE 2 Light Chain (Sequential numbering) (VL1)
Asn5 Thr Leu15 Val Ser39 Lys 3 mutations Heavy Chain (VH1) (VH2)
Gln10 Glu Glu Arg13 Lys Lys Thr16 Ala Ala Pro53 Pro Ala Lys65 Gln
Gln Asp66 Gly Gly Arg74 Glu Glu Ser76 Thr Thr Leu93 Val Val Thr118
Leu Leu 9 mutations 10 mutations
Example 5
Cloning and Generation of Chimeric Anti-IL-13 Clone B-B13
Monoclonal Antibody, a Chimeric Anti-IL-4 Clone 8D4-8 Monoclonal
Antibody and Humanized Variants
[0306] Amino acid sequences of the variable heavy and light chains
of the anti-IL-13 clone B-B13 and the anti-IL-4 clone 8D4-8 were
backtranslated into nucleotide sequence and generated respectively
using a modified protocol of the overlap extension PCR (OE-PCR)
described by Young L. and Dong Q. (Nucl. Acids Res. (2004), 32(7),
e59). PCR products were cloned into the pCR.RTM.4-TOPO using the
Invitrogen TOPO TA cloning kit (Cat #45-0641) and sequenced using
M13forward and M13 reverse primers. The variable domains were fused
together to the constant heavy (IGHG1, Genebank accession number
Q569F4) or light chain (IGKC) Genebank accession number Q502W4)
respectively, digested with NheI and HindIII and each ligated into
the NheI/HindIII sites of the episomal expression vector pXL, an
analogon of the pTT vector described by Durocher et al. (2002),
Nucl. Acids Res. 30(2), E9, creating the plasmids for the mammalian
expression of the chimeric B-B13-heavy and light chains and the
chimeric 8D4-8 heavy and light chains.
[0307] The expression clones encoding the humanized variants of the
anti-IL-13 clone B-B13 and the anti-IL-4 clone 8D4-8 were also
synthetically generated by overlap extension PCR (OE-PCR), based on
the proposed amino acid exchanges of the original sequences.
[0308] The expression plasmids encoding the heavy and light chain
of the antibody were propagated in E. coli DH5a. Plasmids used for
transfection were prepared from E. coli using the Qiagen EndoFree
Plasmid Mega Kit.
[0309] For transfection HEK293FreeStyle cells (Invitrogen) were
seeded at 3.times.10.sup.5 cells/mL in 100 mL volume of serum-free
FreeStyle medium (Invitrogen) in a 500 mL shaker flask. Cells were
cultured in a 37.degree. C. incubator with a humidified atmosphere
of 8% CO.sub.2, on an orbital shaker platform rotating at 110
rpm.
[0310] Three days post-seeding viable and total cell were
determined with a CASY electronic cell counter (Scharfe System
GmbH). Cells with viability greater than 90% were used for
transfection at a cell density of 1-1.5.times.10.sup.6 cells/mL.
100 mL cells were transfected in a 500 mL shaker flask with a mix
of heavy and light chain expression plasmids (5.times.10.sup.-7
.mu.gDNA/cell) using FugeneHD (Roche) at a DNA:FugeneHD ratio of
2:7, at conditions described by the manufacturer. Transfected cells
were cultured for 7 days in a 37.degree. C. incubator (8% CO.sub.2)
on an orbital shaker platform rotating at 110 rpm.
[0311] A Nunc F96-MaxiSorp-Immuno plate was coated with goat
anti-Human IgG (Fc specific) [NatuTcc A80-104A]. The antibody was
diluted to 10 ug/ml in carbonate coating buffer (50 mM sodium
carbonate pH 9.6) and dispensed at 50 uL per well. The plate was
sealed with adhesive tape, and stored overnight at 4 C. The plate
was washed 3 times with Wash buffer (PBS pH 7.4 0.1% Tween20). 150
uL of blocking solution (1% BSA/PBS) was dispensed into each well
to cover the plate. After 1 hour at RT the plate was washed 3 times
with Wash buffer. 100 uL of sample or standards (in a range from
1500 ng/ml to 120 ng/ml) were added and let sit for 1 hour at RT.
The plate was washed 3 times with Wash buffer. 100 uL of goat
anti-Human IgG-FC--HRP conjugate [Natu Tec A80-104P-60] diluted
1:10.000 were added using incubation solution (0.1% BSA, PBS pH
7.4, 0.05% Tween20). After 1 hour incubation at RT, the plate was
washed 3 times with Wash buffer. 100 uL of ABTS substrate (10 mg
ABTS tablet (Pierce 34026) in ml of 0.1 M Na.sub.2HPO.sub.4, 0.05 M
citric acid solution, pH 5.0. Addition of 10 uL of 30%
H.sub.2O.sub.2/10 ml Substrate buffer prior to use) were dispensed
to each well, allow the color to develop. After the color has
developed (approximately 10 to 15 minutes), 50 uL of 1% SDS
Solution were added to stop the reaction. The plate was read at
A405.
[0312] Proteins were purified by affinity chromatography on Protein
A (HiTrap.TM. Protein A HP Columns, GE Life Sciences). After
elution from the column with 100 mM acetate buffer with 100 mM NaCl
pH 3.5, the monoclonal antibodies were formulated in PBS and 0.22
.mu.m filtered. Protein concentration was determined by measurement
of absorbance at 280 nm. Each batch was analyzed using a Protein
200 Plus LabChip kit on the Agilent 2100 bioanalyzer under reducing
and non-reducing conditions to determine the purity and the
molecular weight of each subunit and of the monomer.
Example 6
Characterization of Humanized Anti-IL-13 Clone B-B13 Variants and
Humanized Anti-IL-4 Clone 8D4-8 Variants
[0313] The reagents recombinant human IL-13 and IL-4 were purchased
from Chemicon (USA). The Biacore kinetic analysis was performed as
follows.
[0314] Surface plasmon resonance technology on a Biacore 3000 (GE
Healthcare) was used for detailed kinetic characterisation of
purified anibodics. A capture assay using a species specific
antibody (e.g. human-Fc specific MAB 1302, Chemicon) for capture
and orientation of the investigated antibodies was used. The
capture antibody was immobilied via primary amine groups (10000 RU)
on a research grade CM5 chip (GE Life Sciences) using standard
procedures. The analysed antibody was captured with an adjusted RU
value that would result in maximal analyte bindung of 30 RU at a
flow rate of 10 .mu.l/min. Binding kinetics were measured against
recombinant human IL-4 and IL-13 over a concentration range between
0 to 50 nM in HBS EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA,
0.005% Surfactant P20) at a flow rate of 30 .mu.l/min. Chip
surfaces were regenerated with 10 mM glycine pH2.5. Kinetic
parameters were analysed and calculated in the BIAevaluation
program package using a flow cell without captured antibody as
reference. To investigate additive binding of both antigens, a
wizard-driven co-inject method has been applied in which one
antigen was injected immediately followed by the antigen mix of
IL-13/IL-4.
[0315] The antibodies of the present invention were measured for
biological activity by measuring the inhibition of IL-4 or IL-13
mediated cell proliferation in TF-1 cells. Briefly, Applicants used
IL-4 or IL-13 to stimulate the growth of TF-1 cells. TF-1 is a cell
line that is dependent on cytokines for growth and responds to many
cytokines including TL-4 and IL-13. The induced growth (compared to
baseline conditions in the absence of cytokine) represents the
biological activity of IL-4 or IL-13. Anti-IL-4, anti-IL-13 and
bispecific anti-IL-4/IL-13 antibodies were shown to block IL-4 or
IL-13 induced TF-1 cell growth. In addition, the bispecific
anti-IL-4/IL-13 antibodies were shown to block TF-1 cell
proliferation induced by combined IL-4 and IL-13 stimulation. The
blocking effect was measured in a dose-dependent manner to generate
IC50 (antibody concentration at 50% inhibition) as the antibody
neutralization potency against its target, i.e., IL-4 or IL-13.
Details of the methods employed are described in more detail
below.
[0316] TF-1 cells (ATCC, CRL-2003) were maintained in complete
medium (DMEM with high glucose, 25 mM Hepes buffer and glutamine,
10% FBS, 1.times.P/S, 1 mM sodium pyruvate) containing freshly
added hGM-CSF at final concentration of 4 ng/ml. 24 hrs before
IL-13 (15 ng/ml) or IL-4 (1 ng/ml) treatment. Cells were seeded in
96-well plates at 0.05.times.10.sup.6/ml in complete medium without
hGM-CSF. Serial dilutions of antibody with the corresponding
cytokine were pre-incubated for 30 minutes at 37.degree. C. before
adding to cells. Cells were cultured for 72 hours (37.degree. C.,
5% CO2). MTS/PMS solution of cellTiter 96 Aqueous was added. The
cells were then incubated for 3 hours. After that period,
absorbance at 490 nm using a plate reader was recorded. 1050 values
were calculated using Speed software.
[0317] The binding kinetics and neutralization activity of
humanized B-B13 variants are shown in Table 3. (nt, not
tested).
TABLE-US-00003 TABLE 3 on-rate off-rate KD IC50 antibody (M.sup.-1
.times. S.sup.-1) (S.sup.-1) (M) (M) Murine B-B13 8.64E+05 3.73E-04
5.63E-10 Nt chB-B13 WT 1.76E+06 4.61E-04 2.61E-10 7.4E-9 huB-B13
VL1 .times. VH1 1.74E+06 6.91E-04 3.96E-10 1.57E-8 huB-B13 VL1
.times. VH3 1.93E+06 3.95E-04 2.05E-10 Nt huB-B13 VL2 .times. VH2
1.13E+06 1.77E-04 1.57E-10 Nt huB-B13 VL3 .times. VH1 1.93E+06
3.33E-04 1.72E-10 5.2E-9 huB-B13 VL3 .times. VH2 2.55E+06 1.12E-04
4.39E-11 3.2E-9 huB-B13 VL3 .times. VH3 2.14E+06 4.05E-04 1.89E-10
Nt
[0318] One humanized B-B13 variant, huB-B13 VL3.times.VH2, has
significantly higher affinity compared with the original murine
B-B13 (13 fold) and chimeric B-B13 (6 fold). The improved affinity
may lead to increased potency and efficacy when these humanized
anti-IL-13 antibodies are used to treat asthma patients. In
addition, the humanized antibodies may have reduced immunogenicity
compared with the murine antibody or the chimeric antibody when
used in man.
[0319] The binding kinetics and neutralization activity of
humanized 8D4-8 variants are shown in Table 4.
TABLE-US-00004 TABLE 4 on-rate off-rate KD IC50 antibody (M.sup.-1
.times. S.sup.-1) (S.sup.-1) (M) (M) murine 8D4-8 5.57E+06 2.17E-04
3.77E-11 9.7E-11 ch8D4-8 WT 2.49E+07 1.95E-04 7.83E-12 8.4E-11
Hu8D4-8 VL1 .times. 4.72E+07 1.55E-04 3.29E-12 4.1E-11 VH1 Hu8D4-8
VL1 .times. 2.57E+07 3.48E-04 1.39E-11 1.35E-10 VH2
[0320] One humanized 8D4-8 variant, hu8D4-8 VL1.times.VH1 has
significantly higher affinity compared with the original murine
8D4-8 (11 fold) and chimeric 8D4-8 (2 fold). The improved affinity
may lead to increased potency and efficacy when this humanized
anti-IL-4 antibody is used to treat asthma patients. In addition,
the humanized antibody may have reduced immunogenicity compared
with the murine antibody or the chimeric antibody when used in
man.
Example 7
Cloning and Generation of Humanized Anti-IL-4/IL-13 Bispecific
Antibodies
[0321] The format used for the expression of bispecific antibodies
(BsAb) is an IgG variant of the dual domain double head format
described in U.S. Pat. No. 5,989,830. In this format an IgG
molecule is elongated at its N-terminus on the corresponding heavy
and light chains, by an additional variable domain of a second
antibody. Thus, the resulting IgG molecule is a heterotetramer
composed of two heavy and two light chains. The heavy chains
consist of two variables heavy domains (VH1-VH2) deriving from two
different antibodies joined together by a linker composed of ten
amino acids (G4S).sub.2 and fused to the IgG4 constant domain. The
light chains consist of two variables light domains (VL1-VL2)
deriving from two different antibodies joined together by a linker
composed of ten amino acids (G4S).sub.2 and fused to the constant
kappa region.
[0322] Sequences for the variable heavy and light domains of the
8D4-8 variants were generated by PCR introducing a BamHI
restriction site (GGA TCC) at their respective 5'-ends encoding a
part of the (G4S).sub.2-(GGA TCC)-8D4-8. The 3' sequence of the VH
of the 8D4-8 humanized variants ended with an ApaI restriction site
(encoding the first amino acids of the CH1 domain) for a later
fusion to the IGHG4 sequence (Q569F4, with deletion of the terminal
Lys and a double mutation S241P and L248E). The 3'-end of the
VL8D4-8 ended with a BsiWI restriction site encoding the first two
amino acids of the constant kappa chain for a later fusion to IGKC
(Gene Bank Accession Number Q502W4).
[0323] Sequences for the variable heavy and light domains of the
B-B13 variants were generated by PCR introducing a BamHI
restriction site at their respective 3'-ends encoding a part of the
(G4S).sub.2-(B-B13)-(GGA GGC GGA GGG TCC GGA GGC GGA GGA TCC (SEQ
ID NO: 7)). Both sequences for the VH and VL of the B-B13 variants
were generated with a NheI restriction site at their respective
5'-ends, followed by an ATG start codon and a leader peptide
encoding sequence.
[0324] The VH of B-B13 and 8D4-8 were fused together through their
BamHI sites within the (G4S).sub.2 linker. The VL of B-B13 and
8D4-8 were fused to each other through their BamHI sites within the
(G4S).sub.2 linker. Hence the tandems of heavy and the light chains
generated had the following composition.
[0325] Bispecific antibody heavy chain: NheI-Leader
peptide-VH-B-B13-(G4S).sub.2-VH 8D4-8-ApaI.
[0326] Bispecific antibody light chain: NheI-Leader
peptide-VL-B-B13-(G4S).sub.2-VL 8D4-8-BsiWI.
[0327] All intermediate PCR fragments were cloned into the pCR.RTM.
4-TOPO using the Invitrogen TOPO TA cloning kit (Cat #: 45-0641)
and sequenced using M13 forward and M13 reverse primers.
[0328] After sequence validation the heavy chain tandems were fused
through their ApaI site to the IGHG4 sequence and the variable
light chain tandems were fused through their BsiWI site to IGKC.
The created dual domain heavy chain and light chain were digested
with NheI and HindIII and each ligated into the NheI/HindIII sites
of the episomal expression vector pXL, creating the plasmids for
mammalian expression of the TBTI-heavy and light chains
respectively.
[0329] Four humanized bispecific anti-IL-4/anti-IL-13 constructs
were generated based on the following combinations of humanized VH
and VL versions of B-B13 and 8D4-8 as shown in Table 5.
TABLE-US-00005 TABLE 5 Bispecific anti-IL-4/ anti-IL-13 Ab
Anti-IL-13 Fv Anti-IL-4 Fv huTBTI3_1_1 B-B13 VL3 .times. VH2 8D4-8
VL1 .times. VH2 huTBTI3_2_1 B-B13 VL3 .times. VH2 8D4-8 VL1 .times.
VH1 huTBTI3_1_2 B-B13 VL2 .times. VH2 8D4-8 VL1 .times. VH2
huTBTI3_2_2 B-B13 VL2 .times. VH2 8D4-8 VL1 .times. VH1
Example 8
Characterization of the Humanized Bispecific Antibodies
[0330] Binding and neutralization activity assays were performed as
described in the previous Examples.
[0331] Table 6 depicts the binding kinetics of four humanized
anti-IL-4/IL-13 antibody variants. All four constructs of
bispecific antibodies binds to IL-4 and IL-13 with high
affinities.
TABLE-US-00006 TABLE 6 IL-13 affinity IL-4 affinity on-rate
off-rate KD On-rate off-rate KD BsAB (M.sup.-1 .times. S.sup.-1)
(S.sup.-1) (M) (M.sup.-1 .times. S.sup.-1) (S.sup.-1) (M)
huTBTI3-1_1 2.27E+06 1.70E-04 7.47E-11 2.55E+06 3.78E-04 1.48E-10
huTBTI3-2_1 2.17E+06 1.69E-04 7.80E-11 4.00E+06 1.39E-04 3.47E-11
huTBTI3-1_2 8.50E+05 1.64E-04 1.93E-10 2.23E+06 3.08E-04 1.38E-10
huTBTI3-2_2 8.20E+05 2.12E-04 2.59E-10 3.96E+06 1.32E-04
3.32E-11
[0332] The neutralization activity of humanized anti-IL-4/IL-13
bispecific antibody variants is summarized in Table 7. Both
huTBTI3-1.sub.--1 and huTBTI3-2.sub.--1 completely neutralized
IL-13 or IL-4 induced TF-1 cell proliferation with IC50 shown
below.
TABLE-US-00007 TABLE 7 Antibody IC50 (nM) in IL-13 assay IC50 (nM)
in IL-4 assay huTBTI3-1_1 3.7 1.7 huTBTI3-2_1 4.1 0.32
[0333] It is well known that a mutant IL-13 allele is linked in
high frequency with asthma (Heinzmann A. et al., 2000, Hum Mol
Genet. 9, 4, p549-559). Therefore, the binding kinetics of the
bispecific antibodies to the mutant IL-13 protein (human IL-13
R112Q variant, PeproTech, Rocky Hill, N.J., USA) was studied. The
results indicated that the huTBTI3-1.sub.--1 and huTBTI3-2.sub.--1
bound to the IL-13 variant similarly to the wild type IL-13.
[0334] Table 8 shows the binding kinetics of humanized
anti-IL-4/IL-13 molecules to mutant IL-13 protein.
TABLE-US-00008 TABLE 8 IL13 variant affinity on-rate off-rate KD
BsAB (M.sup.-1 .times. S.sup.-1) (S.sup.-1) (M) huTBTI3-1_1
9.74E+05 1.18E-04 1.21E-10 huTBTI3-2_1 9.48E+05 2.00E-04 2.11E-10
Sequence CWU 1
1
221111PRTArtificial Sequencehumanized mouse/ mouse VL3 region 1Asp
Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10
15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30 Gly Gln Ser Tyr Met His Trp Tyr Gln Gln Lys Ala Gly Gln
Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser
Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp
Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Gln Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Asn Ala 85 90 95 Glu Asp Ser Arg Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 2118PRTArtificial
Sequencehumanized mouse/mouse VH2 region 2Glu Val Gln Leu Lys Glu
Ser Gly Pro Gly Leu Val Ala Pro Gly Gly 1 5 10 15 Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Ser 20 25 30 Ser Ile
Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45
Gly Met Ile Trp Gly Asp Gly Arg Ile Asp Tyr Ala Asp Ala Leu Lys 50
55 60 Ser Arg Leu Ser Ile Ser Lys Asp Ser Ser Lys Ser Gln Val Phe
Leu 65 70 75 80 Glu Met Thr Ser Leu Arg Thr Asp Asp Thr Ala Thr Tyr
Tyr Cys Ala 85 90 95 Arg Asp Gly Tyr Phe Pro Tyr Ala Met Asp Phe
Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115
3108PRTArtificial Sequencehumanized mouse/mouse VL1 region 3Asp Ile
Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15
Asp Thr Ile Thr Leu Thr Cys His Ala Ser Gln Asn Ile Asp Val Trp 20
25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu
Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln
Gln Ala His Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 4124PRTArtificial Sequencehumanized
mouse/mouse VH1 region 4Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Ile Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro
Ser Asp Gly Glu Thr Arg Leu Asn Gln Arg Phe 50 55 60 Gln Gly Arg
Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
100 105 110 Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ala 115 120
5124PRTArtificial Sequencehumanized mouse/mouse VH2 region 5Gln Val
Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20
25 30 Trp Ile His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Met Ile Asp Ala Ser Asp Gly Glu Thr Arg Leu Asn
Gln Arg Phe 50 55 60 Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser
Thr Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Arg Ser Pro Thr Ser Glu
Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Leu Lys Glu Tyr Gly
Asn Tyr Asp Ser Phe Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr
Leu Val Thr Val Ser Ser Ala 115 120 610PRTArtificial Sequencelinker
6Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 730DNAArtificial
Sequenceprimer 7ggaggcggag ggtccggagg cggaggatcc 30815PRTArtificial
SequencehB-B13 VL3 CDR 8Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Gln
Ser Tyr Met His 1 5 10 15 97PRTArtificial SequencehB-B13 VL3 CDR
9Leu Ala Ser Asn Leu Glu Ser 1 5 109PRTArtificial SequencehB-B13
VL3 CDR 10Gln Gln Asn Ala Glu Asp Ser Arg Thr 1 5 1110PRTArtificial
SequencehB-B13 VH2 CDR 11Gly Phe Ser Leu Thr Asp Ser Ser Ile Asn 1
5 10 125PRTArtificial SequencehB-B13 VH2 CDR 12Asp Gly Arg Ile Asp
1 5 1310PRTArtificial SequencehB-B13 VH2 CDR 13Asp Gly Tyr Phe Pro
Tyr Ala Met Asp Phe 1 5 10 1411PRTArtificial Sequenceh8D4-8 VL1 CDR
14His Ala Ser Gln Asn Ile Asp Val Trp Leu Ser 1 5 10
158PRTArtificial Sequenceh8D4-8 VL1 CDR 15Lys Ala Ser Asn Leu His
Thr Gly 1 5 169PRTArtificial Sequenceh8D4-8 VL1 CDR 16Gln Gln Ala
His Ser Tyr Pro Phe Thr 1 5 1710PRTArtificial Sequenceh8D4-8 VH1
CDR 17Gly Tyr Ser Phe Thr Ser Tyr Trp Ile His 1 5 10
189PRTArtificial Sequenceh8D4-8 VH1 CDR 18Ile Asp Pro Ser Asp Gly
Glu Thr Arg 1 5 1914PRTArtificial Sequenceh8D4-8 VH1 CDR 19Leu Lys
Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val 1 5 10
2010PRTArtificial Sequenceh8D4-8 VH2 CDR 20Gly Tyr Ser Phe Thr Ser
Tyr Trp Ile His 1 5 10 219PRTArtificial Sequenceh8D4-8 VH2 CDR
21Ile Asp Ala Ser Asp Gly Glu Thr Arg 1 5 2214PRTArtificial
Sequenceh8D4-8 VH2 CDR 22Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe
Tyr Phe Asp Val 1 5 10
* * * * *